Binding Determinants of Glycoprotein Hormone Receptors

性状（由申请方提供）：糖蛋白激素（GpHs）、促黄体生成素（LH）、促卵泡激素（FSH）和促甲状腺激素（TSH）调节生育力和代谢。它们的受体（GpHR）与大多数G蛋白偶联受体的区别在于存在大的N-末端胞外酶结合结构域（ECD）。每个ECD由9个富含亮氨酸的重复序列（LRR）组成，两侧分别是N-末端和C-末端富含半胱氨酸的区域（分别为NCR和CCR）。与ECD结合的特异性激素的信号通过其CCR传递到七螺旋跨膜区（TM）。突变，激活或失活GpHR功能，已被确定在甲状腺功能亢进症，性早熟和假两性畸形;此外，LH和FSH受体的多态性变体已被证明可以改变女性不孕症，乳腺癌和骨折的风险的易感性。目标：1.确定常见的酶接触位点和关键酶选择性决定因素的LRR的GpHRs，2。鉴定GpHR的富含半胱氨酸的区域中的残基，所述残基对于GpH结合和将结合信号传递至TM是重要的，以及3.确定GpH-GpHR复合物的三维结构。方法：结构域交换/定点诱变、表达、激素结合和cAMP依赖性报告活性作为读数、分子建模、ECD蛋白表达、蛋白纯化、结晶、晶体结构测定。结果：拟议研究的结果将增加我们对GpHR识别“正确”GpH的分子机制以及配体结合后信号逐步传播到TM的知识。这些数据与突变的“功能获得性”GpHR结合，将被塑造成GpHR激活的一般模型，这对于开发更好的治疗方案和合理设计改变受体活性的药物至关重要。此外，这些数据可能会产生一个更好的理解的病因学不同的病理状态已知的一些GpH-GpHR对。

DESCRIPTION (provided by applicant): The glycoprotein hormones (GpHs), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH), regulate fertility and metabolism. Their receptors (GpHRs) are distinguished from most G protein-coupled receptors by the presence of large N-terminal extracellular hormone-binding domains (ECDs). Each ECD consists of nine leucine-rich repeats (LRRs), flanked by N-terminal and C-terminal cysteine-rich regions (NCR and CCR, respectively). The signal of specific hormone binding to the ECD is conveyed by its CCR to the heptahelical transmembrane region (TM). Mutations, activating or inactivating GpHR function, have been identified in hyperthyroidism, precocious puberty and pseudohermaphroditism; moreover, polymorphic variants of LH and FSH receptors have been shown to change the susceptibility to female infertility, breast cancer and risk of bone fractures. AIMS: 1. Identify common hormone-contact sites and key hormone-selective determinants in LRRs of GpHRs, 2. Identify residues in the cysteine-rich regions of GpHRs important for GpH binding and for conveying the binding signal to the TM, and 3. Determine the three-dimensional structure of GpH-GpHR complexes. METHODS: Domain exchange/site-directed mutagenesis, expression, hormone binding and cAMP-dependent reporter activity as readouts, molecular modeling, ECD protein expression, protein purification, crystallization, crystal structure determination. OUTCOME: The results of the proposed studies will increase our knowledge of the molecular mechanisms by which GpHRs recognize the 'correct' GpH, as well as the step-wise propagation of the signal to the TM following ligand binding. These data, in combination with the mutant 'gain-of-function' GpHRs, will be molded into a general model of GpHR activation, essential for the development of better treatment protocols and the rational design of drugs that change receptor activity. Moreover, these data may yield a better understanding of the etiology of different pathological states known for some GpH-GpHR pairs.