Research reportSelective expression of regulators of G-protein signaling (RGS) in the human central nervous system
Introduction
Heterotrimeric G-proteins mediate cellular responses to G-protein coupled receptor (GPCR) activation by serving to activate or inhibit cellular signaling pathways. As such, they constitute a likely point at which sustained responses to extracellular stimuli might be regulated. The regulator of G-protein signaling (RGS) proteins constitute a class of proteins which function as GTPase activating proteins (GAPs) which modify the turnover of G-protein alpha-subunits that are present in the GTP-bound state. They were identified initially in fungi as negative regulators of G-protein signaling pathways where they have prominent roles in promoting desensitization of GPCR signaling [38].
There have been numerous studies performed to establish the Gα subunit preference of mammalian RGS family members, as reviewed by Hollinger and Hepler [12]. Although some degree of preference has been reported for a few RGS species, such as RGS2 for Gαq, in general RGS proteins show broad and overlapping preferences for certain Gα subunits over others. RGS proteins have been shown to modulate Gi- and Gq-mediated signaling from several mammalian GPCRs controlling essential neurological functions. These include serotoninergic [14], dopaminergic [39], metabotropic glutamate [27] and GABAB [28] receptors.
The spatial regulation of RGS expression in a signaling pathway has the potential to dictate its function and specificity by controlling the availability of G-protein species with which the RGS proteins interact. For this reason, knowledge of the tissue expression pattern for RGS proteins can provide insight as to the tissue-specific regulation of physiological functions in which receptor signaling via G-proteins plays a role. Twenty-one RGS genes have been identified in humans to date and 19 of these genes (or their orthologues in other species) have been shown to exhibit GAP activity [12], [31]. Several studies have characterized the gene expression of some of the published rat orthologues of these RGS proteins, showing the existence of different patterns of expression for different RGS family members [8], [10].
At present, little is known about the tissue expression patterns of RGS genes in humans in general and within the human central nervous system. Members of the RGS family are likely to regulate numerous G-protein signaling pathways represented in the nervous system, thereby modulating a variety of physiological functions. Recent studies have provided evidence for a possible role of certain RGS genes in CNS disorders such as schizophrenia [17] and anxiety [19], and in regulating pain signaling and sensitivity to morphine [6]. These and other recent data (reviewed in Ref. [6]), have suggested that RGS proteins can be involved in several disease-specific adaptation processes.
In this study, we have characterized both the tissue distribution and level of mRNA expression for the known human RGS members. We have employed panels of human brain and peripheral tissues and have further examined mRNA expression in discrete regions of the central nervous system for several RGS family members identified as having prominent brain expression. We confirm distinct patterns of RGS mRNA expression and have further identified a central nervous system-specific localization for five RGS family members. These data point to a likely role of specific RGS family members in regulating the function of anatomically discrete physiological pathways.
Section snippets
Materials
The following drugs and chemicals used in the described studies were obtained from the following sources: TRIzol RNA extraction reagent, oligo(dT)12–18 and SuperscriptII Reverse Transcriptase were purchased from GIBCO BRL Life Technologies (San Giuliano Milanese, Italy). All PCR reagents were from Applied Biosystems (Foster City, CA). TaqMan probes and oligonucleotides were synthesized by Chem Progress (Sesto Ulteriano, Italy), and by Applied Biosystems.
Identification of novel human RGS sequences
Human EST and genomic nucleotide sequence
Identification of novel human RGS sequences and phylogenic interrelationship
In order to conduct a comprehensive survey of any gene family, it is first necessary to identify all members of that family from a single organism. To this end, we identified and cloned human othologues of the known RGS genes for which no human orthologue was known at the time. Human RGS8 (AX098425) and RGS18 was identified by full-length sequencing of IMAGE clones 1838553 and 297800, respectively, following initial identification from BLAST searches. During cloning of RGS18, another group
Discussion
The rationale of the present study was to identify human RGS family members with a preferential CNS expression, and further describe their local expression patterns within the CNS. This was viewed as a first step in the identification of therapeutic targets which could control chronic responses of specific functions in the CNS. In this study we have provided quantitative mRNA expression analysis in a broad profile of human tissues for the known 19 members of the human RGS family and the mRNA
Acknowledgements
The authors would like to thank Dr. R. Ravid, Netherlands Brain Bank, The Netherlands, for arrangement/donation of brain tissues.
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