INVESTIGATION OF HETEROTRIMERIC GUANINE NUCLEOTIDE BINDING PROTEIN ACTIVATION

异三聚体(α-β-伽马)Gs介导激动剂诱导的腺酰环化酶(AC)的刺激。α亚基(Gs-α)具有固有的GTP酶活性。当Gs-α与GTP或GTP类似物(例如GTP-伽马-S、GPP[NH]p或GPP[CH2]p)结合时，GS被激活，当GTP被水解时，GS被去激活。抑制Gs活性的自然发生的Gs-α突变会导致一种名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，特别是甲状旁腺激素没有反应。对这些激素无效的结果是，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、皮下骨化和智力低下。我们的研究表明，导致PHP的突变可以1)降低Gs-α与GDP的亲和力，从而破坏蛋白质的稳定性并导致其在生理温度下变性，或者2)缩短Gs激活构象的半衰期，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Gs的激活可以伴随着Gs-α从G蛋白β-伽马亚单位复合体(G-β-Gamma)中解离出来。然而，我们最近的证据表明，亚单位解离是由非生理条件引起的，而不是激活。然而，流行的观点是，尽管缺乏证据，但当Gs被原位激活时，也会发生亚基解离。霍乱毒素(CTX)将ADP-核糖基化Gs-α。GS-α缺乏的环状细胞膜被剥离G-β-伽马。当剥离的Cyc膜与Gs-α和/或G-β-Gamma孵育时，每一种都独立地结合到膜中。膜中必须存在Gs-α和G-β-Gamma，并且它们必须能够形成杂三聚体，才能使CTX生成ADP-核糖基化Gs-α，这表明膜结合的Gs杂三聚体是CTX的底物，但Gs-α亚基本身不是。当Gs-α被GTP-γ-S完全不可逆地激活并掺入剥离的Cyc膜中时，除非同时掺入G-β-γ，否则Gs-α不是CTX的不良底物，也不是AC的弱刺激物。此外，AC的刺激水平与GTP-γ-S激活的Gs-α和G-β-γ在细胞膜上形成的Gs杂三聚体的量相对应，表明AC受到细胞膜上激活的Gs杂三聚体的刺激。异三聚体GI介导激动剂对AC的抑制。表面等离子共振光谱(SPR)被用来确定GI亚基之间的亲和力。当GDP与Gi-α结合时，G-β-γ的平衡解离常数约为10 nm。Gi亚单位亲和力被非水解性GTP类似物降低，并根据与Gi-α结合的类似物的不同而变化。在定性上，GI-α与G-β-伽马的亲和力在GTP-伽马-S结合时小于GPP[NH]p结合时，Gi-α与G-β-伽马结合时又小于GPP[CH2]p结合时。如果GTP符合这种模式，那么它对GI亚单位亲和力的影响就不能像过去广泛做的那样与其类似物等同。-异三聚体G蛋白、腺酰环化酶、霍乱毒素、表面等离子共振

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