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).

以前，我们使用G-Alpha-i1作为骨架，构建了嵌合G-α亚基的集合，并从G-Alpha-Q引入序列，以测试G-Alpha赋予受体选择性中的序列。 这些研究表明，G-Alpha亚基的受体接触表面包括G-Alpha-Q的羧基末端至少50个氨基酸残基的广泛区域。 在这些研究中，我们的限制是由于无法表达嵌合α亚基，其其他序列由G-Alpha-Q贡献。为了确定这些结果是否适用于其他G蛋白类型，并检查了指定选择性接触的其他序列，我们已经构建了一组类似的嵌合结构，从G-Alpha-S蛋白引入了G-Alpha-I1中的序列，并测试与Beta2-辅助受体相互作用。 在这些研究中，我们能够用G-Alpha-S衍生的序列替代G-Alpha-I1的大多数序列，并仍然获得功能性G-Alpha蛋白的表达。 这些对BetA2-肾上腺素能受体和G-Alphapi1/s Chimeras的研究概括了先前针对G-Alpha-I1/Q Chimeras和5HT2C受体发现的结果。 此外，它们排除了所有其他G-Alpha-S结构，将N末端α螺旋保存为提供受体选择性接触（Gutierrez等人，手稿中的手稿）。 通过与Ninds的Reinhard Grisshammer博士合作，我们继续努力使用头足类动蛋白和G-Alpha-Q或G-Alpha-T获得GPCR-G-G蛋白络合物的晶体。 做日期所有实验都无法产生复合物的晶体。 我们都没有成功获得活化的头足动物视紫红质的晶体。 我们与密歇根大学医学院的约翰·特斯默（John Tesmer）合作发起了一个项目，以使用有没有G-Alpha-Q的G-Repeptor激酶2获得头足类动蛋白的晶体。 这些也未能产生晶体。 同样，在与约翰·特斯默（John Tesmer）博士的合作中，我们成功地以2.0埃脉冲分辨率的晶体结构分辨出了90 kDa的头孢半座视网膜磷脂酶C.我们的结构揭示了PLC在CAT CATE型型型型型型型型型型型型型肠上的C-C-末端序列的先前未造成的界面相互作用。 该结构提出了一种通过G-Alpha-Q调节PLC-beta酶的新机制，其中G蛋白可从自动抑制中释放PLC（Lyon等，手稿制备中）。