Nakamura S, Rodbell M
Signal Transduction Section, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709.
Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7150-4. doi: 10.1073/pnas.88.16.7150.
The hydrodynamic behavior of G alpha s, the alpha subunit of the stimulatory guanine nucleotide-binding regulatory protein (G protein), in octyl glucoside extracts of rat liver membranes was investigated. As was previously shown for G proteins similarly extracted from brain synaptoneurosomes, G alpha s behaved as polydisperse structures with S values higher than that of heterotrimeric G proteins. At concentrations of guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) greater than 100 microM, incubation with membranes led to smaller structures having S values in the range of 4-5 S. Incubation of liver membranes with glucagon also caused a marked increase in structures having these S values; glucagon action required the presence of low concentrations of GTP[gamma S] (maximal, 10 microM), was rapid (within 10 sec), and was not observed with vasopressin, angiotensin II, or glucagon-(19-29). When G alpha s in its membrane-bound form was [32P]ADP-ribosylated by cholera toxin and the treated membranes were extracted with octyl glucoside, greater than 35% of the labeled G alpha s was found in material that sedimented through sucrose gradients and contained relatively low levels of immunoreactive G alpha s. Glucagon selectively converted the apparently large molecular weight structures to the 4-5 S structures in the presence of GTP[gamma S], even at 1 mM (the maximal effect of the nucleotide alone), when incubated with the toxin-treated membranes. These findings suggest that the glucagon receptor selectively interacts with polymer-like structures of G alpha s and that activation by GTP[gamma S] results in disaggregation. The role of the beta and gamma subunits of G proteins in the hormone-induced process is not clear since the polymer-like structures extracted with octyl glucoside are devoid of beta and gamma subunits.
研究了大鼠肝细胞膜辛基葡糖苷提取物中刺激性鸟嘌呤核苷酸结合调节蛋白(G蛋白)的α亚基Gαs的流体动力学行为。正如先前对从脑突触神经小体中类似提取的G蛋白所显示的那样,Gαs表现为多分散结构,其S值高于异三聚体G蛋白。当鸟苷5'-[γ-硫代]三磷酸(GTP[γS])浓度大于100μM时,与膜一起孵育会导致形成S值在4-5S范围内的较小结构。用胰高血糖素孵育肝细胞膜也会导致具有这些S值的结构显著增加;胰高血糖素的作用需要低浓度的GTP[γS](最大浓度为10μM),作用迅速(在10秒内),而血管加压素、血管紧张素II或胰高血糖素-(19-29)则未观察到这种作用。当膜结合形式的Gαs被霍乱毒素[32P]ADP核糖基化,并用辛基葡糖苷提取处理过的膜时,在通过蔗糖梯度沉淀且免疫反应性Gαs水平相对较低的物质中发现超过35%的标记Gαs。当与毒素处理过的膜一起孵育时,即使在1mM(单独核苷酸的最大作用浓度)下,胰高血糖素在GTP[γS]存在的情况下也能选择性地将明显的大分子量结构转化为4-5S结构。这些发现表明,胰高血糖素受体选择性地与Gαs的聚合物样结构相互作用,并且GTP[γS]的激活导致解聚。由于用辛基葡糖苷提取的聚合物样结构不含β和γ亚基,因此G蛋白的β和γ亚基在激素诱导过程中的作用尚不清楚。