GASTROINTESTINAL SIGNIFICANCE OF GASTRIN REGULATION

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The acid stimulatory action of gastrin is dependent on its biological activation via post-translational processing and the binding of the active peptide to its receptor on gastric parietal cells. The aims of this proposal focus on elucidation of the biochemical basis for these two crucially important steps at the juncture of hormone-target cell interaction. In previous studies we have explored the biosynthesis of gastrin and developed a model for post-translational processing its precursor. Biological activation of gastrin requires the formation of a carboxyl-terminal amide moiety from a glycine-extended progastrin processing intermediate. To explore the structural requirements for this and other gastrin processing reactions we propose to express, in a variety of endocrine-derived cell lines, gastrin cDNA clones that we and others have previously isolated. By virtue of their synthesis of other peptide hormones, these cell lines are known to be capable of conducting some processing reactions but possible not others. We will examine the expression of gastrin cDNA clones that have site-specific mutations at three processing sites, the Arg57-Arg58 residues that are cleaved to form gastrin-34, the Lys74-Lys75 residues that are cleaved to form gastrin 17, and the carboxyl-terminal Gly93-Arg94-Arg95 complex that signals the amidation process. Because virtually all peptides undergo processing via dibasic cleavage reactions and more than half undergo carboxyl-terminal amidation, the results of these studies may have broad implications relevant to a large variety of hormonally regulated gastrointestinal functions. In other studies we have explored the biochemistry of the parietal cell gastrin receptor by demonstrating a) its selective requirement for amidated gastrins, b) its structure as a single subunit 74kD protein, and c) its linkage to cellular inositol phospholipid turnover and protein kinase C translocation. We propose to extend these studies by purifying the gastrin receptor, determining a portion of its amino acid sequence, and using an oligonucleotide constructed on the basis of this sequence to isolate a cDNA clone encoding the receptor from a parietal cell cDNA library. The library will be screened, in addition, with an antibody generated against the purified gastrin receptor. Another approach that we will pursue is expression cloning of the gastrin receptor in oocytes of Xenopus laevis. After isolating the gastrin receptor cDNA, we will express it in both non-endocrine and endocrine cell lines. The techniques of site-directed mutagenesis will be applied selectively to the receptor's transmembrane and intracytoplasmic domains to explore the structural requirements for, respectively, ligand binding and linkage to signal transduction mechanisms. Through these studies we hope to gain insight into the structure of peptide hormone receptors and their linkage to physiological functions in health and disease.