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Research Articles: Therapeutics, Targets, and Development

Dimerization of CXCR4 in living malignant cells: control of cell migration by a synthetic peptide that reduces homologous CXCR4 interactions

¹ Division of Therapeutic Proteins, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration; ² National Institute of Arthritis and Musculoskeletal and Skin Diseases; and ³ National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland

Requests for reprints: Jinhai Wang or Michael A. Norcross, Division of Therapeutic Proteins, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, NIH Building 29B, Room 4E12, 8800 Rockville Pike, Bethesda, MD 20892. Phone: 301-594-5223; Fax: 301-480-3256. E-mail: Jinhai.wang{at}fda.hhs.gov or michael.norcross{at}fda.hhs.gov

Abstract

Chemokine receptor CXCR4 (CD184) may play a role in cancer metastasis and is known to form homodimers. However, it is not clear how transmembrane regions (TM) of CXCR4 and receptor homotypic interactions affect the function of CXCR4 in living cells. Using confocal microscopy and flow cytometric analysis, we showed that high levels of CXCR4 are present in the cytoplasm, accompanied by lower expression on the cell surface in CXCR4 transfectants, tumor cells, and normal peripheral blood lymphocytes. CXCR4 homodimers were detected in tumor cells, both on the cell surface membrane and in the cytoplasm using fluorescence resonance energy transfer and photobleaching fluorescence resonance energy transfer to measure energy transfer between CXCR4-CFP and CXCR4-YFP constructs. Disruption of lipid rafts by depletion of cholesterol with methyl-ß-cyclodextrin reduced the interaction between CXCR4 molecules and inhibited malignant cell migration to CXCL12/SDF-1. A synthetic peptide of TM4 of CXCR4 reduced energy transfer between molecules of CXCR4, inhibited CXCL12-induced actin polymerization, and blocked chemotaxis of malignant cells. TM4 also inhibited migration of normal monocytes toward CXCL12. Reduction of CXCR4 energy transfer by the TM4 peptide and methyl-ß-cyclodextrin indicates that interactions between CXCR4s may play important roles in cell migration and suggests that cell surface and intracellular receptor dimers are appropriate targets for control of tumor cell spread. Targeting chemokine receptor oligomerization and signal transduction for the treatment of cancer, HIV-1 infections, and other CXCR4 mediated inflammatory conditions warrants further investigation. [Mol Cancer Ther 2006;5(10):2474–83]

Introduction

Heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCR) respond to a variety of different external stimuli or endogenous ligands and activate G proteins. In response to ligands, some GPCRs undergo ligand-dependent endocytosis and dimerization. Recently, heterodimerization of GPCRs was found to occur between GBR1 and GBR2, and between opioid receptors and (1, 2). Hetero-oligomers with enhanced functional activity are formed between receptors for dopamine and somatostatin (3). Receptor oligomerization is a pivotal aspect of the structure and function of GPCRs that has been shown to have implications for receptor trafficking and signaling.

As GPCRs, chemokine receptors also form homodimers or heterodimers. Chemokine receptor heterodimerization of CXCR4 or CCR5 (CD195) with CCR2V64I was suggested to be the mechanism by which individuals with the CCR2V64I allele show delayed AIDS progression (4, 5). CXCR4 is complexed with CCR5 in the presence of CD4 (6) and CCR5 is resistant to ligand-mediated internalization in some cell types (6, 7). Heterodimerization of CCR5 32 with CXCR4 is associated with resistance to X4 HIV type 1 in primary CD4⁺ cells (8). Preformed, antibody-induced, and ligand-stabilized CCR5 homodimers or homo-oligomers (9, 10) have been reported and were associated with inhibition of HIV-1 infection. Constitutive CCR5 and CD4 hetero-associations have been described (11). CXCR4 homodimers are also detected in the presence or absence of ligand (12, 13). Dimer formation may depend on specific receptor transmembrane regions (TM) and on the lipid environment or lipid raft context of the receptors. However, the role of lipid rafts and TMs in CXCR4 dimerization and function is undefined.

CXCR4 is expressed on a variety of cell types, including peripheral blood lymphocytes (PBL) and monocytes. CXCR4 is also highly expressed in human breast cancer cells, malignant breast tumors, prostate cancer, small-cell lung cancer, and pancreatic cancer and metastases (14–18). CXCL12/CXCR4 is implicated in metastasis of tumor cells (14, 19) and CXCR4 is a coreceptor for T-cell tropic (X4) HIV-1 virus infection. Membrane lipid rafts are essential for X4 viral entry (20). Chemokine receptors CXCR4 and CCR5 undergo ligand-dependent internalization in response to ligand binding, as well as ligand-independent sequestration in response to cytokines (21, 22).

In the current study, we use a biophysical approach based on fluorescence resonance energy transfer (FRET) between HIV-1 coreceptors differentially tagged with distinct fluorophores to study factors that affect CXCR4 dimer formation and ligand-directed migration of tumor cells. We now report that dimer formation of CXCR4 occurs in living cells, and that lipid raft microdomains are important in directed migration of cancer cells, possibly through maintaining receptor dimer conformation. An 18-residue long peptide, TM4, encoding the TM4 of CXCR4, modifies the energy transfer between monomers of CXCR4 dimer and blocked CXCR4-mediated cancer cell migration.

Materials and Methods

Cells and Reagents
HEK293 cell line, HeLa cells, and human non–small cell lung carcinoma cell line NCI-H2126 were cultured in DMEM, whereas Sup-T1, Hut-78, and PM1 were cultured in RPMI 1640, with 10% fetal bovine serum, penicillin (100 units/mL), and streptomycin (100 µg/mL). Human PBLs and monocytes were isolated by countercurrent centrifugal elutriation from single-donor preparations of peripheral blood leukocytes, as described previously (23). CXCL12/SDF-1 were purchased from Peprotech (Rocky Hill, NJ), methyl-ß-cyclodextrin was from Sigma (Woburn, MA). Peptide TM4 (VYVGVWIPALLLTIPDFI) and X442_7-23 (IPALLLTIPDFIFANDD) were synthesized by Center for Biologics Evaluation and Research core facility and purified by reverse phase high-performance liquid chromatography. Molecular weight of the peptides was confirmed by mass spectrometry matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis.

Plasmids
Cyan fluorescence protein (CFP)–expressing plasmid peCFP and yellow fluorescence protein (YFP)–expressing plasmid peYFP were purchased from BD Biosciences-Clontech (Palo Alto, CA). peCFP-eYFP fusion construct contains CFP and YFP linked by two amino acids (CFP-2aa-YFP) as described previously (24). pCXCR4-eCFP was constructed as follows: pcDNA3-CXCR4-C9 containing codon optimized human CXCR4 cDNA (kindly provided by Dr. Sodroski, Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA) was digested with KpnI, blunted with mung bean nuclease at 37°C for 10 minutes. CXCR4 cDNA was then released from pcDNA3-CXCR4-C9 by HindIII digestion and purified from agarose gel with Wizard SV gel and PCR clean-up system (Promega, Madison, WI). Purified CXCR4 insert was ligated with pcDNA-eCFP-N1, which was digested with HindIII and SmaI. pCXCR4-eYFP was constructed by ligation of above-purified CXCR4 insert into pcDNA-eYFP-N1, which was digested with HindIII and SmaI as well.

Immunocytochemistry
For cell surface staining, cells were stained with indicated antibodies to cell surface antigens for 30 minutes at 4°C and then subjected to flow cytometry (FACSort, BD Biosciences) as described previously (23). Phycoerythrin-labeled anti-CXCR4 monoclonal antibody (12G5) and phycoerythrin-labeled anti-CCR5 monoclonal antibody (2D7) were purchased from BD PharMingen (San Diego, CA). For intracellular staining, cells were fixed in paraformaldehyde in buffered saline, permeabilized with buffer containing saponin and normal serum (IC Fix/IC Perm buffer kit, BioSource, Camarillo, CA), and then stained with indicated labeled antibodies for 30 minutes at 4°C.

Flow Cytometry and FRET
HeLa and HEK293 cells were grown in six-well plates and transfected with indicated plasmids. Cells were transfected using LipofectAMINE 2000 reagent (Invitrogen, Carlsbad, CA) per the instruction of the manufacturer. Cells were treated with PBS-based enzyme free dissociation buffer and washed with PBS. The influence of methyl-ß-cyclodextrin and TM4 peptide on FRET efficiency was investigated by incubation of cells with indicated reagents, followed by a 30 minutes incubation in the absence or presence of 1 µg/mL SDF-1 at 37°C in protein-free medium before FRET analysis. All flow cytometric FRET data were collected and analyzed using a DakoCytomation Cyan flow cytometer and Summit software (DakoCytomation, Fort Collins, CO). The argon-ion 488 nm (laser 1) and the violet UV 405 nm laser lines (laser 2) at the power fixed by the company were used to excite YFP and CFP, respectively. The YFP signals were collected using 546/10 nm bandpass filter in the primary laser pathway (laser 1). The CFP and FRET signals were collected using 460/20 and 546/10 bandpass filters, respectively, along with a 500 long-pass dichroic splitter filter inserted into the UV laser pathway (laser 2).

Confocal Laser Microscopy
Cells cultured on glass slides were stained with phycoerythrin-labeled anti-CXCR4 antibody with or without permeabilization. Coverslips were mounted with AntiFade Prolong solution with antifade reagents (Molecular Probes, Eugene, OR). Cell surface and intracellular fluorescence was visualized with an LSM5 PASCAL confocal laser-scanning microscope (Carl Zeiss, Thornword, NY). Emission from phycoerythrin (560 nm) was detected after excitation at 543 nm. Nonspecific background fluorescence was determined by staining with labeled isotype control antibodies. For assay of actin polymerization, cells were cultured on glass coverslips overnight and treated as indicated, then stained with Oregon green 514-phalloidin (Molecular Probes) for 30 minutes at 4°C.

Photobleaching FRET with Confocal Laser-Scanning Microscopy
Cells were grown on glass coverslips for 24 hours and transfected with vectors encoding tagged CXCR4 and/or control proteins. Cells were transfected with LipofectAMINE 2000 reagent (Invitrogen) per the instruction of the manufacturer. Coverslips were mounted with AntiFade Prolong solution with antifade reagents (Molecular Probes). Images were collected using a x40 oil objective (1.3 numerical aperture) and Zeiss LSM 510 confocal microscope equipped with a META spectral scanning head. Twelve-bit images for FRET analysis were acquired in the scanning mode (emission range of 478–650 nm in 10-nm steps) with 458-nm excitation. Photobleaching of YFP was done using 514-nm excitation over time periods that lasted up to 39 seconds. Separation of CFP and YFP signals was accomplished using the Zeiss digital linear unmixing algorithm and spectral information collected on individually expressing CFP and YFP samples. For FRET analysis, coverslips were mounted with antifade Prolong solution without antifade reagent. Images were acquired before and after photobleaching.

Migration of Tumor Cells
Cells were washed and resuspended (5 x 10⁶ cells/mL) in RPMI 1640 or DMEM containing 0.5% bovine serum albumin, then cells were incubated at 37°C in a moist atmosphere containing 5% CO₂ for 2 hours. Cells were then treated with indicated reagents for 1 hour at 37°C. Portions (100 µL) of the cell suspension were placed in the upper wells of Transwell chambers (Corning, Acton, MA) containing bare filters with a pore size of 3 µm for Sup-T1 cells, Hut 78, PM1, and monocytes. The same medium (600 µL) supplemented or not with SDF-1 (250 or 300 ng/mL) was placed in the lower chambers. After incubation for 3 hours at 37°C in a moist atmosphere containing 5% CO₂, cells that had migrated through the filter were counted by flow cytometry.

Results

Cell Surface and Intracellular Localization of CXCR4
CXCR4 is a seven-transmembrane GPCR whose expression is routinely assessed by cell surface staining with antibodies, such as monoclonal antibody 12G5. It has been shown previously that CXCR4 translocates from cell surface into cytoplasm either in a ligand-dependent or ligand-independent manner (22, 25). To examine the subcellular localization of CXCR4 in the absence of added stimulators, HEK293 cells were transfected with CXCR4-eCFP or CXCR4-eYFP, then observed using flow cytometry or confocal microscopy. Cell surface CXCR4 (stained by phycoerythrin-labeled anti-CXCR4 monoclonal antibody 12G5, emission at 575 nm) was observed on cells transfected with CXCR4-eCFP (the excitation peak of CFP is 434 nm and emission peak is 476 nm that is out of detection range of FACSort flow cytometry) and CXCR4-eYFP. CXCR4-eYFP was detected in FL1 channel (emission peak 527 nm) and the fluorescence intensity of phycoerythrin-labeled 12G5 staining was positively correlated with that of CXCR4-eYFP (Fig. 1A ). Confocal microscopic analysis of CXCR4-eCFP–transfected cells not only showed cell surface CXCR4, but also high levels of intracellular CXCR4 (Fig. 1B). These data indicate that CXCR4 was localized on the cell surface and inside cells in the absence of ligands or stimulators. To examine subcellular localization of constitutively expressed CXCR4, human primary T cells and HeLa tumor cells were stained with phycoerythrin-labeled anti-CXCR4 antibody 12G5 with or without cell permeabilization. In human PBLs, the geometric mean fluorescence intensity of CXCR4 in permeabilized cells was dramatically increased compared with that of nonpermeabilized cells (Fig. 1C), whereas the geometric mean fluorescences of isotype control and CCR5 antibody were not affected by cell membrane permeabilization. This indicated that CXCR4 was localized mostly inside cells in primary lymphocytes and that a large fraction of cells only expressed CXCR4 intracellularly. In human cervical cancer cells HeLa, the geometric mean fluorescence intensity of CXCR4 was much lower in intact cells compared with that of permeabilized cells (Fig. 1C), whereas the geometric mean fluorescences of controls (isotype and anti-CCR5) were not affected by cell membrane permeabilization, indicating that CXCR4 was also localized inside cells in human tumor cells. Similarly, confocal microscopic analysis revealed that cell surface CXCR4 staining of intact HeLa cells was much weaker compared with intracellular CXCR4 staining of permeabilized HeLa cells (Fig. 1D), confirming that the majority of CXCR4 was located in the cytoplasm of cervical cancer cells. Human non–small cell lung carcinoma cell line NCI-H2126 was also examined by confocal microscopic analysis. CXCR4 was expressed only weakly on the cell surface, whereas strong CXCR4 staining was evident after cell permeabilization (Fig. 1E).

View larger version (37K): [in this window] [in a new window]   Figure 1. Cell surface and intracellular CXCR4 expression. A, HEK293 cells were transfected with indicated plasmids and stained for cell surface CXCR4 24 h later by phycoerythrin-labeled anti-CXCR4 antibody 12G5 and analyzed by FACSort flow cytometer; percentages of positive cells are indicated in quadrant. B, HEK293 cells were transfected with pCXCR4-eCFP and analyzed by confocal microscopy 24 h later. C, fresh PBLs and HeLa cells with/without permeabilization were stained with phycoerythrin-labeled anti-CXCR4 antibody 12G5, anti-CCR5 antibody 2D7, or isotype control, and analyzed by FACSort flow cytometer. Number in each histogram, geometric mean fluorescence intensity. HeLa cells (D) and human non–small cell lung carcinoma cell line NCI-H2126 (E) were cultured in slide chambers and stained with phycoerythrin-labeled anti-CXCR4 antibody 12G5 with/without permeabilization, and mounted with ProLong Gold antifade reagent. Phycoerythrin-labeled isotype control was used as negative control for cells with/without permeabilization. Mean fluorescence intensity of X axis (C).

View larger version (27K): [in this window] [in a new window]   Figure 2. Homodimer formation of CXCR4 in living cells. HEK293 cells were transfected with indicated plasmids and assayed for FRET 24 h later by DakoCytomation Cyan flow cytometer. A, dot plots of peYFP, peCFP, peYFP/peCFP, or peCFP-eYFP–transfected cells. B, dot plots of pCXCR4-eCFP, pCXCR4-eYFP, or both transfected cells.

Homodimerization of Intracellular and Cell Surface CXCR4
To further show whether dimerization of CXCR4 also occurred at the cell surface and inside cells, HEK293 cells were transfected with CXCR4-CFP and CXCR4-YFP and then monitored microscopically for FRET using photobleaching FRET, which has the advantage of examining protein subcellular localization and dimerization at the same time. Because of energy loss, the fluorescence intensity of the donor is simultaneously reduced or quenched. When the acceptor fluorophore is bleached, the energy transfer from donor to acceptor is prevented, and thus the fluorescence intensity of the donor increases in the absence of the acceptor fluorophore in the bleached region. The donor here is the CXCR4-CFP and the acceptor is the CXCR4-YFP. Figure 3A shows a montage of confocal fluorescence micrographs of HEK293 cells coexpressing CXCR4-eCFP and CXCR4-eYFP. The top panels show emissions from CXCR4-eYFP and CXCR4-eCFP, as well as merged images before photobleaching CXCR4-eYFP in a controlled region (outlined by the yellow rectangle). The lower panels show CXCR4-eYFP, CXCR4-eCFP, and merged signals in the same cells following photobleaching. Photobleaching eYFP did not affect eCFP emission in cells expressing noncolocalized eCFP and eYFP (data not shown). In cells expressing colocalized CXCR4-eCFP and CXCR4-eYFP, increased eCFP emission was observed in bleached regions, indicating that substantial energy transfer occurred between CXCR4-eCFP and CXCR4-eYFP before photobleaching. When CXCR4-YFP was bleached, the fluorescence intensity of CXCR4-CFP in the bleached regions, including cell surface and intracellular, was increased, thus demonstrating FRET detection by confocal microscopy.

View larger version (15K): [in this window] [in a new window]   Figure 3. Cell surface and intracellular CXCR4 dimer formation detected by confocal photobleaching of acceptor YFP. HEK293 cells were cotransfected with pCXCR4-eCFP and pCXCR4-eYFP and mounted with mounting solution without ProLong antifade reagent. A, detection of CXCR4 dimer by photobleaching. Split XY and comerged images are shown before and after photobleaching. Yellow frame, photobleaching areas. B, CXCR4 dimer detected on cell surface and cytoplasm. HEK293 cells were transfected with pCXCR4-eCFP and pCXCR4-eYFP and mounted with mounting solution without ProLong antifade reagent. Photobleached intracellular area (region 1, red sphere, ROI 1), cell surface (region 2, green circle, ROI2), and unbleached area (region 3, blue circle, ROI3) are shown at right with their respective multiplicity of intensity analysis at left.

Involvement of Lipid Rafts in the Homodimerization of CXCR4
SDF-1–induced CXCR4 polarization and signaling depends on lipid raft formation (26, 27). We examined the role of lipid rafts in the dimerization of CXCR4 by depletion of cholesterol with methyl-ß-cyclodextrin. Depletion of lipid rafts induced a 15% to 20% reduction in energy transfer between molecules of CXCR4 in the absence or presence of CXCL12 (Fig. 4 ). SDF1/CXCL12 in our hands did not consistently change energy transfer in these cells compared with constitutive FRET levels.

View larger version (10K): [in this window] [in a new window]   Figure 4. Impact of lipid rafts and synthetic peptides on CXCR4 homodimer interaction. HEK293 cells were transfected with indicated plasmids. Twenty-four hours later, cells were cultured in serum-free medium for 2 h, treated with methyl-ß-cyclodextrin (mßCD; 10 mmol/L) or synthetic peptide TM4 (50 µg/mL) for 1 h at 37°C, then treated with CXCL12 as indicated and assayed for FRET by DakoCytomation Cyan flow cytometer. Data are from one of two independent experiments with similar results. Med, medium.

Inhibition of CXCR4-Mediated Migration by Depletion of Cholesterol
Many eukaryotic cells are able to sense external gradients of certain signaling molecules and respond with asymmetric changes in cell morphology and motility. In responding to chemoattractants, lipid rafts and associated proteins redistribute to the leading edge raft and uropod rafts (29, 30). To study the role of lipid rafts in CXCR4-mediated migration, SupT1 cells were starved for 2 hours and then treated with methyl-ß-cyclodextrin for 1 hour to deplete cholesterol. Migration of cancer cells with or without methyl-ß-cyclodextrin treatment was examined in a chemotaxis assay. CXCL12 induced a strong chemotaxis response and this response was completely abolished by methyl-ß-cyclodextrin pretreatment (Fig. 5A ), indicating that lipid rafts are essential for CXCR4-mediated cell migration.

View larger version (9K): [in this window] [in a new window]   Figure 5. Role of lipid rafts and CXCR4 dimerization in SDF-1–directed migration of cancer cells. A, SupT1 cells were incubated in medium for 2 h, then treated with methyl-ß-cyclodextrin (10 mmol/L) or synthetic peptide TM4 (50 µg/mL) for 1 h at 37°C. Hut-78 and PM1 (B), and monocytes (C) were incubated in medium for 2 h, then treated with synthetic peptide TM4 (50 µg/mL) for 1 h at 37°C. Cell migration was examined in response to CXCL12 as described in Materials and Methods. Columns, mean of triplicate samples; bars, SD. One experiment representative of two independent experiments for each cell line.

Blocking SDF-1–Induced Migration of Cancer Cells by a Synthetic Peptide TM4

Blocking CXCL12-Induced Actin Polymerization in Tumor Cells by Synthetic Peptide
The driving force for cell migration is remodeling of the actin cytoskeleton. Chemokine receptor GPCRs transduce signals and induce concurrent changes in actin that enable the cell to move toward the chemoattractant. CXCL12 stimulated actin polymerization in tumor cells (Fig. 6 ) and pretreatment with TM4 peptide blocked chemokine-induced polymerization.

View larger version (12K): [in this window] [in a new window]   Figure 6. Effect of TM4 peptide on CXCL12-induced actin polymerization. HeLa cells were cultured for 2 h, then treated with TM4 peptide for 1 h at 37°C. Cells on coverslips were stimulated with CXCL12 for 10 min at 37°C, stained with Oregon green 514-phalloidin, then observed with a confocal microscope.

The concept that GPCRs, including chemokine receptors, exist as dimers or oligomers has advanced rapidly in recent years based on structural and functional studies. Direct molecular crosstalk between related GPCR subfamilies is associated with enhanced functional activity. In this report, we show that CXCR4 dimers are found on the cell surface and inside cells. Lipid rafts are important for interactions of dimer molecules and ligand-directed migration of cancer cells. Synthetic peptide TM4, which partially reduced energy transfer between molecules of CXCR4 dimer, abolished CXCL12-induced actin polymerization and migration of cancer cells.

There is growing evidence that interactions of GPCRs occur initially during biosynthesis. It was shown that immature forms of GPCRs, such as oxytocin and vasopressin receptors and CCR5, are present as dimers, while transiting through the endoplasmic reticulum (31, 32). Confocal analysis of CXCR4-eCFP–transfected cells showed that CXCR4 was located in vessicle-like structures in the cytoplasm. Intracellular CXCR4 molecules were also detected in human primary T cells, HeLa cervical cancer cells, and NCI 2126 non–small cell lung carcinoma cells, indicating that intracellular CXCR4 observed in transfected cells is not an artifact of transfection of a fusion protein and is a natural form of the protein. Although the function of intracellular CXCR4 is unclear, dimerization of intracellular CXCR4 observed by confocal FRET supports the assertion that CXCR4 proteins may traffic to the cell surface as oligomers and remain oligomeric in the plasma membrane. These results are consistent with a previous study that showed dimerization of immature forms of CXCR4 during biosynthesis (13). Failure of ligands to further enhance dimerization of CXCR4 is consistent with the scenario that CXCR4 was already dimerized inside cells. The role of intracellular CXCR4 in cell surface CXCR4 recycling has yet to be determined. The large pool of intracellular CXCR4 in PBLs and tumor cells that could recycle indicates that continued therapy would be required for treatments, including anti-HIV-1 or antitumor therapy, targeting cell surface CXCR4 expression.

GPCRs share many structural features, including a bundle of seven transmembrane helices connected by six loops of varying lengths. Rhodopsins are the only GPCR with defined crystal structure (28). Paracrystalline arrays of dimers of native rhodopsin were revealed by IR-laser atomic force microscopy in disk membranes of mouse retinae (33). The structure of chemokine receptors has yet to be determined. Two models developed from atomic force microscopy analysis of mouse native rhodopsin (34) and from cryoelectron microscopy analysis of squid rhodopsin (35) show that TM4 plays a central role in dimerization. Further studies using cross-linking of substituted cysteines indicated that residues across the entire TM4 region of dopamine D2 receptor are included in the dimer interface (36).

In this report, we show that TM4 peptide is able to reduce interactions between CXCR4 receptors and completely block migration of cancer cells to CXCL12. A recent report using bioluminiscence resonance energy transfer techniques (37) indicated that CXCL12 may alter receptor conformations to enhance energy transfer between receptors. Transmembrane peptides, including X442, another peptide derived from TM4 of CXCR4, inhibited the CXCL12-induced conformational transitions of the dimer but did not affect constitutive energy transfer. The transmembrane peptides also functionally blocked production of cyclic AMP in response to SDF-1/CXCL12 (37). With respect to ligand-induced changes, one report found that FRET signal increased between rat CXCR4 receptors after SDF-1 stimulation (38), whereas another study did not observe significant increases in bioluminiscence resonance energy transfer in response to CXCL12 (13). It is not clear why these systems differ in effects of ligands and peptides on CXCR4 energy transfer, but possibilities include differences in the presence of carrier protein in the culture medium during stimulation, in dose of stimulants, and in incubation times before energy transfer measurements. Although the mechanism of TM4 action has yet to be determined, it is possible that TM4 peptide may disrupt interactions within CXCR4 homodimers or interfere with CXCR4-mediated signal transduction. As a consequence, SDF-1–induced actin polymerization and migration are inhibited.

Our observation that monocytes are also inhibited by TM4 has implications for tumor therapy. Tumor-associated macrophages release cytokines, chemokines, growth factors, and other proteins that stimulate tumor growth, angiogenesis, and invasion/metastasis (39). Tumor-associated macrophages comprised a major component of tumor mass in both primary and secondary human tumors (40). Coculture with monocytes increases tumor cell invasion. Dramatic reduction of tumor growth and angiogenesis was achieved by small interfering RNA of CSF-1 that reduced the number of tumor-associated macrophages (41). Inhibition of monocyte migration by TM4 peptide may also reduce the number and effects of tumor-associated macrophages on tumor growth.

Chemokine receptors have emerged as attractive targets regulating cancer metastasis in vivo. CXCL12/SDF-1 exhibits peak levels of expression in organs representing the first destinations of breast cancer metastasis. Blocking CXCL12/CXCR4 interaction by anti-CXCL12 or anti-CXCR4 antibodies significantly impairs metastasis of breast cancer cells to regional lymph nodes and lung (14). CXCR4 and CXCL12 stand out as likely targets for therapeutics for lung, liver, bone marrow, and brain metastases. In this report, lipid rafts were also necessary for CXCL12-induced cell migration and had effects on homotypic receptor interactions. We further show that a peptide that has effects on receptor interactions can potently control cancer cell migration. Receptor oligomerization is a pivotal aspect of the structure and function of GPCRs that has been shown to be important in receptor signaling. It may be possible to block cancer metastases through agents that interfere with oligomeric receptor structures and/or receptor signaling. Further studies are warranted to address receptor structure and signaling in immunity, HIV-1 infections, cancer biology and therapy, and various inflammatory or allergic conditions.

Acknowledgments

We thank Drs. Judy A. Beeler and Barbara L. Rellahan for critically reading the manuscript.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7/20/05; revised 8/ 9/06; accepted 8/16/06.

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