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.
Showing the most recent 10 out of 13 publications
