Structure and Function of Carboxypeptidases

描述（由申请人提供）：我们的长期目标是了解调节羧基肽酶在生理和病理过程中的作用。膜结合羧基肽酶（CP） M通过其产生g蛋白偶联的激肽B1受体（B1R）的去精氨酸激肽激动剂的能力，是钾likrein- kinin系统的重要调节因子。激肽是肾脏和心血管功能以及炎症过程的重要调节因子。主要目的是研究一种新的变构蛋白- CPM和B1R之间增强受体信号的蛋白相互作用。具体目的1：探索CPM和B1R在膜上的相互作用，并确定其相互作用的重要残基。假设：CPM表面带正电荷的残基与膜磷脂结合，使CPM在膜上定向，与B1R的基础相互作用是由其c端结构域介导的，而变构调节是由靠近催化结构域的表面介导的。我们将：(i)确定CPM和B1R是否直接相互作用。（ii）研究CPM上的外部正电荷对膜定向和与B1R结合的重要性。（ii）确定B1R上介导其与CPM结合的位点。（iii）确定CPM上与B1R相互作用的站点。（iv）产生干扰CPM/B1R相互作用的肽。具体目的2：阐明CPM与B1R之间相互作用的特点，从而导致受体激活的变构调节。假设：CPM通过结合变构位点增强B1R功能，CPM结合底物导致B1R构象改变，增强受体激活。我们将确定：(i) CPM特异性和肽底物亲和力在B1R激活的变构调节中的作用，以及（ii） CPM变构调节对B1R构象的影响。具体目的3：确定CPM介导的B1R激活的变抗调节在g蛋白偶联、-阻滞蛋白相互作用和下游信号传导中的作用，并确定CPM/B1R相互作用在调节内皮屏障功能中的重要性。我们的假设是CPM与B1R的相互作用通过基础相互作用增强了所有B1R信号，但通过变构调制进一步增强了gq耦合响应。此外，CPM/B1R相互作用的破坏将减轻B2R激动剂对B1R介导的内皮屏障损伤。我们将确定：(i) CPM与B1R相互作用介导的g蛋白偶联与激动剂结合。（ii） -arrestin-2募集参与B1R激动剂激活与CPM介导的变构增强。（iii） B1R通过Gq和Gi介导的途径对激动剂或CPM相互作用的下游信号输出。（iv） CPM在调节内皮/血管通透性的B1R信号的变构调节中的作用。这些研究将阐明CPM对B1R信号变构增强的新机制，CPM也产生其配体。了解这一过程是如何被调节的，可能会导致治疗肾脏和心血管疾病的新药的开发。

DESCRIPTION (provided by applicant): Our long term objective is to understand the roles of regulatory carboxypeptidases in physiological and pathological processes. Membrane-bound carboxypeptidase (CP) M is an important regulator of the kallikrein- kinin system via its ability to generate des-Arg-kinin agonists of the G-protein coupled kinin B1 receptor (B1R). Kinins are important regulators of renal and cardiovascular function and inflammatory processes. The major objective is to investigate a novel allosteric protein-protein interaction between CPM and B1R that enhances receptor signaling. Specific Aim 1: To explore the interaction of CPM and B1R on the membrane and determine the residues important for their interaction. Hypothesis: positively charged residues on CPM's exterior bind to membrane phospholipids to orient CPM on the membrane and basal interaction with the B1R is mediated by its C-terminal domain whereas allosteric modulation is mediated by a surface near the catalytic domain. We will: (i) determine whether CPM and B1R directly interact. (ii) investigate the importance of the exterior positive charges on CPM for proper membrane orientation and binding to the B1R. (ii) determine the site(s) on the B1R that mediates its binding to CPM. (iii) determine the site(s) on CPM that interacts with the B1R. (iv) generate peptides to interfere with CPM/B1R interactions. Specific Aim 2: To elucidate the characteristics of the interaction between CPM and the B1R that results in allosteric modulation of receptor activation. Hypothesis: CPM enhances B1R function by binding to an allosteric site and substrate binding by CPM results in B1R conformational change and enhanced receptor activation. We will determine: (i) the role of CPM specificity and peptide substrate affinity on allosteric modulation of B1R activation and (ii) the effect of CPM allosteric modulation on B1R conformation. Specific Aim 3: To determine the role of CPM-mediated allosteric modulation of B1R activation in G-protein coupling, -arrestin interaction and downstream signaling and to determine the importance of CPM/B1R interaction in regulating endothelial barrier function. Our hypothesis is that CPM interaction with the B1R enhances all B1R signaling via basal interaction but further enhances Gq-coupled responses by allosteric modulation. Furthermore, disruption of CPM/B1R interaction will attenuate B1R-mediated endothelial barrier damage in response to B2R agonist. We will determine: (i) G-protein coupling mediated by CPM interaction with the B1R vs. agonist binding. (ii) the involvement of -arrestin-2 recruitment in B1R agonist activation vs. CPM mediated allosteric enhancement . (iii) the downstream signaling output from the B1R via Gq and Gi mediated pathways in response to agonist or CPM interaction. (iv) the role of CPM in allosteric modulation of B1R signaling in regulating endothelial/vascular permeability. These studies will elucidate a novel mechanism for allosteric enhancement of B1R signaling by CPM which also generates its ligand. Understanding how this process can be regulated could lead to the development of novel drugs to treat renal and cardiovascular diseases. PUBLIC HEALTH RELEVANCE: We are investigating the interaction of two proteins, carboxypeptidase M and B1 receptor, on the cell surface that results in increased production of regulatory molecules that affect cell and organ function. The B1 receptor is known to regulate kidney and cardiovascular function, especially under conditions of infection or inflammation. Understanding how the interaction of these proteins can be enhanced or blocked could lead to the development of novel drugs to treat renal and cardiovascular diseases.