INVESTIGATION OF HETEROTRIMERIC GUANINE NUCLEOTIDE BINDING PROTEIN ACTIVATION

异三聚鸟嘌呤核苷酸结合蛋白激活的研究

• 批准号: 6290640
• 负责人: ROBERT VICTOR REBOIS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 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/6290640
• 关键词: ADP ribosylation; G protein; adenylate cyclase; binding proteins; brain cell; chemical association; cow; enzyme activity; genetic transcription; genetic translation; guanine nucleoside; guanosine diphosphate; guanosine triphosphate; guanosinetriphosphatases; immunoprecipitation; membrane reconstitution /synthesis; nucleotide analog; protein structure; recombinant DNA

Heterotrimeric (alpha-beta-gamma) Gs mediates agonist-induced stimulation of adenylyl cyclase (AC). The alpha subunit (Gs-alpha) has intrinsic GTPase activity. Gs is activated when Gs-alpha binds GTP or a GTP analog (e.g. GTP-gamma-S, Gpp[NH]p, or Gpp[CH2]p) and it is deactivated when GTP is hydrolyzed. Naturally occurring mutations of Gs-alpha that suppress Gs activity lead to a human disease called pseudohypoparathyroidism (PHP). Patients with PHP do not respond to hormones that use Gs-coupled receptors, particularly parathyroid hormone. The outcome of being refractory to these hormones is that patients with PHP have a collection of maladies labeled as Albright hereditary osteodystrophy which include short stature, obesity, subcutaneous ossifications, and mental retardation. Our investigations indicate that mutations which cause PHP either 1) reduce the affinity of Gs-alpha for GDP which destabilize the protein and causes it to be denatured at physiological temperatures, or 2) reduce the half-life of the activated conformation of Gs so that agonist-mediated stimulation of AC is attenuated. For decades it has been known that Gs activation in solution can be accompanied by dissociation of Gs-alpha from the G protein beta-gamma subunit complex (G-beta-gamma). However, our recent evidence suggests that subunit dissociation is caused by non- physiological conditions rather than activation. Nevertheless, the prevalent view is that subunit dissociation also occurs when Gs is activated in situ despite a lack of evidence. Cholera toxin (CTx) will ADP-ribosylate Gs-alpha. Gs-alpha-deficient cyc- membranes were stripped of G-beta-gamma. When the stripped cyc- membranes were incubated with Gs-alpha and/or G-beta-gamma, each was incorporated into the membranes independently of the other. Both Gs-alpha and G-beta- gamma had to be present the in membranes, and they had to be able to form a heterotrimer in order for CTx to ADP-ribosylate Gs-alpha, indicating that the membrane bound Gs heterotrimer is a substrate for CTx, but the Gs-alpha subunit by itself is not. When Gs-alpha was completely and irreversibly activated with GTP-gamma-S and incorporated into stripped cyc- membranes it was a poor substrate for CTx and a weak stimulator of AC unless G-beta-gamma was also incorporated. Furthermore, the level of AC stimulation corresponded to the amount of Gs heterotrimer that was formed in the membranes from GTP-gamma-S- activated Gs-alpha and G-beta-gamma indicating that AC is stimulated by an activated Gs heterotrimer in cell membranes. Heterotrimeric Gi mediates agonist-induced inhibition of AC. Surface plasmon resonance spectroscopy (SPR) is being used to determine the affinity of Gi subunits for each other. When GDP is bound to Gi-alpha the equilibrium dissociation constant for G-beta-gamma is approximately 10 nM. Gi subunit affinity is decreased by non-hydrolyzable GTP analogs, and varies depending upon the analog that is bound to Gi-alpha. Qualitatively, the affinity of Gi-alpha for G-beta-gamma is less when GTP-gamma-S is bound, than when Gpp[NH]p is bound, which is in turn less than when Gpp[CH2]p is bound. If GTP fits the pattern then its effects on Gi subunit affinity can not be equated with those of its analogs as has been done extensively in the past. - heterotrimeric G proteins, adenylyl cyclase, cholera toxin, surface plasmon resonance

异三聚体（α-beta-gamma）GS介导激动剂诱导的刺激腺苷环酶（AC）。 α亚基（GS-Alpha）具有内在的GTPase活性。当GS-Alpha结合GTP或GTP类似物（例如GTP-GAMMA-S，GPP [NH] P或GPP [CH2] P）时，GS被激活。抑制GS活性的GS-Alpha的自然发生的突变导致一种称为假甲型甲状腺功能减退症（PHP）的人类疾病。 PHP患者对使用GS偶联受体，尤其是甲状旁腺激素的激素反应不反应。对这些激素难治性的结果是，PHP患者的疾病收集为疾病，标记为奥尔布赖特遗传性骨肌营养不良，其中包括矮小的身材，肥胖，皮下注射和智力低下。我们的研究表明，引起PHP的突变要么是1）降低GS-α对GDP的亲和力，使蛋白质破坏蛋白质并在生理温度下使其变性，或者2）减少GS活化构象的半衰期，因此ACONIST介导的AC刺激的AC刺激会减弱。几十年来，众所周知，溶液中的GS激活可以伴随着GS-Alpha与G蛋白β-Gamma亚基复合物（G-Beta-Gamma）的解离。但是，我们最近的证据表明，亚基解离是由非生理条件而不是激活引起的。然而，普遍的观点是，尽管缺乏证据，但在原位激活GS时也会发生亚基解离。霍乱毒素（CTX）将ADP-核酰亚酸GS-α。将GS-Alpha缺乏的Cyc-膜剥去了G-Beta-Gamma。当将剥离的Cyc-膜与GS-Alpha和/或G-Beta-Gamma孵育时，每个膜都独立于另一个孵育膜中。 GS-Alpha和G-Beta-Gamma必须存在于膜中，并且它们必须能够形成异构体，以使CTX以ADP-ribosylate GS-Alpha的形式进行ADP-ribosylate GS-Alpha，这表明GS Heterotrimer绑定了GS Heterotrimer的A substx，但GS-Alppha subitpha subitha subiN by by ynot ynot ynot ynot ynot ynot ynot ynot ynot ynot ys sub。当GS-Alpha被GTP-Gamma-S完全且不可逆转地激活并掺入被剥离的Cyc-膜中时，除非G-Beta-Gamma也合并，否则CTX的底物和AC的弱刺激器较差。此外，AC刺激水平对应于GTP-GAMMA-S-S-激活的GS-Alpha和G-Beta-Gamma在膜上形成的GS杂体量，表明AC在细胞膜中被活化的GS异三聚体刺激。异三聚体GI介导激动剂诱导的AC抑制作用。表面等离子体的共振光谱（SPR）用于确定胃肠道亚基的亲和力彼此的亲和力。当GDP与Gi-Alpha结合时，G-Beta-Gamma的平衡分离常数约为10 nm。胃肠道亚基的亲和力通过非氢化糖GTP类似物降低，并且根据与gi-alpha结合的类似物而变化。定性地，与GPP [NH] P结合时，G-Beta-gamma对G-Beta-gamma的亲和力少于GPP [CH2] P被绑定时。如果GTP适合该模式，那么它对GI亚基亲和力的影响就不能像过去一样广泛地等同于其类似物的影响。 - 异三聚体G蛋白，腺苷酸环化酶，霍乱毒素，表面等离子体共振