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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK034306-09
Application #
3232640
Study Section
General Medicine A Subcommittee 2 (GMA)
Project Start
1984-07-01
Project End
1994-06-30
Budget Start
1992-07-01
Budget End
1993-06-30
Support Year
9
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Todisco, A; Ramamoorthy, S; Witham, T et al. (2001) Molecular mechanisms for the antiapoptotic action of gastrin. Am J Physiol Gastrointest Liver Physiol 280:G298-307
Sawada, M; Finniss, S; Dickinson, C J (2000) Diminished prohormone convertase 3 expression (PC1/PC3) inhibits progastrin post-translational processing. Regul Pept 89:19-28
Wang, L D; Wang, M; Todisco, A et al. (2000) The human histamine H(2) receptor regulates c-jun and c-fos in a differential manner. Am J Physiol Cell Physiol 278:C1246-55
Stepan, V M; Tatewaki, M; Matsushima, M et al. (1999) Gastrin induces c-fos gene transcription via multiple signaling pathways. Am J Physiol 276:G415-24
Stepan, V M; Dickinson, C J; del Valle, J et al. (1999) Cell type-specific requirement of the MAPK pathway for the growth factor action of gastrin. Am J Physiol 276:G1363-72
Stepan, V M; Sawada, M; Todisco, A et al. (1999) Glycine-extended gastrin exerts growth-promoting effects on human colon cancer cells. Mol Med 5:147-59
Stepan, V M; Krametter, D F; Matsushima, M et al. (1999) Glycine-extended gastrin regulates HEK cell growth. Am J Physiol 277:R572-81
Nagahara, A; Wang, L; Del Valle, J et al. (1998) Regulation of c-Jun NH2-terminal kinases in isolated canine gastric parietal cells. Am J Physiol 275:G740-8
Todisco, A; Takeuchi, Y; Urumov, A et al. (1997) Molecular mechanisms for the growth factor action of gastrin. Am J Physiol 273:G891-8
Ford, M G; Valle, J D; Soroka, C J et al. (1997) EGF receptor activation stimulates endogenous gastrin gene expression in canine G cells and human gastric cell cultures. J Clin Invest 99:2762-71

Showing the most recent 10 out of 64 publications