Golgi localized β1-adrenergic receptors stimulate Golgi PI4P hydrolysis by PLCε to regulate cardiac hypertrophy.

Increased adrenergic tone resulting from cardiovascular stress leads to development of heart failure, in part, through chronic stimulation of β1 adrenergic receptors (βARs) on cardiac myocytes. Blocking these receptors is part of the basis for β-blocker therapy for heart failure. Recent data demonstrate that G protein-coupled receptors (GPCRs), including βARs, are activated intracellularly, although the biological significance is unclear. Here we investigated the functional role of Golgi βARs in rat cardiac myocytes and found they activate Golgi localized, prohypertrophic, phosphoinositide hydrolysis, that is not accessed by cell surface βAR stimulation. This pathway is accessed by the physiological neurotransmitter norepinephrine (NE) via an Oct3 organic cation transporter. Blockade of Oct3 or specific blockade of Golgi resident β1ARs prevents NE dependent cardiac myocyte hypertrophy. This clearly defines a pathway activated by internal GPCRs in a biologically relevant cell type and has implications for development of more efficacious β-blocker therapies

A Novel Gβγ-Subunit Inhibitor Selectively Modulates μ-Opioid- Dependent Antinociception and Attenuates Acute Morphine-Induced Antinociceptive Tolerance and Dependence

The Gβγ subunit has been implicated in many downstream signaling events associated with opioids. We previously demonstrated that a small molecule inhibitor of Gβγ-subunit-dependent phospholipase (PLC) activation potentiated morphine-induced analgesia (Bonacci et al., 2006). Here, we demonstrate that this inhibitor, M119 (cyclohexanecarboxylic acid [2-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-yl)-(9Cl)]), is selective for μ-opioid receptor-dependent analgesia and has additional efficacy in mouse models of acute tolerance and dependence. When administered by an intracerebroventricular injection in mice, M119 caused 10-fold and sevenfold increases in the potencies of morphine and the μ-selective peptide, DAMGO, respectively. M119 had little or no effect on analgesia induced by the κ agonist U50,488 or δ agonists DPDPE or Deltorphin II. Similar results were obtained in vitro, as only activation of the μ-opioid receptor stimulated PLC activation, whereas no effect was seen with the κ- and δ-opioid receptors. M119 inhibited μ-receptor-dependent PLC activation. In studies to further explore the in vivo efficacy of M119, systemic administration M119 also resulted in a fourfold shift increase in potency of systemically administered morphine. Of particular interest, M119 was also able to attenuate acute, antinociceptive tolerance and dependence in mice treated concomitantly with both M119 and morphine. These studies suggest that small organic molecules, such as M119, that specifically regulate Gβγ subunit signaling may have important therapeutic applications in enhancing opioid analgesia, while attenuating the development of tolerance and dependence

Purification from Sf9 cells and characterization of recombinant Gq alpha and G11 alpha. Activation of purified phospholipase C isozymes by G alpha subunits

Members of the Gq alpha subfamily of heterotrimeric guanine nucleotide-binding proteins (G proteins) activate phospholipase C (PLC). The complementary DNAs (cDNAs) for the G protein alpha subunits Gq alpha and G11 alpha were expressed in insect (Sf9) cells using recombinant baculovirus. Active, nonaggregated, and membrane-associated protein was generated only when the alpha subunit cDNA was expressed together with cDNAs encoding G protein beta and gamma subunits. Recombinant alpha subunits (rGq alpha and rG11 alpha) were purified by three-step procedures, as was a PLC-activating alpha subunit(s) endogenous to Sf9 cells. Guanosine 5'-3-(thio)triphosphate (GTP gamma S) activated rGq alpha and rG11 alpha with an apparent K0.5 of 30 microM; similarly high concentrations of the nucleotide were required to observe [35S]GTP gamma S binding to rGq alpha. Activated rGq alpha and rG11 alpha each stimulated all three isoforms of purified PLC-beta with the rank order of potency PLC-beta 1 = PLC-beta 3 > or = PLC-beta 2; both alpha subunits also stimulated PLC-beta 1 and PLC-beta 3 to a much greater extent (10-fold) than they did PLC-beta 2. In contrast, activated rGq alpha and rG11 alpha failed to stimulate either PLC-delta 1 or PLC- gamma 1. Recombinant Gi alpha 1, Gi alpha 2, Gi alpha 3, Go alpha (A), Gs alpha, and Gz alpha all failed to stimulate any of the isoforms of PLC. The apparent affinities of rGq alpha and rG11 alpha for PLC-beta 1 and their capacities to activate the enzyme were similar to values observed for purified brain Gq alpha/11 alpha. Purified brain beta gamma subunits also stimulated the three isoforms of PLC-beta. The capacities of rGq alpha and rG11 alpha to activate PLC-beta 1 and PLC- beta 3 greatly exceeded those of beta gamma, whereas Gq alpha, G11 alpha and beta gamma were roughly equiefficacious with PLC-beta 2; the alpha subunits were more potent than beta gamma in all cases. The effects of alpha and beta gamma together were nonadditive for both PLC- beta 1 and PLC-beta 2. These results demonstrate that Gq alpha and G11 alpha specifically and selectively stimulate beta isoforms of PLC and confirm the idea that these members of the Gq alpha subfamily of G proteins are physiological regulators of this signaling pathway

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Mechanisms for Phospholipase Cε-Dependent Regulation of Cardiac Hypertrophy

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pharmacology and Physiology, 2012.To explore the mechanisms of phospholipase Cε (PLCε) signaling in cardiomyocyte hypertrophy, PLCε protein was depleted by siRNA in neonatal rat ventricular myocytes (NRVMs). Endothelin-1 (ET-1), norepinephrine (NE), isoproterenol (ISO), and insulin-like growth factor-1 (IGF-1) were chosen as hypertrophic agonists. Expression of Gαq in NRVMs was also testes as a hypertrophic stimulus. We found that depletion of PLCε with siRNA significantly inhibited hypertrophic growth and gene expression induced by all agonists tested as well as Gαq, demonstrating that PLCε plays a convergent role in regulating cardiomyocyte hypertrophy. Our data show that PLC catalytic activity is required for PLCε-dependent regulation of hypertrophy, although PLCε deletion did not change global agonist-induced total inositol phosphate generation, suggesting PLC activity was required locally for PLCε-dependent regulation of hypertrophy. Further, PLCε binds to muscle-specific A kinase anchoring protein (mAKAPβ) – a nuclear membrane scaffold protein in the heart. The binding sites on the interaction surface of PLCε and mAKAPβ were mapped and the domains were expressed in NRVMs to disrupt endogenous binding of PLCε to mAKAPβ, which resulted in significantly reduced agonist or Gq-induced hypertrophy, proving that the perinuclear localization of PLCε was required for its hypertrophic function. Furthermore, we explored downstream effectors of PLCε in regulation of hypertrophy. We found that depletion of PLCε inhibited protein kinase D (PKD) activity stimulated by ET-1 or NE in NRVMs, and disruption of PLCε binding to mAKAPβ also reduces it, demonstrating that nuclear PKD activation required PLCε. Similarly, our data show that PLCε depletion inhibits ET-1 stimulated nuclear Ca2+ release in NRVMs, suggesting that IP3 generated by PLCε-dependent hydrolysis contributed to nuclear calcium signals. To further understand the functional role of PLCε found in cardiomyocyte hypertrophy, inducible and tissue specific PLCε knockout mice were generated. Conditional deletion of PLCε in mouse cardiac myocytes after development protected the heart from pressure overload-induced hypertrophy, which strongly supports our mechanistic data from NRVMs. Collectively, our studies have unveiled a novel and important mechanism of regulating cardiac hypertrophy, which suggest PLCε may be a promising target for pharmacological manipulation

G protein βγ subunit mediated signaling specificity : implications for βγ subtypes and novel mechanisms of activation

Thesis (Ph. D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Biochemistry and Biophysics, 2007.Activators of G protein signaling (AGS proteins) are a group of accessory proteins that can activate G protein signaling independent of a receptor. A new member of this protein family, AGS8, was found to promote Gβγ mediated signaling in transfected Cos-7 cells, but the molecular mechanism for this action is unknown. We show that AGS8 binds to the effector and α subunit binding region (“hot spot”) on Gβγ yet does not interfere with α subunit binding or PLCβ activation. AGS8 stimulates activation of PLCβ2 by a Gαβγ heterotrimer and forms a quaternary complex with, Gα, Gβγ and PLCβ2. Furthermore, AGS8 rescues PLCβ2 binding and regulation by an inactive βγ subunit with a mutation (β1(W99A)γ2) in the “hot spot” that normally prevents binding and activation of PLCβ2. This demonstrates that, in the presence of AGS8, the “hot spot” is not used for Gβγ interactions with PLCβ2 and a new effector-binding interface on Gβγ is used for effector activation without subunit dissociation. Further mutagenic analysis indicates that a new binding and activation surface is created in the amino-terminal coiled-coil region of the βγ complex when AGS8 is bound. These data implicate a mechanism for AGS8, and potentially other Gβγ binding proteins, in directing Gβγ signaling through alternative effector activation sites on Gβγ. What contributes to the specificity of interactions of different βγ subunit with receptors, Gα subunits, and effectors is not clear. We are interested in understanding the molecular basis for Gβγ isoform specific differences in cells and in vitro. We found that differences existed between Gβ1γ2 and Gβ1γ7 in terms of binding to peptides and Gα subunit. Isopreteronol stimulated β-adrenergic receptor signaling was inhibited by knocking down the expression of γ7 with shRNA. However, the inhibition was rescued by re-introducing either γ2 or γ7, indicating that the loss of function of knocking down a single γ isoform may be compensated by over-expression of other isoforms. Thus, though in vitro biochemical studies demonstrated differences existing between individual γ subunit isoforms, the physiological relevance of these differences is hard to pursue because of the complexity of the cellular signaling networks