Molecular Determinants of AGRP Function

描述（由申请人提供）： 肥胖是西方社会面临的最大公共卫生问题之一。这种情况源于能量摄入和消耗之间的不平衡，并导致糖尿病，心血管疾病和癌症。在大脑中，控制能量平衡的关键焦点是黑皮质素信号传导，其中有两种配体，α-黑素细胞刺激激素和Agouti相关蛋白（AGRP）。这些分子通过CNS黑皮质素受体（MCR）以相反的方式起作用，分别促进负能量平衡和正能量平衡。AGRP是一种多结构域蛋白，其MCR活性集中在其不寻常的富含Cys的C-末端结构域。PI的实验室最近确定了该结构域的结构，并鉴定了被认为对AGRP选择中枢神经系统的MCR的能力至关重要的区域。该研究计划的目标是通过确定AGRP的分子特征来显着拓宽对MCR信号传导的理解，AGRP赋予其受体选择性和对MCR功能的控制。提出了四个目标。1)将确定在色素沉着中起作用的同源agglutamine蛋白的结构。AGRP和agglutamine结构的比较将有助于确定这些信号分子中控制其各自MCR选择性的区域。2)蛋白质设计和光交联将用于评价与AGRP直接接触的MC受体区域。这些研究将检验AGRP中的特定环介导MCR识别的假设。3)将确定AGRP N-末端结构域的结构和功能。最近的研究结果表明，该区域通过与细胞表面多配体蛋白聚糖结合来增强MCR拮抗作用。这一概念将使用结构指导的药理学和转基因方法进行测试。4)将评估新型AGRP胱氨酸结结构的稳定性和设计潜力。AGRP C-末端的特异性支架先前在哺乳动物蛋白中未被鉴定。然而，对植物和无脊椎动物胱氨酸结的研究表明，这种支架是理想的药理学设计。热力学研究将检查稳定性，噬菌体展示将用于开发选择MCR类别之外的受体的胱氨酸结。

DESCRIPTION (provided by applicant): Obesity is one of the greatest public health concerns facing Western society. The condition arises from an imbalance between energy intake and expenditure and contributes to diabetes, cardiovascular disease and cancer. In the brain, a key focal point for control of energy balance is melanocortin signaling, in which there are two ligands, alpha-melanocyte stimulating hormone and Agouti-related protein (AGRP). These molecules act in opposite ways through CNS melanocortin receptors (MCRs) to promote negative and positive energy balance, respectively. AGRP is a multi-domain protein with MCR activity concentrated in its unusual Cys-rich, C-terminal domain. The Pl's laboratory recently determined the structure of this domain and identified regions that are postulated to be essential for AGRP's ability to select MCRs of the central nervous system. The goal of this research program is to significantly broaden the understanding of MCR signaling by determining the molecular features of AGRP that confer its receptor selectivity and control over MCR function. Four Aims are proposed. 1) The structure of the homologous agouti protein that functions in pigmentation will be determined. Comparison of the AGRP and agouti structures will help to identify regions within these signaling molecules that control their respective MCR selectivity. 2) Protein design and photo crosslinking will be used to evaluate the MC receptor regions that make direct contact with AGRP. These studies will test the hypothesis that a specific loop in AGRP mediates MCR recognition. 3) The structure and function of the AGRP N-terminal domain will be determined. Recent findings suggest that this region potentiates MCR antagonism by binding to cell surface syndecans. This concept will be tested using structure-guided pharmacological and transgenic methodologies. 4) The stability and design potential of the novel AGRP cystine-knot structure will be evaluated. The specific scaffold of the AGRP C-terminus has not been previously identified in mammalian proteins. However, studies with plant and invertebrate cystineknots suggest that this scaffold is ideal for pharmacological design. Thermodynamic studies will examine stability and phage display will be used to develop a cystine-knot that selects for receptors outside of the MCR class.