Bimolecular interactions of CRF ligands and receptors

性状（由申请方提供）：促肾上腺皮质激素释放因子（CRF）、尿皮质素（UCN）及其受体在下丘脑垂体肾上腺轴、中枢神经系统和各种外周组织的生理学中起着重要作用。受体的什么结构特性负责配体选择性和受体激活？在初步结果中，我们描述了一种抗氧化的放射性标记的sauvagine类似物（YQLS），可以使用交联赖氨酸残基的化学交联剂与CRFR1和CRFR2共价交联。CNBr切割和定点诱变用于表征与结合配体的赖氨酸残基非常接近的受体的赖氨酸残基。此外，开发了几种对苯甲酰基苯丙氨酸（Bpa）取代的YQLS放射性配体，对CRFR1和CRFR2具有高亲和力，并显示在UV照射后与CRFR1和CRFR2交联。 参与配体结合和信号传导的重要功能域的表征是理解配体和受体如何相互作用的重要问题。我们将进行精细作图，以表征参与与配体相互作用的残基（受体上）（特异性目标1）。在具体目标2中，我们将使用BPA取代的放射性标记的YQLS配体和化学交联的放射性标记的YQLS的光亲和交联的组合，对结合配体及其受体之间的邻近点进行详细的映射。CRF受体具有高度保守的6个半胱氨酸残基。我们分析了半胱氨酸残基的单个和成对突变的功能相关性，并提出了一个模型，为他们的二硫键配对。最近，通过研究在细菌和哺乳动物细胞中表达的截短的NT结构域的化学性质，提出了一种新的模型。在具体目标3中，我们将研究所提出的模型在表达天然受体的哺乳动物细胞系中的有效性。成功地实现本项目的目标将增加我们对CRF和尿皮质素结合并激活其受体的机制的理解，并有助于设计和开发受体特异性激动剂和拮抗剂。

DESCRIPTION (provided by applicant): Corticotropin-releasing factor (CRF), urocortins (UCNs) and their receptors play a central role in the physiology of the hypothalamic pituitary adrenal axis, the central nervous system and a variety of peripheral tissues. What structural properties of the receptors are responsible for ligand selectivity and receptor activation? In Preliminary Results we described an oxidation-resistant radio labeled sauvagine analog (YQLS), that can be covalently cross linked to CRFR1 and CRFR2, using a chemical cross linking agent that cross links lysine residues. CNBr cleavage and site-directed mutagenesis were used to characterize lysine residues of the receptor that are in a close proximity to lysine residues of the bound ligand. Additionally, several p-benzoylphenylalanine (Bpa)-substituted YQLS radioligands, with a high affinity to CRFR1 and CRFR2, were developed and shown to cross-link to CRFR1 and CRFR2 after UV irradiation. Characterization of the important functional domains that are involved in ligand binding and signaling is an important question for understanding how ligand and receptor interact. We shall perform a fine mapping to characterize the residues (on the receptor) that are involved in the interaction with the ligand (Specific Aim 1). In Specific Aim 2 we shall perform a detailed map of the points of proximity between the bound ligand and its receptor using a combination of photo-affinity cross linking of Bpa-substituted radio labeled YQLS ligands and chemically-cross linked radio labeled YQLS. The CRF receptors have highly conserved 6 cysteine residues. We have analyzed the functional relevance of single and paired mutations of the cysteine residues and proposed a model for their disulfide bridge pairing. Recently, a new model was proposed from studying the chemical properties of a truncated NT- domain expressed in bacteria and in mammalian cells. In Specific Aim 3 we will examine the validity of the proposed model in mammalian cell lines expressing native receptors. Success in pursuing the objectives of this project will increase our understanding of the mechanisms by which CRF and urocortins bind to and activate their receptors and shall help the design and the development of receptor-specific agonists and antagonists.