Elsevier

Gene

Volume 278, Issues 1–2, 31 October 2001, Pages 63-79
Gene

Human GABABR genomic structure: evidence for splice variants in GABABR1 but not GABABR2

https://doi.org/10.1016/S0378-1119(01)00678-3Get rights and content

Abstract

The type B γ-aminobutryic acid receptor (GABABR) is a G protein coupled receptor that mediates slow pre- and post-synaptic inhibition in the nervous system. We find that the human GABABR2 gene spans greater than 350 kb and contains 2.8 kb of coding region in 19 exons. The overall similarity in genomic structure with regard to conservation of intron position and exon size between human or Drosophila GABABR1 and GABABR2 genes suggests a common ancestral origin. Multiple transcripts GABABR1a–c and GABABR2a–c have been described and alternative splicing has been proposed to result in GABABR1c, GABABR2b and GABABR2c. The results described here provide support for the existence of GABABR1c but not for GABABR2b and GABABR2c. Splice junctions present in the GABABR1 gene sequence are consistent with the formation of GABABR1c by exon skipping of one sushi domain module. The GABABR2 gene lacks canonical splice junctions for the reported variants. Consistent with this, RNA analysis demonstrates the presence of GABABR1c and GABABR2 transcripts in fetal and adult human brain RNA but GABABR2b and GABABR2c transcripts are not detected. These results provide insight into the evolution and transcript diversity of the mammalian GABABR genes.

Introduction

GABA is a neurotransmitter that mediates most fast and slow inhibitory synaptic transmission in the nervous system. Fast inhibitory synaptic transmission generally occurs through ionotropic type A GABA receptors (Rabow et al., 1995) whereas slow transmission occurs through metabotropic type B GABA receptors (GABABRs) (Hill and Bowery, 1981). Although the GABABR agonist baclofen has been used for over 30 years as a treatment for spasticity resulting from multiple sclerosis, spinal cord injuries and other spinal cord diseases (Jones et al., 1970), the molecular site of its action has only recently been delineated with the cloning of two mammalian genes, GABABR1 and GABABR2 (Kaupmann et al., 1997, Kaupmann et al., 1998, White et al., 1998, Jones et al., 1998, Kuner et al., 1999, Martin et al., 1999, Ng et al., 1999a). In addition, three Drosophila GABABR genes have been cloned (Mezler et al., 2001). The GABABR system is believed to be a key target for drugs that modulate epilepsy, pain and cognitive processes (Couve et al., 2000). An understanding of the receptor isoforms encoded by the GABABR genes is important for future development of subtype selective agonists and antagonists.

Two transcripts, GABABR1a and GABABR1b, were initially described for the GABABR1 gene, encoding proteins of 100k and 130k Mr that differ only in their N-terminal sequence (Kaupmann et al., 1997). The proteins share 797 C-terminal amino acids. The N-terminal sequence of the GABABR1a protein is 165 amino acids with a signal peptide and two sushi domains. The GABABR1b protein contains a different N-terminal sequence of 47 amino acids as well as a different signal sequence.

A single GABABR2 transcript was initially described (White et al., 1998, Kaupmann et al., 1998, Jones et al., 1998, Kuner et al., 1999, Martin et al., 1999, Ng et al., 1999a). Two additional transcripts, GABABR2b and GABABR2c, have been proposed to arise from alternative splicing of the GABABR2 message (Clark et al., 2000). The GABABR2b cDNA lacks 78 bp, encoding a protein with a 26 amino acid C-terminal deletion but maintaining the same C-terminal tail as the originally described receptor. The GABABR2c cDNA lacks 88 bp, resulting in the deletion of 13 C-terminal amino acids and the addition of 46 amino acids, coded for by nucleotides that were part of the 3′ untranslated sequence in the primary transcript. This alternative use of nucleotides is predicted to generate a different C-terminal tail for the protein. HEK293 cells transfected with GABABR2b and GABABR1a cDNAs inhibit adenylyl cyclase in response to GABA and baclofen (Ng et al., 1999b). Each GABABR transcript displays a remarkably similar expression pattern (Calver et al., 2000). Although no difference in expression or function has been detected for the reported GABABR2b or GABABR2c variants, it has been proposed that a difference in pharmacology may emerge from further research (Couve et al., 2000, Billinton et al., 2001).

The human GABABR1 gene on chromosome 6p21.3 (Grifa et al., 1998) is in the genetic locus for neurobehavioral disorders that include schizophrenia, juvenile myoclonic epilepsy and dyslexia. In addition, the human GABABR2 gene on chromosome 9q22.1 is a candidate gene for hereditary sensory neuropathy type 1 (Martin et al., 1999, Ng et al., 1999a). Our ability to determine whether these diseases involve a mutation in the GABABR genes is enhanced by an understanding of their genomic organization. This is based on the fact that disease mutations can arise in 5′ regulatory regions, at splice junctions, in introns as well as in coding sequences (Cooper and Mattox, 1997).

We have analyzed the sequence organization of the human and Drosophila GABABR2 genes and compared this organization to that of human GABABR1. This analysis provides a starting point for the examination of these genes as candidate disease genes and provides an analysis of the potential of the GABABR gene to encode distinct receptor isoforms. The presence of alternative GABABR transcripts, predicted from the GABABR genomic sequence analysis, was experimentally tested using human adult and fetal brain mRNA. Introns in the GABABR gene family were compared and used as markers to establish evolutionary relationships.

Section snippets

Human GABABR2

The GABABR2 genomic sequence was determined by direct sequencing of PAC genomic clones, PAC 16946 and PAC 16947 (Martin et al., 1999), and by computer searches of GenBank databases. PAC DNA was prepared using the KB-100 Magnum kit (Genome Systems, St. Louis, MO), sequenced with an Applied Biosystems 3773A DNA sequencer (Foster City, CA) and compiled using the program Sequencher (Gene Codes Corporation, Ann Arbor, MI). Sequence programs Seqed, Map and Gap were from the Genetics Computer Group

Organization of the human GABABR2 gene

We have shown that human GABABR2 maps to chromosome 9q22.1 (Martin et al., 1999). Here we report that an analysis of two PAC and five BAC genomic clones reveals the GABABR2 gene organization (Fig. 1). The PAC clones span 150 kb and include the sequence encoding the GABABR2 C-terminal but not N-terminal sequences. Five BAC clones that together include the entire GABABR2 transcript were derived from a GenBank database search (Fig. 1). The human GABABR2 gene spans greater than 350 kb and is split

Discussion

GABABRs are potential targets for therapeutic agents for the treatment of pain, depression, drug addiction, epilepsy, and the enhancement of cognition (Couve et al., 2000). Defining the GABABR transcripts and the proteins they encode is a necessary part of understanding GABABR pharmacology. In this report, we use computational analysis to examine the potential of human genomic GABABR sequences to encode human GABABR transcripts. The results provide support for the recently described GABABR1

Acknowledgements

This research was supported by grants from the National Institute of Alcohol Abuse and Alcoholism (NIAAA11697) and the National Institute of Child Health and Human Development (NICHHD22539).

References (38)

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