Xiang Yang, Devic Eric, Kobilka Brian
Department of Molecular and Cellular Physiology, Howard Hughes Medical Institution, B157 Beckman Center, Stanford Medical Center, Palo Alto, CA 94043, USA.
J Biol Chem. 2002 Sep 13;277(37):33783-90. doi: 10.1074/jbc.M204136200. Epub 2002 Jul 3.
Beta(1) and beta(2) adrenergic receptors (AR) regulate the intrinsic contraction rate in neonatal mouse cardiac myocytes through distinct signaling pathways. It has been shown that stimulation of beta(1)ARs leads to a protein kinase A-dependent increase in contraction rate. In contrast, stimulation of beta(2)ARs has a biphasic effect on contraction rate, with an initial protein kinase A-independent increase followed by a sustained decrease that is blocked by pertussis toxin. The beta(2)AR undergoes agonist-induced endocytosis in cardiac myocytes while the beta(1)AR remains on the cell surface. It has been shown that a PDZ domain binding motif at the carboxyl terminus of beta(1)AR interacts with the postsynaptic density protein PSD-95 when both are expressed in HEK293 cells. We found that mutation of this PDZ binding motif in the beta(1)AR (beta(1)AR-PDZ) enabled agonist-induced internalization in cardiac myocytes. Moreover, stimulation of beta(1)AR-PDZ had a biphasic effect on the myocyte contraction rate similar to that observed following stimulation of the beta(2)AR. The secondary decrease in the contraction rate was mediated by G(i) and could be blocked by pertussis toxin. Furthermore, a non-selective endocytosis inhibitor, concanavalin A, inhibited the internalization of wild type beta(2)AR and the mutated beta(1)AR-PDZ, and blocked the coupling of both receptors to G(i). Finally, treating myocytes with a membrane-permeable peptide representing beta(1)AR PDZ motif caused the endogenous beta(1)AR to behave like beta(1)AR-PDZ. These studies suggest that association of the beta(1)AR with PSD-95 or a related protein dictates signaling specificity by retaining the receptor at the cell surface and preventing interaction with G(i).
β1和β2肾上腺素能受体（AR）通过不同的信号通路调节新生小鼠心肌细胞的内在收缩速率。已表明，刺激β1AR会导致蛋白激酶A依赖性的收缩速率增加。相比之下，刺激β2AR对收缩速率具有双相作用，最初是蛋白激酶A非依赖性的增加，随后是持续的降低，百日咳毒素可阻断这种降低。β2AR在心肌细胞中经历激动剂诱导的内吞作用，而β1AR则保留在细胞表面。已表明，当β1AR和突触后密度蛋白PSD-95在HEK293细胞中共同表达时，β1AR羧基末端的一个PDZ结构域结合基序会与PSD-95相互作用。我们发现，β1AR中该PDZ结合基序的突变（β1AR-PDZ）使激动剂诱导的心肌细胞内吞作用成为可能。此外，刺激β1AR-PDZ对心肌细胞收缩速率具有双相作用，类似于刺激β2AR后观察到的情况。收缩速率的二次降低由G(i)介导，可被百日咳毒素阻断。此外，一种非选择性内吞抑制剂伴刀豆球蛋白A可抑制野生型β2AR和突变型β1AR-PDZ的内吞作用，并阻断这两种受体与G(i)的偶联。最后，用代表β1AR PDZ基序的膜渗透性肽处理心肌细胞，可使内源性β1AR表现得像β1AR-PDZ。这些研究表明，β1AR与PSD-95或相关蛋白的结合通过将受体保留在细胞表面并防止与G(i)相互作用来决定信号特异性。
