SECRETIN RECEPTOR STRUCTURE, FUNCTION, AND REGULATION

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With the long-term goal of better understanding the roles of the gastrointestinal peptide hormone secretin in health and disease, the current proposal focuses on structure, function, and regulation of the secretin receptor in the exocrine pancreas. Secretin is important physiologically and is potentially important in the pathogenesis, diagnosis, and treatment of pancreatic disease. Our general hypothesis is that structural and regulatory themes active for the secretin receptor will be distinct from those of the majority of G protein- coupled receptors, and will provide Insights into the molecular basis for integrated function of the exocrine pancreas. We propose to follow complementary pathways directed toward the secretin receptor molecule and the cellular mechanisms for regulating receptor function. In preparation, the pancreatic secretin receptor cDNA has been cloned, sequenced, and expressed; fusion proteins which incorporate tags for efficient affinity purification of the recombinant receptor have been constructed and validated; and novel radioligands have been developed. The secretin receptor cDNA sequence has established its close relationship to receptors for VIP, PACAP, PTH, and GHRF, and the absence of structural motifs typical of the majority of G protein- coupled receptors. The predicted large and complex, cysteine-rich ectodomains of these receptors interestingly complement the large pharmacophoric domains of their natural ligands. In this work, we will characterize the structure of this domain of the receptor, postulating its key role in agonist binding. This includes analysis of the patterns of disulfide bonding and glycosylation, and the functional impact of each. The agonist-binding domain will be defined both directly using affinity labeling with novel probes incorporating photolabile residues "intrinsic to" and "spanning" the pharmacophore, and indirectly using molecular constructs which include deletions, receptor chimeras, and site-specific mutants. The effects of receptor phosphorylation will be studied in model recombinant receptor-bearing cell systems, and mutagenesis approaches will be utilized to define key domains and residues. Finally, the secretin receptor on the pancreatic acinar cell, a well-established model for stimulus-activity coupling, will be studied to define receptor regulatory processes in a prototypic native cellular environment An understanding of structural and regulatory themes for the secretin receptor should have broad implications for this new important receptor family, as well as providing the basis for exploring the roles of this receptor in normal physiology, in development, and in the pathogenesis of disease.