Neural control of the gastrointestinal (GI) track function is mediated through neurotransmitters and neuropeptides, released from extrinsic and intrinsic nerve fibers in the gut, which interact with cell surface receptors that transmit intracellular signals. Neuropeptides such as secretin, glucagon, and vasoactive intestinal peptide (VIP) are structurally related peptides which regulate digestive processes such as absorption, secretion and peristaltic function. Receptors for these respective neuropeptides are capable of recognizing with different affinities secretin, glucagon, and VIP suggesting that the receptors may be homologous in primary sequence. I have cloned the VIP receptor of HT29 colonic adenocarcinoma cells, and propose to clone cDNA encoding the secretin and glucagon receptors from cultured cell lines selectively bearing the respective receptors. Identification of these receptors and pathways of signal transduction will be determined by transfection of the receptor cDNAs into mammalian cells, and analyzing binding characteristics and the effects of guanine nucleotides and analogues. Common structural motifs involved in ligand binding, and interaction with specific G proteins, kinases and regulatory proteins will be examined by site-directed mutagenesis, insertion/deletion analyses and receptor chimeras. Antibodies to peptides derived from the deduced sequence of the receptors will be used to examine expression in the various cells and tissue of the GI tract. Message distribution will be examined by Northern analyses and in situ hybridization, and the genomic organization will be elucidated. The availability of full length cDNAs for these closely related receptors will permit a careful analysis of the mechanism of ligand interaction with the receptor binding site, resulting in the future design of specific antagonists and agonists for many important GI tract functions. A second objective is to examine the very efficient signalling pathway for VIP in HT29 cells, which can result in an induction of cAMP amounting to 3 orders of magnitude. I propose to purify the VIP receptor-coupled G alpha protein subunit from HT29 cells, and examine its activity in membrane preparations of S49 cyc- cell transfected with the VIP receptor cDNA. If necessary, the adenylyl cyclase enzyme from HT29 cells will also be purified and its activity will be examined in reconstitution assays containing the purified G alpha protein, and membrane preparations of COS 7 cells transfected with the VIP receptor cDNA.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
1R29DK044876-01
Application #
3464660
Study Section
Biochemical Endocrinology Study Section (BCE)
Project Start
1992-04-01
Project End
1997-03-31
Budget Start
1992-04-01
Budget End
1993-03-31
Support Year
1
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Type
Schools of Medicine
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
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Xia, M; Leppert, D; Hauser, S L et al. (1996) Stimulus specificity of matrix metalloproteinase dependence of human T cell migration through a model basement membrane. J Immunol 156:160-7
Xia, M; Sreedharan, S P; Dazin, P et al. (1996) Integrin-dependent role of human T cell matrix metalloproteinase activity in chemotaxis through a model basement membrane. J Cell Biochem 61:452-8
Sreedharan, S P; Huang, J X; Cheung, M C et al. (1995) Structure, expression, and chromosomal localization of the type I human vasoactive intestinal peptide receptor gene. Proc Natl Acad Sci U S A 92:2939-43
Ichikawa, S; Sreedharan, S P; Owen, R L et al. (1995) Immunochemical localization of type I VIP receptor and NK-1-type substance P receptor in rat lung. Am J Physiol 268:L584-8
Patel, D R; Kong, Y; Sreedharan, S P (1995) Molecular cloning and expression of a human secretin receptor. Mol Pharmacol 47:467-73
Sreedharan, S P; Patel, D R; Xia, M et al. (1994) Human vasoactive intestinal peptide1 receptors expressed by stable transfectants couple to two distinct signaling pathways. Biochem Biophys Res Commun 203:141-8
Ichikawa, S; Sreedharan, S P; Goetzl, E J et al. (1994) Immunohistochemical localization of peptidergic nerve fibers and neuropeptide receptors in Peyer's patches of the cat ileum. Regul Pept 54:385-95
Goetzl, E J; Patel, D R; Kishiyama, J L et al. (1994) Specific recognition of the human neuroendocrine receptor for vasoactive intestinal peptide by anti-peptide antibodies. Mol Cell Neurosci 5:145-52

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