Pi3k Gs Signal Control During B2-adrenergic stimulation

GPCR信号转导的一个基本问题是无数的GPCRs和大量的同源G蛋白如何引起高度特异的生理反应。受体信号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在其上传递的细胞室。B-肾上腺素能受体(AR)是一种典型的G-腺苷环化酶(AC)-cAMP-蛋白激酶A(PKA)信号转导通路。在心脏，蛋白激酶A随后磷酸化了许多与心肌收缩有关的调节蛋白，包括肌膜L型钙离子通道、肌浆网钙泵调节因子磷蛋白(PLB)和肌丝蛋白，从而增加了收缩能力(正性变力作用)和加速心脏松弛(正性松弛作用)。 然而，大量证据表明，β2-AR诱导的cAMP/PKA信号紧密定位于L钙通道附近的细胞表膜微区，不能传递到非肌膜靶蛋白，而β1-AR介导的cAMP/PKA信号可分布于整个细胞。具体地说，β1-AR可以激活肌膜L型钙通道，增加多种细胞内蛋白的磷酸化，如肌浆网膜蛋白B和肌丝肌钙蛋白I和C蛋白，从而产生正性收缩和松弛反应。相反，刺激b2-AR选择性地激活钙通道，而不影响上述细胞内PKA靶蛋白，从而在没有松弛作用的情况下产生正性变力作用。此外，对膜片钳单通道记录的研究表明，在心肌细胞和海马神经元上，刺激b2-AR仅以局部模式(包括尖端直径~1 mm的激动剂)调制单个L型钙通道的活动，而不以远程模式(将激动剂灌流到斑片外)调制单一的L型钙通道活动，而刺激b1-AR则增加这两种模式的通道活动。 B2-AR/Gs诱导的cAMP/PKA信号的空间和功能受限可能是通过受体与Gi蛋白的额外偶联来解释的。用百日咳毒素(Ptx)抑制Gi信号允许刺激b2-AR诱导PLB磷酸化，并在远程模式下调制单个L类型的钙通道。虽然GI通路参与了b2-AR介导的cAMP/PKA信号的功能区隔，但b2-AR-GI通路的下游事件仍然很大程度上是未知的。 在本研究中，我们证明了磷脂酰肌醇3-激酶(PI3K)在限制b2-AR-PKA信号转导中起着重要作用。用LY294002或wortmannin抑制PI3K可以使b2-AR-PKA信号到达细胞内底物，表现为磷酸化蛋白(肌浆网钙泵的主要调节因子)的磷酸化显著增加，并显著增强受体介导的心肌细胞正性收缩和松弛反应。PI3K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的多肽抑制剂Bark-ct阻断GI或GBG信号通路，可完全阻断PI3K抑制引起的增强效应，表明参与b2-AR-PKA信号功能区划的通路依次涉及GI、GBG和PI3K。因此，除了在细胞生长和存活中起关键作用外，PI3K还构成了b2-AR-GI信号的关键下游事件，限制和否定了b2-AR/Gs介导的PKA信号。

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.