Seidel M G, Klinger M, Freissmuth M, Höller C
Institute of Pharmacology, University of Vienna, Währinger Strasse 13a, A-1090 Vienna, Austria.
J Biol Chem. 1999 Sep 3;274(36):25833-41. doi: 10.1074/jbc.274.36.25833.
The A(2A)-adenosine receptor, a prototypical G(s)-coupled receptor, activates mitogen-activated protein (MAP) kinase in a manner independent of cAMP in primary human endothelial cells. In order to delineate signaling pathways that link the receptor to the regulation of MAP kinase, the human A(2A) receptor was heterologously expressed in Chinese hamster ovary (CHO) and HEK293 cells. In both cell lines, A(2A) agonist-mediated cAMP accumulation was accompanied by activation of the small G protein rap1. However, rap1 mediates A(2A) receptor-dependent activation of MAP kinase only in CHO cells, the signaling cascade being composed of G(s), adenylyl cyclase, rap1, and the p68 isoform of B-raf. This isoform was absent in HEK293 cells. Contrary to CHO cells, in HEK293 cells activation of MAP kinase by A(2A) agonists was not mimicked by 8-bromo-cAMP, was independent of Galpha(s), and was associated with activation of p21(ras). Accordingly, overexpression of the inactive S17N mutant of p21(ras) and of a dominant negative version of mSos (the exchange factor of p21(ras)) blocked MAP kinase stimulation by the A(2A) receptor in HEK 293 but not in CHO cells. In spite of the close homology between p21(ras) and rap1, the S17N mutant of rap1 was not dominant negative because (i) overexpression of rap1(S17N) failed to inhibit A(2A) receptor-dependent MAP kinase activation, (ii) rap1(S17N) was recovered in the active form with a GST fusion protein comprising the rap1-binding domain of ralGDS after A(2A) receptor activation, and (iii) A(2A) agonists promoted the association of rap1(S17N) with the 68-kDa isoform of B-raf in CHO cells. We conclude that the A(2A) receptor has the capacity two activate MAP kinase via at least two signaling pathways, which depend on two distinct small G proteins, namely p21(ras) and rap1. Our observations also show that the S17N version of rap1 cannot be assumed a priori to act as a dominant negative interfering mutant.
A(2A)-腺苷受体是一种典型的与G(s)偶联的受体，它在原代人内皮细胞中以一种不依赖于环磷酸腺苷(cAMP)的方式激活丝裂原活化蛋白(MAP)激酶。为了阐明将该受体与MAP激酶调节联系起来的信号通路，人A(2A)受体在中国仓鼠卵巢(CHO)细胞和人胚肾293(HEK293)细胞中进行了异源表达。在这两种细胞系中，A(2A)激动剂介导的cAMP积累都伴随着小G蛋白rap1的激活。然而，rap1仅在CHO细胞中介导A(2A)受体依赖性的MAP激酶激活，其信号级联由G(s)、腺苷酸环化酶、rap1和B-raf的p68亚型组成。HEK293细胞中不存在这种亚型。与CHO细胞相反，在HEK293细胞中，A(2A)激动剂对MAP激酶的激活不能被8-溴-cAMP模拟，不依赖于Gα(s)，且与p21(ras)的激活有关。因此，无活性的p21(ras) S17N突变体和mSos(一种p21(ras)交换因子)的显性负性形式的过表达阻断了HEK 293细胞中A(2A)受体对MAP激酶的刺激，但在CHO细胞中没有阻断。尽管p21(ras)和rap1之间有密切的同源性，但rap1的S17N突变体并不是显性负性的，原因如下：(i) rap1(S17N)的过表达未能抑制A(2A)受体依赖性的MAP激酶激活；(ii)在A(2A)受体激活后，rap1(S17N)与包含ralGDS的rap1结合结构域的谷胱甘肽S-转移酶(GST)融合蛋白一起以活性形式被回收；(iii) A(2A)激动剂促进了rap1(S17N)与CHO细胞中B-raf的68-kDa亚型的结合。我们得出结论，A(2A)受体有能力通过至少两条信号通路激活MAP激酶，这两条信号通路依赖于两种不同的小G蛋白，即p21(ras)和rap1。我们的观察结果还表明，不能先验地认为rap1的S17N形式可作为显性负性干扰突变体。
