STRUCTURE/FUNCTION STUDIES OF ALPHA ADRENERGIC RECEPTORS

α/1-肾上腺素能受体(α/1-AR)介导的重要作用 交感神经系统，如小动脉收缩。三 Alpha/1-Ars已被克隆。然而，生理学意义在于 这种亚型多样性仍然不清楚，因为它们在功能上是 在配基识别和信号特性方面相似，并且是 经常共表达。这项提议的目标是更明确地界定 并研究它们的生理功能和可能的差异 这些子类型。在我们努力理解信号转导的过程中 阿尔法/1-AR，我们打算专注于一些突变体，这些突变体可以澄清 哪些残基与亚型选择性有关，以及如果特定的 残基(S饰)参与将信号传递到G蛋白。 其次，将通过开发一个模型系统来进行研究 基因打靶使Alpha/1-AR亚型失活。FRTL-5细胞株 是一个理想的模型系统，因为它的α/1-AR信号特征 受体对甲状腺代谢的调节作用已经得到了很好的表征。 在该细胞系中，似乎只有一个α/1-AR(α/1B) 它可以通过两个不同的G- 蛋白质。然而，另一个亚型(S)的表达在低水平上不能 被排除在外。为了检验这一假设，我建议停用 然后评估其在甲状腺调节中的作用。这个 产生α/1B-AR缺陷细胞系也将是有利的 在分析α/1B-AR突变体产生的特定目的1。 具体来说，我们的目标如下： I)利用现场指导进行结构-功能研究 决定亚型涉及哪些残基的诱变 选择性。 二)充分描述和理解 结构性激活的α/1B-AR，已在第三个 跨膜结构域。 三)通过以下方式实现α/1B-AR基因的双等位基因中断 在体细胞FRTL-5中进行同源重组并产生α/1B- 用于突变研究的AR缺乏细胞系。

Alpha/1-Adrenergic receptors (alpha/1-AR) mediate important effects f the sympathetic nervous system, such as arteriolar constriction. Three alpha/1-ARs have been cloned. However, the physiological significance of this subtype diversity remains unclear, since they are functionally similar in their ligand recognition and signaling properties, and are often coexpressed. The goal of this proposal is to more clearly define their physiological function and investigate possible differences between these subtypes. In our efforts to understand signal transduction by alpha/1-ARs, we intend to focus on a number of mutants that can clarify which residues are involved in subtype selectivity, and if a particular residue(s) is involved in transducing a signal to the G-protein. Secondly, studies will be performed by developing a model system to study alpha/1-AR subtype inactivation by gene targeting. The FRTL-5 cell line is an ideal model system since its alpha/1-AR signaling characteristics and receptor-mediation of thyroid metabolism have been well characterized. In this cell line only a single alpha/1-AR (alpha/1B) appears to be expressed that can couple to its effector pathways via two distinct G- proteins. However, expression of another subtype(s), at low level, cannot be excluded. To test this hypothesis, I propose to inactivate the alpha/1B-AR and then assess its role in thyroid regulation. The generation of an alpha/1B-AR deficient cell line will also be advantageous in the analysis of the alpha/1B-AR mutants generated in Specific Aim 1. Specifically, our aims are the following: i) To undertake structure-function studies using site-directed mutagenesis in determining which residues are involved in subtype selectivity. ii) To fully characterize and understand the properties of a constitutively activated alpha/1B-AR that has been mutated in the third transmembrane domain. iii) To achieve a double allele disruption of the alpha/1B-AR gene by homologous recombination in somatic FRTL-5 cells and generate an alpha/1B- AR deficient cell line for mutational studies.