QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.103.064956v1 310/1/8    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Toth, P. T. Articles by Miller, R. J. Search for Related Content PubMed PubMed Citation Articles by Toth, P. T. Articles by Miller, R. J.

CELLULAR AND MOLECULAR

Regulation of CXCR4 Receptor Dimerization by the Chemokine SDF-1 and the HIV-1 Coat Protein gp120: A Fluorescence Resonance Energy Transfer (FRET) Study

Department of Molecular Pharmacology and Biological Chemistry, The Feinberg School of Medicine, Northwestern University, Chicago, Illinois

Received December 26, 2003; accepted March 10, 2004.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Both the chemokine SDF-1 and the human immunodeficiency virus-1 (HIV-1) coat protein gp120 can bind to CXCR4 chemokine receptors but with different signaling consequences. To understand the molecular basis for these differences, we tagged the rat CXCR4 receptor with enhanced cyan (ECFP) and yellow (EYFP) derivatives of the green fluorescent protein and investigated CXCR4 receptor dimerization in human embryonic kidney (HEK)-tsA201 cells using fluorescence resonance energy transfer (FRET). Elevated FRET was detected under basal conditions from EYFP-CXCR4 and ECFP-CXCR4 receptor-transfected cells indicating a high level of CXCR4 receptor dimerization. In comparison, EYFP-CXCR4 and ECFP-µ-opioid receptor-cotransfected cells displayed a much lower FRET signal. The FRET signal resulting from EYFP-CXCR4- and ECFP-CXCR4-expressing cells could be attenuated by coexpressing nontagged CXCR4 receptors suggesting competition with fluorophore-tagged receptors in the membrane. Nontagged µ-opioid, -opioid, and muscarinic receptors also decreased the FRET between the tagged CXCR4 receptor pairs but to a lesser extent. Application of the CXCR4 receptor agonist SDF-1 (50 nM) further increased the FRET signal from tagged CXCR4 receptors, an effect that was inhibited by the CXCR4 antagonist AMD3100. SDF-1 had no effect when EYFP-CXCR4 and ECFP-µ-opioid receptors were coexpressed. The effect of gp120IIIB on CXCR4 FRET was dependent on the coexpression of human CD4 (hCD4) when it increased the FRET signal, and this was decreased by AMD3100 pretreatment. FRET analysis of tagged hCD4 constructs demonstrated that there was significant association of hCD4 and CXCR4, as well as hCD4 dimerization. These data suggest that CXCR4 dimerization is involved in SDF-1- and gp120-induced signaling events.

In addition to their role as chemokine receptors, CXCR4 receptors also play a central role in the pathogenesis of AIDS (Berger et al., 1999). CXCR4 serves as a coreceptor for gp120, the major coat protein of human immunodeficiency virus-1 (HIV-1), allowing attachment of the virus to its target cell and insertion of viral DNA into its host. According to the most prevalent view, gp120 initially binds to the human CD4 (hCD4) molecule. This produces a conformational change in the gp120 molecule enabling it to bind to the CXCR4 receptor with high affinity (Doms, 2000). However, it is interesting to note that gp120 has been reported not only to bind to CXCR4 receptors but also to produce a variety of agonist- and antagonist-like effects as a result of this interaction (Miller and Oh, 2002). Moreover, some of these effects appear to be independent of hCD4. For example, gp120 exerts toxic effects on neurons and on endothelial cells which express CXCR4 but not hCD4 (Huang et al., 1999; Ullrich et al., 2000; Miller and Oh, 2002; Bodner et al., 2003). Thus, it has been suggested that some effects of gp120 might involve interaction with the CXCR4 receptor in a "CD4-independent" fashion, although the truth of this suggestion remains to be determined (Hesselgesser et al., 1997; Bodner et al., 2003).

Fluorescence resonance energy transfer (FRET) is a phenomenon that occurs between closely associated fluorophore molecules (Stryer, 1978). FRET can only take place if there is spectral coincidence and optimal separation between the participating fluorescent partners. The emission spectrum of the donor molecule must overlap with that of the acceptor molecule's excitation spectrum. The energy transfer also depends on the distance between the donor and the acceptor molecules, as well as their correct orientation. Since FRET efficiency decreases as the inverse sixth power of distance, the acceptor must be in close proximity to the donor (<10 nm) (Stryer, 1978). Monitoring energy transfer between the enhanced cyan (ECFP) and yellow (EYFP) versions of the green fluorescent protein has frequently been used to detect molecular interactions between proteins, including both cytoplasmic (Ruehr et al., 1999) and membrane proteins (Zhou et al., 2003) such as GPCRs (Dinger et al., 2003). Using FRET, bioluminescence resonance energy transfer (BRET), and related methods, it has become clear that many GPCRs can dimerize or exist as higher order arrays and that this association can greatly alter their signaling properties (Angers et al., 2002). Furthermore, in many instances, GPCR dimerization is at least partially driven by agonist binding (Angers et al., 2002). Consequently, in the present series of experiments, we have used FRET to determine the effects of different types of ligands on the molecular state of the CXCR4 receptor and, in particular, to compare the effects of SDF-1 and gp120. Our results are consistent with the existence of both agonist-dependent and -independent dimerization of the CXCR4 receptor as well as constitutive association of CXCR4 and hCD4.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Synthesis of ECFP and EYFP Constructs. The rat CXCR4 was cloned from rat brain total RNA to pCDNA3.1 plasmid (Oh et al., 2001). PCR amplification of CXCR4 DNA from the CXCR4/pCDNA3.1 construct was carried out with plaque-forming unit DNA polymerase (Promega, Madison, WI). Subsequently, a HindIII/AgeI enzyme site was added using the forward primer 5'-cgg cta aag ctt gcc gcc acc atg gaa ata tac-3' and the reverse primer 5'-atc agc ggg tac cgg tct gga gtg aaa act tga-3' by PCR. After heating to 96°C for 5 min, PCR amplification was carried out for 30 cycles: 96°C for 30 s, 56°C for 1 min, and 72°C for 2 min. The PCR product was digested with HindIII/AgeI followed by a gel purification (QIAGEN, Valencia, CA) and was ligated into pEYFP-N1 (BD Biosciences Clontech, Palo Alto, CA) as well as into pECFP-N1 (BD Biosciences Clontech). The EYFP or ECFP were fused to the C-terminal end of the rat CXCR4. The resulting clone was verified by restriction enzyme analysis and fully sequenced using the dRhodamine Terminator cycles sequencing kit (Applied BioSystems, Foster City, CA).

The rat µ-opioid receptor was amplified from rat brain cDNA and cloned to pEYFP-N1 and pECFP-N1 (Bushell et al., 2002). The mouse -opioid receptor was a gift from Dr. Graeme Bell (Howard Hughes Medical Institute, University of Chicago, Chicago, IL).

The human CD4 receptor (in pMV7 plasmid) was obtained through the AIDS Research and Reference Reagent Program (Bethesda, MD; contributor, Dr. Richard Axel). The hCD4 DNA was PCR-amplified (PCR parameters are the same as described above) from the hCD4/pMV7 with plaque-forming unit turbo DNA polymerase (Stratagene, La Jolla, CA) using the following primer 5'-tc ctc gga att ccc aca atg aac cgg-3' and 5'-cgt ggg atc cat ggg gct aca tgt ctt-3', which contained the EcoRI or BamHI site. The hCD4 EcoRI and BamHI fragment was subcloned into the pEYFP-N1 or pCFP-N1 (BD Biosciences Clontech). The construct was verified by sequencing. The human muscarinic receptors (m1 and m3) were kindly provided by Dr. Marlene Hosey (Northwestern University).

Cell Culture and Transfection. HEK-tsA201 cells were grown in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA), 5% fetal bovine serum (Cellgro; Mediatech Inc., Herndon, VA), and 1% penicillin-streptomycin (Cellgro) at 37°C in 5% CO₂. One day before transfection, cells were replated onto poly-L-lysine-coated (Sigma-Aldrich, St. Louis, MO) 25-mm circular microscope cover-glasses. Transient transfections were performed using polyethyleneimine (Boussif et al., 1996) keeping the total DNA concentration constant (5 µg/well) and the DNA ratio of ECFP/EYFP construct = 3:2 throughout every transfection. Cells were treated 48 h after the transfection in the tissue culture medium with the relevant compounds for the time period indicated in the figures. The cells were fixed in 4% paraformaldehyde after the treatment, washed with PBS three times, and the coverslips were mounted with the ProLong Antifade kit (Molecular Probes, Eugene, OR) onto a glass microscope slide. Samples were analyzed the following day.

SDS-Polyacrylamide Gel Electrophoresis and Western Blotting. Ten percent polyacrylamide/SDS gels were prepared using mini-PROTEAN 3 cell (Bio-Rad, Hercules, CA) equipment. The total cellular protein of cells scraped from each culture dish well was measured by a protein microplate assay kit (Bio-Rad). For each transfected cDNA, total protein of equal quantity (10 µg/lane) was loaded onto an SDS gel, which was run at 150 V for 1.5 h. The proteins were electroblotted to polyvinylidene difluoride membranes (Bio-Rad) in 25 mM Tris, 192 mM glycine, and 20% methanol. After the electrotransfer, the membranes were blocked in 5% skimmed milk in PBS and immunostained with anti-GFP antibody (BD Biosciences Clontech) 1:1000 for 1.5 h. After washing, blots were incubated with horseradish peroxidase-conjugated secondary antibody (1:2000; Biochemicals Inc., Santa Cruz, CA) for 1 h at room temperature. Using the enhanced chemiluminescence detection kit, a membrane was developed and exposed to the hyperfilm.

FRET Analysis. Images were collected with an upright fluorescence microscope (Olympus BX50WI) equipped with an intensified Penta-MAX CCD camera (Princeton Instruments, Trenton, NJ) and controlled by MetaMorph software (Universal Imaging Corp., Downingtown, PA; version 4.5) using a 100x NA1.3 objective. The following filter sets were used for the fluorescence channels: EYFP (excitation filter 500 ± 20 nm, beam splitter 515 nm long pass, and emission filter 535 ± 15 nm), ECFP (440 ± 20 nm, 455 nm, and 485 ± 20 nm), and FRET (440 ± 20 nm, 455 nm, 535 ± 15 nm). Four consecutive images (image set) were collected from the same field using the filter settings for EYFP, FRET, ECFP, and the 340-nm excitation filter to obtain the background image. Emissions from ECFP or EYFP constructs were not detectable using the 340-nm excitation filter; however, in addition to the background noise, the background image also contained the autofluorescence signal from the cells. Due to spectral overlap, the acquired FRET image also contains emissions from ECFP and EYFP besides the FRET signal. The following formula has been used to obtain the corrected FRET where the three filter sets have been applied for image acquisition: F^c = Ff - Df(Fd/Dd) - Af(Fa/Aa) (Gordon et al., 1998) where Ff is a background subtracted FRET image obtained with the FRET filter set, Df is the background subtracted ECFP image obtained with the ECFP filter set, and Af is the background subtracted EYFP image obtained with the EYFP filter set. The Fa/Aa ratio represents the percentage of EYFP contribution into the FRET channel in preparations that only express EYFP-tagged construct. Similarly Fd/Dd ratio represents the percentage of ECFP contribution to the FRET image in preparations where only ECFP is expressed. In a separate set of experiments, we determined the values for Fa/Aa and Fd/Dd as 5 and 83%, respectively. The magnitude of detected energy transfer also depends on the amount of donor and acceptor fluorophores expressed in the cells. The literature offers various means to normalize the FRET signal to the expression of fluorophores in the cells (Gordon et al., 1998; Xia and Liu, 2001). The corrected FRET signal has been normalized to the donor fluorescence (F^c/Df), to the product of the donor and the acceptor fluorescence (F^c/Df · Af), or to the square root of the product of the donor and acceptor fluorescence (F^c/(Df · Af)^1/2). We compared the results obtained by all three various ways of normalizations on a repeated set of experiments using identical conditions (data not shown) and found, in agreement with Xia and Liu (2001), that normalizing to the square root of the product of the donor and acceptor provides the smallest difference between means of the results originating from separate sets of experiments and the smallest standard error within one experiment when the intensities from the regions of interests were averaged. The square root normalized data are displayed in our figures with the term "FRET ratio" throughout our paper. To calculate the FRET ratio, we took four to six image sets (each consisting of a CFP, YFP, FRET, and background image) from each coverslip. From each image set containing 1 to 10 cells (depending on the expression density), we assigned one to five membrane ROI per cell (depending on the expression levels) using the Metamorph software (see Fig. 1C for an example that displays three regions of interest). From these ROI, the intensity values of each background subtracted FRET, EYFP, and ECFP ROI were inserted into the FRET ratio equation. This way we obtained 15 to 60 ROI intensity values in each of the experimental conditions. If a cell did not display emission from either the EYFP or the ECFP construct, the cell was discarded from analysis. Only 1 to 2% of the cells expressed only one of the fluorophores. Experimental conditions were repeated two to six times, and we included the "10-min SDF-1 treatment" of EYFP-CXCR4 ECFP-CXCR4 receptor pair and its control in every set of experiments as an internal control because this treatment consistently gave us an increased FRET ratio. In the initial set of experiments, we applied the algorithm described above on a pixel-by-pixel basis to entire images using the built-in functions of Metamorph, Matlab, or NIH ImageJ to calculate the FRET ratios. Subsequently, we incorporated the FRET ratio equation into a Microsoft Excel table and applied the FRET, ECFP, and EYFP average values from the same ROIs. After a thorough comparison using the same data set with the two methods, we chose to use the second method because we found that the difference between the results obtained by the two methods was negligible, and the second method was considerably more efficient.

View larger version (28K): [in this window] [in a new window]   Fig. 1. FRET between different tagged receptor pairs. A, FRET ratio values under various experimental conditions. The ratio of the expressed constructs (EYFP to ECFP) and the plasmid concentrations used for transfections were kept constant throughout these experiments (see Materials and Methods). The normalized FRET ratio is defined under Materials and Methods. Data are displayed as mean ± S.E.M. The calculated FRET ratios are from cells expressing different protein pairs as indicated. The C-Y concatemer is a construct in which ECFP and EYFP fluorophores are directly linked (Zhou et al., 2003). CC + YY denotes the FRET ratio from cells in which ECFP-ECFP dimers and EYFP-EYFP dimers were coexpressed (column 2, n = 61 ROIs). Columns 4 to 7 display FRET ratios from cells in which the EYFP- and ECFP-tagged CXCR4 receptors were coexpressed. The basal CXCR4 FRET ratio is high (column 4, n = 24 ROIs) and was further increased by a 10-min SDF-1 (50 nM) application (column 6, n = 26 ROIs). The FRET ratio resulting from EYFP- and ECFP-tagged µ-opioid receptor expressing cells is displayed in column 8. Note that the basal FRET ratio originating from the fluorophore-tagged µ-opioid receptor (MOP) pairs (µ-opioid receptors; column 8, n = 31 ROIs) is much lower compared with that of the fluorophore-tagged CXCR4 receptor pairs. Columns 9 to 10 display FRET ratio from EYFP-tagged MOP and ECFP-tagged CXCR4 receptor coexpressing cells (n = 16–19 ROIs for each condition). B, superimposed nonoverlapping CXCR4-ECFP (green) and cytoplasmic EYFP-EYFP (red) expression. C, the collected EYFP (top panel), ECFP (middle panel), and FRET (bottom panel) images from tsA201 cells (100x NA1.3 objective). Typical regions of interests (ROI) are marked with red. Membrane areas that contained saturated pixels (such as the membrane area where the three ROIs would converge on this image) were not included as ROI and excluded from analysis. Emissions from out of focus membrane areas appeared as a blur on the image. Colors are arbitrarily assigned to indicate signal strength. Calibration bar, 10 µm.

Source of Drugs. SDF-1 was purchased from R&D Systems (Minneapolis, MN). Lyophilized proteins were reconstituted in 0.1% bovine serum albumin/PBS, and aliquots were stored at -20°C. gp120-, HIV-1IIIB, and AMD3100 were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program (Bethesda, MD). Gp120IIIB was reconstituted in 0.1% bovine serum albumin/PBS solution (100 µg/ml) and stored at -70°C. SDF-1 and gp120IIIB were diluted to the final concentration in the tissue culture medium.

Statistics. The results were analyzed using one-way analysis of variance with Bonferroni's (Figs. 2, 4, and 6) and Dunnett's (Figs. 1, 3, 5, 7, and 8) post tests.

View larger version (26K): [in this window] [in a new window]   Fig. 2. Effect of plasmid ratio on CXCR4 receptor associated FRET signals. A, 5 µg/dish cDNA was used for transfection in each condition and the EYFP to ECFP ratio were kept constant throughout the experiments (see Materials and Methods). Decreasing the amount of CXCR4 cDNA was substituted by empty pCMV7 vector. Note that the basal CXCR4 FRET ratio was still high, and the effect of a 10-min SDF-1 (50 nM) application on FRET became more pronounced with decreased receptor construct concentrations. Further reduction in the concentration of tagged receptor cDNA impaired the reliability of cotransfection (n = 39–78 ROIs for each condition). B, Western blot analysis of EYFP- and ECFP-tagged CXCR4 and hCD4 expression. Lysates from tsA201 cells show the expression of fluorophore-tagged proteins. cDNAs encoding ECFP- and EYFP-tagged constructs were applied to cells in 10-cm culture dishes during transient transfection at a concentration which corresponds to 2 µg/dish and 1.35 µg/dish, respectively. Specificity of the antibody was confirmed by the lack of staining in untransfected tsA-201 cell membranes.

View larger version (12K): [in this window] [in a new window]   Fig. 4. Effect of hCD4 expression on CXCR4-associated FRET signals. Coexpression of hCD4 did not change the basal FRET between fluorophore-tagged CXCR4 receptors (columns 1–2). However, it significantly decreased the effect of a 10-min SDF-1 (50 nM) application (columns 3–4). n = 299 to 350 ROIs for each condition.

View larger version (25K): [in this window] [in a new window]   Fig. 6. Effect of the CXCR4 antagonist AMD3100 on CXCR4-associated FRET. In the absence of coexpressed hCD4 (columns 1–4), AMD-3100 slightly inhibited the basal FRET (column 2) and completely inhibited the effect of a 10-min SDF-1 (50 nM) application (column 4). The effect of AMD-3100 on gp120IIIB-enhanced FRET was also tested in cells in which hCD4 was coexpressed (columns 5–7). The gp120IIIB effect was also reduced by AMD-3100 (column 7). n = 79 to 129 ROIs for each condition.

View larger version (15K): [in this window] [in a new window]   Fig. 3. Competition between tagged CXCR4 receptors and untagged GPCRs. EYFP- and ECFP-tagged CXCR4 receptors were coexpressed with various third constructs as indicated. Nontagged CXCR4 receptor (column 3) was the most effective in decreasing the FRET between fluorophore-tagged CXCR4 receptor pairs (n = 159–215 ROIs for each condition; KOP, -opioid receptors, MOP, m1, and m3).

View larger version (17K): [in this window] [in a new window]   Fig. 5. Effect of gp120IIIB on CXCR4-associated FRET. Gp120IIIB was applied to EYFP- and ECFP-tagged CXCR4 receptor-expressing cells for the time periods indicated in the presence and absence of coexpressed hCD4. The last two columns (columns 7 and 8) display the effect of a 10-min SDF-1 (50 nM) application to the cells for comparison (n = 104–136 ROIs for each condition).

View larger version (22K): [in this window] [in a new window]   Fig. 7. Interactions between hCD4 and CXCR4. Coexpression of hCD4-Y with CXCR4-C resulted in a larger FRET signal than that resulting from hCD4-Y and µ-opioid receptor coexpression (columns 1 and 2, n = 62–67 ROIs). The FRET signal between hCD4-Y and CXCR4-C was not modified by SDF-1 or gp120IIIB (columns 3–6, n = 29–47 ROIs for each condition).

View larger version (16K): [in this window] [in a new window]   Fig. 8. Dimerization of tagged hCD4 subunits. HEK-tsA201 cells coexpressing hCD4-Y and hCD4-C receptors displayed an elevated FRET signal. Application of gp120IIIB (200 pM, 2 h) increased the FRET signal significantly (n = 40–60 ROIs for each condition).

	Results

Top Abstract Materials and Methods Results Discussion References

 
"Calibration" of CXCR4-Associated FRET Signals. We have previously demonstrated that CXCR4 receptors, C-terminally tagged with GFP derivatives, are fully functional in a variety of assays (Bodner et al., 2003). We used three control constructs to carry out the initial FRET measurements, set the plasmid concentrations used for subsequent transfections, and approximate the range of the normalized FRET values obtainable using the current paradigm. We expressed concatenated EYFP-ECFP and also coexpressed concatenated ECFP-ECFP and EYFP-EYFP in HEK-tsA201 cells. A 9-amino acid linker was used to join the two fluorophores (Zhou et al., 2003). Coexpression of EYFP-EYFP and CXCR4-ECFP resulted in a nonoverlapping expression pattern (Fig. 1B). Calculating the normalized FRET from membrane areas under these conditions did not yield positive FRET values. The FRET ratio obtained from the EYFP-ECFP construct was much larger than that resulting from EYFP-EYFP and ECFP-ECFP coexpression (Fig. 1A, columns 1 and 2). In addition, if equal amounts of EYFP DNA and ECFP DNA were expressed in cells (or in the case of the concatenated EYFP-ECFP construct where the expressed protein contained the fluorophores in the 1:1 ratio), the ratio of signal intensities for ECFP/EYFP was 1:10 from cells with EYFP-ECFP concatemer expression and 1:13 from cells expressing EYFP-EYFP and ECFP-ECFP. This would be expected from the fact that ECFP has lower quantum efficiency than EYFP. Since our measurements required the separate acquisition of ECFP and EYFP intensities, in addition to the FRET signal, we slightly altered the ratio of the receptor DNAs used for transfections to increase the detection of the ECFP signal and decrease the saturation of the EYFP signal using the same illumination and camera amplification settings for image acquisition. Thus, in subsequent experiments, we expressed the ECFP- and EYFP-tagged receptor constructs in the ratio of 3:2.

FRET between EYFP- and ECFP-Tagged CXCR4 Receptors. Coexpression of EYFP- and ECFP-tagged CXCR4 receptors resulted in a FRET ratio that was significantly greater than that obtained with coexpression of EYFP and ECFP control constructs. Application of the CXCR4 receptor agonist SDF-1 (50 nM) further increased the FRET ratio in a time-dependent manner. The maximum FRET ratio was observed at 10 min, after which the signal decreased again (Fig. 1A, columns 4–7).

FRET between EYFP- and ECFP-Tagged µ-Opioid Receptors. As a comparison with the results for CXCR4 receptors, we coexpressed EYFP- and ECFP-tagged µ-opioid receptors. The basal FRET ratio associated with this receptor pair was much lower than that associated with tagged CXCR4 receptors.

Interestingly, coexpression of EYFP-CXCR4 receptors and ECFP-µ-opioid receptors resulted in a basal FRET ratio that was higher than the FRET ratio obtained from EYFP- and ECFP-tagged µ-opioid receptor expressing cells, but lower than that resulting from EYFP- and ECFP-tagged CXCR4 receptor coexpression (Fig. 1, column 9). Application of SDF-1 (50 nM) did not significantly change the FRET ratio from EYFP-CXCR4 receptor and ECFP-µ-opioid receptor expressing cells (Fig. 1A, column 10).

Optimization of cDNA Concentration Used for Transfection. Since we intended to use a third cDNA construct in some of the following experiments, we had to optimize the plasmid concentrations used for these transfections. We gradually decreased the tagged CXCR4 receptor cDNA (keeping the same ratio between the EYFP-CXCR4 and the ECFP-CXCR4) and introduced an empty control vector cDNA into the transfection mixture leaving the total cDNA concentration constant (5 µg/dish). Lowering the amount of the tagged CXCR4 receptor, cDNA decreased the basal FRET ratio although it still remained well above the background. SDF-1 was effective in increasing the FRET ratio at all concentrations of receptor used (Fig. 2A). Western blotting confirmed that tagged proteins were expressed in approximately equal amounts, and the tagged constructs were not degraded (Fig. 2B).

Coexpression of Nontagged Receptors Decreases the Basal FRET Ratio. The elevated FRET ratio associated with tagged CXCR4 receptors suggests a relatively high affinity interaction between these tagged proteins. Therefore, it would be predicted that untagged receptors might compete for binding interactions between tagged receptors. In keeping with this prediction, coexpression of nontagged CXCR4 receptors with the EYFP-CXCR4 and ECFP-CXCR4 receptor pair reduced the basal FRET ratio (Fig. 3); however, the presence of untagged receptors did not reduce the effect of SDF-1 (not shown). Interestingly, the expression of untagged - or µ-opioid receptors also decreased the basal FRET ratio significantly but to a lesser extent than untagged CXCR4 receptor. SDF-1 was still effective under these conditions as well (data not shown). -Opioid or µ-opioid receptors are considered to couple to the G_o/G_i class of G proteins. We wondered whether the ability of GPCRs to interact with CXCR4 was specific for G_o/G_i-linked GPCRs. Therefore, we also expressed the CXCR4 receptor together with the muscarinic 1 (m1) and 3 (m3) receptors, which are thought to couple to the G_q class of G proteins (Budd et al., 2003). Although the m3 receptor was able to reduce CXCR4-associated FRET to some extent, the m1 muscarinic receptor was ineffective in this regard (Fig. 3). Thus, we conclude that the structural determinants that govern GPCR interactions with CXCR4 receptors are not obviously correlated with the class of G protein they normally activate.

Effect of hCD4 on FRET between Tagged CXCR4 Receptors. As discussed above, the HIV-1 coat protein gp120 utilizes the CXCR4 receptor to facilitate its binding and infection of target cells. In addition, the hCD4 molecule is frequently used as a coreceptor; however, effects of gp120 that are hCD4-independent have also been reported (Hesselgesser et al., 1997; Miller and Oh, 2002). To probe the interactions of gp120 with the CXCR4 receptor, we began by testing the effect of hCD4 coexpression on the FRET originating from tagged CXCR4 pairs. Empty vector was coexpressed in control experiments to maintain the transfected cDNA concentration constant. hCD4 coexpression did not modify the basal FRET ratio; however, it significantly reduced the effect of SDF-1 (Fig. 4). It is interesting to note in this context that the interaction of hCD4 with the CCR5 chemokine receptor similarly reduces the activation of that receptor by its agonist MIP-1 (Wang and Staudinger, 2003).

Effect of gp120IIIB on FRET between Tagged CXCR4 Receptors. Gp120IIIB is a version of gp120 that is able to bind to CXCR4 receptors and produce a large number of typical effects including widely described neurotoxicity (Miller and Oh, 2002). This has included reports of hCD4-independent effects (Miller and Oh, 2002). In the next set of experiments, we investigated the effects of gp120IIIB on FRET resulting from the expression of tagged CXCR4 receptors. The results from this series of experiments are displayed in Fig. 5. The effect of SDF-1 under the same conditions is redrawn from Fig. 4 as a comparison. The nature of the effect of gp120IIIB on the FRET ratio proved to be dependent on hCD4 expression and followed a different time course from that seen with SDF-1. In the absence of hCD4 expression, gp120IIIB (200 pM) actually decreased the FRET ratio between tagged CXCR4 receptors. The SDF-1-induced increase in the FRET ratio was not affected by gp120 either in a short (10 min) or longer (2 h) incubation period in the absence of hCD4 expression (not shown). However, in the presence of hCD4 expression, gp120IIIB increased the FRET ratio in a time-dependent fashion. The most pronounced gp120IIIB effect in hCD4-expressing cells was observed after 2 h of treatment (Fig. 5), which is substantially slower than the effect produced by SDF-1. Furthermore, the gp120IIIB-evoked increase in FRET ratio never reached the magnitude produced by SDF-1 application.

AMD3100 has been widely demonstrated to antagonize the effects of SDF-1 on CXCR4 receptors (Hatse et al., 2002). Interestingly, AMD3100 produced a significant decrease in the FRET ratio resulting from tagged CXCR4 expression. In addition, the effects of SDF-1 and gp120IIIB were completely inhibited (Fig. 6).

Association of CXCR4 Receptors and hCD4. As discussed above, there is considerable evidence that the hCD4 molecule acts as a "coreceptor" together with CCR5 and/or CXCR4 receptors to facilitate HIV-1 entry. We therefore used the FRET paradigm to examine the degree to which hCD4 normally associates with the CXCR4 receptor. As can be seen in Fig. 7, a significant degree of FRET was observed when tagged CXCR4 and hCD4 were coexpressed. In contrast, we observed that only very low levels of FRET were observed between hCD4 and the µ-opioid receptor. Thus, as is the case of CXCR4 receptors, it appears that CXCR4 and hCD4 are associated constitutively to a significant extent. The interaction between these two proteins was not altered by either SDF-1 or by gp120IIIB. Interestingly, the hCD4 molecule also appeared to self-associate. When CFP- and YFP-tagged hCD4 were coexpressed, we observed a significant degree of FRET (Fig. 8). Furthermore, the FRET signal was enhanced following a protracted incubation with gp120IIIB.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The CXCR4 chemokine receptor has been of great interest for several reasons. First, signaling through this receptor by its unique agonist, the chemokine SDF-1, controls many important processes ranging from chemotaxis of leukocytes to the migration and maturation of stem cells in different tissues (Kim and Broxmeyer, 1999). Second, CXCR4 receptors are also of central importance in the regulation of inflammatory responses and in the pathogenesis of AIDS (Berger et al., 1999; Horuk, 2001). Hence, it is critical for us to understand the precise pharmacology of these receptors and how different types of ligands elicit or antagonize CXCR4-mediated signaling. To this end, there are several key questions to be considered. For example, it is unclear how SDF-1 normally activates the receptor. In this instance, a central role for agonist-driven receptor dimerization has been suggested (Rodriguez-Frade et al., 2001). Furthermore, the precise signaling consequences resulting from the interaction of gp120 with the receptor are not clear. In this regard, it has become increasingly apparent that the interaction of gp120 with the receptor can elicit or antagonize signaling events that greatly influence the survival of target cells (Miller and Oh, 2002). Furthermore, the precise role of the hCD4 molecule in gp120-mediated effects is also unclear in many instances (Bodner et al., 2003). The results reported in this paper suggest that the CXCR4 molecule and the hCD4 molecule exist, to a significant extent, as preformed complexes which contain homomeric and heteromeric interactions between the different subunits. Furthermore, the results suggest that dimerization of CXCR4 receptors occurs in an agonist-dependent and -independent manner.

In keeping with the proposed role of dimerization in CXCR4 signaling, numerous studies have demonstrated that GPCRs exist not only as monomers but can also form homo- and heterodimers as well as higher order structures (Angers et al., 2002). In some cases, dimerization has been shown to fundamentally transform the properties of GPCRs (Gomes et al., 2001). In addition to biochemical studies using immunoprecipitation to suggest the existence of GPCR dimers (Hebert et al., 1996), recent studies have used biophysical techniques to support the existence of dimers in live cells (Angers et al., 2000; Ramsay et al., 2002; Dinger et al., 2003). In the present article, we have used FRET to monitor energy transfer between the EYFP-tagged CXCR4 receptor and the ECFP-tagged CXCR4 receptors expressed in HEK-tsA201 cells.

Initially, we used control ECFP and EYFP constructs to test the sensitivity of the present paradigm. Predictably, the largest FRET signal was associated with expression of the concatenated EYFP-ECFP pair, in which a 9-amino acid linker provided a very short fixed distance between the two fluorophores. A very low, but still appreciable, FRET signal was associated with cells where EYFP-EYFP and ECFP-ECFP constructs were coexpressed. In contrast, we did not obtain any measurable FRET in cases where we coexpressed two tagged proteins one of which had a nuclear and the other a cytoplasmic localization (data not shown). Thus, it is likely that some low degree of FRET results from random interactions between any tagged protein pair expressed in the same cellular compartment. This observation prompted us to design a series of experiments to differentiate FRET signals resulting from specific interactions between CXCR4 receptors in dimers versus those resulting from increased CXCR4 receptor "concentration" in the membrane (see below).

GPCR homodimerization has been described in the case of 2-adrenergic (Angers et al., 2000), dopamine (Ng et al., 1996), - and -opioid (Ramsay et al., 2002), M3 muscarinic (Zeng and Wess, 1999), mGluR5 (Romano et al., 1996), and NPY1,2,5 (Dinger et al., 2003), including the CXCR4 (Vila-Coro et al., 1999; Babcock et al., 2003) and CCR5 (Issafras et al., 2002) chemokine receptors. However, the degree of constitutive relative to agonist-induced association has been reported to differ widely in each instance. In some cases, receptor ligand interactions are absolutely necessary to produce dimer formation (Angers et al., 2002). In other instances, receptor dimers are formed under basal conditions and ligand-receptor interactions may enhance the energy transfer between the receptors—suggesting either agonist-induced facilitation of dimer formation or agonist-induced reorientation of dimer subunits (Angers et al., 2002). In our experiments, we found a high level of basal FRET resulting from CXCR4 receptor homodimerization. This is in agreement with Babcock et al. (2003) who also observed ligand-independent dimerization of CXC4 receptors. We also observed that the FRET ratio could be modestly enhanced by the addition of the CXCR4 receptor agonist SDF-1 in a time-dependent manner displaying a maximum effect around 10 min. This might suggest a model whereby monomeric and dimeric receptors exist in equilibrium with each other and that this can be influenced by agonist binding. On the other hand, SDF-1-induced conformational changes may enhance the interactions between the subunits of existing dimers. In the studies of Babcock et al. (2003), SDF-1 also tended to produce an increase in FRET although this did not reach significance. However, this apparent difference may be explained by the fact that, in the present series of studies, CXCR4 association was measured only in the cell membrane (see Fig. 1C) whereas in Babcock et al. (2003), interactions covering the entire cell were included. Indeed, it has frequently been demonstrated that CXCR4 molecules constantly cycle between cell membrane and intracellular membrane compartments, and that at steady state there is a considerable pool of receptors within the cell (Gillard et al., 2002). Presumably SDF-1 would only regulate receptors that are expressed in the plasma membrane. Thus, it would be important to concentrate solely on this subset of receptors if one wished to fully appreciate the effects of the agonist.

The specificity of CXCR4 dimer formation is supported by the receptor competition experiments. Coexpressing nontagged CXCR4 receptors with tagged CXCR4 dimers decreased the basal FRET. Competition between tagged and nontagged receptors would be predicted to reduce the number of tagged dimers formed and reduce FRET. Interestingly, the nontagged µ-opioid (G_i-linked), -opioid (G_i-linked), and m3 muscarinic (G_q-linked) receptors decreased FRET between tagged CXCR4 receptor pairs although not to the same extent as untagged CXCR4. On the other hand, the untagged m1 muscarinic receptor (G_q-linked) was ineffective in this regard. Such a result is also consistent with our observation that coexpressed tagged CXCR4 and µ-opioid receptors produced a significant amount of FRET. These data suggest two things. The first is that the "competition" between tagged and untagged CXCR4 receptors is not just the result of dilution of tagged molecules in the membrane, and second, there is a degree of specific association between CXCR4 and other types of GPCRs such as opioid receptors. Thus, the motifs that govern the interaction between CXCR4 receptors are presumably preserved to some degree among other GPCRs although this is not correlated with the ability of a GPCR to interact with a particular type of G protein. It is interesting to note that m3 receptors, which have been reported to be able to dimerize (Zeng and Wess, 2000), were able to compete with the CXCR4 receptor dimer formation. In contrast, m1 muscarinic receptors have been reported to be unable to form heterodimers either with the m3 (Zeng and Wess, 2000) or the CXCR4 receptor (present study), and no reports of m1 homodimerization exist. Hence, there is clearly some selectivity associated with CXCR4 heterodimerization.

How do our data help us to understand the interactions of gp120 with the CXCR4 receptor? There are several issues here. The first is that it is clear that gp120 does not just bind to the receptor but that this binding also has some "agonist"-like consequences. In the presence of hCD4, gp120IIIB was able to produce an increase in tagged CXCR4-associated FRET. However, the degree and rate of the gp120IIIB effect was smaller and slower than that produced by SDF-1. This might suggest that SDF-1-induced conformational changes in the CXCR4 receptor are not fully reproduced by gp120 and that it is an agonist of lower efficacy than SDF-1. Consequently, binding of gp120 to CXCR4 might not fully reproduce the repertoire of signaling events normally produced by SDF-1, and this may have deleterious consequences for target cell survival. There have been several reports in the literature suggesting that this is the case (Miller and Oh, 2002).

Our data also further illustrate that there are mutual interactions between CXCR4 and hCD4. It is interesting to note that not only does hCD4 associate with CXCR4 in the membrane but this interaction reduces the ability of SDF-1 to increase FRET. This result appears analogous to parallel studies on the interaction of hCD4 with the CCR5 receptor, the other major coreceptor for HIV-1. In that case, it has been demonstrated that not only does hCD4 directly interact with CCR5 but also reduces the ability of MIP-1 to activate the receptor (Wang and Staudinger, 2003). In addition to this modulatory effect of hCD4, we also demonstrated dimerization of hCD4, something that has also been suggested in other investigations (Moldovan et al., 2002). It has been demonstrated in several studies that CXCR4 and hCD4 are colocalized in the plasma membrane and that this association may be enhanced by gp120 (Ugolini et al., 1997). Thus, it appears that CXCR4 and hCD4 can interact at several levels including in multicomponent membrane patches as well as in multisubunit complexes. The relative degree to which these various complexes form may well be under the control of diverse influences including, but not limited to, gp120.

In summary, the data reported here support previous suggestions that the CXCR4 receptor exists primarily as a homodimer. SDF-1 and gp120 can alter the degree of homodimerization. It is therefore possible that CXCR4 dimerization is a sine qua non for its productive signaling through association with JAKs, G proteins, and other downstream signaling partners.

	Acknowledgements

 
We appreciate the expert technical assistance of Christopher Mauer and Hong Ma.

	Footnotes

 
This work was supported by National Institutes of Health Grants NS43095, DA13141, MH40165, NS33826, and NS21442 to R.J.M. The generous support from the National Institutes of Health AIDS Research and Reference Reagent Program is greatly appreciated. The following reagents were obtained through the National Institutes of Health AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: human CD4 receptor (contributor Dr. Richard Axel) AMD3100 (contributor AnorMED, Inc.), and HIV-1 IIIB gp120.

Preliminary results have been presented in abstract format: Program 831.8. 2002 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2002. Online.

DOI: 10.1124/jpet.103.064956.

ABBREVIATIONS: GPCR, G protein-coupled receptor; JAK, Janus kinase; HIV-1, human immunodeficiency virus-1; hCD4, human CD4; FRET, fluorescence resonance energy transfer; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; ECFP, enhanced CFP; EYFP, enhanced YFP; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; ROI, region of interest; MOP, µ-opioid receptor; m1, muscarinic 1 receptor; m3, muscarinic 3 receptor; BRET, bioluminescence resonance energy transfer; HEK, human embryonic kidney.

Address correspondence to: Dr. Richard J. Miller, Department of Molecular Pharmacology and Biological Chemistry, Northwestern University, 303 E. Chicago Ave, Chicago, IL 60611. E-mail: r-miller10{at}northwestern.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Angers S, Salahpour A, and Bouvier M (2002) Dimerization: an emerging concept for G protein-coupled receptor ontogeny and function. Annu Rev Pharmacol Toxicol 42: 409-435.[CrossRef][Medline]

Angers S, Salahpour A, Joly E, Hilairet S, Chelsky D, Dennis M, and Bouvier M (2000) Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc Natl Acad Sci USA 97: 3684-3689.[Abstract/Free Full Text]

Babcock GJ, Farzan M, and Sodroski J (2003) Ligand-independent dimerization of CXCR4, a principal HIV-1 coreceptor. J Biol Chem 278: 3378-3385.[Abstract/Free Full Text]

Baggiolini M, Dewald B, and Moser B (1997) Human chemokines: an update. Annu Rev Immunol 15: 675-705.[CrossRef][Medline]

Bajetto A, Bonavia R, Barbero S, and Schettini G (2002) Characterization of chemokines and their receptors in the central nervous system: physiopathological implications. J Neurochem 82: 1311-1329.[CrossRef][Medline]

Berger EA, Murphy PM, and Farber JM (1999) Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism and disease. Annu Rev Immunol 17: 657-700.[CrossRef][Medline]

Bodner A, Toth PT, Oh SB, Lu M, Tran PB, Chin RK, Ren D, and Miller RJ (2003) CD4 dependence of gp120IIIB-CXCR4 interaction is cell-type specific. J Neuroimmunol 140: 1-12.[CrossRef][Medline]

Boussif O, Zanta MA, and Behr JP (1996) Optimized galenics improve in vitro gene transfer with cationic molecules up to 1000-fold. Gene Ther 3: 1074-1080.[Medline]

Budd DC, McDonald J, Emsley N, Cain K, and Tobin AB (2003) The C-terminal tail of the M3-muscarinic receptor possesses anti-apoptotic properties. J Biol Chem 278: 19565-19573.[Abstract/Free Full Text]

Bushell T, Endoh T, Simen AA, Ren D, Bindokas VP, and Miller RJ (2002) Molecular components of tolerance to opiates in single hippocampal neurons. Mol Pharmacol 61: 55-64.[Abstract/Free Full Text]

Dinger MC, Bader JE, Kobor AD, Kretzschmar AK, and Beck-Sickinger AG (2003) Homodimerization of neuropeptide y receptors investigated by fluorescence resonance energy transfer in living cells. J Biol Chem 278: 10562-10571.[Abstract/Free Full Text]

Doms RW (2000) Beyond receptor expression: the influence of receptor conformation, density and affinity in HIV-1 infection. Virology 276: 229-237.[CrossRef][Medline]

Gillard SE, Lu M, Mastracci RM, and Miller RJ (2002) Expression of functional chemokine receptors by rat cerebellar neurons. J Neuroimmunol 124: 16-28.[CrossRef][Medline]

Gomes I, Jordan BA, Gupta A, Rios C, Trapaidze N, and Devi LA (2001) G protein coupled receptor dimerization: implications in modulating receptor function. J Mol Med 79: 226-242.[CrossRef][Medline]

Gordon GW, Berry G, Liang XH, Levine B, and Herman B (1998) Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys J 74: 2702-2713.[Medline]

Hatse S, Princen K, Bridger G, De Clercq E, and Schols D (2002) Chemokine receptor inhibition by AMD3100 is strictly confined to CXCR4. FEBS Lett 527: 255-262.[CrossRef][Medline]

Hebert TE, Moffett S, Morello JP, Loisel TP, Bichet DG, Barret C, and Bouvier M (1996) A peptide derived from a beta2-adrenergic receptor transmembrane domain inhibits both receptor dimerization and activation. J Biol Chem 271: 16384-16392.[Abstract/Free Full Text]

Hesselgesser J, Halks-Miller M, DelVecchio V, Peiper SC, Hoxie J, Kolson DL, Taub D, and Horuk R (1997) CD4-independent association between HIV-1 gp120 and CXCR4: functional chemokine receptors are expressed in human neurons. Curr Biol 7: 112-121.[CrossRef][Medline]

Horuk R (2001) Chemokine receptors. Cytokine Growth Factor Rev 12: 313-335.[CrossRef][Medline]

Huang MB, Hunter M, and Bond VC (1999) Effect of extracellular human immunodeficiency virus type 1 glycoprotein 120 on primary human vascular endothelial cell cultures. AIDS Res Hum Retrovir 15: 1265-1277.[CrossRef][Medline]

Issafras H, Angers S, Bulenger S, Blanpain C, Parmentier M, Labbe-Jullie C, Bouvier M, and Marullo S (2002) Constitutive agonist-independent CCR5 oligomerization and antibody-mediated clustering occurring at physiological levels of receptors. J Biol Chem 277: 34666-34673.[Abstract/Free Full Text]

Kim CH and Broxmeyer HE (1999) Chemokines: signal lamps for trafficking of T and B cells for development and effector function. J Leukoc Biol 65: 6-15.[Abstract]

Miller RJ and Oh SB (2002) Why do neurons express chemokine receptors?, in Universes in Delicate Balance: Chemokines and the Nervous System (Ransohoff RM, Suzuki K, Proudfoot AEI, Hickey WF, and Harrison JK eds) pp 273-288, Elsevier Science B.V., Amsterdam.

Moldovan MC, Yachou A, Levesque K, Wu H, Hendrickson WA, Cohen EA, and Sekaly RP (2002) CD4 dimers constitute the functional component required for T cell activation. J Immunol 169: 6261-6268.[Abstract/Free Full Text]

Ng GY, O'Dowd BF, Lee SP, Chung HT, Brann MR, Seeman P, and George SR (1996) Dopamine D2 receptor dimers and receptor-blocking peptides. Biochem Biophys Res Commun 227: 200-204.[CrossRef][Medline]

Oh SB, Tran PB, Gillard SE, Hurley RW, Hammond DL, and Miller RJ (2001) Chemokines and glycoprotein120 produce pain hypersensitivity by directly exciting primary nociceptive neurons. J Neurosci 21: 5027-5035.[Abstract/Free Full Text]

Ramsay D, Kellett E, McVey M, Rees S, and Milligan G (2002) Homo- and hetero-oligomeric interactions between G-protein-coupled receptors in living cells monitored by two variants of bioluminescence resonance energy transfer (BRET): hetero-oligomers between receptor subtypes form more efficiently than between less closely related sequences. Biochem J 365: 429-440.[CrossRef][Medline]

Rodriguez-Frade JM, Mellado M, and Martinez AC (2001) Chemokine receptor dimerization: two are better than one. Trends Immunol 22: 612-617.[CrossRef][Medline]

Romano C, Yang WL, and O'Malley KL (1996) Metabotropic glutamate receptor 5 is a disulfide-linked dimer. J Biol Chem 271: 28612-28616.[Abstract/Free Full Text]

Ruehr ML, Zakhary DR, Damron DS, and Bond M (1999) Cyclic AMP-dependent protein kinase binding to A-kinase anchoring proteins in living cells by fluorescence resonance energy transfer of green fluorescent protein fusion proteins. J Biol Chem 274: 33092-33096.[Abstract/Free Full Text]

Stantchev TS and Broder CC (2001) Human immunodeficiency virus type-1 and chemokines: beyond competition for common cellular receptors. Cytokine Growth Factor Rev 12: 219-243.[CrossRef][Medline]

Stryer L (1978) Fluorescence energy transfer as a spectroscopic ruler. Annu Rev Biochem 47: 819-846.[CrossRef][Medline]

Tran PB and Miller RJ (2003) Chemokine receptors; signposts to brain development and disease. Nat Rev Neurosci 4: 444-455.[CrossRef][Medline]

Ugolini S, Moulard M, Mondor I, Barois N, Demandolx D, Hoxie J, Brelot A, Alizon M, Davoust J, and Sattentau QJ (1997) HIV-1 gp120 induces an association between CD4 and the chemokine receptor CXCR4. J Immunol 159: 3000-3008.[Abstract]

Ullrich CK, Groopman JE, and Ganju RK (2000) HIV-1 gp120- and gp160-induced apoptosis in cultured endothelial cells is mediated by caspases. Blood 96: 1438-1442.[Abstract/Free Full Text]

Vila-Coro AJ, Rodriguez-Frade JM, Martin De Ana A, Moreno-Ortiz MC, Martinez AC, and Mellado M (1999) The chemokine SDF-1alpha triggers CXCR4 receptor dimerization and activates the JAK/STAT pathway. FASEB J 13: 1699-1710.[Abstract/Free Full Text]

Wang X and Staudinger R (2003) Interaction of soluble CD4 with the chemokine receptor CCR5. Biochem Biophys Res Commun 307: 1066-1069.[CrossRef][Medline]

Xia Z and Liu Y (2001) Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes. Biophys J 81: 2395-2402.[Medline]

Zeng F and Wess J (2000) Molecular aspects of muscarinic receptor dimerization. Neuropsychopharmacology 23: S19-S31.[CrossRef][Medline]

Zeng FY and Wess J (1999) Identification and molecular characterization of m3 muscarinic receptor dimers. J Biol Chem 274: 19487-19497.[Abstract/Free Full Text]

Zhang XF, Wang JF, Matczak E, Proper JA, and Groopman JE (2001) Janus kinase 2 is involved in stromal cell-derived factor-1alpha-induced tyrosine phosphorylation of focal adhesion proteins and migration of hematopoietic progenitor cells. Blood 97: 3342-3348.[Abstract/Free Full Text]

Zhou JY, Toth PT, and Miller RJ (2003) Direct interactions between the heterotrimeric G protein subunit Gbeta 5 and the G protein gamma subunit-like domain-containing regulator of G protein signaling 11: gain of function of cyan fluorescent protein-tagged Ggamma 3. J Pharmacol Exp Ther 305: 460-466.[Abstract/Free Full Text]

This article has been cited by other articles:

				K. E. Luker, M. Gupta, and G. D. Luker Imaging chemokine receptor dimerization with firefly luciferase complementation FASEB J, March 1, 2009; 23(3): 823 - 834. [Abstract] [Full Text] [PDF]

				R. Sengupta, S. Burbassi, S. Shimizu, S. Cappello, R. B. Vallee, J. B. Rubin, and O. Meucci Morphine Increases Brain Levels of Ferritin Heavy Chain Leading to Inhibition of CXCR4-Mediated Survival Signaling in Neurons J. Neurosci., February 25, 2009; 29(8): 2534 - 2544. [Abstract] [Full Text] [PDF]

				L. Navarro-Borelly, A. Somasundaram, M. Yamashita, D. Ren, R. J. Miller, and M. Prakriya STIM1-Orai1 interactions and Orai1 conformational changes revealed by live-cell FRET microscopy J. Physiol., November 15, 2008; 586(22): 5383 - 5401. [Abstract] [Full Text] [PDF]

				H. J. Harris, M. J. Farquhar, C. J. Mee, C. Davis, G. M. Reynolds, A. Jennings, K. Hu, F. Yuan, H. Deng, S. G. Hubscher, et al. CD81 and Claudin 1 Coreceptor Association: Role in Hepatitis C Virus Entry J. Virol., May 15, 2008; 82(10): 5007 - 5020. [Abstract] [Full Text] [PDF]

				F. A. White, H. Jung, and R. J. Miller Chemokines and the pathophysiology of neuropathic pain PNAS, December 18, 2007; 104(51): 20151 - 20158. [Abstract] [Full Text] [PDF]

				W. Chun and G. V. W. Johnson Activation of Glycogen Synthase Kinase 3beta Promotes the Intermolecular Association of Tau: THE USE OF FLUORESCENCE RESONANCE ENERGY TRANSFER MICROSCOPY J. Biol. Chem., August 10, 2007; 282(32): 23410 - 23417. [Abstract] [Full Text] [PDF]

				A. Guyon and J.-L. Nahon Multiple actions of the chemokine stromal cell-derived factor-1{alpha} on neuronal activity J. Mol. Endocrinol., March 1, 2007; 38(3): 365 - 376. [Abstract] [Full Text] [PDF]

				G. Gaibelet, T. Planchenault, S. Mazeres, F. Dumas, F. Arenzana-Seisdedos, A. Lopez, B. Lagane, and F. Bachelerie CD4 and CCR5 Constitutively Interact at the Plasma Membrane of Living Cells: A CONFOCAL FLUORESCENCE RESONANCE ENERGY TRANSFER-BASED APPROACH J. Biol. Chem., December 8, 2006; 281(49): 37921 - 37929. [Abstract] [Full Text] [PDF]

				J. Wang, L. He, C. A. Combs, G. Roderiquez, and M. A. Norcross Dimerization of CXCR4 in living malignant cells: control of cell migration by a synthetic peptide that reduces homologous CXCR4 interactions. Mol. Cancer Ther., October 1, 2006; 5(10): 2474 - 2483. [Abstract] [Full Text] [PDF]

				M. Zaitseva, T. Romantseva, J. Manischewitz, J. Wang, D. Goucher, and H. Golding Increased CXCR4-dependent HIV-1 fusion in activated T cells: role of CD4/CXCR4 association J. Leukoc. Biol., December 1, 2005; 78(6): 1306 - 1317. [Abstract] [Full Text] [PDF]

				G. Badr, G. Borhis, D. Treton, C. Moog, O. Garraud, and Y. Richard HIV Type 1 Glycoprotein 120 Inhibits Human B Cell Chemotaxis to CXC Chemokine Ligand (CXCL) 12, CC Chemokine Ligand (CCL)20, and CCL21 J. Immunol., July 1, 2005; 175(1): 302 - 310. [Abstract] [Full Text] [PDF]

				Y. Percherancier, Y. A. Berchiche, I. Slight, R. Volkmer-Engert, H. Tamamura, N. Fujii, M. Bouvier, and N. Heveker Bioluminescence Resonance Energy Transfer Reveals Ligand-induced Conformational Changes in CXCR4 Homo- and Heterodimers J. Biol. Chem., March 18, 2005; 280(11): 9895 - 9903. [Abstract] [Full Text] [PDF]

				B. Ahr, M. Denizot, V. Robert-Hebmann, A. Brelot, and M. Biard-Piechaczyk Identification of the Cytoplasmic Domains of CXCR4 Involved in Jak2 and STAT3 Phosphorylation J. Biol. Chem., February 25, 2005; 280(8): 6692 - 6700. [Abstract] [Full Text] [PDF]

				M.-B. Huang, L. L. Jin, C. O. James, M. Khan, M. D. Powell, and V. C. Bond Characterization of Nef-CXCR4 Interactions Important for Apoptosis Induction J. Virol., October 15, 2004; 78(20): 11084 - 11096. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.103.064956v1 310/1/8    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Toth, P. T. Articles by Miller, R. J. Search for Related Content PubMed PubMed Citation Articles by Toth, P. T. Articles by Miller, R. J.