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

异源三聚体（α-β-γ）Gs介导激动剂诱导的腺苷酸环化酶（AC）刺激。α亚基（Gs-alpha）具有内在的GT3活性。当Gs-α结合GTP或GTP类似物（例如GTP-γ-S、Gpp[NH]p或Gpp[CH 2]p）时，Gs被激活，而当GTP水解时，Gs被失活。抑制Gs活性的天然发生的Gs-α突变导致称为假性甲状旁腺功能减退症（PHP）的人类疾病。PHP患者对使用GS偶联受体的激素没有反应，特别是甲状旁腺激素。这些激素难以治疗的结果是PHP患者患有一系列被标记为奥尔布赖特遗传性骨营养不良的疾病，包括身材矮小、肥胖、皮下骨化和智力迟钝。我们的研究表明，导致PHP的突变1）降低Gs-alpha对GDP的亲和力，使蛋白质不稳定并导致其在生理温度下变性，或2）降低Gs活化构象的半衰期，从而减弱激动剂介导的AC刺激。几十年来，人们已经知道，溶液中的Gs活化可以伴随着Gs-α从G蛋白β-γ亚基复合物（G-β-γ）中的解离。然而，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。然而，普遍的观点是，尽管缺乏证据，亚基解离也发生在Gs原位激活时。霍乱毒素（CTx）可使Gs-alpha发生ADP-核糖基化反应。将Gs-a缺陷的细胞膜去除G-beta-gamma。当将剥离的cyc-膜与Gs-α和/或G-β-γ孵育时，每一种都独立于另一种掺入膜中。Gs-alpha和G-beta- gamma都必须存在于膜中，并且它们必须能够形成异源三聚体，以便CTx将ADP-核糖基化Gs-alpha，这表明膜结合的Gs异源三聚体是CTx的底物，但Gs-alpha亚基本身不是。当Gs-alpha被GTP-γ-S完全且不可逆地激活并掺入剥离的细胞膜时，除非也掺入G-β-γ，否则它是CTx的不良底物和AC的弱刺激物。此外，AC刺激的水平对应于在膜中由GTP-γ-S-活化的Gs-α和G-β-γ形成的Gs异源三聚体的量，表明AC被细胞膜中活化的Gs异源三聚体刺激。异源三聚体Gi介导激动剂诱导的AC抑制。表面等离子体共振光谱（SPR）用于确定Gi亚基彼此的亲和力。当GDP与Gi-α结合时，G-β-γ的平衡解离常数约为10 nM。Gi亚基亲和力被不可水解的GTP类似物降低，并且取决于与Gi-α结合的类似物而变化。定性地，当GTP-γ-S结合时，Gi-alpha对G-β-γ的亲和力小于当Gpp[NH]p结合时的亲和力，Gpp[NH]p结合时的亲和力又小于当Gpp[CH 2]p结合时的亲和力。如果GTP符合该模式，则其对Gi亚基亲和力的影响不能等同于其类似物，如过去广泛进行的那样。- 异源三聚体G蛋白，腺苷酸环化酶，霍乱毒素，表面等离子体共振