Peptide hormones are the chemical messengers that provide the medium of communication between one part of the body and another and thus play a crucial role in the integrative function of the body. Study of the regulation of peptide hormone biosynthesis, therefore, can provide important insight into the control of major physiological functions in health and disease. As with most proteins and peptides, the regulation of hormones can occur at the level of transcription, translation, and secretion. In addition to these well studied processes, in the case of peptide hormones there is another potential level of regulation, that of post-translational processing. Of the various processing reactions, the most important is the carboxyl terminal amidation reaction catalyzed by the enzyme Peptidyl-glycyl Alpha-amidating Monooxygenase (PAM) since it confers biological activity to more than half of the known peptide hormones and is the rate-limiting step in the post-translational processing of gastrin. The proposed studies will focus on the exploration of the biochemical and physiological implications of PAM in regulating the synthesis of gastrin as it relates to functional control of gastric acid secretion. Preliminary studies have characterized PAM in the antrum and pituitary as well as during development and related alterations in PAM activity to changes in gastrin post-translational processing and gastric acid secretion.
The first aim of the proposal will be to characterize specific differences in PAM can account for the tissue -specific differences in the post- translational processing of gastrin in the antrum and pituitary. Second, the polymerase chain reaction will be used to amplify reversed transcribed PAM mRNA sequences in the gastrointestinal tract. The amplified full length rat PAM cDNA will subsequently be cloned and sequenced and can be used as a probe to measure PAM at sequence, synthetic PAM polypeptide fragments will be made to generate an anti-PAM antibody. This antibody will then be used to quantitate PAM protein by western blots and to localize PAM in the gastrointestinal tract with immunohistochemistry. Fourth, utilizing the above tools, PAM expression will be examined in vivo, during development, in isolated gastrin producing G cells in primary culture, and in PAM containing endocrine tumor cell lines. Finally, it will be of critical importance of relate changes in PAM gene expression, synthesis, and/or activity not only to changes in the post-translational processing of gastrin but also to alterations of physiologic functions such as gastric acid secretion, gastrin receptor binding, and gastrointestinal cell growth. The University of Michigan, and specifically Dr. T. Yamada's laboratory, provides an ideal environment for the proposed project since all of the necessary equipment and technical resource personnel and collaborators are readily available.
