Intron 4 containing novel GABAB1 isoforms impair GABAB receptor function.
BACKGROUND
Gamma-aminobutyric acid type B (GABAB) receptors decrease neural activity through G protein signaling. There are two subunits, GABAB1 and GABAB2. Alternative splicing provides GABAB1 with structural and functional diversity. cDNA microarrays showed strong signals from human brain RNA using GABAB1 intron 4 region probes. Therefore, we predicted the existence of novel splice variants.


RESULTS
Based on the probe sequence analysis, we proposed two possible splice variants, GABAB1j and GABAB1k. The existence of human GABAB1j was verified by quantitative real-time PCR, and mouse GABAB1j was found from a microarray probe set based on human GABAB1j sequence. GABAB1j open reading frames (ORF) and expression patterns are not conserved across species, and they do not have any important functional domains except sushi domains. Thus, we focused on another possible splice variant, GABAB1k. After obtaining PCR evidence for GABAB1k existence from human, mouse, and rat, it was cloned from human and mouse by PCR along with three additional isoforms, GABAB1l, GABAB1m, and GABAB1n. Their expression levels by quantitative real-time PCR are relatively low in brain although they may be expressed in specific cell types. GABAB1l and GABAB1m inhibit GABAB receptor-induced G protein-activated inwardly rectifying K(+) channel (GIRK) currents at Xenopus oocyte two-electrode voltage clamp system.


CONCLUSIONS
This study supports previous suggestions that intron 4 of GABAB1 gene is a frequent splicing spot across species. Like GABAB1e, GABAB1l and GABAB1m do not have transmembrane domains but have a dimerization motif. So, they also could be secreted and bind GABAB2 dominantly instead of GABAB1a. However, only GABAB1l and GABAB1m are N- and C-terminal truncated splicing variants and impair receptor function. This suggests that the intron 4 containing N-terminal truncation is necessary for the inhibitory action of the new splice variants.
Section of Neurobiology and Institute for Cellular and Molecular Biology, Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Austin, Texas, USA.
Changhoon
C
Lee
R Dayne
RD
Mayfield
R Adron
RA
Harris
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005260:Female; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000235:genetics / Q000502:physiology; D020869:Gene Expression Profiling; D006801:Humans; D007438:Introns / Q000235:genetics*; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D009865:Oocytes / Q000502:physiology; D020033:Protein Isoforms / Q000235:genetics / Q000502:physiology; D051381:Rats; D018080:Receptors, GABA-B / Q000235:genetics* / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D014981:Xenopus; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D020033:Protein Isoforms; D018080:Receptors, GABA-B; D005680:gamma-Aminobutyric Acid