Selectivity in G-protein-receptor coupling

G 蛋白-受体偶联的选择性

• 批准号: 7966972
• 负责人: John K Northup
• 金额: $ 27.89万
• 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/7966972
• 关键词: Amino Acids; Asthma; Binding Sites; C-terminal; Cardiovascular Diseases; Cattle; Cell Surface Receptors; Chemicals; Chimera organism; Complex; Crystallization; Disease; Family; Future; G alpha q Protein; G(q) Alpha; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Genes; Hormones; Human; Human body; Light; Marketing; Measurable; Mediating; Molecular; Neurotransmitters; Odors; Opsin; Pathway interactions; Peptides; Pharmacologic Substance; Physiological; Publishing; Research; Retinal; Rhodopsin; Sepia; Signal Transduction; Signaling Molecule; Smell Perception; Solutions; Structure; Taste Disorders; Temperature; Testing; Therapeutic Agents; Therapeutic Intervention; Toxic effect; Transducin; Variant; Vertebral column; Visual; Work; base; depression; design; drug development; insight; mutant; novel; octylglucopyranoside; protein aminoacid sequence; receptor; response

We have examined the significance of the recent crystal structure of bovine opsin complexed with an 11-mer peptide sequence derived from the carboxyl terminus of the retinal G-protein transducin (Gt) alpha subunit chain. We synthesized the L341 mutant sequence of the published crystal structure as well as the wild-type sequence for the C-terminal 11 residues of Gt-alpha to test for crystallization of the rhodopsin from the cephalopod mollusc Sepia officinales. Neither peptide stabilized Sepia rhodopsin in dodecylmaltoside or octylglucoside solution. In contrast, the C-terminal sequence of Gq protein, the cognate G-protein for cephalopod visual response, increased the rate of thermal denaturation of Sepia rhodopsin. Thus, these sequences were not pursued for obtaining crystals with Sepia rhodopsin. Additionally, we tested the functional interactions of the C-terminal peptides with rhodopsins. The L341 peptide displayed a Ki of 3 10-5 M for inhibiting bovine rhodopsin activation of Gt while the wild-type sequence inhibited with a Ki of 6 10-4 M. These values were unchanged with variation of the concentration of Gt-alpha, indicating a non-competitive mechanism for the inhibition. Further, neither peptide displayed the expected competitive interaction when Gt-alpha was varied at several fixed concentrations of the peptides. Saturation analyses of the inhibition as a function of temperature revealed that the L341 mutant and K341 wild-type sequences inhibit by different chemical interactions, the L341 peptide saturating with a shallow profile (Hill slope 0.6) and being strongly hydrophobic while the K341 wild-type sequence saturates with a steep slope (Hill slope 2.4) and dominantly enthalpic. Thus, neither peptide interacts at the Gt-alpha binding site of rhodopsin; and the L341 mutant does not reflect the same interaction as the native K341 sequence. Therefore, we will not pursue these sequences further for structural studies. 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 or G-alpha-s to test for the sequences within G-alpha conferring receptor-selectivity. Because of the crystal of opsin with the C-terminal 11-mer mutant sequence from G-alpha-t, we examined the G-alpha-i1/q chimera containing the terminal 11 amino acid residues contributed by G-alpha-i1/q11. Tested with the human 5HT2c receptor, the results we have obtained are ambiguous as to the contribution of this sequence for receptor recognition. While this chimeric G-alpha-i1/q11 is not recognized as being quantitatively identical to G-alpha-q, we can not rule out some enhancement of recognition compared with G-alpha-i1. Our future work will test this and a similar G-alpha-i1/s11 construct using receptors that show no measurable activation of G-alpha-i1, as opposed to the human 5HT2c receptor which recognizes G-alpha-i1 poorly, but measurably.

我们研究了牛视蛋白与源自视网膜 G 蛋白转导蛋白 (Gt) α 亚基链羧基末端的 11 聚体肽序列复合的最新晶体结构的重要性。 我们合成了已发表的晶体结构的 L341 突变序列以及 Gt-α C 端 11 个残基的野生型序列，以测试来自头足类软体动物乌贼的视紫红质的结晶。 这两种肽都不能稳定十二烷基麦芽糖苷或辛基葡萄糖苷溶液中的棕褐色视紫红质。 相反，Gq 蛋白（头足类视觉反应的同源 G 蛋白）的 C 端序列增加了棕褐色视紫红质的热变性速率。 因此，这些序列不是为了获得具有棕褐色视紫红质的晶体而进行的。 此外，我们还测试了 C 端肽与视紫红质的功能相互作用。 L341 肽显示出抑制牛视紫红质 Gt 激活的 Ki 为 3·10-5 M，而野生型序列的抑制 Ki 为 6·10-4 M。这些值随着 Gt-α 浓度的变化而变化。表明抑制的非竞争性机制。 此外，当 Gt-α 在肽的几个固定浓度下变化时，两种肽都没有表现出预期的竞争性相互作用。 作为温度函数的抑制饱和分析表明，L341 突变体和 K341 野生型序列通过不同的化学相互作用进行抑制，L341 肽以浅轮廓（希尔斜率 0.6）饱和并且具有强疏水性，而 K341 野生型则具有强疏水性。序列以陡峭的斜率（Hill 斜率 2.4）饱和并且主要是焓。 因此，这两种肽都不与视紫红质的 Gt-α 结合位点相互作用。并且L341突变体不反映与天然K341序列相同的相互作用。 因此，我们不会进一步对这些序列进行结构研究。 此前，我们构建了一个嵌合 G-α 亚基的集合体，使用 G-α-i1 作为主链，并引入 G-α-q 或 G-α-s 的序列来测试 G-α 内赋予受体选择性的序列。 由于视蛋白晶体具有来自 G-alpha-t 的 C 端 11 聚体突变序列，我们检查了含有 G-alpha-i1/q11 贡献的末端 11 个氨基酸残基的 G-alpha-i1/q 嵌合体。 用人类 5HT2c 受体进行测试，我们获得的结果对于该序列对受体识别的贡献是不明确的。 虽然这种嵌合 G-alpha-i1/q11 不被认为在数量上与 G-alpha-q 相同，但我们不能排除与 G-alpha-i1 相比识别能力有所增强。 我们未来的工作将测试这个和类似的 G-alpha-i1/s11 构建体，使用没有显示出可测量的 G-alpha-i1 激活的受体，而不是人类 5HT2c 受体，它识别 G-alpha-i1 很差，但可测量。