LOCALIZATION/SIGNAL TRANSDUCTION

描述(取自应用程序) 这一建议旨在定义涉及到的分子机制 对信号转导和细胞生长的调节 七螺旋，G蛋白偶联，PACAP受体(PACAP-R)。PACAP-R是一种 研究受体与细胞内效应器偶联的有用模型，因为 激动剂刺激后，这些受体能够激活这两种受体 腺苷环化酶(AC)和磷脂酶C(PLC)。一些肿瘤细胞 表达PACAP-Rs还会分泌PACAP，提示可能存在自分泌 激素对肿瘤生长的影响。人类PACAP-R的基因是 最近克隆并显示包含两个外显子，编码第三个 可交替剪接成四个不同cDNA胞内环 剪接变体。尽管每个剪接变体都能够激活 两个(AC)和(PLC)、两个(SV2和SV-3)对PLC显示出更好的效果 激活和诱导即刻早期基因。 因此，这项提案的第一个具体目标是描述 PACAPR剪接变异体的组织分布及其信号转导途径 它们是结合在一起的。将使用RT-PCR来确定组织分布 并使用最近开发的PACAP-R抗体进行免疫组织化学。 初步研究表明，PACAP受体剪接变异体是偶联的 结合特定的G-蛋白，如Gas和Gaq。PACAP-R的区域 是否参与G蛋白偶联将由受体决定 诱变。参与PACAP-R剪接变异体偶联的G蛋白 将通过检测配体诱导的PLC在NIH/3T3中的反应来研究 HPACAP-R剪接变异体与GAS共转染细胞 或者是Gaq。用第二种方法研究重组反义基因的作用 GAs和Gaq受体介导的PLC和AC刺激的构建 将会被研究。第二个具体目标是表征PACAP介导的 即刻早期基因c-fos、c-myc和c-jun在NIH/3T3中的表达 稳定表达hPACAP-R剪接变异体的细胞。激动剂诱导效应 每个hPACAP-R剪接变异体的即刻早期基因表达 采用RT/PCR和Northern印迹分析。这些变化将是 与观察到的生物反应的差异相关联 化验。这里提出的这些研究将增进我们对 PACAP受体的定位，有助于更好地了解其 独特的信号转导特性及其对细胞生长和生长的作用 去分化。

DESCRIPTION (Taken from application) This proposal is directed at defining the molecular mechanisms involved in the regulation of signal transduction and cellular growth by the heptahelical, G-protein-coupled, PACAP receptor (PACAP-R). PACAP-Rs are a useful model to study receptor coupling to intracellular effectors because following agonist stimulation these receptors are capable of activating both adenylate cyclase (AC) and phospholipase C (PLC). Some tumor cells expressing PACAP-Rs also secrete PACAP suggesting a potential autocrine effect of the hormone on tumor growth. The gene for the human PACAP-R was recently cloned and shown to contain two exons encoding the 3rd intracellular loop that can be alternatively spliced into four distinct cDNA splice variants. Although each splice variants is capable of activating both (AC) and (PLC), two (SV2, and SV-3) show greater efficacy for PLC activation and the induction of immediate early genes. Therefore, the first specific aim of this proposal is to characterize the tissue-distribution of PACAPR splice variants and the signaling pathways to which they are coupled. Tissue distribution will be determined using RT-PCR and by using recently developed PACAP-R antibodies for immunohistochemistry. Preliminary studies suggest that PACAP receptor splice variants are coupled to specific G-proteins such as Gas and Gaq. The regions of the PACAP-R that are involved in G-protein coupling will be determined by receptor mutagenesis. The G-proteins involved in coupling to PACAP-R splice variants will be studied by examining the ligand-induced PLC response in NIH/3T3 cells cotransfected with the cDNA of hPACAP-R splice variants and either Gas or Gaq. By a second approach the effects of recombinant antisense gene constructs of Gas and Gaq on receptor-mediated stimulation of PLC and AC will be studied. The second specific aim is to characterize PACAP-mediated expression of the immediate early genes, c-fos, c-myc and c-jun in NIH/3T3 cells stably expressing hPACAP-R splice variants. Agonist induced effects on immediate early gene expression for each hPACAP-R splice variant will be performed using RT/PCR and northern blot analysis. These changes will be correlated with observed differences in biological response by using growth assays. These studies presented here will advance our understanding of the localization of PACAP receptors, lead to a greater understanding of their unique signal transduction properties and their role on cellular growth and de-differentiation.