Mechanism of agonist and antagonist binding to .alpha.2 adrenergic receptors: evidence for a precoupled receptor-guanine nucleotide protein complex

Publisher Website
Google Scholar
Abstract
The .alpha.2 adrenergic receptor (AR) inhibits adenylate cyclase via an interaction with Ni, a guanine nucleotide binding protein. The early steps involved in the activation of the .alpha.2 AR by agonists and the subsequent interaction with Ni are poorly understood. In order to better characterize these processes, we have studied the kinetics of ligand binding to the .alpha.2 AR in human platelet membranes on the second time scale. Binding of the .alpha.2 antagonist [3H]yohimbine was formally consistent with a simple bimolecular reaction mechanism with an association rate constant of 2.5 .times. 105 M-1 s-1 and a dissociation rate constant of 1.11 .times. 10-3 s-1. The low association rate constant suggests that this is not a diffusion-limited reaction. Equilibrium binding of the .alpha.2 adrenergic full agonist [3H]UK 14304 was characterized by two binding affinities: Kd1 = 0.3-0.6 nM and Kd2 = 10 nM. The high-affinity binding corresponds to approximately 65% and the low-affinity binding to 35% of the total binding. The kinetics of binding of [3H]UK 14304 were complex and not consistent with a mass action interaction at one or more independent binding sites. The dependence of the kinetics of [3H]UK 14304 concentration revealed a fast phase with an apparent bimolecular reaction constant k+ of 5 .times. 106 M-1 s-1. The rate constants and amplitudes of the slow phase of agonist binding were relatively independent of ligand concentration. These results were analyzed quantitatively according to several variants of the "ternary complex" binding mechanism. In the model which best accounted for the data, (1) approximately one-third of the .alpha.2 adrenergic receptor binds agonist with low affinity and is unable to couple with a guanine nucleotide binding protein (N protein), (2) approximately one-third is coupled to the N protein prior to agonist binding, and (3) the remainder interacts by a diffusional coupling of the .alpha.2 AR with the N protein or a slow, ligand-independent conformational change of the .alpha.2 AR-N protein complex. The rates of interaction of liganded and unliganded receptor with N protein are estimated.