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.

我们已经检查了牛opsin的最新晶体结构的重要性，该牛蛋白与视网膜G蛋白转丁蛋白（GT）Alpha亚基链的11-MER肽序列复合了。 我们合成了已发表的晶体结构的L341突变序列以及GT-Alpha的C末端11残基的野生型序列，以测试来自头孢藻Mollusc sepia officine的Rhopopsin结晶。 肽既不稳定在十二烷基马尔替剂或辛基葡萄糖苷溶液中的棕褐色蛋白质。 相反，GQ蛋白的C末端序列，用于头足动物视觉响应的同源G蛋白增加了棕褐色淡紫蛋白的热变性速率。 因此，这些序列没有用于获得棕褐色紫红蛋白的晶体。 此外，我们测试了C末端肽与视紫红蛋白的功能相互作用。 L341肽的Ki为3 10-5 m，用于抑制GT的牛Rhopopsin激活GT，而野生型序列则以6 10-4的ki抑制。这些值与GT-AlphA的浓度无变化，表明抑制性不相交的机制。 此外，当GT-Alpha在几个固定浓度的肽中变化时，肽都不显示预期的竞争相互作用。 对抑制作用作为温度的饱和分析表明，L341突变体和K341野生型序列通过不同的化学相互作用抑制，L341肽具有浅层剖面（Hill Slope 0.6）的饱和（山坡0.6），并且强烈疏水量，而K341 Wild-type序列却非常饱和，而hill splope则饱满。 因此，肽都在视紫红质的GT-Alpha结合位点相互作用。 L341突变体不反映与天然K341序列相同的相互作用。 因此，我们不会进一步追求这些序列进行结构研究。 以前，我们使用G-Alpha-i1作为骨架构建了一个嵌合G-α亚基合奏，并从G-Alpha-Q或G-Alpha-S引入序列，以测试G-Alpha Condring受体选择性中的序列。 由于Opsin的晶体具有G-Alpha-T的C末端11-Mer突变序列，因此我们检查了含有G-Alpha-I1/Q11贡献的末端11氨基酸残基的G-Alpha-I1/Q Chimera。 用人的5HT2C受体测试，我们获得的结果对该序列对受体识别的贡献含糊不清。 尽管该嵌合G-Alpha-I1/Q11并未被认为与G-Alpha-Q相同，但与G-Alpha-I1相比，我们不能排除对识别的一些增强。 我们未来的工作将使用没有显示G-Alpha-i1的受体的受体来测试这一点，并且与人类5HT2C受体相似，与人类5HT2C受体相似，与人类5HT2C受体相比，该受体识别G-Alpha-i1差，但可以测量。