Real-time visualization of the cellular redistribution of G protein-coupled receptor kinase 2 and beta-arrestin 2 during homologous desensitization of the substance P receptor

The substance P receptor (SPR) is a G protein-coupled receptor (GPCR) that plays a key role in pain regulation. The SPR desensitizes in the continued presence of agonist, presumably via mechanisms that implicate G protein cou pled receptor kinases (GRKs) and beta-arrestins, The temporal relationship of these proposed biochemical events has never been established for any GPC R other than rhodopsin beyond the resolution provided by biochemical assays . We investigate the real-time activation and desensitization of the human SPR in live HEK293 cells using green fluorescent protein conjugates of prot ein kinase C, GRK2, and beta-arrestin 2. The translocation of protein kinas e C beta II-green fluorescent protein to and from the plasma membrane in re sponse to substance P indicates that the human SPR becomes activated within seconds of agonist exposure, and the response desensitizes within 30 s, Th is desensitization process coincides with a redistribution of GRK2 from the cytosol to the plasma membrane, followed by a robust redistribution of bet a-arrestin 2 and a profound change in cell morphology that occurs after I m in of SPR stimulation. These data establish a role for GrRKs and beta-arres tins in homologous desensitization of the SPR and provide the first visual and temporal resolution of the sequence of events underlying homologous des ensitization of a GPCR in living cells.