Research report
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

https://doi.org/10.1016/S0169-328X(01)00038-9Get rights and content

Abstract

Regulators of G-protein signaling (RGS) proteins are a novel family of GTPase-activating proteins that interact with Gα subunits of the Gi/o, Gz, Gq and G12/13 subfamilies to dampen G-protein-coupled receptor (GPCR)-mediated signaling by accelerating intrinsic Gα-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 (Gα 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.

Introduction

G-protein-coupled receptor (GPCR) signaling is a ubiquitous process that occurs across both vertebrate and invertebrate species. In general, neurotransmitter/hormonal-associated membrane receptor activation induces conformational changes in heterotrimeric G proteins that favor exchange of GDP for GTP on the Gα subunit. This exchange facilitates the dissociation of Gα and Gβγ components and initiates intracellular signal propagation via a number of second messenger pathways. The intrinsic GTPase activity of the Gα subunit results in the hydrolysis of Gα-associated GTP to GDP to terminate agonist-mediated signaling. An essential function of regulators of G-protein signaling (RGS) proteins is to serve as GTPase activating proteins (GAPs) that accelerate the Gα-GTP hydrolytic turnover by up to 1000-fold [25] specifically for Gαi/o and Gαq, but not Gαs subunits. A number of in vitro studies have described the potent GAP properties of several RGS proteins, including RGS2 [17], [18], [21], RGS16 [3], [6] and GAIP [11], [20], to attenuate either Gαi/o- and/or Gαq-mediated signaling in mammalian cells. Thus far over 20 mammalian genes containing the RGS domain have been identified [13], [23], [26] including splice variants of RGS9 [31], [34] and RGS12 [32]. In addition to the RGS domain, other modular domains, structural motifs, and diverse protein binding partners other than the Gα subunit have been identified [10], [33], [36]; suggesting undiscovered and complex functional actions for RGS proteins on neurotransmitter-dependent pathways [1], [9], [16].

Information elucidating the regulatory specificity of RGS proteins on Gαi/o- and Gαq-coupled receptor-mediated signal transduction is accumulating. For example, the N-terminal sequence of RGS4 is required for receptor-selective inhibition of Gαq-mediated signaling [35]. In chick dorsal root ganglion neurons, recombinant GAIP and RGS4 were shown to selectively accelerate the desensitization rates of Gαi- and Gαo-mediated pathways and alter the time-courses of norepinephrine-mediated effects [12]. A number of studies have demonstrated a dynamic and differential regulation of RGS expression as a function of either drug or physical intervention suggesting brain region-specific and long-term neuroadaptive responses in animals [5], [21], [28]. The supporting evidence for involvement of RGS proteins in a variety of cellular mechanisms, such as RGS2 upregulation by seizure [21] as well as a general role in immune system responses [22], provides scope for this family of signaling regulators as novel therapeutic targets.

To date, the distribution of RGS3 through RGS11 has been reported for rat brain by use of in situ hybridization techniques, in which RGS4, RGS5, RGS7 and RGS8 demonstrated a broad regional distribution pattern, and RGS3, RGS6, RGS9, RGS10 and RGS11 displayed a localized distribution [15]. In the present study, we describe a comparative examination of RGS1, RGS2, RGS13, RGS14, RGS16 and GAIP mRNA expression levels in rat brain by similar in situ techniques. This information will extend our knowledge of RGS mRNA distribution patterns in the central nervous system and will be valuable for the study of relationships between these important GPCR signaling modulators and their functional and temporal roles in specific pathway networks, as well as their potential use as therapeutic targets.

Section snippets

In situ hybridization

Adult male Wistar rats (n=2; 250–400 g) were euthanized with CO2 (National Institute of Health Guide for the Care and Use of Laboratory Animals). The brains were removed, immediately frozen on a bed of pulverized dry-ice and stored at −70°C. Coronal sections (10 μm thick) were thaw-mounted onto chilled (−20°C) slides pretreated with Vectabond reagent (Vector Labs., Burlington, CA, USA), and in situ hybridization was performed as described previously [2] with minor modifications. In brief,

General features

RGS1 mRNA was not detected in rat brain by in situ hybridization; however, Northern blot analysis confirmed that RGS1 was expressed in lung, liver, skeletal muscle and placenta (not shown). RGS2 was detected at moderate to high levels in most brain regions examined, including cerebral cortex (neocortical layers and piriform cortex), striatum, hippocampus, amygdala, several thalamic and hypothalamic nuclei (Fig. 1A–C), and hindbrain regions such as the pontine, interpeduncular and dorsal raphe

Discussion

RGS proteins constitute a novel and diverse group of GTPase-activating proteins that display brain region-specific expression patterns. Described here is the first detailed identification of brain regions that express RGS13, RGS14, RGS16 and GAIP. We also provide additional information about the distribution of RGS2. These findings complement previous in situ hybridization studies by Gold et al. describing the mRNA expression patterns of RGS3 through RGS11 in rat brain [15], and enhance the

Acknowledgements

The authors thank Dr. Ken Rhodes for helpful discussions and Mr. Brian Strassle for technical imaging assistance.

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    Present address: Bristol-Myers Squibb, Department of Applied Genomics, 311 Pennington-Rocky Hill Road, Pennington, NJ 08534, USA.

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