Our long-term objective is to understand the important roles of regulatory carboxypeptidases in physiological and pathological processes. We will emphasize the role of membrane-bound carboxypeptidase (CP) M as a regulator of signaling through cytokine-regulated B1 kinin receptor as follows. Hypothesis 1: CPM is an important regulator of the B1 kinin system via its ability to generate the B1 receptor agonist and efficiently deliver it to the receptor on the membrane.
Specific Aim 1 : Elucidate the role of CPM in generating des-Arg kinins for activation of B1 receptors by determining: (i) whether CPM and the B1 receptor are co-localized on the cell surface; (ii) the ability of CPM to generate and deliver the des-Arg kinin B1 agonists to the receptor and stimulate a corresponding signal; (iii) the relationship of the B1 response to the ability of CPM to hydrolyze B2 receptor agonists on the cell surface. Hypothesis 2: The C-terminal transthyretin-like domain of CPM contains a unique disulfide bond that tethers the enzyme, orienting it to alter access to substrates on the cell surface and allow more efficient delivery of products to membrane receptors.
Specific Aim 2 : Determine the role of the C-terminal domain of CPM in regulating its hydrolysis of peptide substrates on the cell membrane, its localization and shedding. We will: (i) express recombinant forms of CPM in which the C-terminal domain is replaced with a flexible tether, the two C-terminal cysteines have been mutated or a chimeric form with the C-terminal transmembrane region of angiotensin I converting enzyme; (ii) investigate, for each form of CPM: kinetic parameters for peptide substrates ranging in size from 2 to 53 amino acids, inhibitor affinity, pH optimum and stability; (iii) determine the rate and mode of release from the membrane; (iv) determine the effect on CPM's membrane localization.
Specific Aim 3 : Determine the functional importance of the C-terminal domain of CPM on its ability to generate and deliver des-Arg-kinins to the B1 kinin receptor by: (i) investigating the effect of C-terminal domain mutations on the co-localization of CPM and B1 receptor on the membrane; (ii) determining the effect of mutations on its ability to convert B2 agonists and generate the B1 receptor responses by measuring increases in intracellular calcium, arachidonic acid release and nitric oxide production. These studies will provide novel information on the structure and function of CPM that can be involved in physiological and pathophysiological processes. For example, CPM regulation of bradykinin activity, which controls salt and water excretion in the kidney, and generation of agonists for the B1 receptor, which is upregulated by inflammatory cytokines, resulting in prolonged receptor signaling and production of important cardiovascular mediators such as nitric oxide.
