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J. Biol. Chem., Vol. 279, Issue 14, 13514-13521, April 2, 2004

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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^*

Peter C. Simons, Sean M. Biggs, Anna Waller, Terry Foutz, Daniel F. Cimino, Qing Guo¶, Richard R. Neubig||, Wei-Jen Tang¶, Eric R. Prossnitz, and Larry A. Sklar**

From the Cancer Center, Department of Pathology and Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131, ^¶Ben-May Cancer Research Institute, University of Chicago, Chicago, Illinois, 60637, and ^||University of Michigan School of Medicine, Ann Arbor, Michigan 48109

We developed a novel and generalized approach to investigate G protein-coupled receptor molecular assemblies. We solubilized a fusion protein consisting of the ₂-adrenergic receptor and green fluorescent protein (GFP) for bead-based flow cytometric analysis. ₂-Adrenergic receptor GFP bound to dihydroalprenolol-conjugated beads, providing a K_d for the fusion protein and, in competition with 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 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.

Received for publication, September 17, 2003 , and in revised form, December 22, 2003.

^* This work was supported by National Institutes of Health Grants GM60799 and EB00264 (to L. A. S.), HL-476417 (to R. R. N.), and GM53459 (to W.-J. Tang). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed. Tel.: 505-272-6892; Fax: 505-272-6995; E-mail: lsklar{at}salud.unm.edu.

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