Anxiolytic effect of the GPR103 receptor agonist peptide P550 (homolog of neuropeptide 26RFa) in mice. Involvement of neurotransmitters

The GPR103 receptor is a G protein-coupled receptor, which plays a role in several physiological functions. However, the role of the GPR103 receptor in anxiety has not been clarified. The first aim of our study was to elucidate the involvement of the GPR103 receptor in anxious behavior. Mice were treated with peptide P550, which is the mouse homolog of neuropeptide 26RFa and has similar activity for the GPR103 receptor as neuropeptide 26RFa. The anxious behavior was investigated using an elevated plus-maze paradigm. The second aim of our study was to investigate the underlying neurotransmissions. Accordingly, mice were pretreated with a nonselective muscarinic acetylcholine receptor antagonist, atropine, a γ-aminobutyric acid subunit A (GABA_A) receptor antagonist, bicuculline, a non-selective 5-HT₂ serotonergic receptor antagonist, cyproheptadine, a mixed 5-HT₁/5-HT₂ serotonergic receptor antagonist, methysergide, a D₂, D₃, D₄ dopamine receptor antagonist, haloperidol, a nonselective α-adrenergic receptor antagonist, phenoxybenzamine and a nonselective β-adrenergic receptor antagonist, propranolol. Our results demonstrated that peptide P550 reduces anxious behavior in elevated plus maze test in mice. Our study shows also that GABA_A-ergic, α- and β-adrenergic transmissions are all involved in this action, whereas 5-HT₁ and 5-HT₂ serotonergic, muscarinic cholinergic and D₂, D₃, D₄ dopaminergic mechanisms may not be implicated.     

Anti-allodynic effects of intrathecally and intracerebroventricularly administered 26RFa, an intrinsic agonist for GRP103, in the rat partial sciatic nerve ligation model

26RFa and QRFP are endogenous ligands of GPR103. 26RFa binding sites are widely distributed in the brain and the spinal cord where they are involved in processing pain. In the present study, the effects of intrathecal and intracerebroventricular applications of 26RFa on the level of mechanical allodynia induced by partial sciatic nerve ligation were examined in rats. The level of mechanical allodynia was measured using von Frey filaments. Intrathecal and intracerebroventricular injection of 26RFa attenuated the level of mechanical allodynia. 26RFa has been reported to activate not only GPR103 but also neuropeptide FF2 receptor and the effect of intrathecally and intracerebroventricularly administered 26RFa was not antagonized by BIBP3226, an antagonist of neuropeptide FF receptor. Immunohistochemical examination revealed that QRFP-like immunoreactivity (QRFP-LI) was expressed mainly in the small to medium sized neurons in the L5 dorsal root ganglion (DRG) and that partial sciatic nerve injury increased the percentage of QRFP-LI positive neurons. 7 days after the nerve injury, QRFP-LI positive neurons in the L5 DRG ipsilateral to the partial sciatic nerve injury were larger than those in the L5 DRG ipsilateral to the sham operation. These data suggest that (1) exogenously applied 26RFa modulates nociceptive transmission at the spinal and the supraspinal brain in the neuropathic pain model, (2) the mechanism 26RFa uses to produce an anti-allodynic effect may be mediated by the activation of GPR103, and (3) partial sciatic nerve ligation affects the expression of QRFP-LI in the dorsal root ganglion.     

26RFa, a novel orexigenic neuropeptide, inhibits insulin secretion in the rat pancreas

26RFa is a novel orexigenic neuropeptide identified as the endogenous ligand of the orphan G protein-coupled receptor GPR103. GPR103 shares sequence identity with the receptors for neuropeptide-Y and galanin, two peptides known to inhibit insulin secretion. We have investigated the effect of 26RFa on insulin and glucagon secretion in the perfused rat pancreas. 26RFa dose-dependently reduced glucose-induced insulin release, inhibited the insulin responses to both arginine and exendin-4 and did not affect glucagon output. The inhibitory effect of 26RFa on exendin-4-induced insulin secretion was not observed in pancreata from pertussis toxin-treated rats, thus suggesting that 26RFa may inhibit insulin secretion, at least in part, via a pertussis toxin-sensitive G(i) protein coupled to the adenylyl cyclase system.     

Intracerebroventricular administration of 26RFa produces an analgesic effect in the rat formalin test

GPR103 is one of the orphan G protein-coupled receptors. Recently, an endogenous ligand for GPR103, 26RFa, was identified. Many 26RFa binding sites have been observed in various nuclei of the brain involved in the processing of pain such as the parafascicular thalamic nucleus, the locus coeruleus, the dorsal raphe nucleus, and the parabrachial nucleus. In the present study, the effects of intracerebroventricular injection of 26RFa were tested in the rat. Intracerebroventricular injection of 26RFa significantly decreased the number of both phase 1 and phase 2 agitation behaviors induced by paw formalin injection. This analgesic effect of 26RFa on the phase 1 response, but not phase 2 response, was antagonized by BIBP3226, a mixed antagonist of neuropeptide Y Y1 and neuropeptide FF receptors. Intracerebroventricular injection of 26RFa has no effect in the 52.5 °C hot plate test. Intracerebroventricular injection of 26RFa had no effect on the expression of Fos-like immunoreactivity induced by paw formalin injection in the superficial layers of the spinal dorsal horn. These data suggest that (1) 26RFa modulates nociceptive transmission at the supraspinal site during a formalin test, (2) the mechanism 26RFa uses to produce an analgesic effect on the phase 1 response is different from that on the phase 2 response, and (3) intracerebroventricularly injected 26RFa dose not directly inhibit the nociceptive input to the spinal cord.     

Structural studies on 26RFa, a novel human RFamide-related peptide with orexigenic activity

A novel hypothalamic neuropeptide of the RFamide family, comprising 26 amino acids residues and thus termed 26RFa, has been recently characterized in human, and was found to be the endogenous ligand for the orphan G protein-coupled receptor GPR103. Intracerebroventricular injection of 26RFa provokes a robust increase in food intake in rodents. In the present study, we have investigated the solution conformation of 26RFa by using two-dimensional NMR spectroscopy in different media. In water, 26RFa exhibits mainly a random coil conformation although the presence of a nascent helix was detected between residues 6 and 15. In methanol, 26RFa adopts a well-defined conformation consisting of an amphipathic alpha-helical structure (Pro4-Arg17), flanked by two N- and C-terminal disordered regions. The strong conservation, from amphibians to mammals, of the amino acid sequence corresponding to the amphipathic helix and to the C-terminal flexible octapeptide of 26RFa, suggests that these two domains are crucial for the interaction of the peptide with its receptor.     

Molecular evolution and functional characterization of the orexigenic peptide 26RFa and its receptor in vertebrates

Several neuropeptides possessing the RFamide motif at their C-termini (designated RFamide peptides) have been characterized in the hypothalamus of a variety of vertebrates. To date, five groups of the RFamide peptide family have been shown to exert several important neuroendocrine, behavioral, sensory, and autonomic functions. Since the discovery of the 26-amino acid RFamide peptide (termed 26RFa) from the frog brain, 26RFa has been shown to exert orexigenic activity in mammals and to be a ligand of the previously identified orphan G-protein-coupled receptor GPR103. Recently, 26RFa and its cognate receptor GPR103 have been identified in the brain of birds. This mini-review summarizes the advances in the identification, localization, and functions of 26RFa and its cognate receptor GPR103 in vertebrates and highlights recent progress made in birds.     

Disruption of the Plasma Membrane Integrity by Cholesterol Depletion Impairs Effectiveness of TRH Receptor-Mediated Signal Transduction via Gq/G11α Proteins

We monitored the radioligand-binding characteristics of thyrotropin-releasing hormone (TRH) receptors, functional activity of G_q/11α proteins, and functional status of the whole signaling cascade in HEK293 expressing high levels of TRH receptors and G₁₁α. Our analyses indicated that disruption of plasma membrane microdomains by cholesterol depletion did not markedly influence the binding parameters of TRH receptors, but it altered efficacy of signal transduction. The functional coupling between TRH receptor and G_q/11α was assessed by agonist-stimulated [³⁵S]GTPγS binding, and results of these measurements pointed out to significantly lower potency of TRH to mediate G protein activation in the plasma membrane fraction isolated from cholesterol-depleted cells; there was a shift in sensitivity by one order of magnitude to the higher concentrations. A markedly lower sensitivity to stimulation with TRH was also observed in our experiments dealing with determination of hormone-induced Ca²⁺ response. These data suggest that the intact structure of plasma membranes is an important optimum signal transduction initiated by TRH receptors and mediated by G_q/11α proteins.     

Activation of NPFF2 receptor stimulates neurite outgrowth in Neuro 2A cells through activation of ERK signaling pathway

Neurite outgrowth is an important process in neural regeneration and plasticity, especially after neural injury, and recent evidence indicates that several G_αi/o protein-coupled receptors play an important role in neurite outgrowth. The neuropeptide (NP)FF system contains two G_αi/o protein-coupled receptors, NPFF₁ and NPFF₂ receptors, which are mainly distributed in the central nervous system. The aim of the present study was to determine whether the NPFF system is involved in neurite outgrowth in Neuro 2A cells. We showed that Neuro 2A cells endogenously expressed NPFF₂ receptor, and the NPFF₂ receptor agonist dNPA inhibited cyclic adenosine monophosphate (cAMP) production stimulated by forskolin in Neuro 2A cells. We also demonstrated that NPFF and dNPA dose-dependently induced neurite outgrowth in Neuro 2A cells, which was completely abolished by the NPFF receptor antagonist RF9. Pretreatment with mitogen-activated protein kinase inhibitors PD98059 and U0126 decreased dNPA-induced neurite outgrowth. In addition, dNPA increased phosphorylation of extracellular signal-regulated kinase (ERK) in Neuro 2A cells, which was completely antagonized by pretreatment with U0126. Our results suggest that activation of NPFF₂ receptor stimulates neurite outgrowth in Neuro 2A cells through activation of the ERK signaling pathway. Moreover, NPFF₂ receptor may be a potential therapeutic target for neural injury and degeneration in the future.     

G protein-coupled Receptor 30 (GPR30) Forms a Plasma Membrane Complex with Membrane-associated Guanylate Kinases (MAGUKs) and Protein Kinase A-anchoring Protein 5 (AKAP5) That Constitutively Inhibits cAMP Production

GPR30, or G protein-coupled estrogen receptor, is a G protein-coupled receptor reported to bind 17β-estradiol (E₂), couple to the G proteins G_s and G_i/o, and mediate non-genomic estrogenic responses. However, controversies exist regarding the receptor pharmacological profile, effector coupling, and subcellular localization. We addressed the role of the type I PDZ motif at the receptor C terminus in receptor trafficking and coupling to cAMP production in HEK293 cells and CHO cells ectopically expressing the receptor and in Madin-Darby canine kidney cells expressing the native receptor. GPR30 was localized both intracellularly and in the plasma membrane and subject to limited basal endocytosis. E₂ and G-1, reported GPR30 agonists, neither stimulated nor inhibited cAMP production through GPR30, nor did they influence receptor localization. Instead, GPR30 constitutively inhibited cAMP production stimulated by a heterologous agonist independently of G_i/o. Moreover, siRNA knockdown of native GPR30 increased cAMP production. Deletion of the receptor PDZ motif interfered with inhibition of cAMP production and increased basal receptor endocytosis. GPR30 interacted with membrane-associated guanylate kinases, including SAP97 and PSD-95, and protein kinase A-anchoring protein (AKAP) 5 in the plasma membrane in a PDZ-dependent manner. Knockdown of AKAP5 or St-Ht31 treatment, to disrupt AKAP interaction with the PKA RIIβ regulatory subunit, decreased inhibition of cAMP production, and St-Ht31 increased basal receptor endocytosis. Therefore, GPR30 forms a plasma membrane complex with a membrane-associated guanylate kinase and AKAP5, which constitutively attenuates cAMP production in response to heterologous agonists independently of G_i/o and retains receptors in the plasma membrane.     

Atypical Responsiveness of the Orphan Receptor GPR55 to Cannabinoid Ligands

The cannabinoid receptor 1 (CB₁) and CB₂ cannabinoid receptors, associated with drugs of abuse, may provide a means to treat pain, mood, and addiction disorders affecting widespread segments of society. Whether the orphan G-protein coupled receptor GPR55 is also a cannabinoid receptor remains unclear as a result of conflicting pharmacological studies. GPR55 has been reported to be activated by exogenous and endogenous cannabinoid compounds but surprisingly also by the endogenous non-cannabinoid mediator lysophosphatidylinositol (LPI). We examined the effects of a representative panel of cannabinoid ligands and LPI on GPR55 using a β-arrestin-green fluorescent protein biosensor as a direct readout of agonist-mediated receptor activation. Our data demonstrate that AM251 and SR141716A (rimonabant), which are cannabinoid antagonists, and the lipid LPI, which is not a cannabinoid receptor ligand, are GPR55 agonists. They possess comparable efficacy in inducing β-arrestin trafficking and, moreover, activate the G-protein-dependent signaling of protein kinase CβII. Conversely, the potent synthetic cannabinoid agonist CP55,940 acts as a GPR55 antagonist/partial agonist. CP55,940 blocks GPR55 internalization, the formation of β-arrestin GPR55 complexes, and the phosphorylation of ERK1/2; CP55,940 produces only a slight amount of protein kinase CβII membrane recruitment but does not stimulate membrane remodeling like LPI, AM251, or rimonabant. Our studies provide a paradigm for measuring the responsiveness of GPR55 to a variety of ligand scaffolds comprising cannabinoid and novel compounds and suggest that at best GPR55 is an atypical cannabinoid responder. The activation of GPR55 by rimonabant may be responsible for some of the off-target effects that led to its removal as a potential obesity therapy.The CB₁² and CB₂ cannabinoid receptors comprise a two-member subfamily of G-protein-coupled receptors (GPCRs) that are notable as the targets of the tetrahydrocannabinol (THC) derivatives found in marijuana. More recently CB₁ receptors along with other GPCRs have been promoted as therapeutic pharmacological targets in the billion dollar weight loss market for controversial drugs such as rimonabant (SR141716A) and Fen-phen. Thus, an important utility of cannabinoid family receptors to society appears to arise from their role in regulating a broad spectrum of addiction-based behaviors, and the addition of new members to the cannabinoid receptor family may have social and economic implications that reach far beyond the initial scientific discovery. As a consequence, the re-classification of an orphan GPCR as a cannabinoid family member should be done with caution requiring strict criteria of receptor activation by THC derivatives or endogenous cannabinoid compounds and a widespread agreement of the results by the scientific community.Marijuana, one of the most widely abused substances (1), mediates many of its psychotropic effects by targeting CB₁ receptors in the central nervous system, but studies with CB₁ and CB₂ knock-out mice indicate that the complex pharmacological properties on pain, mood, and memory exhibited by exogenous cannabinoids and the endogenous arachidonic acid-based endo-cannabinoids, including anandamide and 2-arachidonoylglycerol (2-AG), are not fully explained by their activation of CB₁ and CB₂ (2–4). The CB₁ and CB₂ receptors are 44% identical and signal through G_i/o-mediated pathways. Activation of either receptor is inhibitory for cAMP production via adenylyl cyclase and stimulatory for mitogen-activated protein kinase (MAPK) (extracellular-regulated protein kinase 1/2 (ERK1/2)) activation (5). However, the failure of these two receptors to account for the full complement of physiological effects observed with cannabinoid ligands has led to the hypothesis that additional cannabinoid-like receptors exist.The orphan GPCR, GPR55, which exhibits only 10–15% homology to the two human cannabinoid receptors (6), is one of a number of plausible cannabinoid family member candidates (7). GPR55 was first identified and mapped to human chromosome 2q37 a decade ago (8). In the human central nervous system, it is predominantly localized to the caudate, putamen, and striatum (8), coupling to Gα₁₃ (9, 10), Gα₁₂, or Gα_q (11).GPR55 has been tested against a number of cannabinoid ligands with mixed results. Observations using a GTPγS functional assay indicate that GPR55 is activated by nanomolar concentrations of the endocannabinoids 2-AG, virodhamine, noladin ether, and palmitoylethanolamine (10) and the atypical cannabinoids Abn-CBD and O-1602 (12) as well as by the drugs CP55,950, HU210, and Δ⁹-THC (11). Exposure of GPR55 to the cannabinoids THC and JWH015 in dorsal root ganglion neurons and in receptor-transfected HEK293 cells correlates with increases of intracellular Ca²⁺ (11). In contrast, GPR55 is insensitive to the CB₁ inverse agonist AM281 and the potent cannabinoid agonist WIN55212-2 but is antagonized by the marijuana constituent CBD (9, 10). However, Oka et al. (13) reported that GPR55 is not a typical cannabinoid receptor, as numerous endogenous and synthetic cannabinoids, including many mentioned above, had no effect on GPR55 activity. They present compelling data suggesting that the endogenous lipid LPI and its 2-arachidonyl analogs are agonists at GPR55 as a result of their abilities to phosphorylate extracellular-regulated kinase and induce calcium signaling (13, 14). Further studies indicate that LPI and the rimonabant-like CB₁ inverse agonist AM251 induce oscillatory Ca²⁺ release through Gα₁₃ and RhoA (9). These reports were all performed in HEK 293 cells, yet each documented a distinct and conflicting chemical space of agonists that recognized GPR55. To resolve these inconsistencies in classification, an alternative approach for identifying GPR55 ligands that is insensitive to the endogenous complement of cellular receptors could circumvent many of the challenges that have arisen in the measurements of G-protein signaling.β-Arrestins are intracellular proteins that bind and desensitize activated GPCRs and in the process form stable receptor/arrestin signaling complexes (15, 16). β-Arrestin redistribution to the activated membrane-bound receptor represents one of the early intracellular events provoked by agonist binding and, consequently, is less prone to a false positive or negative readout as compared with studying a downstream signaling event as a readout of receptor activation. β-arrestin-green fluorescent chimeras can make this process attractive to monitor by forming remarkably sensitive and specific probes of GPCR activation that are independent of downstream G-protein-mediated signaling (17–19). We have determined GPR55 responsiveness to a representative panel of cannabinoid ligands and LPI in the presence (and absence) of a β-arrestin2-green fluorescent protein (βarr2-GFP) biosensor. Our data demonstrate that LPI, the CB₁ inverse agonist/antagonists SR141716A, and AM251 are GPR55 agonists, and the CB₁ agonist CP55940 is a GPR55 antagonist/partial agonist. These data together with our inability to observe activation of GPR55 by Δ⁹-THC and endocannabinoids indicate that GPR55 should be classified as an atypical cannabinoid receptor at best.     

Identification of a neuropeptide modified with bromine as an endogenous ligand for GPR7

We isolated a novel gene in a search of the Celera data base and found that it encoded a peptidic ligand for a G protein-coupled receptor, GPR7 (O'Dowd, B. F., Scheideler, M. A., Nguyen, T., Cheng, R., Rasmussen, J. S., Marchese, A., Zastawny, R., Heng, H. H., Tsui, L. C., Shi, X., Asa, S., Puy, L., and George, S. R. (1995) Genomics 28, 84-91; Lee, D. K., Nguyen, T., Porter, C. A., Cheng, R., George, S. R., and O'Dowd, B. F. (1999) Mol. Brain Res. 71, 96-103). The expression of this gene was detected in various tissues in rats, including the lymphoid organs, central nervous system, mammary glands, and uterus. GPR7 mRNA was mainly detected in the central nervous system and uterus. In situ hybridization showed that the gene encoding the GPR7 ligand was expressed in the hypothalamus and hippocampus of rats. To determine the molecular structure of the endogenous GPR7 ligand, we purified it from bovine hypothalamic tissue extracts on the basis of cAMP production-inhibitory activity to cells expressing GPR7. Through structural analyses, we found that the purified endogenous ligand was a peptide with 29 amino acid residues and that it was uniquely modified with bromine. We subsequently determined that the C-6 position of the indole moiety in the N-terminal Trp was brominated. We believe this is the first report on a neuropeptide modified with bromine and have hence named it neuropeptide B. In in vitro assays, bromination did not influence the binding of neuropeptide B to the receptor.     

UROTENSIN II MEDIATES ERK1/2 PHOSPHORYLATION AND PROLIFERATION IN GPR14-TRANSFECTED CELL LINES

ABSTRACT

Urotensin-II (U-II), a vasoactive cyclic neuropeptide, was recently identified as the natural ligand for the G-protein coupled receptor GPR14. The expression pattern of U-II and GPR14 are consistent with a role as a neurohormonal regulatory system in cardiovascular homeostasis. Urotensin-II induces a rapid and short-lasting rise in intracellular calcium in recombinant GPR14 expressing cells. In the present study we show that U-II induces signal transduction pathways leading to the long-lasting activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in chinese hamster ovary cells expressing human GPR14 (CHO-GPR14). Furthermore, we observed a growth-stimulating and PD98059 sensitive activity of U-II in CHO-GPR14 cells, but not CHO-K1 cells. The investigation of the GPR14 induced signal transduction pathways leading to ERK1/2 phosphorylation revealed a previously unsuspected role for G_i/o-protein coupling and showed an involvement of phospatidylinositol-3-kinase, phospholipase C and calcium channel mediated mechanisms. Our results suggest that U-II and its receptor GPR14 may be involved in long-lasting physiological effects such as cardiovascular remodeling.     

Diabetes-related defects in sarcoplasmic Ca2+ release are prevented by inactivation of Gα11 and Gαq in murine cardiomyocytes

Neurohumoral stimulation of Gq-coupled receptors has been proposed as a central mechanism in the pathogenesis of diabetic heart disease. The resulting contractile dysfunction is closely related to abnormal intracellular Ca²⁺ handling with functional defects of the sarcoplasmic reticulum (SR). The present study was therefore designed to determine the role of G_q-protein signaling via Gα₁₁ and Gα_q in diabetes for the induction of functional and structural changes in the Ca²⁺ release complex of the SR. An experimental type 1-diabetes was induced in wild type, Gα₁₁ knockout, and Gα_11/q-knockout mice by injection of streptozotocin. Cardiac morphology and function was assessed in vivo by echocardiography. SR Ca²⁺ leak was tested in vitro based on a ⁴⁵Ca²⁺ assay and protein densities as well as gene expression of ryanodine receptor (RyR2), FKBP12.6, sorcin, and annexin A7 were analyzed by immunoblot and RT-PCR. In wild type animals 8 weeks of diabetes resulted in cardiac hypertrophy and SR Ca²⁺ leak was increased. In addition, diabetic wild type animals showed reduced protein levels of FKBP12.6 and annexin A7. In Gα₁₁- and Gα_11/q-knockout animals, however, SR Ca²⁺ release and cardiac phenotype remained unchanged upon induction of diabetes. Densities of the proteins that we presently analyzed were also unaltered in Gα₁₁-knockout mice. Gα_11/q-knockout animals even showed increased expression of sorcin and annexin A7. Thus, based on the present study we suggest a signaling pathway via the G_q-proteins, Gα₁₁ and Gα_q, that could link increased neurohumoral stimulation in diabetes with defective RyR2 channel function by regulating protein expression of FKBP12.6, annexin A7, and sorcin.     

Selective Increases in Phosphoinositide Signaling Activity and G Protein Levels in Postmortem Brain from Subjects with Schizophrenia or Alcohol Dependence

Abstract: Comparisons of the activity of the G protein-mediated phosphoinositide signal transduction system and of G protein levels were made in two regions of frontal cortex from eight schizophrenic, alcohol-dependent, and control subjects. G protein-mediated phosphoinositide hydrolysis was measured by stimulating cortical membranes incubated with [³H]phosphatidylinositol with 0.3–10 µM guanosine 5′-O-(3-thio)triphosphate (GTPγS). In frontal cortex areas 8/9, GTPγS-induced phosphoinositide hydrolysis was 50% greater in schizophrenic than control or alcohol-dependent subjects, whereas there were no differences among these groups of subjects in the response to GTPγS in frontal cortex area 10. Agonists for dopaminergic, cholinergic, purinergic, serotonergic, histaminergic, and glutamatergic receptors coupled to the phosphoinositide signaling system increased [³H]phosphatidylinositol hydrolysis in a GTPγS-dependent manner. Responses to most agonists were similar in all three subject groups in both cortical regions, with the largest difference being a 40% greater response to dopaminergic receptor stimulation in frontal cortex 8/9 from schizophrenic subjects. Measurements of the levels of phospholipase C-β, and of α-subunits of G_q, G_o, G_i1, G_i2, and G_s, made by immunoblot analyses revealed no differences among the groups of subjects except for increased Gα_o in schizophrenic subjects and increased Gα_o and Gα_i1 in alcohol-dependent subjects. These results demonstrate that schizophrenia is associated with increased activity of the phosphoinositide signal transduction system and increased levels of Gα_o, whereas the phosphoinositide system was unaltered in alcohol dependence, but Gα_o and Gα_i1 were increased.