Regulation of Agonist-Receptor Binding by G Proteins and Divalent Cations in Spermatozoa Solubilized A1 Adenosine Receptors

Abstract

Solubilized A1 adenosine receptor (A1AR) was used to investigate the effect of several cations on agonist-binding characteristics and GTP hydrolysis. It was shown by Western blot with Gβ-M14 that this preparation contains both G proteins and receptor. The role of the receptor molecule is to facilitate the activation of G proteins as α-GTP complex, and GTP hydrolysis has important consequences for the basic deactivation mechanism. Divalent cations, such as Mn2+, Ca2+, and Mg2+, potentiated the agonist-specific binding: Mn2+had the highest apparent affinity with half-maximal effect at 50 μM. Binding assays, performed in the presence of 100 μM Mn2+, showed an increase in the apparent affinity of the binding sites, whereas, in the presence of 1 mM Mg2+, significant alteration of the apparent affinity, but not of the number of sites, was detected. Concentrations of 1 mM Mg2+and 100 μM Mn2+enhanced GTPase activity, whereas 5 mM Ca2+resulted in the increase ofVmaxvalues without significant alterations ofKm. In the presence of A1-specific agonists, Mn2+and Mg2+caused a decrease ofVmaxvalues and an increase of GTP affinity. Other cations, such as Co2+, Cd2+, Cu2+, and Zn2+, inhibited the binding capacity but caused almost no changes in GTP hydrolysis kinetics.