Subunit stoichiometry of oligomeric membrane proteins: GABAA receptors isolated by selective immunoprecipitation from the cell surface.
GABAA receptors are hetero-oligomeric proteins of unknown subunit stoichiometry. In this study alpha 1 beta 3 GABAA receptor channels were functionally expressed in Xenopus oocytes. Direct immunoprecipitation from the oocyte surface was used to exclusively isolate mature GABAA receptors. The subunit ratio was determined by quantitation of the amount of [35S]methionine incorporated into individual receptor subunits. Antibody released from the antigen or antibody not reacted was prevented from reassociation with labeled antigen by addition of excess unlabeled antigen. Variation of the alpha 1 beta 3 ratio of injected cRNAs only slightly affected the subunit ratio in mature receptors. This indicates that the subunit stoichiometry generated is independent of the pools of newly synthesized subunit monomers and supports the view that the receptor assembly is a regulated process. The ratio of alpha 1/beta 3 subunits was found to be 1.1 +/- 0.1 (SEM, n = 6). Our data are in best agreement with a tetrameric receptor with the composition 2 alpha 2 beta. For a pentameric receptor the ratio found slightly favors a receptor with the composition 3 alpha 2 beta. The method developed here is applicable to the determination of the subunit stoichiometry of other recombinant oligomeric membrane proteins.
Kellenberger
S
S
Department of Pharmacology, University of Bern, Switzerland.
Eckenstein
S
S
Baur
R
R
Malherbe
P
P
Buhr
A
A
Sigel
E
E
D000818:Animals; D000911:Antibodies, Monoclonal; D001345:Autoradiography; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis*; D008715:Methionine / Q000378:metabolism; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D011233:Precipitin Tests; D011963:Receptors, GABA-A / Q000096:biosynthesis*; D014982:Xenopus laevis
D000911:Antibodies, Monoclonal; D008565:Membrane Proteins; D011963:Receptors, GABA-A; D008715:Methionine