SOMATOSTATIN RECEPTOR STRUCTURE AND FUNCTION

描述（改编自申请人的摘要）：SS是一种神经肽 它可以作为一种激素，神经递质或自分泌调节剂， 因子，以抑制各种内分泌，外分泌 和神经靶细胞。 Schonbrunn博士已经证明SS的行为 通过质膜受体抑制垂体激素分泌， 与一种或多种百日咳毒素敏感性G蛋白偶联。 SS受体 激活多种转导途径，包括抑制 腺苷酸环化酶，钾传导的刺激和 钙电导 然而，SS受体的结构是未知的。 事实上，尚不清楚SS受体的亚型是否存在，如果存在， 无论它们激活相同还是不同的膜效应器。 解决 针对这些问题，研究者提出克隆SS受体的cDNA 并在哺乳动物表达系统中单独检查它们的功能。 最初的研究将集中在克隆编码85，000道尔顿的cDNA。 她最近通过光亲和性鉴定了这种蛋白质作为SS受体 标签。 所采用的一般战略将涉及净化 85 kDa受体蛋白，获得肽的氨基酸序列 片段，并使用这些序列来合成 用于筛选cDNA文库的合适探针。 验证 推定的SS受体克隆的鉴定将通过（a）以下步骤完成： 对克隆进行测序，并比较来自 从纯化的受体获得的肽序列的cDNA，和 (b)通过在受体阴性哺乳动物细胞系中表达cDNA 包含所需的转导机制，并证明 所产生的蛋白质的功能。 在确定了 从cDNA序列中获得这个85 kDa SS受体的结构，她将 在低严格条件下通过同源克隆鉴定相关cDNA。 表达测定将再次确定这些cDNA是否编码功能性的 具有不同类似物特异性或转导的SS受体亚型 机制等 如果是这样的话，这些cDNA将被测序以阐明 SS受体亚型的结构，并用于确定分布 编码这个受体家族的各个成员的mRNA。 到 使她能够测量SS受体蛋白，她将产生抗体， SS受体通过使用适当的表达系统产生大的 用于免疫的每种受体和受体片段的量。 的 抗体和cDNA将用于定量SS水平 受体蛋白和mRNA后处理垂体细胞 先前通过结合测量显示影响受体密度的药剂。 此外，她还将确定受体磷酸化是否是 参与SS受体功能的调节。 所有这些 研究将首次阐明结构，功能和 SS受体在分子水平上的调节。

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.