STRUCTURE OF THE BETA ADRENERGIC RECEPTOR CYCLASE SYSTEM

这一提议的重点是理解分子 在受体的不同区域发生的相互作用- G蛋白-效应器偶联系统通过使用多个 接近了。放射性碘标记的新型拮抗剂和激动剂 对邻苯二酚具有选择性的光活性化合物 将开发B2AR配体结合位点的一部分。 这些化合物将被用于光亲和标记配体。 杆状病毒表达的Sf9 b2-AR的结合区。部分 将获得[125I]的N-末端或完整序列 利用多种光探针对多肽进行光标记化 B2AR的结合域。B2AR上的“外星人”， 它参与长效b2的作用机制。 激动剂(即沙美特罗)将使用新的 沙美特罗衍生物。腺苷的可溶性胞质结构域 将环化酶在大肠杆菌中进行表达和纯化。这个 可溶性腺苷、三磷酸腺苷和抑制性“p”位 将使用可光激活的化合物和 光标记肽的纯化。光标记法 实验还将在完整的腺酰环化酶上进行， 该基因将在Sf9细胞中过表达。可光激活的 来源的特定结构域相互作用肽的衍生物 将利用B2AR和GAS的序列来识别 B2AR与GAS之间、GAS与GAS之间的相互作用域 腺苷环化酶，以及视紫红质和α转导蛋白之间。 催化活性可溶性IC1的晶体结构 和IIC2腺酰环化酶的测定。结合部位 Forskolin、核苷酸和Gas的结构域也将是 在催化活性的可溶性IC1和IIC2中测定 腺酰环化酶通过产生共晶体。这其中的一个组成部分 工作包括表达和纯化大量的 IC1结构域和/或催化活性结构 混合IC1。IIC2分子适合于 结晶学。这些实验将增加我们的 了解受体-G蛋白-效应器偶联系统， 例如儿茶酚胺β受体，它们的功能是控制 自主神经功能，如心率、血压和 肝脏、脂肪和脑组织的神经功能和代谢状态 肌肉。

The focus of this proposal is to understand the molecular interactions which occur in the various domains of the receptor- G protein-effector coupling system through the use of multiple approaches. Novel antagonist and agonist radioiodinated photoactive compounds which are selective for the catechol portion of the b2AR ligand binding site will be developed. These compounds will be used to photoaffinity label the ligand binding domain of the baculovirus-expressed Sf9 b2-AR. Partial N-terminal or complete sequence will be obtained for [125I] photolabeled peptides using a variety of photoprobes to "map" the binding domain of the b2AR. The "exosite" on the b2AR, which is involved in the mechanism of action of long-acting b2 agonists (i.e., salmeterol), will be identified using novel salmeterol derivatives. Soluble cytoplasmic domains of adenylyl cyclase will be expressed in E. coli and purified. The forskolin, ATP, and inhibitory "p" site on the soluble adenylyl cyclase will be identified using photoactivatable compounds and purification of photolabeled peptides. Photolabeling experiments will also be performed on intact adenylyl cyclase, which will be overexpressed in Sf9 cells. Photoactivatable derivatives of specific domain-interacting peptides derived from the sequences of the b2AR and Gas will be utilized to identify interacting domains between b2AR and Gas, between Gas and adenylyl cyclase, and between rhodopsin and alpha transducin. The crystal structure of the catalytically active soluble IC1 and IIC2 adenylyl cyclase will be determined. The binding site domains for forskolin, nucleotides, and Gas will also be determined in the catalytically active soluble IC1 and IIC2 adenylyl cyclase by generating co-crystals. A component of this work involves expression and purification of large quantities of the IC1 domain and/or construction of catalytically active hybrid IC1. IIC2 molecules which are suitable for crystallography. These experiments will increase our understanding of receptor-G protein-effector coupling systems, such as catecholamine beta-receptors, which function to control autonomic functions, such as heart rate, blood pressure, and neuronal function and metabolic state of liver, adipose, and muscle.