Research reportDistribution of the GABAB receptor subunit gb2 in rat CNS
Introduction
GABAB receptors, originally identified as a distinct subclass of GABA receptors by Bowery et al. 3, 4, 11are well established as members of the G protein-coupled receptor (GPCR) super family. They mediate a variety of inhibitory cellular processes through modulation of ion channels and adenylyl cyclase activity 6, 17, 22. Kaupmann et al. [14]have described the first primary sequence for a GABAB receptor which was isolated from rat brain using expression cloning techniques. The rat GABAB receptors identified, rgb1a and rgb1b, are alternatively spliced variants of the same gene product which differ in their amino terminal sequence and are most closely related to metabotropic glutamate receptors. They exhibit a reduced affinity for GABAB receptor compounds as compared with native receptors yet the rank order of potency for these compounds is similar to that described in the literature for GABAB receptors in tissue preparations. Recent work by Couve et al. [10]has shown that recombinant gb1 receptors expressed in heterologous systems do not reach the cell surface and are trapped in the endoplasmic reticulum. These data suggest that the gb1 receptors require additional information for functional targeting to the plasma membrane that appears to be missing in the cells used to study receptor function thus far. Until recently there had been no meaningful study of the function of these receptors. We and others have recently reported that coexpression of gb1a or gb1b with a related protein, gb2, in various heterologous expression systems results in the expression of functional GABAB receptors 12, 15, 19, 23, 29. The results of these studies suggest that the native GABAB receptor may be a heterodimer of gb1 and gb2 subunits.
Here we describe the isolation of human and rat cDNAs for the gb2 GABAB receptor subunit and the CNS distribution of gb2 mRNA in the rat. This is only the second GABAB receptor primary sequence to be identified. In situ hybridization histochemistry revealed that gb2 and gb1 mRNA exhibit overlapping expression patterns in many rat brain regions with transcripts for both receptors found in the same cells in a vast majority of cases. The colocalization data are supportive of our previous studies showing that coexpression of gb2 with gb1 in heterologous expression systems is necessary to observe GABAB receptor function [23]. In addition, rgb2 mRNA exhibits a more discrete expression pattern in some rat brain regions than rgb1 and appears to be absent from white matter suggesting that it is predominantly localized to neurons. If functional GABAB receptors are composed of heteromers then the finding that rgb1 message is abundant in many brain regions and in white matter where rgb2 is barely detectable suggests that there may be additional, as yet unidentified GABAB receptors.
Section snippets
Cloning of hgb2
A tblastn search of the Genbank EST database using the amino acid sequence of the rat gb1 receptor as query detected three ESTs, R76139, R80448, and Z43654, which showed distant homology to transmembrane domains 5–7 and another EST, T07261, which showed similarity to transmembrane domains 1–3. Primers designed from these sequences (JC136 gcgggatccCTTCGGCACGAATACCAGGCAGAGGGT for 5′ extension; JC137 ccggaattcCAGAAGCTCATAAAGATGTCGAGTCC and JC135 ccggaattcTTCTTAGCTTTGGGAGACCCGCA ACGT for 3′
hgb2 and rgb2 amino acid sequences
Through EST database searches, polymerase chain reaction, and library screening as described in Section 2we have identified and isolated human and rat homologues of a novel receptor, hgb2 and rgb2, with homology to the GABAB receptors, rgb1a and rgb1b, identified by Kaupmann et al. [14]. Fig. 1 shows a schematic of the hgb2 and rgb2 receptor nucleotide sequences denoting the placement of oligonucleotide primers and restriction enzyme sites used to generate each of these constructs and the
Discussion
There are several clinical situations in which GABAB receptors may prove to be potential sites for therapeutic intervention. Disrupted GABAergic neurotransmission has been implicated in a number of neurological and psychiatric disorders, including epilepsy, schizophrenia, and affective disorders. Due to the potential therapeutic value of GABAB receptors, it is important to identify the molecular structures of GABAB receptors and understand how they function. Recent work by Kaupmann et al. [14]
Acknowledgements
J.A.C. was supported by a Young Investigator Award from the National Alliance for Research on Schizophrenia and Depression.
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