CONICET | Buscador de Institutos y Recursos Humanos

Histamine H2 receptors (H2r) belong to the large family of G-protein coupled receptors (GPCRs). By coupling to Gs protein, H2r stimulation triggers adenylyl cyclase activation, and in turn rapid cAMP accumulation. Following H2r phosphorylation and desensitization by GRK2, H2r are internalized and recycled back to the cell membrane. It is widely assumed that specific inhibition of GPCRs response occurs by â arrestin binding to the phosphorylated receptor. In this context, GRK major function is to enable â arrestin binding to the receptor and uncoupling from heterotrimeric G protein. In the present study, we investigated whether H2r phosphorylation was necessary for the desensitization and/or internalization/resensitization of the H2r. Therefore the effect of GRK2K220R (a phosphorylation-inactive mutant with disrupted kinase activity) was evaluated on these processes in COS7-H2r and U937 transfected cells. In COS7-H2r cells, transfection with either GRK2K220R mutant or GRK2 wild type, led to a 30% decrease in cAMP response to amthamine due to enhanced H2r desensitization. When H2r internalization and recycling were determined by binding assays, we found that GRK2K220R dampened H2r internalization and therefore resensitization behaving as the GRK2 antisense construct. Similar results were obtained in U937 cells stably transfected with the GRK2K220R mutant. These findings support that receptor phosphorylation results crucial for H2r internalization and resensitization. However, kinase activity is not necessary to achieve H2r desensitization. Furthermore, the GRK2K220R mutant attenuates H2r signaling to a similar extent as the GRK2 wild type kinase, revealing a phosphorylation-independent mechanism of H2r regulation by GRK2.