Roles of phosphorylation dependent and independent mechanisms in the regulation of Histamine H2 receptor by G Protein-coupled Receptor Kinase 2

FERNANDEZ N; GOTTARDO FL; ALONSO N; MONCZOR F; SHAYO C; DAVIO C

JOURNAL OF BIOLOGICAL CHEMISTRY (ONLINE)

AMER. SOC. Biochemistry Molecular Biology INC

Año: 2011

1083-351X

It is widely assumed that G proteincoupled receptor kinase 2 (GRK2) mediated specific inhibition of G protein-coupled receptors (GPCRs) response involves GRK-mediated receptor phosphorylation followed by b 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 Gas-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 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. In order 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 plekstrin-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 coimmunoprecipitates with the H2R, reversed GRK2K220Rmediated 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 domainmediated regulation of GPCRs beyond Gaq-coupled receptors. On the otherhand, GRK2 kinase activity proved to be necessary for receptor internalization and the resulting resensitization.