Zamah A Musa, Delahunty Martha, Luttrell Louis M, Lefkowitz Robert J
Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710, USA.
J Biol Chem. 2002 Aug 23;277(34):31249-56. doi: 10.1074/jbc.M202753200. Epub 2002 Jun 12.
While classically viewed as a prototypic G(s) and adenylyl cyclase-coupled G protein-coupled receptor, recent studies have indicated that some aspects of beta(2)-adrenergic receptor (beta(2)-AR) signaling are inhibited by pertussis toxin, indicating that they are mediated by G(i)/G(o) proteins. These signals include activation of ERK MAPKs and Akt activation, as well as hypertrophic and anti-apoptotic pathways in cardiac myocytes. Studies in cultured cells have suggested the hypothesis that protein kinase A (PKA)-mediated phosphorylation of the beta(2)-AR regulates its coupling specificity with respect to G(s) and G(i). Using a Chinese hamster ovary cell system, we show that mutant beta(2)-ARs with Ala substituted for Ser at consensus PKA sites stimulate robust cyclic AMP accumulation (G(s)) but are unable to activate ERK (G(i)). In contrast, Ser --> Asp mutants are dramatically impaired in their ability to activate adenylyl cyclase but are significantly more active than wild type receptor in activating ERK. Activation of adenylyl cyclase by wild type and Ser --> Ala mutant receptors is not altered by pertussis toxin, whereas adenylyl cyclase stimulated through the Ser --> Asp mutant is enhanced. Activation of ERK by wild type and Ser --> Asp receptors is inhibited by pertussis toxin. To further rigorously test the hypothesis, we utilized a completely reconstituted system of purified recombinant wild type and PKA phosphorylation site mutant beta(2)-ARs and heterotrimeric G(s) and G(i). G protein coupling was measured by receptor-mediated stimulation of GTPgammaS binding to the G protein. PKA-mediated phosphorylation of the beta(2)-AR significantly decreased its ability to couple to G(s), while simultaneously dramatically increasing its ability to couple to G(i). These results are reproduced when a purified recombinant Ser --> Asp mutant beta(2)-AR is tested, whereas the Ser --> Ala receptor resembles the unphosphorylated wild type. These results provide strong experimental support for the idea that PKA-mediated phosphorylation of the beta(2)-adrenergic receptor switches its predominant coupling from G(s) to G(i).
虽然传统上认为β₂肾上腺素能受体（β₂-AR）是一种典型的与G(s)和腺苷酸环化酶偶联的G蛋白偶联受体，但最近的研究表明，百日咳毒素可抑制β₂-AR信号传导的某些方面，这表明它们是由G(i)/G(o)蛋白介导的。这些信号包括ERK MAPK的激活、Akt的激活，以及心肌细胞中的肥大和抗凋亡途径。在培养细胞中的研究提出了这样一个假设，即蛋白激酶A（PKA）介导的β₂-AR磷酸化调节其与G(s)和G(i)的偶联特异性。利用中国仓鼠卵巢细胞系统，我们发现，在PKA共有位点将丝氨酸替换为丙氨酸的突变型β₂-AR能刺激强烈的环磷酸腺苷积累（G(s)），但无法激活ERK（G(i)）。相反，丝氨酸→天冬氨酸突变体激活腺苷酸环化酶的能力显著受损，但在激活ERK方面比野生型受体活性明显更高。野生型和丝氨酸→丙氨酸突变体受体对腺苷酸环化酶的激活不受百日咳毒素的影响，而通过丝氨酸→天冬氨酸突变体刺激的腺苷酸环化酶则增强。野生型和丝氨酸→天冬氨酸受体对ERK的激活受到百日咳毒素的抑制。为了进一步严格检验这一假设，我们利用了一个完全重组的系统，该系统包含纯化的重组野生型和PKA磷酸化位点突变型β₂-AR以及异源三聚体G(s)和G(i)。通过受体介导的GTPγS与G蛋白结合的刺激来测量G蛋白偶联。PKA介导的β₂-AR磷酸化显著降低了其与G(s)偶联的能力，同时显著增加了其与G(i)偶联的能力。当测试纯化的重组丝氨酸→天冬氨酸突变型β₂-AR时，可重现这些结果，而丝氨酸→丙氨酸受体类似于未磷酸化的野生型。这些结果为PKA介导的β₂-肾上腺素能受体磷酸化将其主要偶联从G(s)转换为G(i)这一观点提供了有力的实验支持。
