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

Maria Waldhoer,Alan Wise,Graeme Milligan,Michael Freissmuth,Christian Nanoff

doi:10.1074/jbc.274.43.30571

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 A(1)-adenosine receptor to that of Galpha(i-1) (A(1)/Galpha(i-1)) and of Galpha(o) (A(1)/Galpha(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 (-)-N(6)-3[(125)I](iodo-4-hydroxyphenylisopropyl)adenosine; these were similar in the wild-type receptor and the fusion proteins over the temperature range of 10 to 30 degrees C. Agonist dissociation may be limited by the stability of the ternary complex. This assumption was tested by creating fusion proteins in which the Cys(351) of Galpha(i-1) was replaced with glycine (A(1)/Galpha(i-1)C351G) or isoleucine (A(1)/Galpha(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.

Highlights

Signaling by G protein-coupled receptors is initiated by the formation of a ternary complex which consists of agonist (H),1 receptor (R), and G protein (G) [1, 2]; upon activation of the receptor by an agonist, the activated receptor (R*) associates with its cognate G protein(s)
High Affinity Agonist Binding to the A1-Adenosine Receptor in the Presence of Varying Amounts of rG␣i-1 Proteins—High affinity agonist binding to G protein-coupled receptors depends on the formation of a ternary complex HRG between agonist (H), receptor (R), and G protein (G) [1]
A fusion construct of the ␤2-adrenergic receptor and G␣s was originally shown to yield a functional protein capable of efficiently activating its prototypic effector adenylyl cyclase [33, 34]; this approach has more recently been extended to other receptor/G protein tandems and several aspects of receptor-G protein coupling have been investigated with these constructs (29, 35; for review, see Ref. 36)

Summary

The abbreviations used are

H, agonist; R and R*, inactive and active conformation, respectively, of a G protein-coupled receptor; G, G protein; [125I]HPIA, (Ϫ)-N6-3[125I](iodo-4-hydroxyphenylisopropyl)adenosine; CHAPS, 3-[3-cholaminpropyl)dimethylammonio]-1-propanesulfonic acid; CPA, N6-cyclopentyladenosine; A1/G␣i-1 and A1/G␣o, fusion proteins composed of the human A1-adenosine receptor and G␣i-1 or G␣o; DPCPX, 8-cyclopentyl-1,3-[3H]dipropylxanthine

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION