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J Biol Chem, Vol. 274, Issue 43, 30571-30579, October 22, 1999

Kinetics of Ternary Complex Formation with Fusion Proteins Composed of the A₁-Adenosine Receptor and G Protein -Subunits

Maria Waldhoer, Alan Wise, Graeme Milligan^§, Michael Freissmuth, and Christian Nanoff

From the Institute of Pharmacology, University of Vienna, Währinger Straße 13a, A-1090 Vienna, Austria, the  Receptor Systems Unit, Glaxo-Wellcome Research and Development, Stevenage, Hertfordshire SG1 2NY, United Kingdom, and the ^§ Institute of Biomedical & Life Sciences, Glasgow University, Glasgow G128QQ, United Kingdom

High affinity agonist binding to G protein-coupled receptors depends on the formation of a ternary complex between agonist, receptor, and G protein. This process is too slow to be accounted for by a simple diffusion-controlled mechanism. We have tested if the interaction between activated receptor and G protein is rate-limiting by fusing the coding sequence of the human A₁-adenosine receptor to that of G_i-1 (A₁/G_i-1) and of G_o (A₁/G_o). Fusion proteins of the expected molecular mass were detected following transfection of HEK293 cells. Ternary complex formation was monitored by determining the kinetics for binding of the high affinity agonist ()-N⁶-3[¹²⁵I](iodo-4-hydroxyphenylisopropyl)adenosine; these were similar in the wild-type receptor and the fusion proteins over the temperature range of 10 to 30 °C. Agonist dissociation may be limited by the stability of the ternary complex. This assumption was tested by creating fusion proteins in which the Cys³⁵¹ of G_i-1 was replaced with glycine (A₁/G_i-1C351G) or isoleucine (A₁/G_i-1C351I) to lower the affinity of the receptor for the G protein. In these mutated fusion proteins, the dissociation rate of the ternary complex was accelerated; in contrast, the rate of the forward reaction was not affected. We therefore conclude that (i) receptor activation per se rather than its interaction with the G protein is rate-limiting in ternary complex formation; (ii) the stability of the ternary complex is determined by the dissociation rate of the G protein. These features provide for a kinetic proofreading mechanism that sustains the fidelity of receptor-G protein coupling.

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