SOMATOSTATIN RECEPTOR STRUCTURE AND FUNCTION

描述(摘自申请者的摘要)：SS是神经肽 它们可以作为激素、神经递质或自分泌调节 抑制多种内分泌、外分泌分泌过程的因子 和神经元靶细胞。Schonbrunn博士已经证明了党卫军的作用 通过质膜受体抑制垂体激素的分泌 与一个或多个百日咳毒素敏感G蛋白偶联。SS受体 激活多个转导通路，包括抑制 腺苷酸环化酶对钾电导的刺激和抑制 钙电导。然而，生长抑素受体的结构尚不清楚。 事实上，目前还不清楚SS受体的亚型是否存在，如果存在， 它们是否激活相同或不同的膜效应器。致信地址 针对这些问题，研究人员建议克隆SS受体的cDNA 并分别研究它们在哺乳动物表达系统中的功能。 初步研究将集中在克隆编码85,000道尔顿的cDNA 她最近通过光亲和力鉴定为SS受体的蛋白质 贴标签。要采用的一般策略将包括净化 85 kDa受体蛋白，获得多肽的氨基酸序列 来自受体的片段，并使用这些序列来合成 筛选cdna文库的合适探针。核实 推测的SS受体克隆的鉴定将通过(A)完成 对克隆进行测序，并将推导出的氨基酸序列与 从纯化的受体获得的多肽序列的cDNA，以及 (B)在受体阴性的哺乳动物细胞系中表达cDNA 包含所需的转换机械，并展示了 所产生的蛋白质的功能能力。在确定了 这个85 kDa SS受体的结构，她将 在低严格性条件下通过同源克隆鉴定相关的cDNA。 表达分析将再次确定这些cDNA是否编码功能 SS受体亚型具有不同的模拟特异性或转导 机械装置。如果是这样的话，这些cDNAs将被测序以阐明 生长抑素受体亚型的结构及其分布 编码这个受体家族的单个成员的mRNAs。至 使她能够测量SS受体蛋白，她就会产生抗体 SS受体通过使用适当的表达系统产生大鼠 用于免疫的每个受体和受体片段的数量。这个 抗体和cDNAs将被用来定量SS水平 受体蛋白和信使核糖核酸在垂体细胞中的作用 以前被证明通过结合测量来影响受体密度的药物。 此外，她还将确定受体的磷酸化是否 参与SS受体功能的调节。加在一起，这些 研究将首次阐明该蛋白的结构、功能和 生长抑素受体在分子水平的调控。

DESCRIPTION (adapted from the applicant's abstract): SS is a neuropeptide which can act as a hormone, a neurotransmitter or an autocrine regulatory factor to inhibit secretory processes in a variety of endocrine, exocrine and neuronal target cells. Dr. Schonbrunn has shown that SS acts to inhibit pituitary hormone secretion via a plasma membrane receptor which is coupled to one or more pertussis toxin sensitive G proteins. SS receptors activate multiple transduction pathways, including inhibition of adenylylate cyclase, stimulation of potassium conductance and inhibition of calcium conductance. However, the structures of SS receptors are unknown. Indeed, it is not clear whether subtypes of SS receptors exist and, if so, whether they activate the same or different membrane effectors. To address these problems, the investigator propose to clone cDNAs for SS receptors and examine their function individually in mammalian expression systems. Initial studies will focus on cloning the cDNA encoding the 85,000 dalton protein which she has recently identified as a SS receptor by photoaffinity labelling. The general strategy to be employed will involve purifying the 85 kDa receptor protein, obtaining amino acid sequences for peptide fragments from the receptor, and using these sequences to synthesize appropriate probes for screening a cDNA library. Verification of the identity of the putative SS receptor clones will be accomplished (a) by sequencing the clones and comparing the deduced amino acid sequence from the cDNA to the peptide sequences obtained from the purified receptor, and (b) by expressing the cDNAs in receptor negative mammalian cell lines containing the required transduction machinery and demonstrating the functional capabilities of the resulting protein. Having determined the structure of this 85 kDa SS receptor from the cDNA sequence, she will identify related cDNAs by homology cloning under low stringency conditions. Expression assays will again determine if these cDNAs code for functional SS receptor subtypes with different analog specificities or transduction mechanisms. If so, these cDNA's will be sequenced to elucidate the structures of SS receptor subtypes and used to determine the distribution of mRNAs coding for the individual members of this receptor family. To enable her to measure SS receptor proteins, she will generate antibodies to SS receptors by using appropriate expression systems to produce large amounts of each receptor and receptor fragments for immunization. The antibodies and the cDNA's will be used to quantitate the level of SS receptor protein and mRNA following treatment of pituitary cells with agents previously shown to affect receptor density by binding measurements. In addition, she will determine whether receptor phosphorylation is involved in the regulation of SS receptor function. Together, these studies will elucidate for the first time the structure, function and regulation of SS receptors at a molecular level.