CRF and urocortins and their receptors

我们正在探索一种假设，即促肾上腺皮质激素释放因子(CRF)/urocortin配体家族及其同源受体在调节对应激和其他环境的适应性反应中发挥着特定和关键的作用。在之前的授权期，该项目与整个计划的合作者一起鉴定了CRF，克隆了CRF受体CRFR1和CRFR2，并鉴定了与这两种受体都有高亲和力的urocortin(UCN)1。在过去的一段时间里，我们和Sawchenko项目发现了另外两个对CRFR2具有高度选择性的配体UCN 2和3。我们的重点是探索CRFR的N末端第一胞外域(ECD1)的作用，它包括多肽激动剂和拮抗剂的关键结合部位。我们设计并表达了猪ECD1‘S的可溶性形式 CRFR1和CRFR2beta对适当的配体保持亲和力，并与Salk研究所的Roland Riek合作，通过核磁共振解析CRFR2beta-ECD1的结构。在接下来的一段时间里，我们将与R.Riek、Core C和Rivier项目密切合作，解决CRFR1、CRFR2pha和CRFR2beta的受体ECD1配体复合体的结构，以及主要由R2α的ECD1组成的新型可溶性剪接变异体。ECD1中蛋白质-蛋白质相互作用基序的发现产生了一个可检验的假说，即我们在心肌细胞中看到的CRFR2β和erbB2之间的功能相互作用可能部分依赖于这两个受体之间的相互作用。在小鼠中CRFR2基因缺失的结果表明，该受体及其配体在中枢神经系统、心血管系统和胰岛发挥着多种作用。这3种尿皮质素缺乏的小鼠是与最初的Lee项目合作开发的，将在该项目中分析垂体/肾上腺和胰腺激素的调节以及CRF相关受体和剩余配体的表达。最后，我们将研究UCN 3(以及后来的UCN 2)的意义，它被证明是在β细胞中产生的，并对刺激 胰岛素和胰升糖素的分泌，作为胰岛功能的局部调节。在该计划的背景下，该项目的进展应继续提供有关CRF家族配体和受体的分子性质、调节和生理作用的信息，并提供关于内分泌、代谢和应激相关疾病的见解。

We are exploring the hypothesis that the Corticotropin Releasing Factor (CRF)/urocortin family of ligands and their cognate receptors play specific and critical roles in the modulation of adaptive responses to stress and other circumstances. In previous granting periods, this Project, with collaborators throughout the Program, characterized CRF, cloned 2 CRF receptors, CRFR1 and CRFR2 and identified urocortin (Ucn) 1, which had high affinity for both receptors. During the last period, we and the Sawchenko Project have found two additional ligands, Ucn 2 and 3, which are highly selective for CRFR2. We have focused on exploring the roles of the N-terminal, first extracellular domain (ECD1) of the CRFRs, which include critical binding sites for peptide agonists and antagonists. We have designed and expressed soluble forms of the ECD1's of CRFR1 and CRFR2beta with retained affinity for appropriate ligands and collaborated with Roland Riek, Salk Institute, to solve the structure of the CRFR2beta-ECD1 by NMR. In the next period, we will work closely with a new project headed by R. Riek, Core C and the Rivier Project to solve the structures of receptor ECD1- ligand complexes for CRFR1, CRFR2alpha and CRFR2beta as well as a novel soluble splice variant comprising mainly the ECD1 of R2alpha. The identification of a protein-protein interaction motif in the ECD1 spawned the testable hypothesis that the functional interaction that we had seen between CRFR2beta and erbB2 in cardiomyocytes may in part depend upon an interaction between the two receptors. Results with mice null for CRFR2 have suggested a variety of roles for this receptor and its ligands in the CNS, cardiovascular system and pancreatic islets. Mice deficient in each of the 3 urocortins have been developed in collaboration with the original Lee project and will be analyzed in this project with respect to the regulation of pituitary/adrenal and pancreatic hormones as well as the expression of CRF-related receptors and remaining ligands. Finally, we will examine the significance of Ucn 3 (and later, Ucn 2), shown to be produced in beta cells and to stimulate insulin and glucagon secretion, as local regulators of islet functions. In the context of the Program, progress in this Project should continue to yield information concerning the molecular nature, regulation and physiologic roles of ligands and receptors of the CRF family and provide insight regarding endocrine, metabolic and stress-related diseases.