Real-time analysis of ternary complex on particles: direct evidence for partial agonism at the agonist-receptor-G protein complex assembly step of signal transduction.
We developed a novel and generalized approach to investigate G protein-coupled receptor molecular assemblies. We solubilized a fusion protein consisting of the beta(2)-adrenergic receptor and green fluorescent protein (GFP) for bead-based flow cytometric analysis. beta(2)-Adrenergic receptor GFP bound to dihydroalprenolol-conjugated beads, providing a K(d) for the fusion protein and, in competition with beta(2)-adrenergic receptor ligands, K(d) values for agonists and antagonists. Beads displaying chelated nickel bound purified hexahistidine-tagged G protein heterotrimers and, subsequently, the binary complex of agonist with beta(2)-adrenergic receptor GFP. The dose-response curves of ternary complex formation revealed maximal assembly for ligands previously classified as full agonists and reduced assembly for ligands previously classified as partial agonists. Guanosine 5'-3-O-(thio)triphosphate-induced dissociation rates of the ternary complex were the same for full and partial agonists. Soluble G protein, competing with ternary complexes on beads provided an affinity estimate of agonist-receptor complexes to G protein. When performed simultaneously, the two assemblies discriminated between agonist, antagonist or inactive molecule in a manner appropriate for high throughput, small volume drug discovery. The assemblies can be further generalized to other G protein coupled receptor protein-protein interactions.
Simons
Peter C
PC
Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131, USA.
Biggs
Sean M
SM
Waller
Anna
A
Foutz
Terry
T
Cimino
Daniel F
DF
Guo
Qing
Q
Neubig
Richard R
RR
Tang
Wei-Jen
WJ
Prossnitz
Eric R
ER
Sklar
Larry A
LA
D058666:Adrenergic beta-2 Receptor Agonists; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D003199:Computer Systems; D004082:Dihydroalprenolol / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008164:Luminescent Proteins / Q000235:genetics; D008863:Microspheres; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D020298:U937 Cells
D058666:Adrenergic beta-2 Receptor Agonists; D000318:Adrenergic beta-Agonists; D008024:Ligands; D008164:Luminescent Proteins; D018343:Receptors, Adrenergic, beta-2; D049452:Green Fluorescent Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004082:Dihydroalprenolol; D019204:GTP-Binding Proteins