Selectivity in G-protein-receptor coupling

G 蛋白-受体偶联的选择性

• 批准号: 8148597
• 负责人: John K Northup
• 金额: $ 30.52万
• 依托单位: NATIONAL INSTITUTE ON DEAFNESS AND OTHER COMMUNICATION DISORDERS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8148597
• 关键词: G-Protein-Coupled Receptors

Previously, we had constructed an ensemble of chimeric G-alpha subunits using G-alpha-i1 as the backbone and introducing sequence from G-alpha-q to test for the sequences within G-alpha conferring receptor-selectivity. These studies revealed that the receptor contact surface of the G-alpha subunit comprises an extensive region of at least 50 amino acid residues of the carboxyl terminus of the G-alpha-q. We were limited in those investigations by inability to express chimeric alpha subunits with additional sequence contributed by G-alpha-q. To determine if these results apply to other G-protein types and to examine for additional sequence(s) specifying selective contact, we have constructed a similar set of chimeric structures introducing sequence from the G-alpha-s protein into G-alpha-i1 and testing for interaction with the beta2-adrenergic receptor. We were able in these studies to replace the majority of the sequence of G-alpha-i1 with G-alpha-s derived sequence and still obtain expression of functional G-alpha protein. These studies with the beta2-adrenergic receptor and G-alphapi1/s chimeras recapitulated the results found previously for G-alpha-i1/q chimeras and the 5HT2c receptor. Further, they rule out all other G-alpha-s structure save the N-terminal alpha helix as providing receptor-selective contact (Gutierrez et al, manuscript in preparation). In collaboration with Dr. Reinhard Grisshammer, NINDS, we have continued efforts to obtain crystals of GPCR-G-protein complexes using cephalopod rhodopsin and G-alpha-q or G-alpha-t. Do date all experiments have failed to yield crystals of the complexes. Neither have we succeeded in obtaining crystals of the activated cephalopod rhodopsin. We initiated a project in collaboration with Dr. John Tesmer, University of Michigan School of Medicine, to obtain crystals of the complexes of cephalopod rhodopsin with G-receptor kinase 2 with and without G-alpha-q. These have also failed to yield crystals to date. Also in collaboration with Dr. John Tesmer, we have succeeded in obtaining the crystal structure at 2.0 angstrom resolution of a 90 kDa fragment of the cephalopod retinal phospholipase C. Our structure reveals a previously unkown interdomain interaction between C-terminal sequence of the PLC acting as an auto-inhibitor of the catalytic domain. This structure suggests a novel mechanism for regulation of PLC-beta enzymes by G-alpha-q in which the G-protein releases the PLC from auto-inhibition (Lyon et al, manuscript in preparation).

此前，我们构建了一个嵌合 G-α 亚基的集合，使用 G-α-i1 作为主链，并引入 G-α-q 的序列来测试 G-α 内赋予受体选择性的序列。 这些研究表明，G-α 亚基的受体接触表面包含 G-α-q 羧基末端至少 50 个氨基酸残基的广泛区域。 我们在这些研究中受到限制，因为无法表达具有 G-alpha-q 贡献的附加序列的嵌合 α 亚基。为了确定这些结果是否适用于其他 G 蛋白类型并检查指定选择性接触的其他序列，我们构建了一组类似的嵌合结构，将 G-alpha-s 蛋白的序列引入 G-alpha-i1并测试与β2-肾上腺素能受体的相互作用。 在这些研究中，我们能够用 G-alpha-s 衍生序列替换 G-alpha-i1 的大部分序列，并且仍然获得功能性 G-alpha 蛋白的表达。 这些针对 β2-肾上腺素受体和 G-alphapi1/s 嵌合体的研究概括了之前针对 G-alpha-i1/q 嵌合体和 5HT2c 受体发现的结果。 此外，他们排除了除 N 端 α 螺旋之外的所有其他 G-α-s 结构，因为它提供了受体选择性接触（Gutierrez 等人，手稿正在准备中）。 我们与 NINDS 的 Reinhard Grissammer 博士合作，继续努力使用头足类视紫红质和 G-alpha-q 或 G-alpha-t 获得 GPCR-G-蛋白复合物晶体。 截至目前，所有实验均未能产生复合物晶体。 我们也没有成功获得活化的头足类视紫红质晶体。 我们与密歇根大学医学院的 John Tesmer 博士合作发起了一个项目，以获得头足类视紫红质与 G 受体激酶 2（含和不含 G-alpha-q）的复合物晶体。 迄今为止，这些方法也未能产生晶体。 同样与 John Tesmer 博士合作，我们成功获得了头足类视网膜磷脂酶 C 90 kDa 片段的 2.0 埃分辨率的晶体结构。我们的结构揭示了 PLC 作用的 C 端序列之间以前未知的域间相互作用。作为催化结构域的自动抑制剂。 这种结构提出了一种通过 G-α-q 调节 PLC-β 酶的新机制，其中 G 蛋白将 PLC 从自动抑制中释放出来（Lyon 等人，手稿正在准备中）。