The CC chemokine receptor 5 (CCR5) is a 7 membrane-spanning domain, G protein-coupled receptor (GPCR), which regulates chemotaxis and effector functions of T lymphocytes, macrophages, and dendritic cells (reviewed in Oppermann 1 ).

Upon activation by its cognate chemokines, CCL5 (formerly regulated upon activation, normal T cell expressed and secreted [RANTES]), CCL3, and CCL4 (formerly macrophage inflammatory proteins 1␣ and 1␤, respectively), 2 CCR5 leads to cellular signaling through G pro- teins 3 as well as G protein-independent pathways.

4 In addition to its physiologic functions, CCR5 is also the main coreceptor of HIV, in association with CD4, 5 a cell-surface glycoprotein that partici- pates in molecular complexes involved in both T-cell development and antigen recognition by T cells.

CD4 is a single-membrane spanning domain receptor interacting with class II major histocom- patibility complex (MHC) molecules 6 and with its cognate ligand interleukin-16 (IL-16).

So far, no specific physiologic function has been attributed to the CCR5-CD4 complex.


In the context of immune cell chemotaxis in vivo, recent studies have shown that the sustained (hours) lymphocyte motility within lymph nodes is driven by the lymphoid chemokine-rich environment and mediated by the activation of chemokine receptors.

7 Paradoxically, studies in reconstituted models have shown that stimulated chemokine receptors, including CCR5, are rapidly desensitized (seconds to min- utes) via phosphorylation and ␤-arrestin translocation before being internalized and eventually degraded.

1,8-10 In fact, an adaptive mecha- nism to escape inactivation and maintain prolonged reactivity in vivo upon sustained stimulation has been reported for other GPCRs, based on the retention of important stocks of functional receptors within intracel- lular compartments (reviewed in Achour et al 11 ).

Initially described for protease-activated receptors (such as the thrombin receptor 12 ), this phenomenon was subsequently reported for dopamine D1 receptors in tubular renal cells 13 and for ␦-opioid receptors in neurons.

14 The list of GPCRs displaying a predominant intracellular localization is rapidly growing.

11 A common feature for most of these prevalent intracellular GPCRs is their capacity of interacting with a vast array of membrane- associated or cytoplasmic proteins that facilitate their translocation to the cell surface.

15 Often, these proteins possess their own functions, in addition to aiding GPCR expression at the cell surface.

11 Among them, type I single-transmembrane domain proteins with a large N-terminal extracellular domain and a short C terminus are particularly frequent.

This group includes receptor activity-modifying proteins (RAMPs), which facilitate cell-surface expression of a class B GPCRs 16 ; the receptor transporting proteins (RTPs) and receptor expression- enhancing protein 1 (REEP1), which permit functional cell-surface targeting of odorant 17 and taste receptors 18 ; the M10 MHC class I molecules, which are necessary for the correct trafficking of pheromone receptors to the plasma membrane 19 ; and the MC2R accessory protein (MRAP), which interacts with the melanocortin 2 receptor, regulating its trafficking from the endoplasmic reticulum (ER) to the cell surface.

20 In the current study we investigated the subcellular distribution of endogenous CCR5 receptor in human T lymphocytes and in a monocytic cell line and found that this receptor is predominantly intracellular.

We also report that CCR5 and CD4 can associate within intracellular compartments and that this association en- hances the export of CCR5 to the cell surface.


THP-1 and HeLa-P4R5 cells were kind gifts from Philip Strange (School of Pharmacy, University of Reading, Reading, United Kingdom) and Florence Submitted February 26, 2008; accepted December 1, 2008.

Prepublished online as Blood First Edition paper, December 8, 2008; DOI 10.1182/blood-2008-02- 141275.


The online version of this article contains a data supplement.


The publication costs of this article were defrayed in part by page charge payment.

Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734.

Margottin (Institut Cochin, Paris, France), respectively.

Cell-culture media were from Invitrogen (Carlsbad, CA) and chemicals from Sigma-Aldrich (St Louis, MO) unless otherwise specified.

Chinese hamster ovary K1 (CHO-K1) cells were cultured in F12 medium, HeLa-P4R5 in Dulbecco modified minimal essential medium (DMEM) and THP-1 cells in RPMI 1640, in an atmosphere of 5% CO 2 .

Media were supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 g/mL streptomy- cin.

Transfections of CHO-K1 and HeLa-P4R5 cells were performed using GeneJuice-Transfection Reagent (Novagen, Gibbstown, NJ).


Constructs for bioluminescence resonance energy transfer (BRET) experi- ments were described previously.

21 Briefly, CCR5 and CXCR4 coding regions were amplified from their respective cDNAs using appropriate sense and antisense primers, containing unique HindIII or BamH1 sites, respectively.

The fragments were then subcloned in frame between the HindIII and BamH1 sites of a plasmid for YFP (Clontech/BD Biosciences, Mountain View, CA) or for a humanized form of Renilla luciferase (Biosignal; PerkinElmer Life Sciences, Waltham, MA).


The plasmid for CCR5⌬Cter-YFP was obtained by the amplification of a shorter fragment (corresponding to met1-gly301 CCR5) using a more proximal antisense primer.

For CD4-Luc and CD4-YFP constructs, the stop codon of the CD4 sequence was suppressed and replaced by a restriction site using site- directed mutagenesis (QuickChange site-directed mutagenesis kit; Strat- agene, La Jolla, CA).

The coding regions of all constructs were entirely sequenced.


The GABABR1-YFP and OBR-YFP constructs were kind gifts from Michel Bouvier (Institut de Recherche en Immunologie et Cancérolo- gie, Université de Montréal, Montreal, QC) and Ralf Jockers (Institut Cochin, Paris, France), respectively.

The constructs for GFP-tagged wild- type VPU and phosphorylation mutant VPU 2/6 22 were kind gifts from Florence Margottin.

The plasmid for ER-DsRed was purchased from Clontech/BD Biosciences.


Mouse monoclonal antibodies were as follows: 2D7 anti-human CCR5 (555991; BD Pharmingen, San Diego, CA) directed against the second extracellular loop of CCR5, OKT4 anti-human CD4 (for fluorescence- activated cell sorting [FACS] and immunofluorescence, 140048; eBio- science, San Diego, CA), 12G5 anti-human CXCR4 (ab 21 555; Abcam, Cambridge, MA), 1F6 anti-human CD4 (for immunoblot experiments, NCL-L-CD4-1F6; Novocastra, Newcastle, United Kingdom), anti-GFP (11-814-460-001; Roche Applied Science, Indianapolis, IN).

Rat monoclo- nal antibodies were as follows: 1/85a Alexa Fluor 647-conjugated anti- human CCR5 (313712; Biolegend, San Diego, CA), which recognizes the N-terminal extremity of CCR5, 23 Alexa Fluor 647-conjugated rat IgG2a, k isotype control (400526; Biolegend), 3F10 anti-hemagglutinin (anti-HA) (1-867-423; Roche).

Rabbit polyclonal antibodies were as follows: anti- giantin (ab24586; Abcam) anti-TGN46 (ab16052; Abcam), anti-GPR78/ BiP (ab21685; Abcam), anti-phospho-p44/42 (Thr202/Tyr204) MAP kinase (9101; Cell Signaling, Danvers, MA), anti ERK 1-2 (06-182; Cell Signaling), Cy3-conjugated anti-goat immunoglobulin (305-167-003; Jack- son Immunoresearch, West Grove, PA), peroxidase-conjugated anti-mouse IgG (315-035-003; Jackson Immunoresearch), peroxidase-conjugated anti- goat IgG (28 169; Anaspec, San Jose, CA).

Goat polyclonal antibodies were as follows: anti-calnexin (C-20; sc-6465; Santa Cruz Biotechnology, Santa Cruz, CA), anti-actin (I-19; sc-1616; Santa Cruz Biotechnology), peroxidase- conjugated anti-rabbit IgG (111-035-003; Jackson Immunoresearch), Cy5- conjugated anti-mouse IgG (610-110-121; Rockland, Gilbertsville, PA).

Donkey polyclonal antibodies were as follow: Cy3-conjugated anti-rabbit IgG (711-167-003; Jackson Immunoresearch), Cy3-conjugated anti-rat IgG (712-166-153; Jackson Immunoresearch), Alexa-Fluor R 488-conjugated anti- mouse IgG (HϩL; A-21202; Molecular Probes/Invitrogen, Eugene, OR).


Human T cells were isolated from the peripheral blood of healthy volun- teers by Ficoll density gradient centrifugation, followed by negative depletion on magnetic beads (Human T lymphocyte Enrichment set DM; Becton Dickinson).

T cells were activated by incubation for 48 hours with anti-CD3 and anti-CD28 antibody-coated beads (Dynabeads Human CD3/ CD28 T-Cell Expander Kit; Invitrogen), at a bead:cell ratio of 1:3, in RPMI 1640 medium supplemented with pooled human AB serum and antibiotics.


The proportion of surface CCR5, and CD4 on resting or activated T lymphocytes, THP-1 cells, and CHO-K1 cells expressing exogenous receptors was determined by analyzing in parallel, intact and permeabilized cells.

For total staining, 10 6 cells were fixed in 1% paraformaldehyde for 20 minutes, washed with phosphate-buffered saline (PBS), and permeabilized for 1 hour at 4°C in PBS, containing 2% FBS, and 0.2% Triton X-100, as described.

24 Cells were then incubated on ice for 1 hour in 100 L PBS, 2% FBS with the 1/85a Alexa Fluor 647-conjugated anti-human CCR5 antibody (1:50) or the OKT4 anti-human CD4 (1:100) and control isotypes at the same dilutions.

For CD4, this step was followed by 1 hour incubation with Cy5-conjugated anti-mouse IgG (1:100).

For extracellular staining, cells were incubated on ice for 1 hour with PBS, 2% FBS without Triton X-100 and subsequently incubated with the appropriate antibodies, as above.

After washing with PBS, cells were fixed in 2% paraformaldehyde and processed on a Cytomycs FC500 FACS analyzer (Beckman Coulter, Fullerton, CA).


To study the impact of CD4 coexpression on CCR5 targeting at the cell surface, CHO-K1 cells were cotransfected with plasmids coding for CCR5-Luc (at fixed concentration) and CD4-YFP (at increasing concentra- tions).

Thirty-six hours after transfection, cells were incubated on ice for 1 hour with 0.5 g/mL of the 2D7 anti-human CCR5 antibody, followed by incubation with Cy5-conjugated anti-mouse IgG (1:100).


To determine the effect of CD4 coexpression on CXCR4 targeting at the cell surface, CHO-K1 were cotransfected with plasmids coding for CXCR4-Luc (at fixed concentration) and CD4-YFP (at increasing concen- trations).

FACS experiments were conducted, as described above for CCR5, by incubating cells for 1 hour with 0.5 g/mL 12G5 anti-human CXCR4 antibody, followed by incubation with Cy5-conjugated anti-mouse IgG (1:100).


The level of endogenous CCR5 at the cell surface of THP-1 cells after CD4 silencing was measured with the 1/85a Alexa Fluor 647-conjugated anti-CCR5 antibody as described above.

The proportion of surface CCR5 was evaluated by analyzing both intact and permeabilized cells.


For each analysis, 10 000 cells were sorted and the data analyzed using the Cytomics RXP software.


CHO-K1 cells were seeded on glass cover slips in 6-well plates (2 ϫ 10 5 per well), transfected with the indicated plasmids, and processed for immunofluorescence 48 h after transfection.

Subsequently cells were fixed in 4% paraformaldehyde for 20 minutes at 4°C or in methanol for 5 minutes at Ϫ20°C, according to the antibody used.

After quenching with 50 mM NH 4 Cl in PBS for 15 minutes, cells were washed with PBS and then incubated with PBS-bovine serum albumin (BSA) 1% with or without 0.1% saponin and labeled with the indicated antibodies.


The subcellular localization of endogenous CCR5 and CD4 in T lympho- cytes and THP-1 cells was analyzed by immunofluorescence in cells maintained in suspension.

After fixation in 4% paraformaldehyde for 20 minutes at room temperature and quenching (as described above), cells were incubated with PBS-BSA1% or PBS-BSA1% containing 0.05% of Tween 20, for permeabilization.

25 Cells were then incubated for 1 hour with the 2D7 anti-human CCR5 or the OKT4 anti-human CD4 antibodies.

After incubation with appropriate secondary antibodies, cells were washed with PBS and mounted on slides.


All images were obtained using a confocal Leica TCS SP2 AOBS microscope, 63ϫ/1.4 NA oil objective, and analyzed using Metamorph 7 software (Molecular Devices, Sunnyvale, CA).


Cells growing in 100-mm dishes were lysed in 50 mM HEPES (N-2- hydroxyethylpiperazine-NЈ-2-ethanesulfonic acid), pH 7.4, 250 mM NaCl, 2 mM EDTA (ethylenediaminetetraacetic acid), 0.5% NP40, 10% glycerol, 0.5% deoxycholic acid (lysis buffer), containing Complete protease inhibi- tors (Roche Applied Science), on ice for 1 hour.

After centrifugation at 13 000g for 20 minutes, the supernatant was collected and protein concentration determined (BCA protein assay; Pierce, Rockford, IL).

Supernatants were incubated with anti-GFP antibody (2 g) for 3 hours at 4°C and then incubated with protein G-Sepharose beads for 1 hour at 4°C.

After centrifugation, the immunobead-bound material was washed 3 times with lysis buffer.

The immunoprecipitates were resolved in 10% SDS- PAGE gels.

After transfer on nitrocellulose, blots were probed with the appropriate antibodies, all used at 1:1000 dilution.

Immunoblots were revealed by luminescence (ECL; GE Healthcare Life Sciences, Piscataway, NJ).


CHO-K1 grown in 6-well plates were transfected with the plasmid encoding CCR5-Luc with or without the plasmid for CD4-YFP.

After overnight starvation, cells were detached with 10 mM EDTA in PBS, centrifuged (1400g for 5 minutes) and resuspended in Hank-balanced salt solution.

Aliquots were subjected to luciferase activity measurements to determine average CCR5-Luc concentration.

After addition of 5 M coelenterazine h (Interchim, Montluçon, France), emitted light was mea- sured in a Mithras LB940 reader (Berthold Instruments, Oak Ridge, TN).

Other aliquots of cells were stimulated with MIP-1␤ (Peprotech, Rocky Hill, NJ) for various periods of time, chilled on ice, centrifuged (8000g for 1 minute at 4°C) and analyzed for ERK activation as described.

26 Briefly, after resuspension in ice-cold 50 mM Tris-HCl pH 6.8, containing 2.5 mM orthovanadate, 2.5 mM EDTA, and a cocktail of protease inhibitors, cells were lysed in 2ϫ Laemmli sample buffer.

Proteins were denatured (5 minutes at 95°C) separated by 10% SDS-PAGE and transferred on nitrocellulose membranes.

Membranes were probed with phospho-p44/42 MAPK or MAPK 1-2 antibodies and autoradiograms quantified using the ImageJ 1.38X software (National Institutes of Health, Bethesda, MD).


CHO-K1 cells (5 ϫ 10 5 per well of a 6-well plates) were transfected with 30 ng plasmid DNA coding for the BRET donor (CD4-Luc) and increasing amounts of BRET acceptor plasmids (CCR5-YFP, CCR5⌬Cter-YFP, GABABR1-YFP; 10-1000 ng per well) or YFP (0.5-100 ng per well).

Twenty-four hours after transfection, cells were washed in PBS, detached using 10 mM EDTA in PBS, centrifuged (1400g for 5 minutes), resus- pended in Hank-balanced salt solution and distributed in 96-well plates (PerkinElmer plates; 10 5 cells per well).

After addition of the luciferase substrate, coelenterazine h (5 M final concentration), luminescence and fluorescence were measured simultaneously (at 485 and 530 nm, respec- tively) in a Mithras LB940 plate reader

The BRET ratio was calculated as: ([emission at 530 nm/emission at 485 nm] - [background at 530 nm/ background at 485 nm]), where background corresponds to signals in cells expressing the Rluc fusion protein alone under the same experimental conditions.

For better readability, results were expressed in milli-BRET units (mBRET), 1 mBRET corresponding to the BRET ratio multiplied by 1000.

BRET ratios were plotted as a function of ([YFP-YFP 0 ]/YFP 0 )/(Rluc/ Rluc 0 ), where YFP is the fluorescence signal at 530 nm after excitation at 485 nm, and Rluc the signal at 485 nm after addition of coelenterazine h. YFP 0 and Rluc 0 correspond to the same values in cells expressing the Rluc fusion protein alone.

In some experiments, cells were incubated 14 hours before the BRET assay with brefeldin A (BFA) at 2 g/mL.


THP-1 cells were transfected using the Amaxa nucleofector system, in Amaxa solution V (program U010), with 0.5 M siRNA oligonucleotides (S1: 5Ј-GAACUGACCUGUACAGCUUUU-3Ј; S2: 5Ј-GAGCGGAUGU- CUCAGAUCAUU-3Ј; S3: 5Ј-UAACUAAAGGUCCAAUCCAAUU-3Ј) targeting human CD4 or an unrelated oligonucleotide (5Ј-UAGCGAC- UAAACACAUCAA-3Ј, all from Dharmacon, Lafayette, CO).

For each experiment, 2 rounds of transfection were performed: 2 ϫ 10 6 cells were transfected a first time, cultured for 72 hours, resuspended and transfected again using the same protocol.

Assays were conducted 7 days after the first transfection.

For RNA interference experiments on human T cells, 4 ϫ 10 6 freshly purified T lymphocytes were transfected using the Amaxa nucleofec- tor system, in Amaxa solution T (program U14) with 1 M S1 and S3 oligonucleotides, as indicated above.

After nucleofection, cells were cultured in RPMI 1640 medium supplemented with 10% FBS, antibiotics and interleukin-7 (5 ng/mL; R&D Systems, Minneapolis, MN) for 72 hours and then activated for 48 hours with anti-CD3 and anti-CD28 antibody- coated beads as described above.


Data are expressed as a mean value plus or minus SEM.

Data were analyzed using Student t test.

P values less than .05 were considered statistically significant.


To determine the cellular distribution of CCR5, both purified T lymphocytes from healthy donors and cells of the human THP-1 monocytic cell line, expressing endogenous CCR5 were analyzed by FACS (Figure 1A and Table S1, available on the Blood website; see the Supplemental Materials link at the top of the online article).

In freshly prepared unstimulated T lymphocytes, the proportion of surface CCR5 was very low, with approximately 95% of total receptors being intracellular, as measured by fluorescence analysis of intact or permeabilized cells with a CCR5-specific antibody.

As shown in previous studies, 27 surface CCR5 increased after stimula- tion with anti-CD3 and anti-CD28 antibodies (from 5% to Ϸ11%).

In THP-1 cells, CCR5 was also principally present within intracel- lular compartments, with approximately 90% of the receptor being found within internal compartments.

These observations were obtained with 2 different antibodies recognizing distinct epitopes (Table S1).

The same procedure was used to measure in parallel the distribution of the CD4 molecule.


As expected, in both T lympho- cytes and monocytes, CD4 was predominantly expressed at the cell surface (up to 92% in activated T lymphocytes).

To confirm these findings, immunofluorescence confocal microscopy studies were conducted on the same cells subjected (P) or not (NP) to membrane permeabilization (Figure 1B).

Whereas the CD4 staining was comparable in permeabilized and nonpermeabilized cells, the major proportion of CCR5 appeared to be intracellular in both T lymphocytes and THP-1 cells.

The Jurkat T-cell line, which does not express CCR5, was used as negative control for staining.

Colocalization studies were also conducted and demonstrated that endogenous CCR5 colocalized with both Golgi (Giantin) and ER (Bip) markers in THP-1 cells, although additional subcellular localizations cannot be excluded.


CCR5 distribution was finally analyzed in transiently transfected CHO cells, which will be used in this study as a reconstitution model.

As found for the endogenous receptor in T lymphocytes and THP-1 cells, exogenous CCR5 was predominantly intracellular in CHO cells under our experimental conditions, whether wild-type receptor, or a form tagged at the C terminus with a yellow variant of the green fluorescent protein (YFP), were expressed (Figure S1 and Table S1).

Confocal microscopic analyses of transfected CHO cells confirmed that both wild-type CCR5 and CCR5-YFP colocalized with a panel of both Golgi and ER markers (Figure S2).

From the data above, it appears that both endogenous and exogenous CCR5 are prone to intracellu- lar retention.


As indicated in the introduction, the cell surface expression of many predominantly intracellular GPCRs can be markedly en- hanced by the association with other proteins in the endoplasmic reticulum (ER) and/or the Golgi apparatus, and remain associated with them, once the receptors have reached the cell surface.

11,15 We thus investigated whether CD4, in addition to its intrinsic immuno- logic functions, might also interact with CCR5 in the ER and facilitate its export to the cell surface.

For this purpose, we made use of a natural mutant of CCR5, lacking its C-terminal intracellu- lar tail, 28 that is completely retained in the ER, 29 as supported by the complete absence of cell-surface detection by antibodies in immu- nofluorescence and FACS experiments (Figure 2A,B).

CHO cells were transiently transfected with a construct encoding CD4, in addition to plasmids for either wild-type or truncated CCR5, harboring a N-terminal HA epitope and a C-terminal YFP (HA- CCR5-YFP and HA-CCR5⌬Cter-YFP constructs, respectively).


In these cells, similar amounts of CD4 were coimmunoprecipitated with both the full-length and truncated forms of CCR5 (Figure 2D) and both HA-CCR5⌬Cter-YFP and HA-CCR5-YFP colocalized with CD4 within intracellular compartments (Figure 2C).

We confirmed that the CD4-CCR5 association in CHO cells, evidenced by coimmunoprecipitation studies, was not due to nonspecific hydrophobic interaction, by showing that mixing cell lysates expressing either molecule did not allow coimmunoprecipitation (Figure S3).


CD4-CCR5 interaction was further examined in live CHO cells using bioluminescence resonance energy transfer (BRET).

CD4 fused to Renilla luciferase (CD4-Luc) was expressed in the presence of increasing concentrations of BRET acceptors, consisting of either HA-CCR5-YFP or HA-CCR5⌬Cter-YFP.


In both cases, hyperbolic saturation curves were obtained, indicating specific interactions (Figure 2E).

BRET signals markedly depend on the relative distance of donor and acceptor, 30 explaining why maximal BRET values were different in wild-type CCR5 and the truncated mutant.

In contrast, the value of the YFP:Luc ratio, for which half-maximal BRET was obtained, was comparable, indicat- ing that these 2 forms of CCR5 display the same propensity to associate with CD4.


Free YFP and the GABABR1 receptor fused to YFP (GABABR1-YFP) were used as negative controls.

Although GABABR1 is constitutively retained in the ER 31 like HA- CCR5⌬Cter-YFP, only a weak nonsaturable interaction with CD4- Luc could be measured.

Finally, in the presence of free YFP, only a linear nonspecific BRET signal was obtained.


Bystander BRET signals may occur upon random collisions in cells expressing Downloaded from http://ashpublications.org/blood/article-pdf/113/9/1938/1485891/zh800909001938.pdf by guest on 16 May 2024 nonphysiologic high levels of BRET pairs.

30 To rule out this possibility, we verified that the levels of BRET donor and acceptor proteins at BRET saturating concentration were comparable or below those of endogenous CD4 and CCR5 in human monocytic THP-1 cells (Figure S4A).


To confirm that the measured BRET signals are a result of interactions occurring in the ER, cells were pretreated with BFA, a drug which induces Golgi apparatus disassembly and redistribution of Golgi-derived vesicles to the ER.

The Golgi apparatus was indeed dismantled after BFA treatment (Figure 3A), and FACS analysis could not detect almost any surface receptor in cells expressing HA-CCR5-YFP (Figure 3B).

Despite these effects, BRET values remained comparable whether or not cells were treated with BFA (Figure 3C).


Together, these data are consistent with the hypothesis that CCR5 and CD4 associate within intracellular compartments, likely in the ER, before they are exported to the cell surface.


We next examined whether CD4 might enhance the proportion of surface CCR5 in a reconstituted CHO-K1 cell model.

A fixed concentration of a plasmid coding for luciferase-tagged CCR5 (CCR5-Luc) was cotransfected with increasing amounts of a CD4-YFP construct.

Surface CCR5 values, measured by FACS with an anti-CCR5 antibody in cells coexpressing CD4-YFP, were plotted as a function of the CD4-YFP-associated fluorescence (Figure 4).


In cotransfected cells, surface CCR5 progressively increased with increasing levels of CD4-YFP to reach a maximum, corresponding to an augmentation of more than 100%, and then returned gradually to basal values for higher levels of CD4-YFP (Figure 4B).

The descending portion of the curve was not due to a decreased number of CD4 molecules that could reach the cell surface, since in the same cells, surface CD4 increased linearly with increasing amounts of total CD4-YFP (Figure S4B).

Through- out the experiments, we verified that the amount of CCR5-Luc remained constant by measuring luciferase-associated activity.


In control experiments, with either free YFP or with the single transmembrane domain leptin receptor fused to YFP (OBR-YFP), no enhancement of surface CCR5 was observed (Figure 4A,B).

As for BRET experiments, the effect of CD4 on surface CCR5 expression was achieved at physiologic concentrations of the 2 proteins (Figure S4A).


HIV can use a second chemokine Downloaded from http://ashpublications.org/blood/article-pdf/113/9/1938/1485891/zh800909001938.pdf by guest on 16 May 2024 coreceptor, in conjunction with CD4, to infect target cells.

32 This receptor, CXCR4, also displays a predominant intracellular distri- bution in human T lymphocytes and THP-1 monocytes 33 (and data not shown) and can form stable complexes with CD4, which are detectable by coimmunoprecipitation experiments 34 (Figure 4C).

However, contrasting with what was observed for CCR5, its surface expression was not positively modulated by CD4 in transfected cells (Figure 4D).


The functional significance of the enhancement of surface CCR5, due to the presence of CD4, was estimated by measuring ERK1-2 activation.

CHO cells expressing identical amounts of CCR5-Luc, determined by comparing luciferase activity, and a concentration of CD4-YFP corresponding to its maximal effect on CCR5 export, were stimulated or not with nonsaturating concentra- tions of MIP-1␤ for increasing periods of time up to 10 minutes (Figure 5).

The maximal activation of the MAP kinase doubled in cells coexpressing CD4 and, in addition, it was also reached much faster (compare lanes 3 [time ϭ 1 minute] in Figure 5A and corresponding histograms in Figure 5B).


We next examined whether the effect of CD4 on CCR5 surface targeting also occurs in cells expressing endogenous receptors.

We reasoned that a substantial decrease of CD4 molecules in these cells might affect the density of CCR5 at the cell surface.

A panel of 3 interfering RNAs (siRNAs) was used, which targeted the CD4 sequence.

THP-1 cells were submitted to 2 consecutive nucleofec- tions and tested after a week to achieve sufficient inhibition of CD4 synthesis and decay of CCR5 molecules already present at the cell surface before the effect of siRNAs.


A significant reduction of 70 to 80% of total CD4 was obtained with 2 specific siRNAs, compared with the control (Figure 6A,B).

In the cells showing reduced CD4, surface CCR5 was significantly decreased by 25% to 50%, compared with the control and to cells expressing the ineffective siRNA (Figure 6C,D).

Unexpectedly, cells treated with the 2 siRNAs active on CD4 expression showed a significant enhance- ment of 20% to 60% of total CCR5 (Figure S5) suggesting a possible feedback regulation.

These results support the hypothesis that CD4 molecules can modulate the proportion of surface CCR5 in cells expressing both molecules.


In a physiopathologic context, the HIV-1-encoded Vpu protein was reported to induce rapid degradation of CD4 shortly after its synthesis 35 via the ER- associated protein degradation pathway.

36 In line with our findings above, the specific targeting of CD4 molecules to the proteasome by overexpressed Vpu should affect the surface expression of CCR5.

A stable HeLa cell line, expressing both CCR5 and CD4 (HeLa-P4R5), was transiently transfected with either wild-type GFP-tagged Vpu or a GFP-fused mutant Vpu that had lost its capacity for down-regulating CD4 36 or free GFP (Figure S6).

After 48-hour transfection, surface expression of CCR5 was quantified by FACS in cells expressing comparable levels of green fluores- cence.

Expression of Vpu-GFP induced comparable and significant reduction of both total CD4 and surface CCR5, whereas the control mutant Vpu was ineffective.


Finally, the contribution of CD4 to the cell-surface expression of CCR5 was explored in primary human cells.

T cells purified from peripheral blood mononuclear cells (PBMCs) were nucleo- fected with appropriate siRNAs, activated to enhance the cell- surface expression of CCR5 and analyzed for both total CD4 content and proportion of cell-surface CCR5 (Figure 6E).

In line with the results obtained with THP-1 cells, for all 3 tested donors, the siRNA-mediated reduction of CD4 was associated with a decreased proportion of CCR5 at the cell surface compared with the control, ranging from 30% to 65%.


Although previously unreported, the predominant intracellular localization observed here for CCR5 was described for other GPCRs and might constitute a more general feature than previously appreciated.

The physiologic significance of this phenomenon may vary as a function of the receptor and the tissue where it occurs.

For example, thrombin receptors are irreversibly activated by cleavage, internalized and degraded in lysosomes, whereas intracellular receptors are protected from activation by the protease.

Replenish- ment of plasma membrane thrombin receptors is thus correlated with recovery of thrombin responsiveness.

12 In neurons, only a small fraction of ␦-opioid receptors is localized at the neuronal plasma membrane, 37 consistent with their low physiologic involve- ment in acute pain response.

38 Cell-surface translocation of ␦-opi- oid receptors from intracellular compartments might account for the enhanced effect of drugs, specifically targeting this receptor, during chronic pain.

37 Similarly, what we describe here for CCR5 may represent a mechanism for maintaining a sustained sensitivity of leukocytes to chemokine guidance within tissues.


From a mechanistic point of view, 2 important issues must be addressed to fully understand this phenomenon, namely, what are the molecular tethers, which retain receptors within intracellular compartments, and what are the intracellular events that allow their release.

So far, only 2 proteins directly and specifically involved in the control of GPCR cell-surface export via intracellular retention have been identified.


The second extracellular loop of the protease- activated PAR2 receptor was shown to interact with the N-terminal domain of the Golgi-resident type I transmembrane protein p24A, and to be retained in the Golgi because of this interaction.

Upon activation of cell-surface PAR2, the small G protein ARF1 is recruited in its GDP-bound form, to Golgi membranes, where a specific exchange factor activates ARF1.

This process results in the dissociation of PAR2 from p24A and receptor sorting to the plasma membrane.


In a different context, during development, a GPCR- retaining protein was reported to control the surface receptor availability of Frizzled, a GPCR, which promotes caudalizing signals.

This ER-resident protein, Shisa, is specifically expressed in head ectoderm, where it binds to and inhibits cell-surface traffick- ing of Frizzled.

Shisa-mediated receptor retention thus constitutes a mechanism to control head-tail polarity.


Other recent examples of regulatory retaining proteins have been reported for growth-factor receptors, 41 the gamma secretase complex, 42 and the glutamate transporter, 43 suggesting that this field may rapidly evolve.


A clear picture of the cellular mechanisms, which allow the release of intracellular GPCRs and their cell-surface translocation, is still missing.

Various cellular signals were reported to elicit "acute" effects, such as the rise in intracellular calcium by either release from intracellular stores or direct opening of ion channels, in the case of ␦-opioid receptors.

44 Similarly, in tubular renal cells, dopamine D1 receptors can be recruited from cytosolic stores to the plasma membrane upon agonist activation of cell-surface recep- tors 13 or via atrial natriuretic peptide-dependent heterologous Downloaded from http://ashpublications.org/blood/article-pdf/113/9/1938/1485891/zh800909001938.pdf by guest on 16 May 2024 proteins engaged in the secretory pathway, 47 a vast array of membrane-associated or cytoplasmic proteins has been identified, which interact with GPCRs within intracellular compartments and facilitate their cell-surface expression.

The list of these proteins, which also includes RAMPs, RTPs, REEP, MRAP, and M10 MHC class I molecules, already mentioned in the introduction, is rapidly growing.

11 Based on the data reported in the present study, the CD4 molecule may be a new member of this list.


CCR5-CD4 association has been known for a long time, as these molecules combine to form a receptor for HIV at the cell surface of mononuclear blood cells.

5 Although still a matter of debate, studies based on different experimental approaches have suggested the existence of a physical, constitutive, virus- independent, CCR5-CD4 association at the cell surface of both transfected 34,48 and primary cells.

49,50 The association of HIV coreceptors is likely mediated by interactions involving the extra- cellular globular domain of CD4 and the extracellular regions of CCR5, 34,48,51 but it has not yet been determined whether these interactions occur once the HIV coreceptors have reached the plasma membrane or if they are established within intracellular compartments before their export to the cell surface.

Our data strongly support the hypothesis that CCR5-CD4 interaction occurs within intracellular compartments, likely in the ER.


Facilitating the export of CCR5 to the cell surface is clearly not the principal biologic function of CD4 and it is possible that the surface expression of CCR5 is enhanced by the interaction with other proteins, which eventually interact with CCR5 along the biosynthetic pathway.

In addition, it remains to be established whether CCR5 might also be rapidly mobilized by cell signaling events, as for the dopaminergic and opioid receptors mentioned above.

From a mechanistic point of view, CD4-CCR5 association in the ER might facilitate further interactions with components of the biosynthetic pathway, likely via the carboxyterminal tail of CCR5.

Indeed, although not involved in the interaction with CD4, the CCR5 tail was reported to contain a bipartite motif critical for CCR5 cell-surface expression, 52 and we confirmed that the CCR5⌬Cter mutant was completely retained within intracellular compartments, even in the presence of excess CD4 (not shown).

Interestingly, the effect of CD4 on surface CCR5 was concentration- dependent, displaying a bell-shaped pattern comparable with that observed for calnexin and the ER-membrane-associated protein, DRIP78, on the maturation and trafficking of dopaminergic recep- tors.

46,53 The existence of an optimal concentration of chaperones or escort proteins likely reflects the requirement of a specific stoichiometry between these proteins, the receptors, and other components of the biosynthetic machinery.

In the case of CD4, an alternative possible explanation may be that, at high concentra- tions, CD4 itself can saturate the export machinery.


For many years, no functional explanation was proposed of how HIV may have selected a composite receptor to infect human immune cells.

Recently, a plausible explanation for the formation of the second major HIV receptor, constituted by CD4 and CXCR4 on T lymphocytes, came from the observation that CXCR4 associ- ates with the T-cell receptor to signal in T cells.

54 Thus, the stable proximity of CXCR4 and CD4 at the cell surface of lymphocytes likely arises from the bridging of these proteins via a common interactor, the T-cell receptor.

Interestingly, based on our findings, a different explanation emerges for CCR5: the physiologic constitu- tive association of CCR5 and CD4 in the ER of blood mononuclear cells, and the subsequent translocation of the resulting complexes at the plasma membrane might be promoted by the escort properties of CD4 for CCR5.


CC Chemokine Receptor 5 (CCR5) is an important mediator of chemotaxis and the primary coreceptor for HIV-1.

A recent report by other researchers suggested that primary T cells harbor pools of intracellular CCR5.

With the use of a series of complementary techniques to measure CCR5 expression (antibody labeling, Western blot, quantitative reverse transcription polymerase chain reaction), we established that intracellular pools of CCR5 do not exist and that the results obtained by the other researchers were false-positives that arose because of the generation of irrelevant binding sites for anti-CCR5 antibodies during fixation and permeabilization of cells.

[ 1, 2, 3] The G protein–coupled receptor (GPCR) CC Chemokine Receptor 5 (CCR5) recruits and activates leukocytes by responding to its chemokine ligands.

[ 1, 2] It is also the primary coreceptor for HIV-1.

[ 1, 3] Like other GPCRs, CCR5 is desensitized after ligation through clathrin-dependent endocytosis leading to intracellular sequestration before receptor recycling.

[ 4, 5] Receptor down-modulation is an important component of the anti-HIV activity of both native chemokines [6, 7] and highly potent chemokine analogues.

[ 8, 9] Certain GPCRs are stored in intracellular pools to provide rapid, renewable resources for surface expression.

[ 4, 10] Most studies of CCR5 suggest the receptor is predominantly localized at the cell surface.

[ 1, 2, 6, 7] On the basis of a series of experiments in which anti-CCR5 antibodies were used to detect CCR5 in fixed and permeabilized cells, Achour et al. [ 11] recently reported that CCR5 is predominantly intracellular in T cells, proposing that receptor storage is a mechanism for maintaining sustained sensitivity of leukocytes to chemokines within tissue.

[ 11] To investigate this new concept, we studied the expression of CCR5 protein and CCR5 RNA.

Like Achour et al. [ 11], we found apparent high levels of intracellular CCR5 with the use of flow cytometry in fixed, permeabilized T cells, but these results were not consistent with the low levels of CCR5 mRNA and the results of Western blotting.

We conclude that large intracellular pools of CCR5 are not present within circulating human T cells.


In initial flow cytometric experiments, human PBMCs were gated for expression of both CD3 and CD4 or CD8 and were stained with 3 monoclonal anti-CCR5 antibodies recognizing different domains.

[ 14-17] In agreement with the findings of Achour et al. [ 11], we found that, although cells from each donor indicated variable but universally low levels of CCR5 staining on nonpermeabilized cells, uniformly strong signals were obtained on fixed and permeabilized cells.

Control GHOST (3) parental cell lines that do not express CCR5, however, also gave positive results when the same anti-CCR5 antibodies were used to stain fixed and permeabilized cells.

Only human T cells with detectable cell surface CCR5 have CCR5 RNA by quantitative PCR and protein by Western blot.

We flow-sorted PBMCs into CD3+CCR5+ and CD3+CCR5- populations, with gates set to population extremes to minimize contamination.

The CCR5- population had 0.1%-2.6% contamination with surface CCR5+ cells.

Consistent with this low level of contamination, median CCR5 copy number in the CCR5- population determined by quantitative PCR was between 40 and 95 times lower than that measured in the positive population.

Total cell lysates were also prepared and analyzed by Western blot.

As negative and positive controls, lysates were prepared from parental GHOST (3) cells and CCR5-transfected GHOST (3) cells.

A band corresponding to CCR5 (~42 kDa) was visible in the positive control sample but not in the negative control.

Under the same conditions, the corresponding band was apparent in the cell surface CCR5+ but not in the cell surface CCR5- populations of sorted T cells from each of 3 donors.

The cell surface CCR5- populations remained negative for CCR5 by Western blot up to the highest loaded dose tested (120 µg protein per lane).


Fixing and permeabilization can lead to false-positive signals with anti-CCR5 antibodies.

To investigate how apparently high levels of intracellular CCR5 can be detected by anti-CCR5 antibodies on fixed and permeabilized cells, we carried out labeling experiments on either unmodified Chinese hamster ovary (CHO) cells (CHO-WT) or CHO cells stably transduced to express CCR5 (CHO-CCR5).

[ 18] Under mild fixation and permeabilization conditions, CHO-WT cells are negative for CCR5, and CHO-CCR5 cells are labeled at the cell surface as has been seen previously.

[ 1, 2, 6, 7] However, when the same cells are fixed and permeabilized under the same conditions as those used by Achour et al. [ 11] in the flow cytometric experiments, the CHO-WT cells become positive, with significant staining in the nucleus, and the CHO-CCR5 cells remain positive at the cell surface but show similar staining in the nucleus.

Presumably this strong off-target staining, which occurs under certain fixation/permeabilization conditions, is the explanation as to why both Achour et al. [ 11] and our group found intracellular CCR5 signals in fixed and permeabilized T cells.


The report of high-level intracellular stores of CCR5 in human T cells by Achour et al. [ 11] was surprising for several reasons.

First, induction of CCR5 surface expression on activated CD4+ T cells is delayed for 5-8 days after activation, implying that intracellular pools are not readily mobilized to the cell surface.

[ 19, 20] Second, recovery of surface CCR5 is very slow after ligand-induced receptor internalization, [9] implying that surface ligation also saturates the intracellular pool that is accessible to the surface.

We conclude that intracellular pools of CCR5 in human T cells do not exist and that instead, in agreement with several previous studies, [1, 2, 6, 7] CCR5 expression is restricted to a limited population of circulating T cells and predominantly located at the cell surface.

Our findings indicate that the results obtained by Achour et al. [ 11] were almost certainly because of the generation of irrelevant antibody binding sites during the fixing and permeabilization procedure, a phenomenon that is a recognized source of false-positive results in antibody-based experiments.


A recent study by Pilch-Cooper et al. [ 1] refuting the existence of intracellular pools of CCR5 in human T cells challenged some of our observations reported in Blood [2].

Although these authors observed the same intracellular accumulation of CCR5 using our cell permeabilization and fixation conditions before FACS analysis, this intracellular labeling was likely nonspecific binding.

This was based on the observations that our experimental conditions in their hands led to nonspecific signals in osteosarcoma cells, which do not express CCR5, and that confocal immunofluorescence (IF) microscopy studies in untransfected Chinese hamster ovary (CHO) cells showed an off-target nuclear signal [1].

Correct permeabilization conditions are indeed critical to observe intraluminal epitopes while avoiding background.

However, we do not believe that these results challenge our previous conclusions.


First, antibody combinations showing background, such as fluochrome-conjugated anti-CCR5 2D7 antibodies used in FACS studies or nonconjugated 1/85a antibody in IF experiments [1] were not used in Achour et al. [ 2].

In addition, our multiple controls were not considered.

In our Figure 1A [2] the strong signal obtained in permeabilized T lymphocytes with 2 different anti-CCR5 antibodies was not found for the anti-CD4 antibody, excluding a general nonspecific signal [2].

Our confocal IF studies in T cells and THP-1 monocytes, revealed abundant intracellular CCR5, whereas no signal was visible in control CCR5-negative Jurkat T cells (Figure 1B) [2] and IF studies on CCR5-expressing CHO cells did not reveal any nuclear staining with the CCR5 antibody (supplemental Figure 2) [2].

Moreover, the authors apparently misinterpreted their own confocal IF experiments, which clearly showed abundant intracellular CCR5 (supplemental Figure 1 top panels in Pilch-Cooper et al. [ 1]).


Nevertheless, we conducted new FACS and IF experiments in THP-1 and CHO cells (Figure 1), using the “mild” permeabilization procedure recommended by the authors [1].

Confirming the specificity of CCR5 labeling, YFP-tagged CCR5 expressed in CHO cells was labeled by antibodies, and no off-target signal was visible by confocal IF, including in CHO cells transfected with control plasmid.

Under these conditions, intracellular endogenous CCR5 was visible in THP-1 cells and again no staining was observed in CCR5-negative Jurkat cells.

After mild permeabilization, FACS studies performed with the antibody combinations used in our previous study did not show any background in CHO cells and confirmed the presence of endogenous intracellular CCR5 in THP-1 cells (Figure 1C).


The existence of abundant intracellular stores of CCR5 is consistent with similar observations for many G protein-coupled receptors, the family of receptors CCR5 belongs to [3].

In particular, intracellular stores of the chemokine receptor CXCR4 were reported in human T lymphocytes [4].

Interestingly, L-selectin stimulation of T cells induced cell surface mobilization of CXCR4 within 10 minutes.

Because such a fast effect, incompatible with receptor neosynthesis, represents major evidence for the existence of internal stores, we performed a similar experiment using THP-1 monocytes (Figure 1D-F).

Adhesion of THP-1 cells on fibronectin-coated slides for 10 minutes was sufficient to increase 5-fold the fluorescence signal of surface CCR5, whereas surface CD4 was not affected.

Golgi apparatus disassembly, induced by pretreating cells with brefeldin A, inhibited the phenomenon.

These experiments were conducted without cell permeabilization.

This finding and our previous data [2] confirm that intracellular pools of CCR5 do exist in blood cells.

Rapidly mobilizable stores of chemokine receptors may represent a physiologic mechanism to escape desensitization on sustained activation and/or to facilitate blood cell extravasation into peripheral tissues.

Internal pools of CCR5 also represent a challenging problem to solve for the development of anti-HIV drugs targeting this coreceptor.

Studying the regulation of these intracellular pools is of primary importance to accelerate the development of such drugs.


In chloroplasts molecular oxygen is reduced to Superoxide at the reducing sides of the photosystems in higher plants.

The Superoxide radical anion is efficiently removed by Superoxide dismutase, and the generated hydrogen peroxide is detoxified to water by ascorbate peroxidase (Fig. 1).

It is well established that the synthesis rate of hydrogen peroxide is significantly increased in stress situations generated by air pollutants, high light, chilling etc.

Asada and co-workers calculated that without recycling of ascorbate H2O2 would deplete the available ascorbate pool via ascorbate peroxidase within min- utes [1].

This necessitates a reducing system to regenerate as- corbate.

The primary product of ascorbate peroxidase is monodehydroascorbate (MDA), which is rapidly returned into the ascorbate pool either by NADPH-dependent MDA reductase or non-enzymatically by reduced ferredoxin (Fig. 1).

DHA, dehydroascorbate; DHA reductase, dehydroas- corbate reductase; MDA, monodehydroascorbate; MDA reductase, monodehydroascorbate reductase of 10 5 M _1 s _1 [2].

In plant extracts a glutathione-dependent DHA reductase activity has been observed [3] which will re- cycle DHA to ascorbate (Fig. 1).


An increase of DHA reduc- tase activity and an accumulation of DHA have been fre- quently implied as biochemical indicators of oxidative stress in plant metabolism [4] but a characterization of DHA reduc- tase has remained elusive because of rapid loss of enzyme activity.

Here we report that plant cells do not possess and do not require a specific DHA reductase.


All chemicals and reagents were of the highest purity available.

Trypsin inhibitor from soybean (Kunitz type) was obtained from Boehringer.

Antibodies raised against thioredoxin m and / from spin- ach chloroplasts were generous gifts from Dr. B. Buchanan, Univer- sity of California, Berkeley, CA, USA.

Samples of spinach thioredox- in m and / were supplied by Dr. P. Schürmann, University of Neuchätel, Switzerland, which is gratefully acknowledged.

Antibodies raised against trypsin inhibitor were produced in cooperation with Dr. Giorgio Tiscar, University of Bari, Italy.

NADP-malate dehydrogen- ase and fructose-1,6-bisphosphatase were isolated from spinach leaves and assayed by published procedures [5,6].


Spinach leaves were pur- chased on local markets and lots of 100 g leaf material were extracted in 200 ml of potassium phosphate buffer (50 mM, pH 7.8) in a Waring blender.

The homogenate was passed through four layers of miracloth and centrifuged at 13000 rpm for 20 min.

Isolation of chloroplasts from spinach leaves followed the method described by Mourioux and Douce [7].

Activity staining of dehydroascorbate reduction following native gel electrophoresis on 1% agarose gels was performed by a method described by De Tullio et al. [ 8], which involves the reactions between the ascorbate formed, cyanoferrate(III), and ferric chloride.

Immobilon P-membrane (Millipore) was used for Western blots and antisera were applied in 1:1000 dilution.

Anti-rabbit IgG peroxidase conjugate and diaminobenzidine (from Sigma) were used to determine the first antibody.


Recently we described the purification of an apparently novel protein with glutathione-dependent DHA reductase ac- tivity from spinach chloroplasts [9].

N-terminal sequence anal- ysis of this protein revealed that it is highly homologous with trypsin inhibitors of the Kunitz type.

Both the isolated protein from spinach chloroplasts and commercially available soy- bean trypsin inhibitor showed DHA reductase activity in the reduced state and acted as trypsin inhibitors in the oxidized state [9].

Their redox behavior must be due to the presence of conserved cysteine or cystine residues, respectively.

Redox changes between a dithiol and disulfide also supply the reduc- ing equivalents for the reduction of DHA to ascorbate.

In the case of a trypsin inhibitor from potato two cysteines are ar- ranged in a C P F C sequence, reminiscent of the redox-active sites found in thioredoxin (C G P C), glutaredoxin (C P Y C), and protein disulfide isomerase (C G P C) [2,9,10].

All the 0014-5793/97/S17.00 © 1997 Federation of European Biochemical Societies.

All rights reserved.

latter proteins have been shown to possess DHA reductase activity.


In mammalian cells proteins with DHA reductase activity have been described which were subsequently charac- terized as glutaredoxin [11].

In contrast, when we isolated glutaredoxin from green plant tissue it was observed that spin- ach glutaredoxin possesses no DHA reductase activity [12].

The structural differences between mammalian and plant glu- taredoxins responsible for their different reactivity are cur- rently under investigation.


Recently, Kato et al. reported the isolation of a new DHA reductase from rice bran.

The deter- mined N-terminal amino acid sequence was not similar to the N-termini of other known DHA reducing proteins [13]. But it must be taken into account that proteins from storage tissue frequently possess extra amino acids at the N-termini which are not present in the corresponding proteins in tissues of developed plants [14].

Unfortunately, it was not investigated whether the rice bran protein with DHA reducing activity possesses additionally thioredoxin, glutaredoxin or protein di- sulfide isomerase activity [13].

Thus, further investigations are necessary to elucidate the real nature of this protein from rice bran.


Millimolar concentrations of DHA have been measured in the stromal compartment [15].

In view of these considerable concentrations and our knowledge that thioredoxins can be oxidized by DHA [9] we investigated whether DHA interferes with the thioredoxin-dependent redox regulation of C 0 2 fix- ation, sulfate assimilation, ATP synthesis, etc.

The thioredox- in system is vital for chloroplast metabolism because redox control of at least 12 different enzymes is achieved by the reductive cleavage of regulatory disulfide bridges in these tar- get enzymes [16].


Indeed, as shown in Fig. 2, the thioredoxin- dependent activity of NADP-malate dehydrogenase is inhib- ited by 50% in presence of 50 μΜ DHA and reduced to less than 10% by 1 mM DHA.

Thus, in the presence of the milli- molar concentrations of DHA reported in stroma NADP-ma- late dehydrogenase would be completely inactive, resulting in serious consequences for the chloroplast metabolism because the enzyme is part of the defence system protecting plant cells against photoinhibition [17].

Another thioredoxin-dependent enzyme, glutamate synthase, is also inhibited by DHA (not shown), but DHA does not inhibit all thioredoxin-dependent stromal target enzymes with the same efficiency.


The Calvin- Benson cycle enzyme fructose-1,6-bisphosphatase is not inhib- ited when the regulatory sites of the enzyme are protected in its protein complex with thioredoxin (Fig. 3) [6].

Here, each subunit of the tetrameric enzyme possesses a single regulatory disulfide bridge whereas in dimeric NADP-malate dehydro- genase each subunit contains two different regulatory disulfide bridges and only one of these sites is shielded by thioredoxin [18,5].

However, when DHA was added to reduced thioredox- in prior to fructose-1,6-bisphosphatase activation, thioredox- in-dependent activation of the target did not occur because of the oxidation of thioredoxin by DHA.


Obviously, DHA must be regarded as a toxic substance which disturbs the various regulatory functions of the thioredoxin system in chloroplast metabolism.

Consequently, chloroplasts should be equipped with a DHA reductase which keeps the DHA concentration at least below 50 μΜ. However, all proteins with DHA re- ductase activity isolated from chloroplasts possess apparent K m values for DHA in the millimolar range [9,13].

The ques- tion arises which other mechanisms would prevent DHA ac- cumulation.


It has been observed repeatedly that DHA reductase activ- ity measured in crude extracts or in enriched fractions of spinach leaves is very unstable [3].

To ensure that DHA re- ductase was not being overlooked throughout a purification procedure as described [9,12] we analyzed DHA reductase activity patterns in cell-free protein extracts from isolated chloroplasts by native gel electrophoresis combined with ac- tivity staining for DHA reduction.

In protein extracts of spin- ach chloroplasts we detected three different DHA reductase activities (Fig. 4).

Western blot analysis revealed that two signals are due to thioredoxin / and thioredoxin m and that the third signal represents a Kunitz-type trypsin inhibitor.

No additional DHA reductase species could be detected by this experimental procedure.

Thus, it is concluded that no specific DHA reductase is present in spinach chloroplasts but all measurable DHA reductase activity is due to these different redox-active proteins.

Lack of a specific detoxifying enzyme would be highly surprising if DHA concentrations were really in the millimolar range.


The origin of DHA is the non-enzymatic disproportiona- tion of MDA.

Thus, the formation of DHA is determined by the half-life of MDA.

Molecular oxygen is reduced to super- oxide at the photosystems, thylakoid-bound Superoxide dis- mutase and ascorbate peroxidase remove Superoxide and hy- drogen peroxide.

The generated MDA is a substrate either of reduced ferredoxin or of thylakoid-bound MDA reductase.

Thus, MDA will be preferentially removed at its formation site.


In case MDA escapes, a stromal MDA reductase pre- vents an accumulation of MDA.

Both MDA reductase and reduced ferredoxin remove MDA with rate constants of 10 7 - 10 8 M _ 1 s _ 1 [19] whereas the disproportionation rate of MDA is only 10 5 M _ 1 s _ 1 [2].

The enzyme equipment of the chloroplasts can, therefore, be regarded as very efficient and it is improbable that significant amounts of DHA will accumulate.

This is supported by the fact that the measured MDA concentration in chloroplasts is about 10~8 M.


It is, therefore, not a priori necessary to require a specific DHA reductase in chloroplasts.

Nevertheless it is thinkable that the capacity of MDA scavenging is overcome under certain stress circumstances and that a DHA reductase would still be desirable.

In these situations it must be taken into account that glutathione non-enzymatically reduces DHA to ascorbate at high rates, especially at alkaline pH which prevails in the light [20].


It is important to consider that in biological solutions as- corbate is rapidly oxidized to DHA within minutes at phys- iological pH. Only under acidic conditions (pH 2) ascorbate is stable enough to be measured which explains why it cannot be quantified enzymatically [21,22].

In studies of oxidative stress in chloroplasts many steps are necessary to isolate the cell organelles and considerable time elapses.

Thus, it is impossible by presently available methods to prepare samples without artifactual generation of DHA during the experimental pro- cedure.

This fact readily explains the high concentrations of DHA measured in chloroplast and plant extracts [21,22].


Increasing DHA concentrations and/or DHA reductase ac- tivity have been regarded as biochemical indicators of increas- ing oxidative stress in chloroplast metabolism [4].

As outlined in this report, both indicators are misleading and falsify the interpretations.

Moreover, in leaf extracts no extra DHA re- ductase was detected outside the chloroplasts (Fig. 4, lane 4).

We have to conclude that plant cells do not possess any spe- cific dehydroascorbate reductase and that the activities meas- ured in total leaf extracts are due to unrelated proteins present in chloroplasts.

It cannot be excluded, however, that addi- tional proteins with ««specific DHA reductase activity exist outside the chloroplasts [3].

Unspecific DHA reductase activ- ity associated with several unrelated proteins may explain why, unexpectedly, DHA reductase activity was not elevated in extracts of stress-treated plants [23].

To circumvent the poor reproducibility of DHA reducíase determination DHA pools have been analyzed.

This approach also has difficulties as in some studies DHA was not detectable at all in plant leaf extracts [21].

These results support our opinion that DHA is not a constitutive metabolite occurring in considerable amounts in plant metabolism.


In conclusion, the regeneration of ascorbate is obviously not coupled to a glutathione-dependent DHA reductase (Fig. 1).

The glutathione reductase present in leaves is not directly involved in the regeneration of ascorbate and is prob- ably not of importance in avoiding oxidative stress as fre- quently discussed [4].

This offers an explanation why increased levels of glutathione reductase in transgenic plants do not confer increased tolerance against oxidative stress [24].

The above arguments should help to avoid future misconceptions about a stress defense in plant biology.


Ascorbate, an essential antioxidant in plant tissues, is oxidised to dehydroascorbate (DHA) via successive reversible transfers with monodehydroascorbate (MDHA) as a free radical intermediate.

Rapid regeneration of ascorbate from its oxidised forms is required to support antioxidant capacity.

MDHA radicals have a relatively short lifetime and disproportionate at neutral pH values and above to DHA and ascorbate.

MDHA radicals can be reduced directly to ascorbate by reduced ferredoxin and by NAD(P)H-dependent monodehydroascorbate reductases, which are found in several cellular locations.

DHA can be reduced directly by reduced glutathione at alkaline pH values and also by enzymes which catalyse this conversion (dehydroascorbate reductases, DHAR).


It has recently been argued that DHA detected in chloroplast extracts is artefactual, that in vivo DHA levels are negligible and that this metabolite is formed purely artefactually during extraction of plant tissues [1].

This hypothesis was largely based on the observation that, at concentrations thought to exist in the chloroplast, DHA could cause oxidative inactivation of two enzymes known to be regulated by the thioredoxin system [1].

The authors therefore concluded that DHA accumulation must be avoided [1].


This conclusion is erroneous, however, since it does not take into account the dynamic nature of the regulation of the thiol-mediated enzymes of the chloroplasts.

It is well established that the stromal enzymes regulated by the thioredoxin system require ongoing reduction to remain active (e.g. [2,3]).

Data obtained by addition of oxidants to enzymes removed from a continuous system of thioredoxin reduction are not relevant to in vivo conditions in the chloroplast, where the activation state of thiol-regulated enzymes reflects differences between reductive and oxidative fluxes.


Hence, it is important that DHA is reduced back to ascorbate.

It does not, however, mean that DHA cannot exist in vivo.

The authors’ data show that DHA can oxidise thioredoxin and thioredoxin-activated enzymes; this simply means that, like ferredoxin, NADPH and reduced glutathione (GSH), thioredoxin can act as an intermediate in re-reducing the oxidised forms of ascorbate.


The argument that glutathione reductase (GR) is unimportant because DHAR may not exist is an unjustified extension of the hypothesis.

The citation of the review of Allen [4] as stating that GR does not contribute to oxidative stress tolerance in transgenic plants is erroneous.

Glutathione has many roles in the chloroplast, including participation in redox reactions other than those directly associated with the ascorbate pool, and in the control of gene expression [5].

Irrespective of the nature of the interactions between ascorbate and glutathione the latter pool can only be kept predominantly in the reduced state (as is found in all organisms) by the action of GR.

Little, if any, oxidised glutathione (GSSG) is found in isolated chloroplasts [6] and the glutathione pool is well over 90% reduced in leaves [7,8].

In animal tissues such as kidney and red blood cells the GSH to GSSG ratio is higher than 500 [9].

Even in the system found to have the highest proportion of GSSG, blood plasma,

the predominant form of glutathione is GSH [10].


While the AA/MDHA redox couple may function effectively without association to the GSH/GSSG redox couple in certain circumstances, the significance of GSH as a reductant in ascorbate regeneration is established in relation to stress tolerance [11-14].

A tropical fig mutant devoid of DHAR activity was found to be sensitive to high light [12].

Further evidence to support critical roles for DHAR, GSH and GR in maintaining the foliar ascorbate pool has been obtained in transformed plants overexpressing GR which have higher foliar ascorbate contents [8] and improved tolerance to oxidative stress [7,8,13,14].

Similarly, compensation of one antioxidant by the other can be observed, for example, decreases in the meristematic activity of Arabidopsis roots caused by GSH depletion were alleviated by adding ascorbic acid [15].


Ascorbate breakdown occurs via DHA which is catabolised to two- and four-carbon products such as oxalate and tartrate [16,17].

These can accumulate to relatively high levels in plant tissues.

While the reactions through which ascorbate is catabolised in plants remain obscure, degradation is considered to occur by enzymic degradation of DHA.

DHA is therefore a fundamental component of ascorbate turnover as well as the ascorbate redox system.

While it is possible that, even under acidic extraction conditions, some over-estimation of tissue DHA contents is inevitable, the above considerations suggest that DHA is formed in vivo and is an important component of ascorbate metabolism.

Further evidence to support this view is the existence of a high affinity DHA transporter on the plasma membrane [18].


DHARs that catalyse the reduction of DHA by GSH have been purified from rice, spinach and potato [19-22].

Several other proteins such as glutaredoxins (thiol transferases), protein disulphide isomerases, and even a Kunitz-type trypsin inhibitor have been shown to have DHAR activity [23,24].

Nevertheless, the amino acid sequence of the rice DHAR is quite distinct from these other enzymes [22].

Kato et al. [ 22] provided convincing proof that a specific DHAR enzyme does exist in a wide range of plant tissues.

The argument put forward that this is artefactual because seed tissue contains many such DHAR-like proteins [1] is specious.

The authors ignore the data by Kato et al. [ 22] showing that the protein is present in both photosynthetic and non-photosynthetic tissues, as well as being detectable in barley and rice.


Furthermore, we have found the presence of near perfect matches of the N-terminal sequence data from Kato et al. [ 22] with sequences from the Arabidopsis EST cDNA database using TFASTA from the GCG suite of programs [25].

The best of a group of very similar matches is as follows:


All significant sequences matches were near N-termini of derived EST protein sequences.


These matching Arabidopsis EST cDNAs were translated and in stretches of derived amino acid sequence of up to 120 residues no matches to trypsin inhibitors, glutaredoxin, peroxiredoxin, thioredoxin or protein disulphide reductase were detected, despite these sequences being well represented from plants in the EMBL and GenBank data bases.

This rules out the authors’ arguments that Kato et al. [ 20] had purified an artefactual DHAR.

So it is likely that DHAR does exist, although the chloroplast isoform may prove elusive for some time.


The absence of detectable DHAR enzyme on non-denaturing polyacrylamide gels stained for DHAR activity and not ascribed to artefacts cannot be taken as definitive proof that a bona fide DHAR does not exist.

The enzyme may simply not withstand this type of extraction and assay protocol or may be too dilute to detect in this assay system.

Similarly, the argument that DHAR activity is often found not to increase in stressed plants is evidence that only incidental activities from other proteins are being measured [26-29].


These considerations support the view that DHA and DHAR exist in leaves and other plant tissues.

The oxidising ability of DHA does not preclude its presence in chloroplasts where redox regulation of photosynthetic enzymes requires the dynamic interaction of reducing and oxidising components.


In our recent contribution [1] we questioned the role of dehydroascorbate (DHA) and DHA reductase as reliable indicators of oxidative stress in plants.

Several lines of evidence were presented to substantiate that the measurement of DHA pools and DHA reductase activity in crude plant extracts is falsified by technical problems and by the fact that plant cells contain several redox-active proteins with unspecific DHA-reducing activity.

We discussed whether the disproportionation of monodehydroascorbate (MDHA) leads to an appreciable formation of DHA in plant cells considering the known chemical and biochemical data of MDHA reductase and other MDHA-reducing components.

Moreover, we questioned whether plant cells necessarily require a specific DHA reductase and concluded that they probably do not possess a specific DHA reductase at all.

It is not surprising that these statements raise a dispute.

Unfortunately, Foyer and Mullineaux [2] in their commentary try to counter our conclusions not by new facts but by remote arguments.


First of all we did not suggest that DHA will not exist in plant cells at all.

We outlined that it is improbable that significant amounts of DHA will accumulate, a conclusion drawn not only from ample biochemical evidence found in the literature [1] but from data showing that a steady-state concentration of 50 WM will substantially inhibit the regulatory action of plant light-dependent regulation of processes such as CO2 fixation, sulfate assimilation, and nitrate assimilation.


Foyer and Mullineaux argue [2] that this observation was derived from assays not describing the situation in vivo because thioredoxin reductase was not added to regenerate reduced thioredoxin.

They assume that the stromal enzymes regulated by the thioredoxin system require ongoing reduction to remain active.

This would imply that in light the reductively activated target enzymes are permanently deactivated by oxidants and require repeated activation by thioredoxins in vivo.

In the last 10 years knowledge about the action of thioredoxin has improved substantially enough (e.g. by demonstration of complex formation between thioredoxin and target enzymes) to indicate that the very opposite is the case.

A detailed explanation is beyond the scope of this comment but the reader’s attention is drawn to a recent review summarizing the biochemical action of thioredoxins [3].


Among the known proteins possessing DHA reductase activity are thioredoxins, a Kunitz-type trypsin inhibitor, glutaredoxins (thiol transferases), and protein disulfide isomerases.

All these proteins are characterized by a very similar redox-active site of the general amino acid sequence Cys-X-X-Cys.

Reversible redox changes between the dithiol form and disulfide form supply the reducing equivalents for DHA reduction.

If we isolated one of these proteins using the common DHA reductase assay and did not know their original function in plant metabolism we, too, would regard it as DHA reductase.

Many more enzymes containing Cys-X-X-Cys motifs might be able to reduce DHA but in the absence of pertinent experiments have not been recognized.

This offers an explanation of why DHA-reducing proteins have been found in unpredictable numbers in plant tissues [4].


In line with these arguments we suggested further experiments to establish unambiguously the identity of the DHA-reducing protein isolated by Kato et al. [ 5].

We point, for example, to the identification of a putative DHA reductase as a 3K-hydroxysteroid dehydrogenase [5,6].

In our contribution, we may not have been precise enough in discussing this point.

We never stated that we consider the activity described by Kato et al. to be artifactual but do maintain that it could be the side activity of some different enzyme.

The sequence similarity mentioned by Foyer and Mullineaux does not prove a DHA reductase proper.


Extraction of plant samples with a phosphate buffer at physiological pH in a Waring blender is certainly not a procedure to denature proteins.

One of the most gentle methods to observe different isoforms of an enzyme is non-denaturing gel electrophoresis when the protein extracts are applied without delay.

Thus, a specific and original DHA reductase should be measurable by activity staining.

That we did observe the side activities of unrelated proteins obviously repudiates the argument that the investigated protein samples were too diluted.

Moreover, different chromatographic methods have been tried to isolate a specific DHA reductase but all these efforts failed.


The link between DHA formation, recycling of DHA to ascorbate by glutathione-dependent DHA reductase, and reduction of GSSG to GSH by glutathione reductase is commonly regarded as essential for the ascorbate regeneration system in plants.

Because of the questionable existence of an original DHA reductase in plants we discussed whether the ascorbate-glutathione cycle is overestimated among processes constituting the defense system against oxidative stress in plants.

In this context it is thinkable that the role of glutathione reductase to avoid oxidative stress is overestimated, too, but this in no way downplays the central role of glutathione reductase to maintain a reducing environment and it does not exclude that glutathione is required in other processes in plant cells.


These considerations support our view that one has to be careful in accepting either changing DHA pools or DHA reductase activities as indicators of oxidative stress in plants.

This is, in fact, in agreement with the statement of Foyer and Mullineaux [2] who remark: “that DHA reductase activity is often found not to increase in stressed plants is evidence that only incidental activities from other proteins are being measured”.

The remaining problem is to distinguish between incidental and specific DHA reductase activities.

A postulated specific, yet elusive and unmeasurable chloroplast enzyme is in our opinion not reliable enough as indicator of oxidative stress in plants.


Hybridization and introgression have long been recognized by botanists as important factors influencing evolution (e.g. Abbott 1992; Arnold 1997; Ellstrand & Schierenbeck 2000), whereas its importance in animal evolution has been a subject of extensive debate (Arnold 1992; Dowling & Secor 1997; Seehausen 2004).

Recently, numerous studies have demonstrated that closely related animal species often share a history of introgressive hybridization (e.g. Melo-Ferreira et al. 2005; Roca et al. 2005; Berthier et al. 2006; Patterson et al. 2006), indicating that hybridization may be frequent in some cases before the completion of reproductive isolation in animals.

Even though it seems that natural hybridization and introgression are more common in animals than previously assumed, it is still not known how widespread this phenomenon is and whether particular taxa or biogeographical regions are more prone to it.


A recent spatially explicit simulation study (Currat et al. 2008) demonstrated that massive introgression of neutral genes can occur during the invasion of an occupied area as long as interbreeding is not severely prevented between the invading and the local species.

Importantly, it was found that introgression occurs almost exclusively from the local to the invading species, regardless of the relative densities of the two species.


Introgressive hybridization can have a variety of consequences, but its greatest evolutionary importance might lie in it being the source of new genetic variability within taxa.

However, under different circumstances it might cause a merging of the hybridizing species (Arnold 1997).

For the latter reason, conservation policy generally discourages hybridization between species because hybridization can jeopardize the continued integrity of the hybridizing species (O'Brien & Mayr 1991; Leonard & Wayne 2008).


The wolves of the Great Lakes region in the USA were almost exterminated at the beginning of the last century due to habitat depletion associated with the spread of agriculture and direct persecution.

However, they have recovered under the protection of the US Endangered Species Act to currently > 3000 individuals, resulting in an extensive debate concerning their potential delisting.


Over the last century, coyotes have invaded this region and hybridized with wolves (Lehman et al. 1991, Leonard & Wayne 2008).

At present, there is still no general consensus about which species of wolf-like canid currently inhabits the Great Lakes region and if the integrity of the population is under threat by hybridization.


The Great Lakes (GL) wolf is morphologically distinct from both western gray wolves (Canis lupus) and coyotes (Canis latrans) (Nowak 2002).

It has been suggested that the GL wolf is either (i) a smaller subspecies of the gray wolf (Canis lupus lycaon), possibly resulting from hybridization between gray wolves (C. lupus) and red wolves (Canis rufus) (Nowak 2002); (ii) a hybrid zone between gray wolves (C. lupus) and coyotes (C. latrans) (e.g. Lehman et al. 1991; Roy et al. 1994); or (iii) a distinct species (C. lycaon) closely related to, and perhaps conspecific with, the red wolf (C. rufus).

This unique wolf- like canid is hypothesized to have evolved from a coyote- like ancestor, thus representing a small wolf indigenous to North America (Wilson et al. 2000; Kyle et al. 2006).


Here we use maternally, paternally and biparentally inherited molecular markers to analyse modern and his- toric pre-bottleneck and pre-coyote invasion GL wolves, western gray wolves and coyotes in order to evaluate (i) alternative hypotheses regarding the evolutionary origin of the GL wolves; (ii) ongoing hybridization between GL wolves, western gray wolves and coyotes in the Great Lakes region; and (iii) population integrity.


The modern samples used in this study included western gray wolves from the Northwest Territories and Alberta, Canada; GL wolves from Ontario and Quebec, Canada and Minnesota, Wisconsin, Michigan, and New York, USA; coyotes from New York, Massachusetts, Nebraska, Colorado, Nevada and Illinois, USA.

Western coyotes from Nebraska, Colorado, Nevada and Illinois correspond to the original distribution of North American coyotes, whereas eastern coyotes from New York and Massachusetts represent the recent northeastward range expansion.

The GL wolf samples from Canada and Minnesota were from Lehman et al. ( 1991) and Roy et al. ( 1994).


Historic samples consisting of skulls from the collection of the National Museum of Natural History, Smithsonian Institution, included 18 GL wolf samples from Ontario and Quebec, Canada and Michigan, Wisconsin, Minnesota and New York and a historic gray wolf from Labrador (Fig. 1, Table 1).

The historic GL wolves were all collected before coyotes became established in the region.


Whole genomic DNA was extracted from recent muscle tissue samples using a modified phenol/chloroform protocol (Sambrook et al. 1989).

Extraction of historic specimens followed Leonard et al. ( 2005).


Amplification of 420-425 bp of the 5′ end of the mito- chondrial control region followed Vilà et al. ( 1999) for the Museum no.


Twenty-six unlinked biparentally inherited autosomal microsatellites were typed for 58 gray wolves, 195 GL wolves, 48 eastern coyotes, 78 western coyotes and 13 historic GL wolves: Ren94K11, C17.402, Ren239K24, C18.460, Ren274F18, Ren181K04, C11.873, Ren73F08, C02.894, Ren204K13, Ren160J02, Ren106I06 (Breen et al. 2001), FH3109, FH2887, FH2914, FH2785, FH2759 (Guyon et al. 2003), Ren37H09, Ren49F22 (Jouquand et al. 2000), c2017 (Francisco et al. 1996), u109, u225, u250, u253 (Ostrander et al. 1993), vWF (Shibuya et al. 1994) and PEZ05 (PerkinElmer, Zoogen; see NHGRI Dog Genome Project at http://research. nhgri.nih.

Amplification of the microsatellite loci followed Björnerfeldt et al. ( 2008).

PCR products were pooled in seven different batches for genotyping.


Allelic dropout, where one allele at a heterozygous locus fails to amplify, is the most common error associated with amplifications from low concentration DNA, such as historic DNA, resulting in a potential misinterpretation of a heter- ozygous individual as being homozygous at that particular locus (Taberlet et al. 1996).

Therefore, we only scored historic genotypes as homozygous when the same single allele was amplified in at least four replicate PCRs.

When only one allele was amplified from a sample fewer than four replicates - due to PCR failure and depletion of DNA extract - we scored the genotype as consisting of the observed allele and one missing allele.

Allele frequencies for differentiation an- alysis (θ ST ; see below) were then calculated relative to the total number of scored alleles.

This is justified by observations that, despite higher rates of allele dropout in loci with large fragments, dropout was random with respect to allele sizes within a locus (Sefc et al. 2003).


A total of six Y-chromosomal microsatellites were typed for all males and samples of unknown sex (final sample size: 30 gray wolves, 111 GL wolves, 25 eastern coyotes, 41 western coyotes, 4 historic GL wolves): 650-79.3, 990-35 (Bannasch et al. 2005), MS41A, MS41B, MS34A, MS34B (Sundqvist et al. 2001).

All amplifications were carried out in 10-μL reactions.


The PCR mix for 650-79.3 and 990-35 included 1× Smart Taq buffer (QIAGEN), 2.5 mm MgCl 2, 0.25 μm of each dNTP, 0.4 μm of each primer, 0.25 U Smart Taq (QIAGEN) and 1 μL of DNA template (~10 ng).

The PCR profile for 650-79.3 included an initial denaturation step at 95 °C for 15 min followed by 38 cycles (30 s annealing temperature at 65 °C, extension at 72 °C for 1 min, followed by denaturation at 95 °C for 30 s) and a final annealing step at 65 °C for 1 min and an extension step at 72 °C for 10 min.

For 990-35 the annealing temperature was 57 °C.


Ampli- fication of MS41A, MS41B, MS34A and MS34B followed Hailer & Leonard (2008).

For genotyping, the PCR products 650-79.3 and 990-35 were pooled.

Genotyping was carried out on a MegaBACE 1000 instrument (Amersham Bio- sciences).

Genotypes were identified using the software Genetic Profiler version 2.2 (Amersham Biosciences).


Genetic diversity indices for mtDNA sequences and Y- chromosomal data [number of haplotypes (H), haplotype diversity (H D ), and, for mtDNA sequences, nucleotide diversity (π)] were calculated in Arlequin version 3.0 (Excoffier et al. 2005).

Microsatellite variability in modern samples was estimated as the number of alleles (N A ), allelic richness (A R ), observed (H O ) and expected (H E ) heterozygosity, using Arlequin.

We used Micro-Checker 2.2 (van Oosterhout et al. 2004) to test for errors due to stuttering, large allelic dropout and the presence of null alleles.

Exact tests of Hardy-Weinberg equilibrium for each microsatellite locus were calculated in Arlequin with Markov chains of 100 000 steps following 1000 dememorization steps.

We also estimated the probability of linkage disequilibrium between loci in Arlequin based on 10 000 permutations.

P values were corrected for multiple testing following the method of Benjamini & Hochberg (1995).


Mitochondrial sequences were checked and aligned manually to minimize the number of indels using the program Sequencher version 4.6 (Gene Codes).

The best-fit model of nucleotide substitution suggested by Model- Generator version 0.85 (Keane et al. 2006) was HKY + I + G (Hasegawa et al. 1985) with base frequencies A = 0.27609, C = 0.29369, G = 0.15267 and T = 0.27755, proportion of invariable sites I = 0.50, and a gamma shape parameter α = 0.43.

Pairwise HKY + I + G distances between individual sequences computed in paup (Swofford 2002) were imported into mega 4 (Tamura et al. 2007) to visualize phylogenetic relationships by means of a neighbour-joining (NJ) tree.


Due to complete linkage of all loci on the Y-chromosome, composite Y-chromosome genotypes represent haplotypes.

Phylogenetic relationships among the Y-chromosomal haplotypes were visualized by a full median-joining network (Bandelt et al. 1995, Bandelt et al. 1999) with max- imum parsimony post-processing (Polzin & Daneschmand 2003) as implemented in Network (version 4.5; available at www.fluxus-engineering.com/sharenet.htm), putting equal weight on each locus.


We analysed the genetic structure based on autosomal microsatellites (excluding the historic samples) with the Bayesian model-based clustering method implemented in Structure 2.2 (Falush et al. 2007).

The log likelihood of our data [ln Pr(X|K)] was estimated, given different numbers of genetic clusters K, using an admixture model with inde- pendent allele frequencies and ignoring prior population information.

To assess the possible range of K, short [20 000 burn-in cycles, 100 000 Markov chain Monte Carlo (MCMC) iterations] runs for K = 1-17 were repeated five times.

Based on the results of these initial runs, 10 long runs (20 000 burn-in cycles, 1 000 000 MCMC iterations) were run for K = 1-7.

Following Evanno et al. ( 2005) we calculated ΔK, which corresponds to the rate of change of the likelihood between successive K values.

The modal value of this dis- tribution was considered as the uppermost level of genetic structuring (Evanno et al. 2005).


To visualize the distribution of genetic variation in the autosomal microsatellites across individuals, we performed a factorial correspondence analysis (FCA) in Genetix 4.05 (Belkhir et al. 1996-2004).

Population bottlenecks are expected to cause hetorozygosity excess compared to expectations based on the observed number of alleles at microsatellite loci (Cornuet & Luikart 1996).

We used Wilcoxon tests to evaluate whether the number of loci with excess heterozygosity was significantly larger than expected assuming a two-phase mutation model with 90% single-step mutation (SSM) and 10% infinite allele model (IAM, Luikart et al. 1998) implemented in the program Bottleneck (Piry et al. 1999).


To assess whether the genetic composition of the historic GL wolves differed significantly from that of the modern samples, we calculated locus-by-locus θ ST (Weir & Cockerham 1984) from allele frequencies at 20 microsatellite loci (the loci with the largest fragment sizes did not amplify in any of the historic samples) and evaluated the significance with 10 000 permutations as implemented in Arlequin 3.

Global θ ST was calculated as the ratio of the average locus specific V a (variances among groups) and the averages of total variances.

Locus-by-locus probabilities were combined to a global P value by means of Stouffer 's Z-transform test (Stouffer et al. 1949; also see Whitlock 2005).

We used this approach because the standard amova in Arlequin is very sensitive to missing data, which is the case in our historic sample.

For comparative purpose, we also estimated microsatellite θ ST between the historic GL samples and the modern gray wolves, eastern and western coyotes, respectively using the locus-by-locus approach as described above and between modern GL samples and modern gray wolves, eastern and western coyotes using standard amova in Arlequin 3.

Furthermore, we estimated θ ST between historic and modern GL wolves from mtDNA haplotypes.


The NJ tree (Fig. 2a) shows two clades with bootstrap support of 100%, of which one contains all western gray wolf haplotypes (the 'wolf clade') and the other all coyote haplotypes (the 'coyote clade').

In contrast, GL wolves from all locations around the Great Lakes were found in both clades, with 75 and 142 individuals possessing haplotype in American gray wolves (Leonard et al. 2005).

Haplotype diversity in GL wolves was much higher than that in western gray wolves, and was highest in eastern and western coyotes (Table 2).

The occurrence of both wolf- and coyote-clade haplotypes in GL wolves resulted in extraordinarily high nucleotide diversity compared to the other taxa (Table 2).

Differentiation between historic and modern GL wolf haplotypes was significant with θ ST = 0.2404 and P < 0.0001.


Y-chromosomal diversity was generally high, but lowest in GL wolves, and highest in western coyotes (Table 2).

Similar to the mitochondrial sequence data, the median- joining network based on Y-chromosomal haplotypes revealed a separation into a wolf and a coyote clade, with GL wolf haplotypes present in both clades (Fig. 2b).

Unlike the mitochondrial data, eastern coyote haplotypes appeared in both clades.

Only one western coyote haplotype from Illinois, the border zone between western and eastern coyotes, was found in the wolf clade, sharing its haplotype with both an eastern coyote and a GL wolf.

Whereas nine haplotypes were shared between GL wolves and western wolves, only three haplotypes were shared between GL wolves and coyotes (two in the coyote clade, one in the wolf clade).


As in the mitochondrial data, no clear geographical substructuring was evident within GL wolves.

Each of the four historic GL wolves which were successfully genotyped at all six Y-chromosome loci had a unique haplotype in the wolf-clade.

Within the coyote clade, two major GL wolf clusters were evident.

The first cluster, which includes the most frequent as well as eight additional GL-specific haplotypes, is closely related to four divergent western coyotes.

In the second cluster, the most frequent haplotype is shared between GL-wolves and eastern coyotes and is closely related to, but not nested within, the majority of the western coyotes.


Microsatellite diversity was similar in the western gray wolves, GL wolves, eastern and western coyotes (Table 3), with slightly less diversity in wolves than in coyotes.

Slight, but significant departure from Hardy-Weinberg expectations at, and linkage disequilibrium between, several loci indicate some degree of population substructure within and introgression into GL wolves.

Extreme hetero- zygote deficiency at loci Ren239K24 and u225 in the eastern coyotes and Ren239K24 in the western coyotes might indicate the presence of null alleles; however, this should not have a large effect on the assignment tests performed (Carlsson 2008).


Structure analyses showed a peak in ΔK (Evanno et al. 2005) for K = 2, corresponding to two clusters, which sepa- rated wolves (western gray wolf plus GL wolf) and coyotes (Fig. 3a-c).

However, the log-likelihood values [ln Pr(X|K)] consistently increased up to K = 4, where the curve reached a plateau such that ln Pr(X|K) for values of K > 4 were almost identical (Fig. 3a).


For K = 3, the three clusters separated gray wolf, GL wolf and coyote, and for K = 4, clusters corresponded to gray wolf, GL wolf, eastern and western coyotes (Fig 3c).

Although ΔK for K of 3 and 4 were lower than for K = 2 (Fig. 3b), the distribution of samples into three and four clusters corresponded well to geographical groupings.


We assume that the drop in ΔK reflects less sub- structure within wolves and within coyotes than between the two species.

This conclusion is also suggested by the FCA analysis where axis 1 explained most of the variance and clearly differentiated wolves from coyotes (Fig. 3d).

Axis 2 separated GL wolves from western gray wolves and eastern from western coyotes.


Both Structure and FCA analyses showed that GL wolves were distinct from western Canadian wolves, but the clear separation may in part be due to the lack of wolf samples from intervening areas.

Supporting this conclusion is the presence of admixed genotypes in Alberta and further east in Ontario that show western wolf and GL wolf influences.

In contrast, the most geographically disparate wolf populations in Northwest Territories and southeast in Minnesota, Wisconsin and Michigan showed little evidence of admixture (Fig. 3c).


Despite the overall distinction between western gray wolves, GL wolves and coyotes, GL individuals with mixed ancestry were identified in Structure based on the autosomal microsatellite data.

These individuals, that are likely F 1 - hybrids, have varied combinations of Y-chromosomal and mtDNA haplotypes, which indicates a prior history of hybridization.

Surprisingly, some GL wolf individuals with high proportions of eastern coyote autosomal DNA had wolf-like Y-chromosomal and mtDNA haplotypes.

Also, some GL wolf individuals with high proportions of western gray wolf autosomal DNA had coyote-clade Y- chromosomal and mtDNA haplotypes.

All these data point to recurrent incidents of hybridization in the ancestry of these individuals.


Although we detect evidence for past episodic and ongoing hybridization between GL wolves, coyotes and western wolves, there is no indication of a unique hybrid origin of the GL wolf.

If the GL wolves owed their origin to a discrete episode of past hybridization, we would expect them to have consistently mosaic genotypes in the structure analysis and an intermediate position between western wolves and coyotes in the FCA plot.

Rather, our results are consistent with previous studies that showed varying degrees of ongoing and historic introgression across the Great Lakes area.

For example, as in Lehman et al. ( 1991), our microsat- ellite results suggest introgression of coyote genetic material into GL wolves is much more prominent north of the Great Lakes (Ontario, Quebec) than in the south (Minnesota, Wisconsin, Michigan).

Thus, the Great Lakes area appears to be a zone of limited hybridization between gray wolves, GL wolves and coyotes.


The test for heterozygosity excess revealed no evidence for a recent genetic bottleneck in the GL wolves (P = 0.945).

Although it is known that GL wolves experienced a severe bottleneck in the last century, the allelic signal has apparently been eroded due to high levels of introgression from both coyotes and other wolf populations.

Alternatively, the bottleneck may have been limited in geographical scope to the US Great Lakes states which were rapidly repopulated by wolves from Canada that have suffered less of a popu- lation decline.


Modern GL wolves differed significantly from the modern western gray wolves (θ ST = 0.0775, P < 0.0001), eastern (θ ST = 0.1419, P < 0.0001) and western coyotes (θ ST = 0.1329, P < 0.0001), indicating genetic isolation between GL wolves and both coyotes and western gray wolves.

The average number of alleles scored per historic sample (at 20 loci) was 33 (SD 6.7, range 17-40).

The average number of alleles used to calculate θ ST per microsatellite locus was 20.5 (SD 2.6, range 16-26).

Using the subset of 20 microsatellite loci which amplified most successfully from the historic material, the genetic composition of the historic GL wolves differed significantly from that of western gray wolves (θ ST = 0.0572, P < 0.0001), eastern (θ ST = 0.1225, P < 0.0001) and western coyotes (θ ST = 0.0898, P < 0.0001).

The majority of the 20 analysed loci were significantly differentiated between the historic GL wolves and the western gray wolves (13 loci), eastern (19 loci) and western coyotes (17 loci) (Appendix).

In contrast, only five loci showed significant differentiation between the historic and modern GL wolves (Appendix), whereas the remaining 15 loci were not differentiated between the historic and modern samples, despite the observed highly significant mitochondrial differentiation (see above and Leonard & Wayne 2008).

Apparently, some degree of genetic continuity has been retained over the last century despite population bottlenecks and current hybridization with coyotes and other wolf populations.

Nonetheless, despite the low number of significantly differentiated loci and the low global θ ST value of 0.0355, differentiation between the historic and the recent GL sample was significant with a global P < 0.0001.


The autosomal microsatellite data clearly indicate that the GL wolves should be considered gray wolves, despite the high proportion of coyote-like mitochondrial and Y- chromosomal haplotypes.

The numerous GL wolf haplotypes (or haplotype groups) in the coyote clade in the mtDNA and Y-chromosomal data, in particular the ones present in the historic samples and not shared with western coyotes, point to recurrent incidents of ancient introgression from coyotes into the GL wolves.

Since western coyotes apparently show no distinct phylogeographical substructuring (Lehman & Wayne 1991), this lack of haplotype sharing between historic GL wolves and western coyotes indicates that sufficient time has passed since introgression for new haplotypes to evolve, rather than being an artefact due to insufficient geographical sampling of coyotes.

We do not find evidence for a unique grouping of GL wolves in microsatellite, mtDNA or Y-chromosome analyses that would support the past presence of a unique species of wolf in the Great Lakes area (Wilson et al. 2000; Kyle et al. 2006), nor do we find evidence for a unique episode of hybridization that might have led to the intermediate phenotype of wolves throughout the region (Nowak 2002).


The Great Lakes wolves constitute a small-sized wolf ecotype that is adapted to the intermediate prey base of the region (Schmitz & Lavigne 1987).


Abundant evidence for ecological based factors explaining phenotypic and genetic diversity of gray wolves in the New and Old World is accumulating (Carmichael et al. 2001, Geffen et al. 2004; Pilot et al. 2006; Musiani et al. 2007; Carmichael et al. 2007; Muñoz-Fuentes et al. 2009).

For example, ecotypes asso- ciated with coastal habitats, Arctic islands, tundra, boreal forest and the Pleistocene megafauna have been identified (Musiani et al. 2007; Carmichael et al. 2007; Leonard et al. 2007; Muñoz-Fuentes et al. 2009).


Many of these ecotypes have evolved recently, in the past 10 000 years with the retreat of the Laurentide and Cordilleran ice sheets.


The GL wolves, however, have likely had a long history, perhaps 300 000 years, based on mtDNA sequence divergence (Leonard & Wayne 2008).

Therefore, the Great Lakes ecotype may have persisted for a long time period despite genetic exchange with coyotes and other gray wolves.


The presence of coyote clade haplotypes in historic GL wolves suggests an ancient history of interbreeding between coyotes and GL wolves.

Hybridization between GL wolves and coyotes may be more likely when the former is relatively rare (Lehman et al. 1991).

This condition may have occurred repeatedly with the recurrent cycles of glaciation and deglaciation in the Pleistocene and the associated change in range and the relative abundance of the two species in eastern North America.

Our results are consistent with the idea that glacial cycles repeatedly pushed the GL wolves southward into the original range of coyotes where they experienced introgression from the resident species.


However, the reason why hybridization between coyotes and wolves is so extensive in the Great Lakes area and not elsewhere in Canada or the USA where the species now co-exist is uncertain, but it may have to do with the smaller disparity in size between GL wolves and coyotes (e.g. Lehman et al. 1991; Roy et al. 1994; Pilgrim et al. 1998; Hailer & Leonard 2008).


In addition to evidence for ancient introgression of coyote mitochondrial and Y-chromosomal DNA into GL wolves, our results suggest extensive recent and ongoing hybridization between GL wolves and both coyotes and other populations of gray wolves.

Microsatellite genotypes and mtDNA haplotypes suggested the presence of recently admixed wolves in the Great Lakes region (Fig. 3c, d).

Similarly, significant mitochondrial differentiation between historic and modern GL wolves is consistent with extensive recent gene flow between GL wolves and eastern coyotes and other wolf populations.

In contrast, little recent gene flow between GL wolves and eastern coyotes is indicated by Y- chromosomal data, suggestive of sex-biased introgression (see Lehman et al. 1991).