Chemokine receptor CCR5 functionally couples to inhibitory G proteins and undergoes desensitization

Abstract

Chemokine receptor CCR5 is not only essential for chemotaxis of leukocytes but also has been shown to be a key coreceptor for HIV-1 infection. In the present study, hemagglutinin epitope-tagged human CCR5 receptor was stably expressed in Chinese hamster ovary cells or transiently expressed in NG108-15 cells to investigate CCR5-mediated signaling events. The surface expression of CCR5 was confirmed by flow cytometry analysis. The CCR5 agonist RANTES stimulated [35S]GTPgammaS binding to the cell membranes and induced inhibition on adenylyl cyclase activity in cells expressing CCR5. The effects of RANTES were CCR5 dependent and could be blocked by pertussis toxin. Furthermore, overexpression of Gialpha2 strongly increased both RANTES-dependent G-protein activation and inhibition on adenylyl cyclase in cells cotransfected with CCR5. These data demonstrated directly that activation of CCR5 stimulated membrane-associated inhibitory G proteins and indicated that CCR5 could functionally couple to G-protein subtype Gialpha2. The abilities of CCR5 to activate G protein and to inhibit cellular cAMP accumulation were significantly diminished after a brief prechallenge with RANTES, showing rapid desensitization of the receptor-mediated responsiveness. Prolonged exposure of the cells to RANTES caused significant reduction of surface CCR5 as measured by flow cytometry, indicative of agonist-dependent receptor internalization. Our data thus demonstrated that CCR5 functionally couples to membrane-associated inhibitory G proteins and undergoes agonist-dependent desensitization and internalization.

Five-transmembrane domains appear sufficient for a G protein-coupled receptor: Functional five-transmembrane domain chemokine receptors

“…3, the cells expressing mCCR5 and mCXCR4 showed increased migration (C) and extracellular calcium influx (D) in response to chemokine stimulation at levels comparable to the wild-type controls. These data indicate that (12). Stimulation of chemokine receptors activate inhibitory G proteins (12), which induce chemotaxis (16).…”

“…The full-length cDNA encoding the wild-type CXCR4 was cloned by reverse transcription-PCR (RT-PCR) from THP-1 cells using primers designed based on the published sequences (GenBank accession no. X71635), and the wild-type human CCR5 was cloned as described (12). A minor PCR product of approximately 800 bp, in addition to the expected product of 1,020 bp, was generated in PCRs to amplify CCR5 by using human leukocyte cDNAs as a template.…”

“…The assay was carried out as described (12). Cells were lysed in 5 mM Tris⅐HCl, pH 7.5͞5 mM EDTA͞5 mM EGTA at 4°C.…”

TNF-α-Induced Secretion of C-C Chemokines Modulates C-C Chemokine Receptor 5 Expression on Peripheral Blood Lymphocytes

“…It is well established that cell surface expression of CCR5 can be modulated by internalization, which occurs as a consequence of binding of the cognate chemokines or the chemokine derivative AOP-RANTES (47)(48)(49)(50)(51)(52). The internalization mechanism is still under investigation, but appears to involve the G protein-coupled receptor kinases (GRKs) (48), of which GRK2 and GRK3 are expressed in human PBLs.…”

Modulation of tumor necrosis factor related apoptosis-inducing ligand (TRAIL) receptors in a human osteoclast model in vitro

“…Similar stimulation of CCR5 in CHO cells, with MIP-1/3 inhibited AC activity (Blanpain et al, 2001). More specifically, overexpression of Gai2 strongly re-enforced this mechanism of action, suggesting that it is a subunit of interest in the case of CCR5 AC inhibition (Zhao et al, 1998). The opposite effect was observed with MIP-la stimulation in human hepatoma cells, with an inhibition of cAMP formation.…”