Monospecific antibodies as probes for the stoichiometry of recombinant GABA(A) receptors.
GABA(A) receptors composed of alpha1beta3 gamma2 and alpha1beta3 subunits were expressed in insect Sf9 cells and solubilized in 1% Triton X100. In sucrose density gradients, [3H]-Ro15-1788 binding activity, in the case of alpha1beta3 gamma2, and [3H]-muscimol binding activity, in the case of alpha1beta3 containing receptors sedimented as a single sharp peak suggesting the formation of receptors containing a defined number of subunits. When alpha1beta3gamma2 -containing receptors were incubated with an alpha-subunit specific antibody (bd24), a single class of antibody receptor complex was formed irrespective of the receptor-antibody ratio. This is consistent with two alpha subunits cross-linked within the receptor by the antibody. Similar results were obtained using a beta-subunit specific antibody (bd17). Several classes of antibody-receptor complex were formed when receptors were pre-incubated with a gamma specific antibody (anti gamma(2) 1-15 Cys). This profile is consistent with the presence of a single gamma subunit in each complex. Experiments with alpha1beta3 subunit containing receptors and antibody bd24 produced a profile similar to that seen with alpha1beta3 gamma2 receptors, consistent with two alpha subunits per receptor complex. In this case, the anti-beta subunit antibody, bd17, produced a unique and complex profile consistent with three beta subunits per receptor. This method permits the rapid determination of subunit stoichiometries of homogeneous receptor populations
Knight
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AR
Neurogen Corporation, 35 Northeast Industrial Rd, Branford CT 06405, USA. t.knight@cerebrus.ltd.uk
Stephenson
F A
FA
Tallman
J F
JF
Ramabahdran
T V
TV
D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D000936:Antigen-Antibody Complex; D000939:Epitopes; D005442:Flumazenil / Q000494:pharmacology; D018755:GABA Agonists / Q000494:pharmacology; D006801:Humans; D015335:Molecular Probes; D009118:Muscimol / Q000494:pharmacology; D011487:Protein Conformation; D021122:Protein Subunits; D011963:Receptors, GABA-A / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D018755:GABA Agonists; D015335:Molecular Probes; D021122:Protein Subunits; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D009118:Muscimol; D005442:Flumazenil