Journal of Cellular Biochemistry 93:753–760 (2004) Constitutive Association of Cell Surface CCR5 and CXCR4 in the Presence of CD4 Jinhai Wang,* Raymond Alvarez, Gregory Roderiquez, Ennan Guan, and Michael A. Norcross Laboratory of Immunology, Division of Therapeutic Proteins, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892 Abstract Chemokine receptors CCR5 and CXCR4 are the major coreceptors of HIV-1 infection and also play fundamental roles in leukocyte trafficking, metastasis, angiogenesis, and embyogenesis. Here, we show that transfection of CCR5 into CXCR4 and CD4 expressing 3T3 cells enhances the cell surface level of CXCR4. In CCR5 high expressing cells, cell surface level of CXCR4 was incompletely modulated in the presence of the CXCR4 ligand CXCL12/SDF-1a. CCR5 was resistant to ligand-dependent modulation with the CCR5 ligand CCL5/RANTES. Confocal laser microscopy revealed that CCR5 was colocalized with CXCR4 on the cell surface. In CD4 expressing CCR5 and CXCR4 double positive NIH 3T3 cells, immunoprecipitation followed by Western blot analysis revealed that CCR5 was associated with CXCR4 and CD4. CXCR4 and CCR5 were not co-immunoprecipitated in cells expressing CCR5 and CXCR4 but without CD4 expression. Compared to NIH 3T3CD4 cells expressing CXCR4, the entry of an HIV-1 X4 isolate (HCF) into NIH 3T3CD4 expressing both CXCR4 and CCR5 was reduced. Our data indicate that chemokine receptors interact with each other, which may modulate chemokine–chemokine receptor interactions and HIV-1 coreceptor functions. J. Cell. Biochem. 93: 753 – 760, 2004. Published 2004 Wiley-Liss, Inc. { Key words: chemokine receptor; chemokine; HIV-1; viral entry HIV-1 isolates are classified as R5, X4, or R5X4 viruses depending on whether CCR5, CXCR4, or both CCR5 and CXCR4 are used for entry. R5 HIV-1 are the dominant isolates replicating in vivo at the time of seroconversion [Cheng-Mayer et al., 1988; Tersmette et al., 1989; Schuitemaker et al., 1992]. In 40–50% of HIV-1 infected individuals, X4 isolates emerge and replace R5 isolates as the major viral popu- lation [Cheng-Mayer et al., 1988; Tersmette et al., 1989; Schuitemaker et al., 1992; Kinter et al., 1998]. It is unclear why R5 HIV-1 is the primary viruses transmitted sexually. CXCR4 has been characterized as the major coreceptor for T tropic (X4) isolates [Feng et al., 1996] and will associate with CD4 in the presence of gpl20 [Lapham et al., 1996]. As a chemokine receptor, CXCR4 is functional in monocytes/macropha- ges as assessed by receptor downmodulation, chemotaxis, calcium mobilization [Wang et al., 2001a]. Chemokines/chemokine receptors play fun- damental roles in leukocyte trafficking, metas- tasis, angiogenesis, and embyogenesis [Berger et al., 1999; Murphy, 2001]. CC and CXC chemokines are two distinct groups of chemo- kines. Chemokines, especially CC chemokines, may interact with multiple chemokine recep- tors, and these cross interactions are strictly limited within either CC or CXC subfamily. In response to ligand some GPCRs undergo homo- dimerization and ligand dependent endocytosis. Cytokines can also regulate receptor endocyto- sis through ligand-independent endocytosis as we and others have reported for the CCR5 and CXCR4 receptors [Wang et al., 1998, 2001a; Lee et al., 1999a; Garzino-Demo et al., 2000]. Re- cently heterodimerization of GPCR was found between GBRl and GBR2, and between opioid receptors k and d [Kaupmann et al., 1998; White et al., 1998; Jordan and Devi, 1999]. Hetero- oligomers with enhanced functional activity were formed in receptors for dopamine and somatostatin [Rocheville et al., 2000a,b]. Published 2004 Wiley-Liss, Inc. { This article is a US Govern- ment work, and as such, is in the public domain of the United States of America. *Correspondence to: Dr. Jinhai Wang, Division of Ther- apeutic Proteins, Center for Drug Evaluation and Research, FDA, NIH Building 29B, Room 4E12, HFM- 541, 8800 Rockville Pike, Bethesda, MD 20892. E-mail: wangj @cber.fda.gov Received 9 March 2004; Accepted 23 April 2004 DOI 10.1002/jcb.20161