Seibold A, Williams B, Huang Z F, Friedman J, Moore R H, Knoll B J, Clark R B
Department of Integrative Biology and Pharmacology, University of Texas-Houston Medical School, Houston, Texas, USA.
Mol Pharmacol. 2000 Nov;58(5):1162-73. doi: 10.1124/mol.58.5.1162.
The human beta(2)-adrenergic receptor (betaAR) is rapidly desensitized in response to saturating concentrations of agonist by G protein-coupled receptor kinases (GRKs) and cAMP-dependent protein kinase A (PKA) phosphorylation of the betaAR, followed by beta-arrestin binding and receptor internalization. betaAR sites phosphorylated by GRK in vivo have not yet been identified. In this study, we examined the role of the carboxyl terminal serines, 355, 356, and 364, in the GRK-mediated desensitization of the betaAR. Substitution mutants of these serine residues were constructed in which either all three (S355,356,364A), two (S355,356A and S356, 364A), or one of the serines (S356A and S364A) were modified. These mutants were constructed in a betaAR in which the serines of the PKA consensus site were substituted with alanines (designated PKA(-)) to eliminate any PKA contribution to desensitization, and they were stably transfected into human embryonic kidney 293 cells. Treatment of the PKA(-) mutant with 10 microM epinephrine for 5 min caused a 3. 5-fold increase in the EC(50) value and a 42% decrease in the V(max) value for epinephrine stimulation of adenylyl cyclase. Substitution of all three serines completely inhibited the epinephrine-induced shift in the EC(50). Both double mutants, S355,356A and S356,364A, showed a nearly complete loss of the EC(50) shift, whereas the single substitutions, S356A and S364A, caused only a slight decrease in desensitization. None of the mutations altered the epinephrine-induced decrease in V(max,) which seems to be downstream of the receptor. The triple mutation caused a 45% decrease in epinephrine-induced internalization and a 90 to 95% reduction in phosphorylation of the betaAR relative to the PKA(-) (1.9+/- 0.2- and 16.6+/-3.8-fold phosphorylation over basal, respectively). The double mutants caused an intermediate reduction in internalization (20-21%) and phosphorylation (43-52%). None of the serine mutations altered the rate of betaAR recycling. Our data demonstrate that the cluster of serines within the 355 to 364 betaAR domain confer the rapid, GRK-mediated, receptor-level desensitization of the betaAR.
人β(2)-肾上腺素能受体（βAR）在激动剂饱和浓度作用下，通过G蛋白偶联受体激酶（GRK）和βAR的环磷酸腺苷依赖性蛋白激酶A（PKA）磷酸化而迅速脱敏，随后β-抑制蛋白结合和受体内化。体内被GRK磷酸化的βAR位点尚未确定。在本研究中，我们研究了羧基末端丝氨酸355、356和364在GRK介导的βAR脱敏中的作用。构建了这些丝氨酸残基的替代突变体，其中三个丝氨酸（S355,356,364A）、两个丝氨酸（S355,356A和S356,364A）或一个丝氨酸（S356A和S364A）被修饰。这些突变体构建在一个βAR中，其中PKA共有位点的丝氨酸被丙氨酸替代（命名为PKA(-)）以消除PKA对脱敏的任何贡献，并将它们稳定转染到人胚肾293细胞中。用10μM肾上腺素处理PKA(-)突变体5分钟，导致肾上腺素刺激腺苷酸环化酶的EC(50)值增加3.5倍，V(max)值降低42%。三个丝氨酸全部替代完全抑制了肾上腺素诱导的EC(50)变化。两个双突变体S355,356A和S356,364A显示EC(50)变化几乎完全丧失，而单个替代S356A和S364A仅导致脱敏略有降低。所有突变均未改变肾上腺素诱导的V(max)降低，这似乎是在受体下游发生的。三重突变导致肾上腺素诱导的内化降低45%，βAR磷酸化相对于PKA(-)降低90%至95%（分别比基础磷酸化高1.9±0.2倍和16.6±3.8倍）。双突变体导致内化（20 - 21%）和磷酸化（43 - 52%）中度降低。丝氨酸突变均未改变βAR再循环速率。我们的数据表明，βAR结构域355至364内的丝氨酸簇赋予βAR快速的、GRK介导的受体水平脱敏作用。
