Regulation of human chemokine receptors CXCR4 - Role of phosphorylation in desensitization and internalization

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 tar; get 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 (Delta Cyto CXCR4) of CXCR4. The ligand, stromal cell derived factor-1 (SDF-1) stimulated higher G-protein activation, inositol phosphate generation, add a more sustained calcium elevation in cells expressing Delta Cyto 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 Delta Cyto CXCR4 was not detected under any of these conditions. Despite lack of receptor phosphorylation, calcium mobilization by SDF-1 in Delta Cyto 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 ill Delta Cyto CXCR4, cAMP also partially inhibited these responses in both, Wt CXCR4, and Delta Cyto CXCR4. SDF-1, PMA, and cAMP caused phosphorylation of phospholipase C beta 3 in Wt add Delta Cyto CXCR4 cells. Both SDF-1 as well as PMA induced rapid internalization of Wt CXCR4. SDF-1 but not PMA induced internalization of Delta Cyto CXCR4 albeit at reduced levels relative: td 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 C beta 3.