Bimolecular interactions of CRF ligands and receptors

描述(申请人提供)：促肾上腺皮质激素释放因子(CRF)、尿皮质激素(UCNs)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中起核心作用。受体的哪些结构特性与配体的选择性和受体的激活有关？在初步结果中，我们描述了一种标记为苏云金类似物(YQLS)的抗氧化放射性物质，它可以使用一种使赖氨酸残基交联的化学交联剂，与CRFR1和CRFR2共价交联。使用CNBR裂解和定点突变来表征受体的赖氨酸残基，这些残基与结合配体的赖氨酸残基非常接近。此外，还开发了几个对苯甲酰苯丙氨酸(BPA)取代的YQLS放射性配体，它们与CRFR1和CRFR2具有高亲和力，并在紫外光照射后与CRFR1和CRFR2发生交联。鉴定参与配体结合和信号传递的重要功能结构域是理解配体和受体如何相互作用的重要问题。我们将进行精细的定位，以表征参与与配体相互作用的(受体上的)残基(特定目标1)。在具体目标2中，我们将使用BPA取代的标记YQLS配体的光亲和交联标记的YQLS配体和化学交联标记的YQLS的组合来绘制结合配体与其受体之间的接近点的详细图谱。CRF受体高度保守6个半胱氨酸残基。我们分析了半胱氨酸残基的单突变和成对突变的功能相关性，并提出了其二硫键配对的模型。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，提出了一个新的模型。在特定的目标3中，我们将在表达天然受体的哺乳动物细胞系中检验所提出的模型的有效性。成功地实现该项目的目标将增加我们对CRF和urocortins与其受体结合并激活其受体的机制的了解，并将有助于受体特异性激动剂和拮抗剂的设计和开发。

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.