ACTION OF HORMONES ON ADENYLYL CYCLASE SYSTEMS

本研究的总体目标是了解 受体启动的信号转导，主要但不排他地作为 它们涉及腺苷酸环化酶调节。 其中包括环化酶 通过Rs型受体的刺激和通过Ri型受体的抑制。 在上一次资助期间，我们纯化并鉴定了亚基 N_s和Ni组成， 受体占有率改变腺苷酸环化酶活性。 我们也 描述了N-介导的调节酶-受体相互作用， 重组RN相互作用的磷脂囊泡，使用纯Rs和 纯Ns和视紫红质（Ri类似物）和纯Ni。 我们似乎克隆了 编码N蛋白的Mr = 35，000 β亚基的cDNA，尽管 最后的确认仍然没有。 在即将到来的这段时间里，我们 愿望： 1.克隆N_s和Ni的α和γ亚基的cDNA。 2.为了表达细菌N蛋白的α、β和γ亚基， 研究它们各自的功能，如重组后所见，得到N 蛋白质，使用磷脂囊泡中的重组作为测定， Rs-Ns和视紫红质-Ni相互作用。 3.研究编码Ns和Ni亚基基因的遗传结构 并确定这些蛋白质的生物合成模式。 4.为了探索在受体介导的细胞内是否发生亚基解离， 在GTP存在下活化N蛋白，并确定Ni 作用，如在Ns、Ni和Ni的磷脂囊泡中重构后所见， Rs，Ri和腺苷酸环化酶。 5.在可能的序列同源性的基础上， 视紫红质和Ri型受体的克隆和鉴定方法 R1型受体如α 2-肾上腺素能受体的一级结构， 毒蕈碱乙酰胆碱和阿片受体，或者， 毒蕈碱乙酰胆碱受体，并在部分初级基础上， 氨基酸序列测定或多克隆抗体产生， 克隆并测定其一级结构，确定与视紫红质的同源性 并继续克隆其他Ri型受体。 6.探讨N蛋白和ras之间可能的结构同源性 基因产物测试它们的亚基组成和它们可能的物理性质， 与不与腺苷酸环化酶偶联的受体偶联。 这项研究应该大大促进我们对 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.