Department of Surgery, Section of Cardiac and Thoracic Surgery, University of Chicago Medical Center, Chicago, Illinois 60637, USA.
J Biol Chem. 2010 Apr 30;285(18):13748-60. doi: 10.1074/jbc.M110.109272. Epub 2010 Mar 1.
G protein-coupled receptor kinase-2 (GRK2) is a critical regulator of beta-adrenergic receptor (beta-AR) signaling and cardiac function. We studied the effects of mechanical stretch, a potent stimulus for cardiac myocyte hypertrophy, on GRK2 activity and beta-AR signaling. To eliminate neurohormonal influences, neonatal rat ventricular myocytes were subjected to cyclical equi-biaxial stretch. A hypertrophic response was confirmed by "fetal" gene up-regulation. GRK2 activity in cardiac myocytes was increased 4.2-fold at 48 h of stretch versus unstretched controls. Adenylyl cyclase activity was blunted in sarcolemmal membranes after stretch, demonstrating beta-AR desensitization. The hypertrophic response to mechanical stretch is mediated primarily through the G alpha(q)-coupled angiotensin II AT(1) receptor leading to activation of protein kinase C (PKC). PKC is known to phosphorylate GRK2 at the N-terminal serine 29 residue, leading to kinase activation. Overexpression of a mini-gene that inhibits receptor-G alpha(q) coupling blunted stretch-induced hypertrophy and GRK2 activation. Short hairpin RNA-mediated knockdown of PKC alpha also significantly attenuated stretch-induced GRK2 activation. Overexpression of a GRK2 mutant (S29A) in cardiac myocytes inhibited phosphorylation of GRK2 by PKC, abolished stretch-induced GRK2 activation, and restored adenylyl cyclase activity. Cardiac-specific activation of PKC alpha in transgenic mice led to impaired beta-agonist-stimulated ventricular function, blunted cyclase activity, and increased GRK2 phosphorylation and activity. Phosphorylation of GRK2 by PKC appears to be the primary mechanism of increased GRK2 activity and impaired beta-AR signaling after mechanical stretch. Cross-talk between hypertrophic signaling at the level of PKC and beta-AR signaling regulated by GRK2 may be an important mechanism in the transition from compensatory ventricular hypertrophy to heart failure.
G 蛋白偶联受体激酶-2（GRK2）是β-肾上腺素能受体（β-AR）信号和心脏功能的关键调节因子。我们研究了机械拉伸（心肌细胞肥大的有力刺激）对 GRK2 活性和β-AR 信号的影响。为了消除神经激素的影响，将新生大鼠心室肌细胞进行周期性的等张双轴拉伸。通过“胎儿”基因的上调来确认肥大反应。与未拉伸对照相比，在拉伸 48 小时后，心肌细胞中的 GRK2 活性增加了 4.2 倍。拉伸后肌浆膜膜中的腺苷酸环化酶活性减弱，表明β-AR 脱敏。机械拉伸对心肌的肥大反应主要通过 Gαq 偶联的血管紧张素 II AT1 受体介导，导致蛋白激酶 C（PKC）的激活。已知 PKC 可使 GRK2 的 N 端丝氨酸 29 残基磷酸化，导致激酶激活。抑制受体-Gαq 偶联的迷你基因的过表达削弱了拉伸诱导的肥大和 GRK2 激活。PKCα 的短发夹 RNA 介导的敲低也显著减弱了拉伸诱导的 GRK2 激活。在心肌细胞中过表达 GRK2 突变体（S29A）可抑制 PKC 对 GRK2 的磷酸化，消除拉伸诱导的 GRK2 激活，并恢复腺苷酸环化酶活性。PKCα 在转基因小鼠中的心脏特异性激活导致β-激动剂刺激的心室功能受损、环化酶活性减弱以及 GRK2 磷酸化和活性增加。PKC 对 GRK2 的磷酸化似乎是机械拉伸后 GRK2 活性增加和β-AR 信号受损的主要机制。PKC 水平的肥大信号与 GRK2 调节的β-AR 信号之间的串扰可能是从代偿性心室肥厚到心力衰竭的重要机制。
