The overall objective is to elucidate the roles of regulatory carboxypeptidases (CPs) in physiological and pathological processes by emphasizing three broad areas of investigation: l) Structure, 2) Cellular targeting and 3) Functions. Hypothesis 1: The structure of CPM contains features that are unique to the regulatory carboxypeptidase family.
Specific Aim 1 : Crystallize recombinant carboxypeptidase (CP) M and determine its three dimensional structure. Hypothesis 2: The three carboxypeptidase homology domains of CPD are unique: Domains 1 and 2 are active CPs with unique specificities whereas domain 3 is inactive, but binds substrates and influences the kinetics of domains 1 and 2.
Specific Aim 2 : Determine the characteristics of the CP homology domains of CPD and their interactions in the intact molecule by (i) expressing recombinant forms of CPD containing 1, 2 or 3 of the active site domains; (ii) investigating, for each form of CPD: substrate specificity, kinetic parameters for synthetic and naturally occurring peptides, inhibitor affmity, metal ion activation/inhibition, pH optimum and stability; (iii) determining the specificity of binding of peptide substrates for domain 3. Hypothesis 3: CPM contains a unique basolateral targeting signal in its extracellular domain.
Specific Aim 3 : Determine whether the extracellular domain of CPM mediates its unusual basolateral sorting in polarized Madin Darby canine kidney (MDCK) cells by transfecting MDCK cells with various CPM deletion mutants and fusion constructs and then investigate their sorting to the apical and basolateral domains. Hypothesis 4: Regulatory carboxypeptidases inhibit plasminogen activation; CPN by direct interaction with plasminogen or tPA and cellular CPM and CPD by downregulating plasminogen binding to cells.
Specific Aim 4 : Investigate CPN inhibition of plasminogen activation in solution and CPM and CPD regulation of plasminogen activation on the cell surface by (i) elucidating the role of the 50 kDa and 83 kDa subunits of CPN and the mechanism(s) by which they act (i.e., binding or hydrolysis); (ii) measuring plasminogen binding and activation on the surface of cells expressing different levels of CPM and CPD. These studies will provide novel information regarding the structure and function of regulatory CPs that can be involved in a variety of physiological and pathophysiological processes such as: (i) regulation of bradykinin activity which controls salt and water excretion in the kidney; (ii) generation of agonists (e.g., des-Arg9-bradykinin) for the B1 receptor which is upregulated by inflammatory cytokines; (iii) regulation of plasminogen activation which is critical to the intravascular dissolution of fibrin clots, wound healing, angiogenesis, tissue remodeling and metastasis of neoplastic cells.
