ACTION OF HORMONES ON ADENYLYL CYCLASE SYSTEMS

这项研究的总体目标是了解人类免疫缺陷的分子基础 受体启动的信号转导，主要但不完全是AS 它们与腺苷环化酶的调节有关。这包括环化酶 R型受体的刺激和RI型受体的抑制。 在上一次资助期间，我们提纯并鉴定了亚基 转导Rs和Ri的偶联蛋白NS和Ni的组成 受体占位转变为腺苷酸环化酶活性改变。我们也 描述了N介导的激素-受体相互作用的调节和 磷脂小泡中重组的RN相互作用，使用纯Rs和 纯NS和视紫红质(Ri类似物)和纯镍。我们似乎克隆了 编码N蛋白MR=35,000β亚基的cDNA，尽管 目前仍未进行最终确认。在即将到来的授权期内，我们 愿望： 1.克隆NS和Niα亚基和γ亚基的cDNA。 2.表达sn细菌N蛋白的α、β、γ亚基和 研究它们在重组后各自的功能，给出N 蛋白质，用来检测磷脂小泡中的重组 RS-NS和视紫红质-镍相互作用。 3.研究NS和Ni亚基编码基因的遗传组织 并定义了这些蛋白质的生物合成模式。 4.探讨受体介导的亚基解离是否发生 在GTP存在下N蛋白的激活以及确定镍如何 ACTs在NS、Ni、An磷脂微囊中重组后的变化 RS，一种Ri和腺酰环化酶本身。 5.在可能的序列同源性的基础上发展 视紫红质和RI型受体的克隆和测定 RI型受体的一级结构，如α2-肾上腺素能， 毒扁豆碱、乙酰胆碱和阿片受体，或者用于纯化 乙酰胆碱受体，在部分初级的基础上 氨基酸序列测定或多克隆抗体产生，以 克隆并确定其一级结构，确定其与视紫红质的同源性 并继续克隆其他Ri-型受体。 6.探索N蛋白和ras之间可能的结构同源性 基因产品检测其亚基组成及其可能的物理 偶联到不偶联到腺苷环化酶的受体。 这项研究将极大地促进我们对 影响cAMP形成的信号转导的分子基础。

The overall aim of this research is to understand the molecular basis of receptor-initiated signal transductions, primarily but not exclusively as they relate to adenylyl cyclase regulation. This includes cyclase stimulaton by Rs-type receptors and inhibition by Ri-type receptors. During the last grant period, we purified and characterized the subunit compositin of Ns and Ni, the coupling proteins that transduce Rs and Ri receptor occupancy into altered adenylyl cyclasle activity. We also described N-mediated regulation of hormone-receptor interactions and reconstituted RN interactions in phospholipid vesicles, using pure Rs and pure Ns and rhodopsin (a Ri analog) and pure Ni. We appear to have cloned the cDNA coding for the Mr = 35,000 beta subunit of N proteins, although final confirmation is still missing. During this coming grant period we wish: 1. To clone the cDNAs for the alpha and gamma subunits of Ns and Ni. 2. To express sn bacteria alpha, beta and gamma subunits of N proteins and study their respective functions as seen after recombination to give N proteins, using as assays the reconstitution in phospholipid vesicles of Rs-Ns and rhodopsin-Ni interactions. 3. To study the genetic organization of genes coding for Ns and Ni subunits and define the mode of biosynthesis of these proteins. 4. To explore if subunit dissociation occurs on receptor-mediated activation of N proteins in the presence of GTP and to determine how Ni acts, as seen after reconstitution in phospholipid vesicles of Ns, Ni, an Rs, an Ri and adenylyl cyclase proper. 5. To develop on the basis of possible sequence homologies between rhodopsin and Ri-type receptors an approach for cloning and determining the primary structure of Ri-type receptors such as alpha2-adrenergic, muscarinic acetylcholine and opioid receptors, or alternatively, to purify the muscarinic acetylcholine receptor and, on the basis of partial primary amino acid sequence determination or polyclonal antibody generation, to clone and determine its primary structure, define homologies with rhodopsin and proceed with cloning of other Ri-type receptors. 6. To explore possible structural homologies between N proteins and ras gene products testing their subunit composition and their possible physical coupling to receptors that do not couple to adenylyl cyclase. This research should significantly advance our understanding of the molecular basis of signal transductions that impinge on cAMP formation.