Cloning and characterization of a GABAA receptor gamma2 subunit variant.
We have cloned a novel gamma-aminobutyric acid type A (GABAA) receptor gamma2 subunit variant named gamma2XL. gamma2XL contains an alternatively spliced exon, resulting in the addition of 40 amino acids to the N-terminal extracellular domain between Ser171 and Tyr172. We show that gamma2XL failed to localize to the cell surface when it was coexpressed with the alpha2 and beta1 subunits in human embryonic kidney 293 cells. Expression of gamma2XL in 293 cells suppressed GABAA receptor binding in a dose-dependent manner by preventing GABAA receptor cell-surface localization. We also generated a gamma2 mutant with Ser171 and Tyr172 converted to glycine and threonine, respectively. We demonstrate that this mutant has a significantly lower affinity for the alpha2 and beta1 subunits and failed to reach the cell surface when coexpressed with these subunits. Together, our results indicate that Ser171 and Tyr172 in the gamma2 subunit constitute a critical motif. When this motif is disrupted by insertion of the alternative exon, access of the gamma2 subunit to the cell surface is prevented.
Jin
Pei
P
Incyte Corporation, Palo Alto, California 94304, USA. pjin@incyte.com
Zhang
Juan
J
Rowe-Teeter
Courtney
C
Yang
Junming
J
Stuve
Laura L
LL
Fu
Glenn K
GK
D017398:Alternative Splicing; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D005091:Exons; D005998:Glycine / Q000737:chemistry; D006801:Humans; D007150:Immunohistochemistry; D007438:Introns; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011963:Receptors, GABA-A / Q000737:chemistry* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D013912:Threonine / Q000737:chemistry; D014018:Tissue Distribution; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry
C546279:GABRG2 protein, human; D011506:Proteins; D012333:RNA, Messenger; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D013912:Threonine; D014443:Tyrosine; D012694:Serine; D005998:Glycine