Harry A, Chen Y, Magnusson R, Iyengar R, Weng G
Department of Pharmacology, Mount Sinai School of Medicine, New York, New York 10029, USA.
J Biol Chem. 1997 Jul 25;272(30):19017-21. doi: 10.1074/jbc.272.30.19017.
Regulation of adenylyl cyclases 1, 2, and 6 by Galphas was studied. All three mammalian adenylyl cyclases were expressed in insect (Sf9 or Hi-5) cells by baculovirus infection. Membranes containing the different adenylyl cyclases were stimulated by varying concentrations of mutant (Q227L) activated Galphas expressed in reticulocyte lysates. Galphas stimulation of AC1 involved a single site and had an apparent Kact of 0.9 nM. Galphas stimulation of AC2 was best explained by a non-interactive two site model with a "high affinity" site at 0.9 nM and a "low affinity" site at 15 nM. Occupancy of the high affinity site appears to be sufficient for Gbetagamma stimulation of AC2. Galphas stimulation of AC6 was also best explained by a two-site model with a high affinity site at 0. 6-0.8 nM and a low affinity site at 8-22 nM; however, in contrast to AC2, only a model that assumed interactions between the two sites best fit the AC6 data. With 100 microM forskolin, Galphas stimulation of all three adenylyl cyclases showed very similar profiles. Galphas stimulation in the presence of forskolin involved a single site with apparent Kact of 0.1-0.4 nM. These observations indicate a conserved mechanism by which forskolin regulates Galphas coupling to the different adenylyl cyclases. However, there are fundamental differences in the mechanism of Galphas stimulation of the different adenylyl cyclases with AC2 and AC6 having multiple interconvertible sites. These mechanistic differences may provide an explanation for the varied responses by different cells and tissues to hormones that elevate cAMP levels.
研究了Gαs对腺苷酸环化酶1、2和6的调节作用。通过杆状病毒感染，所有三种哺乳动物腺苷酸环化酶均在昆虫（Sf9或Hi-5）细胞中表达。含有不同腺苷酸环化酶的膜受到网织红细胞裂解物中表达的不同浓度突变型（Q227L）激活的Gαs刺激。Gαs对AC1的刺激涉及一个单一位点，其表观Kact为0.9 nM。Gαs对AC2的刺激最好用非交互式双位点模型来解释，其中“高亲和力”位点为0.9 nM，“低亲和力”位点为15 nM。高亲和力位点的占据似乎足以使Gβγ刺激AC2。Gαs对AC6的刺激也最好用双位点模型来解释，其中高亲和力位点为0.6 - 0.8 nM，低亲和力位点为8 - 22 nM；然而，与AC2不同，只有一个假设两个位点之间存在相互作用的模型最符合AC6数据。在存在100 μM福斯高林的情况下，Gαs对所有三种腺苷酸环化酶的刺激显示出非常相似的曲线。在福斯高林存在下，Gαs刺激涉及一个单一位点，表观Kact为0.1 - 0.4 nM。这些观察结果表明福斯高林调节Gαs与不同腺苷酸环化酶偶联的保守机制。然而，Gαs刺激不同腺苷酸环化酶的机制存在根本差异，AC2和AC6具有多个可相互转换的位点。这些机制差异可能为不同细胞和组织对升高cAMP水平的激素产生不同反应提供一种解释。
