Palmitoylation of CCR5 is critical for receptor trafficking and efficient activation of intracellular signaling pathways

CCR5 is a CC chemokine receptor expressed on memory lymphocytes, macrophage s, and dendritic cells and also constitutes the main coreceptor for macroph age-tropic (or R5) strains of human immunodeficiency viruses. In the presen t study, we investigated whether CCR5 as palmitoylated in its carboxyl-term inal do. main by generating alanine substitution mutants for the three cyst eine residues present in this region, individually or in combination. We fo und that wild-type CCR5 was palmitoylated, but a mutant lacking all three C ys residues was not. Through the use of green fluorescent fusion proteins a nd immunofluorescence studies, we found that the absence of receptor palmit oylation resulted in sequestration of CCR5 in intracellular biosynthetic co mpartments. By using the fluorescence recovery after photobleaching techniq ue, we showed that the non-palmitoylated mutant had impaired diffusion prop erties within the endoplasmic reticulum. We next stud led the ability of th e mutants to bind and signal in response to chemokines, Chemokines binding and activation of G(i)-mediated signaling pathways, such as calcium mobiliz ation and inhibition of adenylate cyclase, were not affected. However, the duration of the functional response, as measured by a microphysiometer, and the ability to increase [S-35]guanosine 5 ' -3-O(thio)triphosphate binding to membranes were severely affected for the non-palmitoylated mutant. The ability of RANTES (regulated on activation normal T cell expressed and secr eted) and aminooxypentane-RANTES to promote CCR5 endocytosis was not altere d by cysteine replacements. Finally, we found that the absence of receptor palmitoylation reduced the human immunodeficiency viruses coreceptor functi on of CCR5, but this effect was secondary to the reduction in surface expre ssion. In conclusion, we found that palmitoylated cysteines play an importa nt role in the intracellular trafficking of CCR5 and are likely necessary f or efficient coupling of the receptor to part of its repertoire of signalin g cascades.