C-TERMINAL INTERACTIONS OF ALPHA1-ADRENOCEPTOR SUBTYPES

多年来，该计划的重点一直交替研究 肾上腺素能受体的各种亚型。本申请关注于 α-1亚型，而不是目前研究的β-1和β-2亚型， 因为目的是探索C-末端剪接的功能作用， 已针对α-1亚型证明的变体，但尚未针对β亚型证明 亚型总体目标是了解GPCR的功能作用， 信号传导机制超越了与G蛋白的耦合。从 初步考虑，研究者已经确定了 α亚型的C末端作为这种信号传导的潜在介质，和 建议研究一系列假定的介质，包括神经元 立即早期基因产物Homer和神经元NOS的PDZ结构域。的 待探讨的一般假设是：1。有特异性结合 每个α-l亚型的C末端尾的配偶体; 2.这些亚型 具有带有不同结合配偶体的c-末端剪接变体;和3.的 与各伙伴的这些互动具有重要的职能作用。 为此，研究者计划利用C-末端的融合蛋白， 尾部和全长受体在N-末端进行表位标记， 有五个具体目标：1。调查人员将确定 神经元立即早期基因产物Homer结合PPXXFR共有结合 在alpha 1D-AR尾部的基序，并改变其功能。2.他们将决定 神经元型一氧化氮合酶的PDZ结构域是否与GEEV结合 在α 1A-AR的末端C-末端的共有结合基序， 它的功能。3.他们将确定α 1A，α 1B， 使用生化方法和表达文库的a1 D-AR C-末端尾 用尾融合蛋白筛选。4.他们将识别，克隆， 表征α 1D-ARs和α 1D-ARs的剪接变体。5.他们将 使用a1 A、a1 B和a1 D-AR的剪接变体作为天然存在的C-末端 变异，以检查蛋白质相互作用的功能作用， C末端的尾巴这些实验将阐明C-末端的作用 α 1-AR功能中的相互作用蛋白，以及C-末端的意义 剪接变体。

Over the years, the focus of this program has alternated between studies of various subtypes of adrenergic receptors. The present application focuses on alpha-1 subtypes rather than the beta-1 and beta-2 subtypes studied in the current period, because the aim is to explore the functional role of C-terminal splice variants that have been demonstrated for an alpha-1 subtype, but not for a beta subtype. The overall goal is to understand the functional role of GPCRs in signaling mechanisms that reach beyond the coupling to G proteins. From preliminary considerations, the investigator has identified interactions at the C terminus of the alpha subtypes as potential mediators of such signaling, and proposes to investigate a series of putative mediators including the neuronal immediate early gene product Homer, and the PDZ domain of neuronal NOS. The general hypotheses to be explored are: 1. that there are specific binding partners for C-terminal tails of each alpha-1 subtype; 2. that the subtypes have c-terminal splice variants with distinct binding partners; and 3. that these interactions with the various partners have important functional roles. To this end, the investigator plans to utilize fusion proteins of C-terminal tails and full-length receptors epitope-tagged at the N-terminus in order to pursue five Specific Aims: 1. The investigators will determine whether the neuronal immediate-early gene product Homer binds to a PPXXFR consensus binding motif in the alpha1D-AR tail and alters its function. 2. They will determine whether the PDZ domain of neuronal nitric oxide synthase binds to a GEEV consensus binding motif at the extreme C-terminus of the alpha1A-AR and alters its function. 3. They will identify novel binding partners for alpha1A, a1B, a1D-AR C-terminal tails using biochemical approaches and expressions library screening with tail fusion proteins. 4. They will identify, clone, and characterize splice variants of alphalambdapha1B and alpha1D-ARs. 5. They will use splice variants of a1A, a1B, and a1D-ARs as naturally occurring C-terminal variants to examine the functional roles of protein interactions at the C-terminal tails. These experiments will clarify the role of C-terminal interacting proteins in a1-AR function, and the significance of C-terminal splice variants.