application) Secretin modulates gastric acid release, intestinal motility and pancreatic water and bicarbonate secretion leading to neutralization of acidic chyme. Its effects are mediated by elevating intracellular CAMP via a G protein-coupled receptor (GPCR) that is a member of a unique subclass of GPCRs (Class 11). Regulation of this subclass of receptors, which includes the vasoactive intestinal polypeptide and glucagon receptors, is incompletely understood. We have recently demonstrated that members of this subclass utilize regulatory pathways different from those of Class I GPCRs. Elucidation of this path may further our understanding of GPCR regulation and signal transduction. Using heterologous cell systems transiently transfected with cDNA for the secretin receptor and various G protein receptor kinases (GRKs), we have demonstrated that the secretin receptor is phosphorylated by both GRK and second messenger dependent protein kinases. Phosphorylation by GRKs results in secretin receptor desensitization, however, phosphorylation by protein kinase A (PKA), although responsible for half of receptor phosphorylation, does not diminish receptor signaling. PKA inhibitors do decrease secretin receptor sequestration. Also, unlike the beta-2-adrenergic receptor, secretin receptor sequestration is not altered by functionally impaired dynamin or beta-arrestin mutants. These results suggest the secretin receptor may be regulated by a PKA dependent protein and this receptor may utilize a unique pathway for receptor sequestration. Our hypothesis is the secretin receptor, as a member of a unique class of GPCRs, is regulated by a novel PKA phosphoprotein. The goal of this proposal is to determine if novel proteins participate in secretin receptor signal regulation.
The specific aims i nclude: 1) to determine if novel proteins interact with the third intracellular loop and/or the C-terminal tail region of the secretin receptor using Yeast-2 Hybrid technology, and 2) to use 2-dimensional gel electrophoresis to discern phosphoproteins involved in regulation of this receptor. This information may have significant implications for clinical gastroenterology and GI hormone function.
