A major investigative focus of our laboratory continues to be the characterization of transmembrane signalling mechanisms regulating enteric peptide secretion. Our major finding during the previous grant period was that multiple receptors functionally coupled to adenylyl cyclase (beta-adrenergic, PGE1, somatostatin; SRIF, D2-dopamine) regulate the ubiquitous Na-H exchanger (NHE-1), and hence intracellular pH (pH-i), independently of adenylyl cyclase activity and independently of GTP-binding proteins previously described as being coupled to these receptors. The objective of the current proposal is to test the hypothesis that receptors coupled to adenylyl cyclase regulate NHE-1 through a divergent but conserved pathway involving GTP-binding proteins, and that this effector pathway may serve as one of several control points in regulating peptide secretion from enteric endocrine cells. Specifically, we will investigate three components of this signalling pathway; the receptor, the GTP-binding protein, and the NHE-1 effector by studying effects of the somatostatin receptor (SRIF-R) on NHE-1 in enteric endocrine cells. Based on recent findings that the SRIF-R1 subtype, but not SRIF-R2 subtype, regulates NHE-1, the first aim is to identify amino acid sequences unique to SRIF-R1 that confer specificity for regulating the exchanger. These structure-function studies will use chimeric SRIF-Rl/SRIF-R2 receptors and SRIF-R1 constructs containing deletions and substitutions of selected residues. The objective of Aim 2 is to characterize a pertussis toxin-insensitive G protein that couples the SRIF-R to NHE-1. The rationale for this aim is based on our findings that following pertussis treatment, guanine nucleotides still regulate SRIF inhibition of NHE-1 in enteric endocrine cells. To identify a pertussis-insensitive G protein coupled to the receptor, we will immunoprecipitate receptor-G protein complexes with antisera to specific G protein alpha subunits. We will also photoaffinity label GTP-binding proteins coupled to the SRIF-R1. Additional studies described in Aim 2 will confirm the functional role of this G protein in regulating NHE-1. The objective of Aim 3 is to investigate the functional importance of receptor-regulated NHE-1 activity in peptide secretion. Experiments outlined in this aim will also use video-imaging microscopy to characterize subcellular pH and determine whether agonist-induced changes in pH-i are localized and/or polarized. Our studies indicate that in addition to the SRIF-R, other receptors linked to the regulation of adenylyl cyclase also divergently regulate NHE-1. Conservation of this mechanism, therefore, may be as fundamental as the regulation of adenylyl cyclase. The research proposed in this application represents an examination of the molecular components participating in this signalling pathway.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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General Medicine A Subcommittee 2 (GMA)
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University of California San Francisco
Internal Medicine/Medicine
Schools of Dentistry
San Francisco
United States
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