Yuan N, Friedman J, Whaley B S, Clark R B
Graduate School of Biomedical Sciences, Department of Pharmacology, University of Texas Health Science Center at Houston 77225-0334.
J Biol Chem. 1994 Sep 16;269(37):23032-8.
Activation of cAMP-dependent protein kinase (cAPK) or protein kinase C (PKC) causes a rapid desensitization of beta 2-adrenergic receptor (beta AR) stimulation of adenylylcyclase in L cells, which previous studies suggest involves the cAPK/PKC consensus phosphorylation site in the third intracellular loop of the beta AR, RRSSK263. To determine the role of the individual serines in the cAPK- and PKC-mediated desensitizations, wild type (WT) and mutant beta ARs containing the substitutions, Ser261-->Ala, Ser262-->Ala, Ser262-->Asp, and Ser261/262-->Ala, were constructed and stably transfected into L cells. Results showed that serine 262 was the primary site of the cAPK-induced desensitization, whereas either serine 261 or serine 262 was sufficient to confer the 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA)/PKC-mediated desensitization. Coincident stimulation of cAPK and PKC caused an additive desensitization (6-8-fold increase in the EC50) which was significantly reduced (80%) only by the double substitution mutation. Quantitative evaluation of the coupling efficiencies and the GTP-shift of the WT and mutant receptors demonstrated that only one of the mutants, Ser262-->Ala, was partially uncoupled. The Ser262-->Asp mutation did not significantly uncouple, demonstrating that introducing a negative charge did not appear to mimic the desensitized state of the receptor. The beta AR expression level played a critical role in determining the pattern of beta AR desensitization; i.e. while the overall desensitization was unaltered within a large range of beta AR expression level (10-300 fmol/mg), the increase in EC50 and decrease in Vmax were differentially affected by the change in the receptor level.
环磷酸腺苷依赖性蛋白激酶（cAPK）或蛋白激酶C（PKC）的激活会导致L细胞中β2-肾上腺素能受体（βAR）对腺苷酸环化酶刺激的快速脱敏，先前的研究表明，这涉及βAR第三个细胞内环中的cAPK/PKC共有磷酸化位点RRSSK263。为了确定各个丝氨酸在cAPK和PKC介导的脱敏中的作用，构建了包含Ser261→Ala、Ser262→Ala、Ser262→Asp和Ser261/262→Ala替代的野生型（WT）和突变型βAR，并将其稳定转染到L细胞中。结果表明，丝氨酸262是cAPK诱导脱敏的主要位点，而丝氨酸261或丝氨酸262中的任何一个都足以赋予4β-佛波醇12β-肉豆蔻酸酯13α-乙酸酯（PMA）/PKC介导的脱敏。cAPK和PKC的同时刺激导致加性脱敏（EC50增加6-8倍），只有双替代突变才能使其显著降低（80%）。对WT和突变受体的偶联效率和GTP位移的定量评估表明，只有一个突变体Ser262→Ala部分解偶联。Ser262→Asp突变没有显著解偶联，表明引入负电荷似乎并不能模拟受体的脱敏状态。βAR表达水平在决定βAR脱敏模式中起关键作用；即，虽然在βAR表达水平的很大范围内（10-300 fmol/mg）总体脱敏没有改变，但EC50的增加和Vmax的降低受到受体水平变化的不同影响。
