C-TERMINAL INTERACTIONS OF ALPHA1-ADRENOCEPTOR SUBTYPES

多年来，该计划的重点在研究 肾上腺素能受体的各种亚型。本申请侧重于 阿尔法-1亚型而不是目前研究的β-1和β-2亚型 期间，因为目的是探索C-末端剪接的功能作用 已经为阿尔法-1亚型展示的变体，但没有针对测试型 子类型。总体目标是了解GPCRs在 信号机制超越了与G蛋白的偶联。从… 初步考虑，调查人员已经确定了 作为这种信号的潜在介体的α亚型的C末端，以及 建议研究一系列可能的介质，包括神经元 即刻早期基因产物荷马和神经元型一氧化氮合酶的PDZ结构域。这个 要探索的一般假设是：1.有特定的约束力 为每个α-1亚型的C-末端尾巴配对；2.亚型 具有具有不同结合伙伴的c-末端剪接变体；以及3. 这些与各种合作伙伴的互动具有重要的职能作用。 为此，研究人员计划利用C-末端的融合蛋白 在N端标记尾巴和全长受体，以便 追求五个具体目标：1.调查人员将确定 神经元即刻早期基因产物荷马与PPXXFR共识结合 Alpha1D-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GEV结合 Alpha1A-AR末端的共识结合基序及其改变 它的功能。3.他们将确定Alpha1A、A1B、 利用生化方法和表达文库研究A1D-AR C末端尾部 用Tail融合蛋白进行筛选。4.他们将识别、克隆和 鉴定alphalambdapha1B和alpha1D-Ars的剪接变异体。5.他们会的 使用A1a、A1B和a1D-Ars的剪接变体作为自然产生的C-末端 用于研究蛋白质相互作用的功能作用的变体 C-末端反面。这些实验将阐明C-末端的作用 A1-AR功能中的相互作用蛋白及其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.