Secretin Receptor Structure, Function and Regulation

描述(申请人提供)：本申请的总体目标是更好地了解配体诱导的分泌素受体激活状态的分子基础，分泌素受体是一种典型的B类G蛋白偶联受体。这些见解应该会填补当前知识中的关键空白，并促进受体活性药物的最终开发。这个受体家族包括治疗糖尿病、肥胖症、骨质疏松症、偏头痛、焦虑和抑郁的既定靶点；然而，开发作用于这些靶点的小分子激动剂尤其具有挑战性。最近解决的晶体结构展示了两个家族成员独特的开放螺旋束结构域，有助于解释这一挑战。因此，该结构域已成为当前提议的焦点，其组成部分旨在针对螺旋束与(I)生物活性配体、(Ii)受体氨基末端和(Iii)相关膜蛋白相互作用的影响。中心假设是，启动一系列细胞内信号事件的该结构域的构象变化可以不同地受到与其直接相互作用的各种配体的影响，或通过与受体氨基末端的结合间接影响，以及与四元复合体中其他膜蛋白的相互作用。对分泌素受体参与与血管紧张素1a受体的独特的跨类别异源受体复合体的新认识提供了与生理学和治疗学相关的见解，也有强有力的理由使用分泌素受体激动剂来治疗相关的主要健康问题，包括肥胖、糖尿病、高血压、心力衰竭以及胆汁淤积。我们的具体目标将检验三个假设：(I)螺旋束上没有传统对接口袋的开放空间，在那里可以有效地定位正构体激动剂的作用；(Ii)氨基末端和核心域以一种特定和动态的方式相互作用，影响受体的激活状态；以及 (Iii)关键的分子相互作用存在于跨类异受体复合体中，可用于选择性地调节重要的生物活性。第一个目标将通过利用半胱氨酸捕获顶部残基来探索激动剂活性的分子决定因素 跨膜片段和细胞外环内使用包含螺旋N-帽基序的位置的半胱氨酸，并通过连接到可作为与受体氨基末端高亲和力结合的位点选择性锚定的配体来评估指向自然激活口袋的潜在候选药效团。第二个目标将通过利用生化和突变策略来定义和破坏或稳定促胰液素受体氨基末端和核心之间的界面，并检查其功能含义，从而深入了解受体的全息结构。第三个目标将探索促胰液素和血管紧张素1a受体之间的物理联系，确定它们联系的结构基础和功能含义，以及作用于每种受体的全谱药物的影响。 受体，并开发利用这种独特的异源受体复合体的策略。

DESCRIPTION (provided by applicant): The overall objective of this application is to better understand the molecular basis of ligand-induced activation states of the secretin receptor, a prototypic class B G protein-coupled receptor. These insights should fill key gaps in current knowledge and facilitate the ultimate development of receptor-active drugs. This receptor family includes established targets for treatment of diabetes, obesity, osteoporosis, migraine, anxiety, and depression; however, development of small molecule agonists acting at these targets has been particularly challenging. Recently solved crystal structures demonstrating uniquely open helical bundle domains of two family members have helped to explain this challenge. This domain has, therefore, become the focal point of the current proposal, with component aims directed at the impact of helical bundle interactions with (i) biologically active ligands, (ii) th receptor amino terminus, and (iii) associated membrane proteins. The CENTRAL HYPOTHESIS is that conformational changes in this domain that initiate a spectrum of intracellular signaling events can be differentially affected by various ligands that interact with it directly, or indirecly by binding to the receptor amino terminus, and by interactions with other membrane proteins within quaternary complexes. New recognition of secretin receptor involvement in a unique cross-class hetero-receptor complex with the angiotensin 1a receptor provides insights relevant to physiology and therapeutics, and there is also strong rationale for use of a secretin receptor agonist to manage related major health problems, including obesity, diabetes, hypertension, and heart failure, as well as cholestasis. Our SPECIFIC AIMS will test three hypotheses: (i) the open space high in the helical bundle that is devoid of traditional docking pockets where the orthosteric agonist acts can be effectively mapped; (ii) amino-terminal and core domains interact in a specific and dynamic manner that affects the state of activation of the receptor; and (iii) key molecular interactions exist within a cross-class hetero-receptor complex that can be utilized to selectively modulate important biological activities. The first aim will explore the molecular determinants of agonist activity by utilizing cysteine trapping of residues at the top of transmembrane segments and within extracellular loops using cysteines in positions comprising the helix N-capping motif, and evaluating potential candidate pharmacophores directed to the natural activation pocket by attachment to a ligand that can be applied as a site-selective anchor that binds with high affinity to the receptor amino terminus. The second aim will gain insights into the receptor holo- structure by utilizing biochemical and mutagenesis strategies to define and disrupt or stabilize the interfaces between the secretin receptor amino terminus and core, and to examine functional implications. The third aim will explore the physical association between secretin and angiotensin 1a receptors, defining the structural basis and functional implications of their association, the impact of the full spectrum of types of drugs acting at each receptor, and developing strategies to take advantage of this unique hetero-receptor complex.