Determination of the minimal functional ligand-binding domain of the GABAB1b receptor.
In the mammalian central nervous system, slow inhibitory neurotransmission is largely mediated by metabotropic GABA(B) receptors (where GABA stands for gamma-aminobutyric acid), which belong to the G-protein-coupled receptor gene family. Functional GABA(B) receptors are assembled from two subunits GABA(B1) (GABA(B) receptor subtype 1) and GABA(B2). For the GABA(B1) subunit, which binds the neurotransmitter GABA, two variants GABA(B1a) (GABA(B) receptor subtype 1 variant a) and GABA(B1b) have been identified. They differ at the very N-terminus of their large glycosylated ECD (extracellular domain). To simplify the structural characterization, we designed truncated GABA(B1) receptors to identify the minimal functional domain which still binds a competitive radioligand and leads to a functional, GABA-responding receptor when co-expressed with GABA(B2). We show that it is necessary to include all the portion of the ECD encoded by exon 6 to exon 14. Furthermore, we studied mutant GABA(B1b) receptors, in which single or all potential N-glycosylation sites are removed. The absence of oligosaccharides does not impair receptor function, suggesting that the unglycosylated ECD of GABA(B1) can be used for further functional or structural investigations.
Institute of Biochemistry, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Daniela
D
Deriu
Martin
M
Gassmann
Susan
S
Firbank
Dorothee
D
Ristig
Christina
C
Lampert
Johannes
J
Mosbacher
Wolfgang
W
Froestl
Klemens
K
Kaupmann
Bernhard
B
Bettler
Markus G
MG
Grütter
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D019281:Dimerization; D005091:Exons / Q000235:genetics; D058786:GABA-B Receptor Agonists; D006031:Glycosylation; D006801:Humans; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D051381:Rats; D018080:Receptors, GABA-B / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017384:Sequence Deletion / Q000235:genetics; D015398:Signal Transduction
D058786:GABA-B Receptor Agonists; D008024:Ligands; D018080:Receptors, GABA-B