Gastrointestinal peptide hormones undergo extensive post-translational modification before they achieve their biologically relevant forms. It is important to elucidate these processing mechanisms since they result in the tissue-specific generation of distinct bioactive peptides. During the past funding period we characterized two important processing reactions-endoproteolysis and c-terminal amidation. In preliminary data we noted that prosomatostatin (proSS) is cleaved at single and double basic amino acid residues are performed by distinct enzymes leading to the formation of 2 distinct peptides (SS-28 and SS-14, respectively). Although the enzymes and molecular determinants for dibasic processing have been extensively characterized, little is known of monobasic prohormone processing. In other studies, we noted that the immediate processing precursor of amidated gastrin (glycine-extended gastrin, G-Gly) is a distinct end product of gastrin biosynthesis with trophic effects mediated by a receptor distinct from the gastrin/CCK8 receptors that mediate the effects of amidated gastrin. The conversion of G-Gly to amidated gastrin is mediated by a cooper-dependent enzyme, peptidyl-glycine alpha-amidating monooxygenase (PAM). Recently, our collaborators have isolated an important gene encoding a copper transporter (Menkes protein). In the present proposal we will extend our previous observations and examine the role of the Menkes protein in peptide amidation, gastrin biosynthesis and gastrointestinal function. Thus, toward the goal of elucidating the molecular determinants of peptide processing and its relationships to gastrointestinal physiology we will expand upon our previous work and focus on the following specific aims: 1) Characterize the molecular determinants of prohormone endoproteolysis single basic amino acids found in prosomatostatin; and 2) define the role of the Menkes protein in peptide amidation and gastrointestinal function utilizing endocrine cell lines, a well-characterized mouse model of disordered copper transport, and in human sera from patients with Menkes syndrome and related disorders. Since these processing reactions are common to a wide variety of neuroendocrine precursors, the results of these investigations will also aid in our understanding of the molecular mechanisms that regulate prohormone processing in other gut and neuroendocrine tissues.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK047398-06
Application #
6685275
Study Section
General Medicine A Subcommittee 2 (GMA)
Program Officer
May, Michael K
Project Start
1996-09-01
Project End
2006-11-30
Budget Start
2003-12-01
Budget End
2004-11-30
Support Year
6
Fiscal Year
2004
Total Cost
$283,494
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Pediatrics
Type
Schools of Medicine
DUNS #
073133571
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
Yang, Y K; Dickinson, C; Lai, Y M et al. (2001) Functional properties of an agouti signaling protein variant and characteristics of its cognate radioligand. Am J Physiol Regul Integr Comp Physiol 281:R1877-86
Li, J Y; Finniss, S; Yang, Y K et al. (2000) Agouti-related protein-like immunoreactivity: characterization of release from hypothalamic tissue and presence in serum. Endocrinology 141:1942-50
Yang, Y K; Fong, T M; Dickinson, C J et al. (2000) Molecular determinants of ligand binding to the human melanocortin-4 receptor. Biochemistry 39:14900-11
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
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
Yang, Y K; Dickinson, C J; Zeng, Q et al. (1999) Contribution of melanocortin receptor exoloops to Agouti-related protein binding. J Biol Chem 274:14100-6
Yang, Y K; Thompson, D A; Dickinson, C J et al. (1999) Characterization of Agouti-related protein binding to melanocortin receptors. Mol Endocrinol 13:148-55
Yang, Y k; Dickinson, C; Haskell-Luevano, C et al. (1997) Molecular basis for the interaction of [Nle4,D-Phe7]melanocyte stimulating hormone with the human melanocortin-1 receptor. J Biol Chem 272:23000-10