Mark I. Cockett

RGS7 Is Palmitoylated and Exists as Biochemically Distinct Forms

Abstract: Regulator of G protein signaling (RGS) proteins are GTPase‐activating proteins that modulate neurotransmitter and G protein signaling. RGS7 and its binding partners Gα and Gβ5 are enriched in brain, but biochemical mechanisms governing RGS7/Gα/Gβ5 interactions and membrane association are poorly defined. We report that RGS7 exists as one cytosolic and three biochemically distinct membrane‐bound fractions (salt‐extractable, detergent‐extractable, and detergent‐insensitive) in brain. To define factors that determine RGS7 membrane attachment, we examined the biochemical properties of recombinant RGS7 and Gβ5 synthesized in Spodoptera frugiperda insect cells. We have found that membrane‐bound but not cytosolic RGS7 is covalently modified by the fatty acid palmitate. Gβ5 is not palmitoylated. Both unmodified (cytosolic) and palmitoylated (membrane‐derived) forms of RGS7, when complexed with Gβ5, are equally effective stimulators of Gαo GTPase activity, suggesting that palmitoylation does not prevent RGS7/Gαo interactions. The isolated core RGS domain of RGS7 selectively binds activated Gαi/o in brain extracts and is an effective stimulator of both Gαo and Gαi1 GTPase activities in vitro. In contrast, the RGS7/Gβ5 complex selectively interacts with Gαo only, suggesting that features outside the RGS domain and/or Gβ5 association dictate RGS7‐Gα interactions. These findings define previously unrecognized biochemical properties of RGS7, including the first demonstration that RGS7 is palmitoylated.

Distribution of heterotrimeric G-protein β and γ subunits in the rat brain

Abstract Heterotrimeric G-proteins, comprising α, β and γ subunits, have been shown to play a central role in coupling multiple receptors to a variety of enzymes and ion channels. In vitro studies have demonstrated the existence of selective interactions between various α, β and γ subunits, as well as between specific heterotrimers and target receptor and effector proteins. However, little is known of the physiological relevance of such associations, and the determinants of specificity in G-protein signaling within the brain remain largely unidentified. To investigate the possibility that specific heterotrimeric interactions result from discrete localizations of the G-protein subunits within the brain, we have used the technique of in situ hybridization to map the distribution of G-protein β and γ subunits in the rat brain. β1, β2, β3 and β5 subunits were found to be widely expressed throughout the rat brain, whilst β4 and the G-protein γ subunit messenger RNAs generally showed more discrete expression patterns. The expression patterns for these subunits suggest that individual β and γ subunits may be co-expressed in certain cell types and brain regions; a particularly intriguing and striking co-localization was observed in the case of β4 and γ2 subunit messenger RNAs in layer VI of the occipital cortex. The localizations of the G-protein β and γ subunits, and their potential coupling to various receptor/effector systems, are discussed.

Regional distribution of regulators of G-protein signaling (RGS) 1, 2, 13, 14, 16, and GAIP messenger ribonucleic acids by in situ hybridization in rat brain

Regulators of G-protein signaling (RGS) proteins are a novel family of GTPase-activating proteins that interact with Galpha subunits of the Gi/o, Gz, Gq and G(12/13) subfamilies to dampen G-protein-coupled receptor (GPCR)-mediated signaling by accelerating intrinsic Galpha-GTPase activity. In the present study, we report on messenger ribonucleic acid (mRNA) localization in rat brain of six RGS genes by in situ hybridization. The distribution patterns of RGS2, RGS13, RGS14 and GAIP (Galpha interacting protein) overlapped in most brain regions examined. Highest regional expression was observed for RGS2 in the cerebral cortical layers, striatum, hippocampal formation, several thalamic and hypothalamic nuclei and hindbrain regions such as the pontine, interpeduncular and dorsal raphe nuclei. Levels of RGS14 mRNA closely paralleled those of RGS2 expression levels throughout most brain regions. RGS13 mRNA was enriched in the hippocampal formation, amygdala, mammillary nuclei as well as the pontine and interpeduncular nuclei. GAIP expression levels were highest in the hippocampal formation with moderate to low levels present in all other regions studied. Of the six RGS genes probed, RGS16 mRNA displayed a discrete localization predominantly in the thalamic midline/intralaminar and principal relay nuclei, and the hypothalamic suprachiasmatic nucleus. RGS1 mRNA signal was not detected in brain. In conclusion, the in situ hybridization studies for RGS2, RGS13, RGS14, RGS16 and GAIP mRNAs extend our knowledge of the distribution of RGS genes expressed in the rat central nervous system, and indicate overlapping RGS-enriched regions that may be indicative of functional diversification in GPCR signaling pathway modulation.

RGS7 Attenuates Signal Transduction Through the Gαq Family of Heterotrimeric G Proteins in Mammalian Cells

Abstract: The RGS proteins are a recently discovered family of G protein regulators that have been shown to act as GTPase‐activating proteins (GAPs) on the Gαi and Gαq subfamilies of the heterotrimeric G proteins. Here, we demonstrate that RGS7 is a potent GAP in vitro on Gαi1 and Gαo heterotrimeric proteins and that RGS7 acts to down‐regulate Gαq‐mediated calcium mobilization in a whole‐cell assay system using a transient expression protocol. This RGS protein and RGS4 are reported to be expressed predominantly in brain, and in situ hybridization studies have revealed similarities in the regional distribution of RGS and Gαq mRNA expression. Our findings provide further evidence to support a functional role for RGS4 and RGS7 in Gαq‐mediated signaling in the CNS.

Antisense Oligonucleotide‐Induced Reduction in 5‐Hydroxytryptamine7 Receptors in the Rat Hypothalamus Without Alteration in Exploratory Behaviour or Neuroendocrine Function

Abstract: The effect of a 5‐hydroxytryptamine7 (5‐HT7) receptor‐directed antisense oligonucleotide on rat behaviour and neuroendocrine function was investigated. Six days of intracerebroventricular 5‐HT7 antisense oligonucleotide treatment significantly reduced [3H]5‐HT binding to hypothalamic 5‐HT7 receptors, whereas cortical 5‐HT2C density remained unchanged. In rats on a food‐restricted diet, both antisense and mismatch oligonucleotides reduced food intake and body weight compared with that in vehicle‐treated controls by day 4 of administration. 5‐HT7 antisense oligonucleotide administration did not affect exploratory or locomotor activity in photocell activity monitors on day 4 or elevated plus‐maze behaviour on day 6 of intracerebroventricular treatment. 5‐HT7 antisense oligonucleotide did not affect plasma corticosterone or prolactin levels or 5‐HT turnover in either 5‐HT cell body or terminal areas. These data demonstrate that intracerebroventricular 5‐HT7 antisense oligonucleotide administration selectively reduced rat hypothalamic 5‐HT7 receptor density without affecting any of the biochemical or behavioural measures. The results suggest that this antisense protocol could be a valuable tool to investigate central 5‐HT7 receptor functions, and that this receptor is not critical for the control of neuroendocrine function or food intake.

Immunohistochemical localization of G protein β1, β2, β3, β4, β5, and γ3 subunits in the adult rat brain

Abstract: The regional distributions of the G protein β subunits (Gβ1–β5) and of the Gγ3 subunit were examined by immunohistochemical methods in the adult rat brain. In general, the Gβ and Gγ3 subunits were widely distributed throughout the brain, with most regions containing several Gβ subunits within their neuronal networks. The olfactory bulb, neocortex, hippocampus, striatum, thalamus, cerebellum, and brainstem exhibited light to intense Gβ immunostaining. Negative immunostaining was observed in cortical layer I for Gβ1 and layer IV for Gβ4. The hippocampal dentate granular and CA1–CA3 pyramidal cells displayed little or no positive immunostaining for Gβ2 or Gβ4. No anti‐Gβ4 immunostaining was observed in the pars compacta of the substantia nigra or in the cerebellar granule cell layer and Purkinje cells. Immunoreactivity for Gβ1 was absent from the cerebellar molecular layer, and Gβ2 was not detected in the Purkinje cells. No positive Gγ3 immunoreactivity was observed in the lateral habenula, lateral septal nucleus, or Purkinje cells. Double‐fluorescence immunostaining with anti‐Gγ3 antibody and individual anti‐Gβ1–β5 antibodies displayed regional selectivity with Gβ1 (cortical layers V–VI) and Gβ2 (cortical layer I). In conclusion, despite the widespread overlapping distributions of Gβ1–β5 with Gγ3, specific dimeric associations in situ were observed within discrete brain regions.

Immunohistochemical Distribution of RGS7 Protein and Cellular Selectivity in Colocalizing with Gαq Proteins in the Adult Rat Brain

Abstract : Regulators of G protein signaling (RGS) proteins serve as potent GTPase‐activating proteins for the heterotrimeric G proteins αi/o and αq/11. This study describes the immunohistochemical distribution of RGS7 throughout the adult rat brain and its cellular colocalization with Gαq/11, an important G protein‐coupled receptor signal transducer for phospholipase Cβ‐mediated activity. In general, both RGS7 and Gαq/11 displayed a heterogeneous and overlapping regional distribution. RGS7 immunoreactivity was observed in cortical layers I‐VI, being most intense in the neuropil of layer I. In the hippocampal formation, RGS7 immunoreactivity was concentrated in the strata oriens, strata radiatum, mossy fibers, and polymorphic cells, with faint to nondetectable immunolabeling within the dentate gyrus granule cells and CA1‐CA3 subfield pyramidal cells. Numerous diencephalic and brainstem nuclei also displayed dense RGS7 immunostaining. Dual immunofluorescence labeling studies with the two protein‐specific antibodies indicated a cellular selectivity in the colocalization between RGS7 and Gαq/11 within many discrete brain regions, such as the superficial cortical layer I, hilus area of the hippocampal formation, and cerebellar Golgi cells. To assess the ability of Gαq/11‐mediated signaling pathways to modulate dynamically RGS expression, primary cortical neuronal cultures were incubated with phorbol 12,13‐dibutyrate, a selective protein kinase C activator. A time‐dependent increase in levels of mRNA for RGS7, but not RGS4, was observed. Our results provide novel information on the region‐ and cell‐specific pattern of distribution of RGS7 with the transmembrane signal transducer, Gαq/11. We also describe a possible RGS7‐selective neuronal feedback adaptation on Gαq/11‐mediated pathway function, which may play an important role in signaling specificity in the brain.

Cloning and tissue distribution of the human G protein β5 cDNA

Abstract Heterotrimeric G proteins integrate signals between receptors and effector proteins. We have cloned the human β 5 subunit from a human brain cDNA library. The clone has a 1059 bp open reading frame and is highly homologous to the murine clone. In contrast to the brain specific mouse β 5, northern analysis showed it to be expressed in multiple tissues.

Pindolol‐insensitive [3H]‐5‐hydroxytryptamine binding in the rat hypothalamus; identity with 5‐hydroxytryptamine7 receptors

Pindolol‐insensitive [3H]‐5‐hydroxytryptamine ([3H]‐5‐HT) binding to rat hypothalamic membranes was pharmacologically and functionally characterized to resolve whether this procedure selectively labels 5‐HT7 receptors. Consistent with a previous report, 3 μM and not 100 nM pindolol was required to occupy fully 5‐HT1A and 5‐HT1B receptors. Remaining [3H]‐5‐HT binding was saturable (KD, 1.59±0.21 nM; Bmax, 53.8±3.1 fmol.mg protein−1). Displacement of [3H]‐5‐HT with metergoline and 5‐CT revealed shallow Hill slopes (<0.5) but seven other compounds had slopes >0.8 and pKi values and the rank order of affinity were significantly correlated (r=0.81 and 0.93, respectively) with published [3H]‐5‐HT binding to rat recombinant 5‐HT7 receptors. In the presence of pindolol, 5‐HT‐enhanced accumulation of [32P]‐cyclic AMP was unaffected by the 5‐HT4 antagonist RS39604 (0.1 μM) or the 5‐ht6 antagonist Ro 04–6790 (1 μM) but significantly attenuated by mesulergine (250 nM), ritanserin (450 nM) or methiothepin (200 nM) which have high affinity for the 5‐HT7 receptor. Intracerebroventricular pretreatment with the serotonergic neurotoxin 5,7‐dihydroxytryptamine, 5,7‐DHT, elevated the [3H]‐5‐HT Bmax 2 fold, indicating that the hypothalamic 5‐HT7 receptor is post‐synaptic to 5‐HT nerve terminals and regulated by synaptic 5‐HT levels. These results suggest that, in the presence of 3 μM pindolol, [3H]‐5‐HT selectively labels hypothalamic binding sites consistent with functional 5‐HT7 receptors.

New generation dopaminergic agents. 2. Discovery of 3-OH-phenoxyethylamine and 3-OH-N1-phenylpiperazine dopaminergic templates

Described in this report is a systematic study which led to the identification of two new dopamine D2 partial agonists (5 and 17). Phenols 5 and 17 represent prototypes of two new classes of D2 partial agonists as well as templates for the future design of novel dopaminergic agents.