Alternate use of distinct intersubunit contacts controls GABAA receptor assembly and stoichiometry.
GABA(A) receptors are the major inhibitory transmitter receptors in the CNS. Recombinant GABA(A) receptors composed of alpha(1)beta(3)gamma(2) subunits have been demonstrated to assemble as pentamers consisting of two alpha(1), two beta(3), and one gamma(2) subunit. Using truncated and chimeric alpha(1) subunits, we identified the alpha(1)(80-100) sequence as a major binding site for gamma(2) subunits. In addition, we demonstrated its direct interaction with gamma(2)(91-104), a sequence that previously has been identified to form the contact to alpha(1) subunits. The observation that the amino acid residues alpha(1)P96 and alpha(1)H101, which can be photolabeled by [(3)H]flunitrazepam, are located within or adjacent to the alpha(1)(80-100) sequence, indicates that the benzodiazepine binding site of GABA(A) receptors is located close to this intersubunit contact. The observation that alpha(1)(80-100) interacts with gamma(2) but not with beta(3) subunits indicates the existence of an additional beta(3) binding site on alpha(1) subunits. The preferred alternate use of the gamma(2) and beta(3) binding sites in two different alpha(1) subunits of the same receptor ensures the incorporation of only a single gamma(2) subunit and thus, determines subunit stoichiometry of alpha(1)beta(3)gamma(2) receptors. Distinct binding sites and their alternate use can therefore explain how subunits of hetero-oligomeric transmembrane proteins assemble into a defined protein complex.
Klausberger
T
T
Section of Biochemical Psychiatry, University Clinic for Psychiatry, Institute for Theoretical Chemistry Divisions of Biochemistry, Brain Research Institute, University of Vienna, A-1090 Vienna, Austria.
Sarto
I
I
Ehya
N
N
Fuchs
K
K
Furtmuller
R
R
Mayer
B
B
Huck
S
S
Sieghart
W
W
D000595:Amino Acid Sequence / Q000502:physiology; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008970:Molecular Weight; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding / Q000502:physiology; D021122:Protein Subunits; D051381:Rats; D011963:Receptors, GABA-A / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship; D014162:Transfection
D010446:Peptide Fragments; D021122:Protein Subunits; D011963:Receptors, GABA-A; D011994:Recombinant Proteins