LOCALIZATION/SIGNAL TRANSDUCTION

描述（摘自应用程序） 这项建议是针对定义参与的分子机制， 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 这是研究受体与细胞内效应物偶联的有用模型， 在激动剂刺激后，这些受体能够激活 腺苷酸环化酶（AC）和磷脂酶C（PLC）。 一些肿瘤细胞 表达PACAP-Rs的人也分泌PACAP，这表明PACAP可能是自分泌的。 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 可以选择性剪接成四种不同cDNA的细胞内环 剪接变体。 尽管每种剪接变体都能够激活 AC和PLC均有效，其中SV-2和SV-3对PLC有较好的疗效 激活和诱导立即早期基因。 因此，本提案的第一个具体目标是描述 PACAPR剪接变异体的组织分布和 它们被耦合。 将使用RT-PCR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受体剪接变异体与 特定的G蛋白如Gas和Gaq PACAP-R的区域， 参与G-蛋白偶联的受体将由 诱变 参与PACAP-R剪接变体偶联的G蛋白 将通过检查NIH/3T3中配体诱导的PLC反应来研究 用hPACAP-R剪接变体的cDNA和Gas 或盖奇通过第二种方法， Gas和Gaq的构建体对PLC和AC的受体介导的刺激 将被研究。 第二个具体目标是表征PACAP介导的 NIH/3T3细胞即早基因c-fos、c-myc和c-jun的表达 稳定表达hPACAP-R剪接变体的细胞。 激动剂诱导效应 对于每种hPACAP-R剪接变体的立即早期基因表达， 使用RT/PCR和北方印迹分析进行。 这些更改将 通过使用生长与观察到的生物反应差异相关 测定。 这里介绍的这些研究将促进我们对 PACAP受体的定位，导致更好地了解其 独特的信号转导特性及其对细胞生长和 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.