Peptides are implicated in many physiological processes. Along with the endocrine actions of peptides such as ACTH, paracrine and autocrine actions have been proposed. Peptides act as neuromodulators and neurotransmitters at many synapses. Certain steps in the complex post-translational processing pathway leading from inactive preprohormones to bioactive peptides are carried out in a tissue specific fashion. The proposed studies focus on understanding how peptide precursors are processed in a tissue specific manner and how peptide post-translational processing enzymes function in the diverse cell types in which they are found. 1] The properties governing whether potential endoproteolytic cleavage sites are utilized will be determined by comparing the processing of pro-neuropeptide Y (proNPY) and pro-ACTH/beta-endorphin (PAE) molecules containing mutant mono-, di-, tri- and tetrabasic processing sites. Following stable or transient transfection of cDNAs into AtT-20 mouse corticotrope cells, product peptides will be identified by biosynthetic labeling and peptide analyses. Mutations altering sites for N- and O-linked glycosylation close to and further away from the cleavage site will be investigated for effects on routing and processing. 2] The cell-type specificity of posttranslational processing will be compared by expressing wild-type and mutant proNPY and PAE in AtT-20 cells, GH3 rat somatomammotrope cells, primary rat intermediate pituitary melanotropes and primary atrial myocytes. Localization of endogenous and foreign peptides will be compared by immunofluorescence and immunoelectron microscopy. 3] Determining governing the routing of peptidyl-glycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) to the correct subcellular organelles will be examined by expressing the 6 naturally occurring forms of PAM in fibroblasts and MDCK cells. Routing in cells containing substantial levels of endogenous PAM will be examined by replacing the catalytic domain of PAM with a reporter such as NPY or rat serum albumin. Data in the literature would support a role for the cytoplasmic domain of the transmembrane forms of PAM in routing. 4] If transfection data suggest a role for the cytoplasmic domain of PAM in routing, a cytoplasmic domain affinity column will be used to purify a cytoplasmic binding (assembly) protein; any potential assembly proteins will be purified. 5] Antisense cDNAs to PAM will be introduced into several cell types to determine the consequences of lack of PAM. Candidate endoproteases will be expressed in mammalian cells by transfection and the consequence of introducing a novel protease into the secretory pathway or blocking protease expression with antisense cDNA will be examined.
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