title: Mutation of the DRY motif reveals different structural requirements for the CC chemokine receptor 5-mediated signaling and receptor endocytosis.
abstract: CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor that governs migration of leukocytes and serves as a coreceptor for the R5 tropic strains of human immunodeficiency virus (HIV). CCR5-mediated signaling in response to CC chemokines relies on G protein activation. Desensitization, which rapidly turns off G protein-dependent signaling, involves phosphorylation of CCR5 that promotes interaction of the receptor with beta-arrestins for endocytosis. Whether coupling to G proteins, desensitization, and endocytosis of CCR5 require the same structural determinants remains a matter of investigation. Here, we show that CCR5 displayed agonist-independent coupling to G proteins. This constitutive activity of the receptor was abrogated by TAK779 (N,N-dimethyl-N-[4-[[[2-(4-methylphenyl)-6,7-dihydro-5H-benzocyclohepten-8-yl]carbonyl]amino]benzyl]tetrahydro-2H-pyran-4-aminium chloride), a nonpeptidic CCR5 ligand that inhibits HIV infection and was found to depend on the integrity of the Asp-Arg-Tyr (DRY) motif. Changing Arg-126 by the neutral residue Asn (R126N-CCR5 mutant) abolished CCR5-mediated activation of G proteins, either constitutively or in response to agonists. In contrast, R126N-CCR5 not only retained agonist-promoted phosphorylation and beta-arrestin-dependent endocytosis but also displayed a higher basal phosphorylation than wild-type CCR5. Expression of beta-arrestin in R126N-CCR5-expressing cells resulted in receptor down-regulation, thereby suggesting that R126N-CCR5 spontaneously interacts with beta-arrestins. However, although expression of beta-arrestin favored wild-type CCR5-mediated chemotaxis, it failed to promote migration of cells expressing R126N-CCR5. Overall, these data indicate that structural requirements for CCR5-mediated activation of G proteins, albeit not involved in receptor desensitization and internalization, are needed for beta-arrestin-mediated chemotaxis. These results have implications for how distinct biological responses of CCR5 might rely on a different set of receptor conformations.
authors.affiliation: Institut Pasteur, Unité d'Immunologie Virale, 28 rue du Dr Roux, 75015 Paris, France.
authors.forename: Bernard
authors.initials: B
authors.lastname: Lagane
authors.forename: Sébastien
authors.initials: S
authors.lastname: Ballet
authors.forename: Thierry
authors.initials: T
authors.lastname: Planchenault
authors.forename: Karl
authors.initials: K
authors.lastname: Balabanian
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Le Poul
authors.forename: Cédric
authors.initials: C
authors.lastname: Blanpain
authors.forename: Yann
authors.initials: Y
authors.lastname: Percherancier
authors.forename: Isabelle
authors.initials: I
authors.lastname: Staropoli
authors.forename: Gilbert
authors.initials: G
authors.lastname: Vassart
authors.forename: Martin
authors.initials: M
authors.lastname: Oppermann
authors.forename: Marc
authors.initials: M
authors.lastname: Parmentier
authors.forename: Françoise
authors.initials: F
authors.lastname: Bachelerie
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D019390:Arrestins / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D015394:Molecular Structure; D009154:Mutation; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D019713:Receptors, CCR5 / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D019713:Receptors, CCR5; D000071557:beta-Arrestins