This proposal is a continuation of studies on carboxypeptidase M (CPM), which is membrane-bound in many tissues and cells and may regulate peptide hormone activity at the cell surface. The long term objective is to understand the in vivo functions of CPM. Three areas of investigation will be emphasized. A) THE STRUCTURE OF CPM. Wild type and mutant CPM (generated by 3 prime truncation or site-directed mutagenesis), expressed in a baculovirus system, will be characterized to determine: 1. Whether G1n249 is the side- chain binding residue that mediates its specificity for Arg or Lys; 2. If the C-terminal hydrophobic region of CPM is a signal for phosphatidylinositol-glycan (PI-G) anchoring. Biochemical studies on purified enzyme will determine if Ser 406 is the PI-G attachment site and whether an alternate transmembrane form of CPM is present in kidney. Elucidation of the structure of the CPM gene will help identify possible regulatory regions. B) THE LOCALIZATION OF CPM. It is hypothesized that renal CPM is present in both the proximal tubules and the distal nephron and, subcellularly it is enriched in caveolae of the plasma membrane. The localization of CPM in kidney will be studied by both immunofluorescent light microscopy and immunogold staining with electron microscopy. The distribution of renal CPM mRNA will be determined by in situ hybridization. Using biochemical techniques, CPM enrichment in caveolae from MDCK cells will be studied. C) THE REGULATION OF CPM. Hypothesis: release of CPM and other phosphatidylinositol-glycan (PI-G) anchored proteins from the cell by a phospholipase generates a diglyceride signal that upregulates enzyme synthesis via a protein kinase C. This signalling pathway depends on the functional integrity of plasma membrane caveolae and may be specific to apical or basolateral domains in polarized epithelial cells. MDCK cells will be used to determine: I. whether other PI-G anchored protein are upregulated by stimuli that upregulate CPM; 2. whether the stimulus and/or the response is specific to the apical or basolateral domain in MDCK cells. 3. whether the integrity of caveolae is required for the upregulation of CPM. These studies should provide insight into potential functions of CPM in physiological and pathophysiological processes. For example, in the kidney CPM may control the activity of bradykinin to regulate salt and water excretion. In inflammatory conditions, it could generate an agonist (des-Arg9-bradykinin) for the BI receptor which is upregulated by endotoxin and interleukin I. By cleaving C-terminal Arg from proteins or peptides, it may provide the precursor of nitric oxide. The regulation of CPM and other PI-G anchored proteins may be relevant to pathological conditions that result in their increased release into extracellular fluids (e.g., psoriasis).
