Agonist-induced isomerization of the alpha 1-adrenergic receptor: kinetic analysis using broken-cell and solubilized preparations.

Abstract

The affinity of agonists but not antagonists at hepatic membrane alpha 1-adrenergic receptors is temperature dependent; a 100-fold higher affinity is observed at 4 degrees C than at 37 degrees C. The relationship between these two agonist affinity states was investigated by using a strategy that allows the kinetics of this transition to be examined under equilibrium conditions. When competition assays are performed at 37 degrees C for varying intervals and the reaction mixture is then rapidly cooled by freezing, allowed to thaw, and further equilibrated at 4 degrees C, a rapid and progressive decrease (t1/2 of 1-2 min) in agonist affinity occurs, the extent of which is directly related to the incubation time at 37 degrees C. This decrease in agonist affinity is sustained as long as agonist is present but can be reversed by its subsequent removal. In contrast, no change in affinity is seen in identical experiments when antagonists are employed as the competing ligand. High-affinity binding of agonists is also demonstrated in short-term nonequilibrium experiments, indicating that the low-temperature incubations do not induce, but rather stabilize, a receptor conformation of high affinity for agonists. These findings suggest that the predominantly low-affinity binding of agonists to alpha 1-adrenergic receptors demonstrated in equilibrium studies at physiological temperatures may be the result of a ligand-driven decrease in affinity. Since the transition in receptor affinity for agonists occurs not only in broken-cell preparations but also after detergent solubilization of the membrane receptor, it most likely is due to an agonist-induced change in the conformation of the receptor protein per se.(ABSTRACT TRUNCATED AT 250 WORDS)