ROLE OF RECEPTOR PHOSPHORYLATION IN DESENSITIZATION AND INTERNALIZATION OF THE SECRETIN RECEPTOR

The secretin receptor is prototypic of a recently described family of G protein-coupled receptors. We recently demonstrated its phosphorylat ion in response to agonist stimulation and elimination of this covalen t modification by C-terminal truncation (F. Ozcelebi et al, (1995) Mol . Pharmacol. 48, 818-824). Here, we explore the functional impact of r eceptor phosphorylation and structural determinants for desensitizatio n by comparing receptor behavior after agonist exposure in cell lines expressing wild-type and truncated receptor. To characterize receptor internalization, a novel fluorescent full agonist, [rat secretin-27]-G ly-rhodamine, was developed, which bound specifically and with high af finity. Both receptor constructs bound secretin normally, leading to n ormal G protein coupling and cAMP accumulation and prompt receptor int ernalization. Exposure to 10 nM secretin for 5 min or 12 h prior to wa shing and restimulation with a full range of concentrations demonstrat ed absent cAMP responses in wild-type receptor-bearing cells and respo nses 25 to 30% of control and shifted 1 order of magnitude to the righ t in the truncated receptor-bearing cells. Thus, the major mechanism o f desensitization was phosphorylation-independent receptor internaliza tion. Phosphorylation was associated with a distinct process that like ly represents interference with G protein coupling, manifest as a redu ced rate of cAMP stimulation. Thus, dual distinct mechanisms of desens itization exist in the secretin receptor family that should help prote ct receptor bearing cells from overstimulation.