Regulation of Human Chemokine Receptors CXCR4
ROLE OF PHOSPHORYLATION IN DESENSITIZATION AND INTERNALIZATION*
- Bodduluri Haribabu‡§,
- Ricardo M. Richardson‡,
- Ian Fisher‡,
- Silvano Sozzani¶‖,
- Stephen C. Peiper**,
- Richard Horuk‡,
- Hydar Ali‡ and
- Ralph Snydermanत
- From the Departments of ‡Medicine and§§Immunology, Duke University Medical Center, Durham, North Carolina 27710, the ¶Instuito di Ricerche Farmacologiche, “Mario Negri,” Milan, Italy, the **James Graham Brown Cancer Center, Louisville, Kentucky 40292, and the‡Department of Immunology, Berlex Biosciences, Richmond, California 94804
Abstract
Members of the chemokine receptor family CCR5 and CXCR4 have recently been shown to be involved in the entry of human immunodeficiency virus (HIV) into target cells. Here, we investigated the regulation of CXCR4 in rat basophilic leukemia cells (RBL-2H3) stably transfected with wild type (Wt CXCR4) or a cytoplasmic tail deletion mutant (ΔCyto CXCR4) of CXCR4. The ligand, stromal cell derived factor-1 (SDF-1) stimulated higher G-protein activation, inositol phosphate generation, and a more sustained calcium elevation in cells expressing ΔCyto CXCR4 relative to Wt CXCR4. SDF-1 and phorbol 12-myristate 13-acetate (PMA), but not a membrane permeable cAMP analog induced rapid phosphorylation as well as desensitization of Wt CXCR4. Phosphorylation of ΔCyto CXCR4 was not detected under any of these conditions. Despite lack of receptor phosphorylation, calcium mobilization by SDF-1 in ΔCyto CXCR4 cells was partially desensitized by prior treatment with SDF-1. Of interest, the rapid release of calcium was inhibited without affecting the sustained calcium elevation, indicating independent regulatory pathways for these processes. PMA completely inhibited phosphoinositide hydrolysis and calcium mobilization in Wt CXCR4 but only partially inhibited these responses in ΔCyto CXCR4. cAMP also partially inhibited these responses in both Wt CXCR4 and ΔCyto CXCR4. SDF-1, PMA, and cAMP caused phosphorylation of phospholipase Cβ3 in Wt and ΔCyto CXCR4 cells. Both SDF-1 as well as PMA induced rapid internalization of Wt CXCR4. SDF-1 but not PMA induced internalization of ΔCyto CXCR4 albeit at reduced levels relative to Wt CXCR4. These results indicate that signaling and internalization of CXCR4 are regulated by receptor phosphorylation dependent and independent mechanisms. Desensitization of CXCR4 signaling, independent of receptor phosphorylation, appears to be a consequence of the phosphorylation of phospholipase Cβ3.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grants DE-03738, HL-54166, and AI-38910.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‖ Supported by a fellowship from the Italian Association for Cancer Research.
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↵§ To whom correspondence should be addressed: Dept. of Medicine, Duke University Medical Center, Box 3680, Durham, NC 27710. Tel.: 919-684-2280; Fax: 919-684-4390; E-mail: Boddu001{at}mc.duke.edu.
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↵1 The abbreviations used are: HIV, human immunodeficiency virus; RBL, rat basophilic leukemia cells; ΔCyto, cytoplasmic tail deletion mutants; SDF-1, stromal cell derived factor-1; PMA, phorbol 12-myristate 13-acetate; MIP, macrophage inflammatory protein; FACS, fluorescence activated cell sorting; PKC, protein kinase C; PKA, protein kinase A; PLC, phospholipase C; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis; GTPγS, guanosine 5′-3-O-(thio)triphosphate; Wt, wild type; DMEM, Dulbecco’s modified Eagle’s medium.
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↵2 B. Haribabu, S. Sozzani, H. Ali, and R. Snyderman, unpublished results.
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- Received August 27, 1997.
