Pi3k Gs Signal Control During B2-adrenergic stimulation

B2 肾上腺素能刺激期间的 Pi3k Gs 信号控制

• 批准号: 6674194
• 负责人: Rui-Ping Xiao
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6674194
• 关键词: beta adrenergic receptor; biological signal transduction; cyclic AMP; enzyme activity; heart contraction; phosphatidylinositol 3 kinase; phospholamban; protein structure function

A fundamental question of GPCR signal transduction is how a myriad of GPCRs and numerous cognate G proteins can elicit highly specific physiological responses. One of the most notable determinants of the specificity and effector-selectivity of receptor signaling is the cellular compartment over which the signal can transmit. As a prototypical GPCR, b-adrenoceptor (AR) activates the classical Gs-adenylyl cyclase (AC)-cAMP-protein kinase A (PKA) signaling cascade. In the heart, PKA subsequently phosphorylates a multitude of regulatory proteins involved in cardiac muscle contraction, including sarcolemmal L-type Ca2+ channel, the sarcoplasmic reticulum (SR) Ca2+ pump regulator phospholamban (PLB), and myofilament proteins, resulting in increased contractility (a positive inotropic effect) and accelerated cardiac relaxation (a positive relaxant effect). However, a large body of evidence indicates that ?2-AR-induced cAMP/PKA signaling is tightly localized to the cell surface membrane microdomains in the vicinity of L-type Ca2+ channels and cannot transmit to non-sarcolemmal target proteins, while ?1-AR-mediated cAMP/PKA signaling can broadcast throughout the cell. Specifically, ?1-AR can activate sarcolemmal L-type Ca2+ channels and increase phosphorylation of multiple intracellular proteins, such as PLB at SR membrane and troponin I and C proteins of myofilaments, resulting in both positive contractile and relaxant responses. In contrast, b2-AR stimulation selectively activates the Ca2+ channel, without affecting the aforementioned intracellular PKA target proteins, thus leading to a positive inotropic effect in the absence of a relaxant effect. Moreover, studies with patch-clamp single-channel recordings have shown that in both cardiac myocytes and hippocampal neurons, b2-AR stimulation modulates single L-type Ca2+ channel activity only in a local mode (agonist included within the patch pipette with tip diameter ~1.0 mm) and not in a remote mode (agonist perfused outside the patch), whereas b1-AR stimulation increases the channel activity in both modes. The spatial and functional restriction of b2-AR/Gs-induced cAMP/PKA signaling might be explained by the additional coupling of the receptor to Gi proteins. Inhibition of Gi signaling with pertussis toxin (PTX) allows b2-AR stimulation to induce PLB phosphorylation5 and to modulate single L-type Ca2+ channels in the remote mode. Although the Gi pathway is implicated in the functional compartmentation of b2-AR-mediated cAMP/PKA signaling, the downstream events of the b2-AR-Gi pathway remain largely unknown. In the present study, we demonstrate that phosphatidylinositol 3-kinase (PI3K) plays an essential role in confining the b2-AR-PKA signaling. Inhibition of PI3K with LY294002 or wortmannin enables b2-AR-PKA signaling to reach intracellular substrates, as manifested by a robust increase in phosphorylation of phospholamban (a primary regulator of the sarcoplasmic reticulum Ca2+ pump), and markedly enhances the receptor-mediated positive contractile and relaxant responses in cardiac myocytes. These potentiating effects of PI3K inhibitors are not accompanied by an increase in b2-AR-induced cAMP formation. Blocking Gi or Gbg signaling with pertussis toxin or bARK-ct, a peptide inhibitor of Gbg, completely prevents the potentiating effects induced by PI3K inhibition, indicating that the pathway responsible for the functional compartmentation of b2-AR-PKA signaling sequentially involves Gi, Gbg, and PI3K. Thus, in addition to its pivotal roles in cell growth and cell survival, PI3K constitutes a key downstream event of b2-AR-Gi signaling, which confines and negates the concurrent b2-AR/Gs-mediated PKA signaling.

GPCR信号转导的一个基本问题是无数的GPCR和许多同源G蛋白如何引起高度特异性的生理反应。受体信号传导的特异性和效应器选择性的最显著决定因素之一是信号可以传递的细胞区室。β-肾上腺素受体（AR）作为一种典型的GPCR，激活经典的Gs-adenylyl cyclase（AC）-cAMP-protein kinase A（PKA）信号级联反应。在心脏中，PKA随后磷酸化参与心肌收缩的多种调节蛋白，包括肌膜L型Ca 2+通道、肌浆网（SR）Ca 2+泵调节剂受磷蛋白（PLB）和肌丝蛋白，导致收缩力增加（正性肌力作用）和心脏舒张加速（正性舒张作用）。 然而，大量证据表明，？2-AR诱导的cAMP/PKA信号被紧密定位于细胞表面膜微区的L型钙通道附近，不能传递到非肌膜靶蛋白，而？1-AR介导的cAMP/PKA信号可以在整个细胞中传播。具体来说，？1-AR可激活肌膜L型Ca ~（2+）通道，增加SR膜PLB、肌丝肌钙蛋白I和C等多种胞内蛋白的磷酸化，从而产生正性收缩和舒张反应。相比之下，b2-AR刺激选择性激活Ca 2+通道，而不影响上述细胞内PKA靶蛋白，从而在不存在松弛作用的情况下导致正性肌力作用。此外，膜片钳单通道记录的研究表明，在心肌细胞和海马神经元中，b2-AR刺激仅在局部模式下调节单个L型Ca 2+通道活性（激动剂包含在贴片移液管内，尖端直径约为1.0 mm），而不是在远程模式下（激动剂灌注在贴片外部），而b1-AR刺激增加了两种模式下的通道活性。 b2-AR/Gs诱导的cAMP/PKA信号传导的空间和功能限制可能通过受体与Gi蛋白的额外偶联来解释。用百日咳毒素（PTX）抑制Gi信号传导允许b2-AR刺激诱导PLB磷酸化5并以远程模式调节单个L型Ca 2+通道。虽然Gi途径涉及b2-AR介导的cAMP/PKA信号传导的功能区室化，但b2-AR-Gi途径的下游事件在很大程度上仍然未知。 在本研究中，我们证明了磷脂酰肌醇3-激酶（PI 3 K）在限制b2-AR-PKA信号通路中起着重要作用。用LY 294002或渥曼青霉素抑制PI 3 K能够使b2-AR-PKA信号传导到达细胞内底物，如受磷蛋白（肌浆网Ca 2+泵的主要调节剂）磷酸化的稳健增加所示，并显著增强心肌细胞中受体介导的正性收缩和舒张反应。PI 3 K抑制剂的这些增强作用并不伴随着b2-AR诱导的cAMP形成的增加。用百日咳毒素或Gbg的肽抑制剂bARK-ct阻断Gi或Gbg信号传导完全阻止由PI 3 K抑制诱导的增强作用，表明负责b2-AR-PKA信号传导的功能区室化的途径依次涉及Gi、Gbg和PI 3 K。因此，除了其在细胞生长和细胞存活中的关键作用之外，PI 3 K构成b2-AR-Gi信号传导的关键下游事件，其限制和否定同时发生的b2-AR/Gs介导的PKA信号传导。