G protein βγ subunits have potential as a target for therapeutic treatment of a number of diseases. We performed virtual docking of a small-molecule library to a site on Gβγ subunits that mediates protein interactions. We hypothesized that differential targeting of this surface could allow for selective modulation of Gβγ subunit functions. Several compounds bound to Gβγ subunits with affinities from 0.1 to 60 μM and selectively modulated functional Gβγ-protein-protein interactions in vitro, chemotactic peptide signaling pathways in HL-60 leukocytes, and opioid receptor–dependent analgesia in vivo. These data demonstrate an approach for modulation of G protein–coupled receptor signaling that may represent an important therapeutic strategy.

Recently, we identified a novel signaling pathway involving Epac, Rap, and phospholipase C (PLC)⑀ that plays a critical role in maximal ␤-adrenergic receptor (␤AR) stimulation of Ca 2؉ -induced Ca 2؉ release (CICR) in cardiac myocytes. Here we demonstrate that PLC⑀ phosphatidylinositol 4,5-bisphosphate hydrolytic activity and PLC⑀-stimulated Rap1 GEF activity are both required for PLC⑀-mediated enhancement of sarcoplasmic reticulum Ca 2؉ release and that PLC⑀ significantly enhances Rap activation in response to ␤AR stimulation in the heart. Downstream of PLC⑀ hydrolytic activity, pharmacological inhibition of PKC significantly inhibited both ␤AR-and Epac-stimulated increases in CICR in PLC⑀ ؉/؉ myocytes but had no effect in PLC⑀ Phospholipase C (PLC)3 -mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) results in inositol triphosphate (IP 3 )-mediated Ca 2ϩ release from intracellular stores and diacylglycerol-mediated activation of protein kinase C. This ubiquitous signaling pathway plays an integral role in regulating many physiological processes, including those of the cardiovascular system. PLC⑀ is a recently identified bifunctional PLC isoform that possesses both PIP 2 hydrolytic and Rap guanine nucleotide exchange factor (GEF) activity (1-4). The activity of PLC⑀ is uniquely regulated by direct binding of small G-proteins including Ras, Rap, and Rho (5, 6). PLC⑀ activity is also stimulated by the heterotrimeric G-protein subunits G␣ s , G␤␥, and G␣ 12/13 (5, 7, 8) but direct binding of these subunits to PLC⑀ has not been demonstrated. In primary astrocytes isolated from PLC⑀ ϩ/ϩ and PLC⑀ Ϫ/Ϫ mice, multiple G protein-dependent upstream signals rely critically on PLC⑀-dependent generation of IP 3 and diacylglycerol (9).We recently discovered a surprising role for PLC⑀ regulation downstream of the ␤-adrenergic receptor (␤AR) in cardiac myocytes (10). Compared with normal mice, PLC⑀ Ϫ/Ϫ mice exhibit reduced left ventricular developed pressure in response to strong ␤AR stimulation (10). This deficit results from a decrease in isoproterenol (Iso)-dependent stimulation of electrically evoked Ca 2ϩ release from the sarcoplasmic reticulum (SR) in single ventricular cardiac myocytes. ␤AR stimulation increases cardiac Ca 2ϩ release in a cAMP/ protein kinase A (PKA)-dependent mechanism through phosphorylation of multiple targets of the cardiac excitability and Ca 2ϩ handling machinery (11). Recently, we identified a PKA-independent, PLC⑀-mediated pathway that contributes to maximal Iso-dependent enhancement of Ca 2ϩ -induced Ca 2ϩ release (CICR) in cardiac myocytes (12) * This work was supported, in whole or in part, by National Institutes of Health Grants GMR01053536 (to A. V. S.), AR044657 (to R. T. D.), and DK56294 (to G. G. K.). This work was also supported by American Heart Association Scientist Development Grant 045343T (to B. C. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in ac...

Summary Phospholipase Cε (PLCε) is a multifunctional enzyme implicated in cardiovascular, pancreatic and inflammatory functions. Here we show that conditional deletion of PLCε in mouse cardiac myocytes protects from stress-induced pathological hypertrophy. PLCε siRNA in ventricular myocytes decreases endothelin-1 (ET-1)-dependent elevation of nuclear calcium and activation of nuclear protein kinase D (PKD). PLCε scaffolded to muscle-specific A kinase anchoring protein (mAKAP), along with PKCε and PKD, localizes these components at or near the nuclear envelope and this complex is required for nuclear PKD activation. Phosphatidylinositol 4-phosphate (PI4P) is identified as a perinuclear substrate in the Golgi apparatus for mAKAP-scaffolded PLCε. We conclude that perinuclear PLCε, scaffolded to mAKAP in cardiac myocytes, responds to hypertrophic stimuli to generate DAG from PI4P in the Golgi apparatus, in close proximity to the nuclear envelope, to regulate activation of nuclear PKD, and hypertrophic signaling pathways.

Recently we demonstrated that PLC⑀ plays an important role in ␤-adrenergic receptor (␤AR) stimulation of Ca 2؉-induced Ca 2؉ release (CICR) in cardiac myocytes. Here we have reported for the first time that a pathway downstream of ␤AR involving the cAMPdependent Rap GTP exchange factor, Epac, and PLC⑀ regulates CICR in cardiac myocytes. To demonstrate a role for Epac in the stimulation of CICR, cardiac myocytes were treated with an Epacselective cAMP analog, 8-4-(chlorophenylthio)-2-O-methyladenosine-3,5-monophosphate (cpTOME). cpTOME treatment increased the amplitude of electrically evoked Ca 2؉ transients, implicating Epac for the first time in cardiac CICR. This response is abolished in PLC⑀ ؊/؊ cardiac myocytes but rescued by transduction with PLC⑀, indicating that Epac is upstream of PLC⑀. Furthermore, transduction of PLC⑀ ؉/؉ cardiac myocytes with a Rap inhibitor, RapGAP1, significantly inhibited isoproterenol-dependent CICR. Using a combination of cpTOME and PKA-selective activators and inhibitors, we have shown that ␤AR-dependent increases in CICR consist of two independent components mediated by PKA and the novel Epac/PLC⑀ pathway. We also show that Epac/PLC⑀-dependent effects on CICR are independent of sarcoplasmic reticulum loading and Ca 2؉ clearance mechanisms. These data define a novel endogenous PKA-independent ␤AR-signaling pathway through cAMP-dependent Epac activation, Rap, and PLC⑀ that enhances intracellular Ca 2؉ release in cardiac myocytes.Stimulation of adrenergic receptors by either neurohumoral or systemic release of the catecholamines epinephrine and norepinephrine produces acute increases in cardiac contractility during stress and exercise to increase cardiac output and oxygen delivery to tissues. Much of the increase in cardiac output is due to the direct stimulation of the ␤ adrenergic receptor (␤AR) 2 in cardiac myocytes (1, 2). Activation of ␤AR activates G s and adenylyl cyclase resulting in the production of cAMP and subsequent activation of protein kinase A, which phosphorylates key components of the calcium handling and contractile machinery.Analysis of a phospholipase C (PLC)⑀ knock-out mouse model (PLC⑀ Ϫ/Ϫ ) generated in our laboratory indicates that PLC⑀ contributes to ␤AR-dependent regulation of cardiac function (3). PLC⑀ Ϫ/Ϫ mice exhibit significantly decreased left ventricular developed pressure in response to acute stimulation with the ␤AR agonist, isoproterenol. Isolated myocytes from PLC⑀ Ϫ/Ϫ mice exhibit decreased isoproterenol-dependent enhancement of electrically evoked Ca 2ϩ release in the absence of effects on ␤AR density or cAMP generation. These data implicate PLC⑀ as a novel component of ␤AR regulation of Ca 2ϩ release, which had not previously been described in the heart. However, the pathway linking ␤AR stimulation to PLC⑀ activation is unknown.PLC-mediated phosphatidyl-4,5-bisphosphate hydrolysis resulting in intracellular Ca 2ϩ release and protein kinase C activation is an integral signaling component of many physiological processes in a variety of tissu...

Exercise promotes the formation of intracellular junctions in skeletal muscle between stacks of sarcoplasmic reticulum (SR) cisternae and extensions of transverse-tubules (TT) that increase co-localization of proteins required for store-operated Ca2+ entry (SOCE). Here, we report that SOCE, peak Ca2+ transient amplitude and muscle force production during repetitive stimulation are increased after exercise in parallel with the time course of TT association with SR-stacks. Unexpectedly, exercise also activated constitutive Ca2+ entry coincident with a modest decrease in total releasable Ca2+ store content. Importantly, this decrease in releasable Ca2+ store content observed after exercise was reversed by repetitive high-frequency stimulation, consistent with enhanced SOCE. The functional benefits of exercise on SOCE, constitutive Ca2+ entry and muscle force production were lost in mice with muscle-specific loss of Orai1 function. These results indicate that TT association with SR-stacks enhances Orai1-dependent SOCE to optimize Ca2+ dynamics and muscle contractile function during acute exercise.