STRUCTURE/FUNCTION STUDIES OF ALPHA ADRENERGIC RECEPTORS

α/1-肾上腺素能受体（α/1-AR）介导了 交感神经系统，如小动脉收缩。 三 α/1-AR基因已经克隆出来了。 然而， 这种亚型的多样性仍然不清楚，因为它们在功能上是 它们在配体识别和信号传导性质上相似， 常被表达。 该提案的目的是更明确地界定 他们的生理功能，并研究可能的差异， 这些subtypes。 在我们努力理解信号转导的过程中， α/1-AR，我们打算集中在一些突变体，可以澄清 哪些残基参与亚型选择性，如果一个特定的 残基参与将信号转导至G蛋白。 其次，通过开发一个模型系统进行研究， 通过基因靶向使α/1-AR亚型失活。 FRTL-5细胞系 是一个理想的模型系统，因为它的α/1-AR信号特征 和甲状腺代谢的受体介导已经得到很好的表征。 在该细胞系中，只有一个α/1-AR（α/1B）似乎是 表达，可以通过两个不同的G- proteins. 然而，另一种亚型的表达，在低水平，不能 被排除在外。 为了验证这一假设，我建议 α/1B-AR，然后评估其在甲状腺调节中的作用。 的 产生α/1B-AR缺陷细胞系也是有利的 在特异性目标1中产生的α/1B-AR突变体的分析中。 具体而言，我们的目标如下： （一） 利用定点突变技术进行结构功能研究 确定哪些残基参与亚型突变 选择性 （二）充分认识和理解一个 组成性激活的α/1B-AR，在第三个基因中突变， 跨膜结构域 iii）为了实现α/1B-AR基因的双等位基因破坏， 在体细胞FRTL-5细胞中同源重组并产生α/1B- 用于突变研究的AR缺陷细胞系。

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.