title: Roles of phosphorylation-dependent and -independent mechanisms in the regulation of histamine H2 receptor by G protein-coupled receptor kinase 2.
abstract: It is widely assumed that G protein-coupled receptor kinase 2 (GRK2)-mediated specific inhibition of G protein-coupled receptors (GPCRs) response involves GRK-mediated receptor phosphorylation followed by β-arrestin binding and subsequent uncoupling from the heterotrimeric G protein. It has recently become evident that GRK2-mediated GPCRs regulation also involves phosphorylation-independent mechanisms. In the present study we investigated whether the histamine H2 receptor (H2R), a Gα(s)-coupled GPCR known to be desensitized by GRK2, needs to be phosphorylated for its desensitization and/or internalization and resensitization. For this purpose we evaluated the effect of the phosphorylating-deficient GRK2K220R mutant on H2R signaling in U937, COS7, and HEK293T cells. We found that although this mutant functioned as dominant negative concerning receptor internalization and resensitization, it desensitized H2R signaling in the same degree as the GRK2 wild type. To identify the domains responsible for the kinase-independent receptor desensitization, we co-transfected the receptor with constructions encoding the GRK2 RGS-homology domain (RH) and the RH or the kinase domain fused to the pleckstrin-homology domain. Results demonstrated that the RH domain of GRK2 was sufficient to desensitize the H2R. Moreover, disruption of RGS functions by the use of GRK2D110A/K220R double mutant, although coimmunoprecipitating with the H2R, reversed GRK2K220R-mediated H2R desensitization. Overall, these results indicate that GRK2 induces desensitization of H2R through a phosphorylation-independent and RGS-dependent mechanism and extends the GRK2 RH domain-mediated regulation of GPCRs beyond Gα(q)-coupled receptors. On the other hand, GRK2 kinase activity proved to be necessary for receptor internalization and the resulting resensitization.
authors.affiliation: Laboratorio de Farmacología de Receptores, Cátedra de Química Medicinal, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, 1113 Buenos Aires, Argentina,; Consejo Nacional de Investigaciones Científicas y Técnicas, 1033 Buenos Aires, Argentina. Electronic address: natycfernandez@gmail.com.
authors.forename: Natalia
authors.initials: N
authors.lastname: Fernandez
authors.affiliation: Laboratorio de Farmacología de Receptores, Cátedra de Química Medicinal, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, 1113 Buenos Aires, Argentina.
authors.forename: Federico L
authors.initials: FL
authors.lastname: Gottardo
authors.affiliation: Consejo Nacional de Investigaciones Científicas y Técnicas, 1033 Buenos Aires, Argentina; Laboratorio de Farmacología y Patología Molecular, Instituto de Biología y Medicina Experimental, 1426 Buenos Aires, Argentina, and.
authors.forename: Maria N
authors.initials: MN
authors.lastname: Alonso
authors.affiliation: Laboratorio de Farmacología de Receptores, Cátedra de Química Medicinal, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, 1113 Buenos Aires, Argentina,; Consejo Nacional de Investigaciones Científicas y Técnicas, 1033 Buenos Aires, Argentina.
authors.forename: Federico
authors.initials: F
authors.lastname: Monczor
authors.affiliation: Consejo Nacional de Investigaciones Científicas y Técnicas, 1033 Buenos Aires, Argentina; Laboratorio de Farmacología y Patología Molecular, Instituto de Biología y Medicina Experimental, 1426 Buenos Aires, Argentina, and.
authors.forename: Carina
authors.initials: C
authors.lastname: Shayo
authors.affiliation: Laboratorio de Farmacología de Receptores, Cátedra de Química Medicinal, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, 1113 Buenos Aires, Argentina,; Consejo Nacional de Investigaciones Científicas y Técnicas, 1033 Buenos Aires, Argentina.
authors.forename: Carlos
authors.initials: C
authors.lastname: Davio
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D020125:Mutation, Missense; D010766:Phosphorylation / Q000502:physiology; D017434:Protein Structure, Tertiary; D011968:Receptors, Histamine / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D020298:U937 Cells
chemical_list: D011968:Receptors, Histamine; C496567:GRK2 protein, human; D054769:G-Protein-Coupled Receptor Kinase 2