Signal transduction via guanine nucleotide binding proteins (G proteins) is involved in cardiovascular, neural, endocrine, and immune cell function. Regulators of G protein signaling (RGS proteins) speed the turn-off of G protein signals and inhibit signal transduction, but the in vivo roles of RGS proteins remain poorly defined. To overcome the redundancy of RGS functions and reveal the total contribution of RGS regulation at the G␣ i2 subunit, we prepared a genomic knock-in of the RGS-insensitive G184S Gnai2 allele. The G␣ i2 G184S knock-in mice show a dramatic and complex phenotype affecting multiple organ systems (heart, myeloid, skeletal, and central nervous system). Both homozygotes and heterozygotes demonstrate reduced viability and decreased body weight. Other phenotypes include shortened long bones, a markedly enlarged spleen, elevated neutrophil counts, an enlarged heart, and behavioral hyperactivity. Heterozygous G␣ i2 ؉/G184S mice show some but not all of these abnormalities. Thus, loss of RGS actions at G␣ i2 produces a dramatic and pleiotropic phenotype which is more evident than the phenotype seen for individual RGS protein knockouts.Cell-cell communication is fundamental to the maintenance of homeostasis. The G protein-coupled receptor superfamily is arguably the most abundant and diverse protein family in cellular signaling and is tightly regulated. A novel family of Ͼ20 proteins termed regulators of G protein signaling, or RGS proteins, both tonically inhibit G protein function and also serve as signal control points (2,22,34,39,69). RGS-mediated inhibition of G protein signaling occurs through direct binding of the RGS protein to the G␣ subunit, with subsequent GTPase-accelerating protein (GAP) actions to rapidly deactivate G␣ (2). Deactivation may be accelerated up to 1,000-fold and shuts down both G␣ and G␤␥ signals (42, 48). RGS proteins may also competitively inhibit G␣ binding to effectors such as phospholipase C (32). Most of the currently known RGS proteins interact with either Gi or Gq family G proteins and influence cyclic AMP (cAMP), Ca 2ϩ , mitogen-activated protein kinase, and ion channel signaling. There is strong evidence implicating them in the subsecond kinetics of G i -and G o -mediated ion channel activation and deactivation in the heart (10, 21, 36) and neurons (36). In addition, the conserved RGS domain has been found to serve as a multifunctional protein adapter which can recruit many effectors or regulators to the vicinity of activated G proteins (31,53,62). Notable examples include p115rhoGEF (30, 40) and GRK2 (44). There is also emerging interest in RGS proteins as drug targets (9,20,53,72).However, the physiological functions of RGS proteins remain poorly defined. A number of RGS knockouts have been reported (for example, RGS1, -2, -4, and -9). The RGS9-1 knockout shows prolonged visual potentials (7), and RGS9-2 disruption results in markedly enhanced responses to drugs of abuse, such as cocaine, amphetamines, and opiates (56, 71). A human disorder, bradyopsia, with r...

G protein-coupled receptors strongly modulate neuronal excitability but there has been little evidence for G protein mechanisms in genetic epilepsies. Recently, four patients with epileptic encephalopathy (EIEE17) were found to have mutations in GNAO1, the most abundant G protein in brain, but the mechanism of this effect is not known. The GNAO1 gene product, Gαo, negatively regulates neurotransmitter release. Here, we report a dominant murine model of Gnao1-related seizures and sudden death. We introduced a genomic gain-of-function knock-in mutation (Gnao1+/G184S) that prevents Go turnoff by Regulators of G protein signaling proteins. This results in rare seizures, strain-dependent death between 15 and 40 weeks of age, and a markedly increased frequency of interictal epileptiform discharges. Mutants on a C57BL/6J background also have faster sensitization to pentylenetetrazol (PTZ) kindling. Both premature lethality and PTZ kindling effects are suppressed in the 129SvJ mouse strain. We have mapped a 129S-derived modifier locus on Chromosome 17 (within the region 41–70 MB) as a Modifer of G protein Seizures (Mogs1). Our mouse model suggests a novel gain-of-function mechanism for the newly defined subset of epileptic encephalopathy (EIEE17). Furthermore, it reveals a new epilepsy susceptibility modifier Mogs1 with implications for the complex genetics of human epilepsy as well as sudden death in epilepsy.Electronic supplementary materialThe online version of this article (doi:10.1007/s00335-014-9509-z) contains supplementary material, which is available to authorized users.

RGS proteins and G alpha(i2) signaling play important roles in the control of insulin sensitivity and glucose metabolism. Identification of the specific RGS proteins involved might permit their consideration as potential therapeutic targets for obesity-related insulin resistance and type 2 diabetes.

Activation of G protein-coupled ␣ 2 adrenergic receptors (ARs) inhibits epileptiform activity in the hippocampal CA3 region. The specific mechanism underlying this action is unclear. This study investigated which subtype(s) of ␣ 2 ARs and G proteins (G␣ o or G␣ i ) are involved in this response using recordings of mouse hippocampal CA3 epileptiform bursts. Application of epinephrine (EPI) or norepinephrine (NE) reduced the frequency of bursts in a concentration-dependent manner: (Ϫ)EPI Ͼ (Ϫ)NE ϾϾϾ (ϩ)NE. To identify the ␣ 2 AR subtype involved, equilibrium dissociation constants (pK b ) were determined for the selective ␣AR antagonists atipamezole (8.79), rauwolscine (7.75), 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane hydrochloride (WB-4101; 6.87), and prazosin (5.71). Calculated pK b values correlated best with affinities determined previously for the mouse ␣ 2A AR subtype (r ϭ 0.98, slope ϭ 1.07). Furthermore, the inhibitory effects of EPI were lost in hippocampal slices from ␣ 2A AR-but not ␣ 2C AR-knockout mice. Pretreatment with pertussis toxin also reduced the EPImediated inhibition of epileptiform bursts. Finally, using knock-in mice with point mutations that disrupt regulator of G protein signaling (RGS) binding to G␣ subunits to enhance signaling by that G protein, the EPI-mediated inhibition of bursts was significantly more potent in slices from RGS-insensitive G␣ o G184S heterozygous (G␣ o ϩ/GS) mice compared with either G␣ i2 G184S heterozygous (G␣ i2 ϩ/GS) or control mice (EC 50 ϭ 2.5 versus 19 and 23 nM, respectively). Together, these findings indicate that the inhibitory effect of EPI on hippocampal CA3 epileptiform activity uses an ␣ 2A AR/G␣ o protein-mediated pathway under strong inhibitory control by RGS proteins. This suggests a possible role for RGS inhibitors or selective ␣ 2A AR agonists as a novel antiepileptic drug therapy.

Receptors coupled to G proteins have many effects on the heart. Enhanced signaling by Gαs and Gαq leads to cardiac injury and heart failure, while Gαi2 signaling in cardiac myocytes can protect against ischemic injury and β-adrenergic-induced heart failure. We asked whether enhanced Gαi2 signaling in mice could protect against heart failure using a point mutation in Gαi2 (G184S), which prevents negative regulation by regulators of G protein signaling. Contrary to our expectation, it worsened effects of a genetic dilated cardiomyopathy (DCM) and catecholamine-induced cardiac injury. Gαi2G184S/+/DCM double heterozygote mice (TG9+Gαi2G184S/+) had substantially decreased survival compared to DCM animals. Furthermore, heart weight/body weight ratios (HW/BW) were significantly greater in TG9+Gαi2G184S/+ mice as was expression of natriuretic peptide genes. Catecholamine injury in Gαi2G184S/G184S mutant mice produced markedly increased isoproterenol-induced fibrosis and collagen III gene expression vs WT mice. Cardiac fibroblasts from Gαi2G184S/G184S mice also showed a serum-dependent increase in proliferation and ERK phosphorylation, which were blocked by pertussis toxin and a mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor. Gαi2 signaling in cardiac myocytes protects against ischemic injury but enhancing Gαi2 signaling overall may have detrimental effects in heart failure, perhaps through actions on cardiac fibroblasts.