Kinetics of ternary complex formation with fusion proteins composed of the A1-adenosine receptor and G protein α-subunits

Abstract

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 Gα(i-1) (A1/Gα(i-1) and of Gα(o) (A1/Gα(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 Gα(i-1) was replaced with glycine (A1/Gα(i-1)C351G) or isoleucine (A1/Gα(i-1)C351I) 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.