Ahmed A H, Heppel L A
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, USA.
J Cell Physiol. 1997 Mar;170(3):263-71. doi: 10.1002/(SICI)1097-4652(199703)170:3<263::AID-JCP7>3.0.CO;2-M.
The expression of both A1- and A2a-adenosine receptors occurs in human foreskin and lung fibroblasts (Ahmed et al., 1995, Biochem. Biophys. Res. Commun. 208:871-878). Studies with highly specific A1- and A2a-adenosine receptor agonists provide indirect evidence that binding of adenosine activates Gs and Gi, after which Gs alpha interacts with beta gamma subunits released from Gi. The interaction of Gs alpha with beta gamma augments cyclic adenosine monophosphate (cAMP) accumulation, more than does Gs alpha alone. In the present study, we have provided direct evidence for a role of the beta gamma complex in the augmentation of cAMP accumulation by using a recombinant His6 fusion protein containing the carboxyl third of beta ARK1. This portion of beta ARK1 contains G beta gamma binding sequences and acts as a specific beta gamma scavenger (Koch et al., 1994, Proc. Natl. Acad. Sci. USA 91:12706-12710). In permeabilized fibroblasts, the His6 fusion protein inhibited the augmentation of cAMP accumulation resulting from adenosine binding to both A1 and A2a receptors. In addition, the specific G beta gamma scavenger inhibited the further rise in cellular cAMP levels caused by pretreating cells with pertussis toxin before incubation with adenosine. Finally, we observed that specific A1-adenosine receptor agonists augmented the cAMP accumulation stimulated by A2a-receptor agonists, and this cAMP augmentation was also suppressed by the G beta gamma scavenger. Similar results were obtained when the cells were treated with extracellular ATP and lysophosphatidic acid (LPA) to stimulate Gs and release G beta gamma subunits, respectively.
A1和A2a - 腺苷受体在人包皮和肺成纤维细胞中均有表达（艾哈迈德等人，1995年，《生物化学与生物物理研究通讯》208:871 - 878）。使用高度特异性的A1和A2a - 腺苷受体激动剂进行的研究提供了间接证据，表明腺苷结合激活Gs和Gi，之后Gsα与从Gi释放的βγ亚基相互作用。Gsα与βγ的相互作用比单独的Gsα更能增强环磷酸腺苷（cAMP）的积累。在本研究中，我们通过使用包含βARK1羧基末端三分之一的重组His6融合蛋白，为βγ复合物在增强cAMP积累中的作用提供了直接证据。βARK1的这一部分包含Gβγ结合序列，并作为一种特异性的βγ清除剂（科赫等人，1994年，《美国国家科学院院刊》91:12706 - 12710）。在通透化的成纤维细胞中，His6融合蛋白抑制了腺苷与A1和A2a受体结合所导致的cAMP积累的增强。此外，特异性的Gβγ清除剂抑制了在用百日咳毒素预处理细胞后再与腺苷孵育所引起的细胞内cAMP水平的进一步升高。最后，我们观察到特异性的A1 - 腺苷受体激动剂增强了A2a - 受体激动剂刺激的cAMP积累，并且这种cAMP的增强也被Gβγ清除剂所抑制。当用细胞外ATP和溶血磷脂酸（LPA）分别刺激Gs和释放Gβγ亚基来处理细胞时，也获得了类似的结果。
