Pharmacological analysis of dopamine stimulation of [35S]‐GTPγS binding via human D2short and D2long dopamine receptors expressed in recombinant cells

Abstract

1 The activation of G-proteins by agonist-occupied D₂ or D₃ dopamine receptors in membranes from recombinant cells expressing the cloned receptors has been analysed by a [³⁵S]-guanosine 5′-[γ-thio] triphosphate ([³⁵S]-GTPγS) binding assay. 2 The rate of [³⁵S]-GTPγS binding was increased by dopamine in a dose-dependent manner in membranes from CHO cells stably expressing either the D_2short or D_2long dopamine receptor. 3 The dopamine-induced stimulation of [³⁵S]-GTPγS binding could be inhibited by a range of antagonists. Affinities for antagonists derived from the inhibition of the dopamine stimulation of [³⁵S]-GTPγS binding correlated very well with affinities derived from radioligand binding studies. 4 When the maximum [³⁵S]-GTPγS binding responses stimulated by dopamine acting at different receptor subtypes were compared, there was a tendency for the stimulation via the D_2short receptor to be greater than via the D_2long receptor and for the stimulation via the D₃ dopamine receptor to be less than for either D₂ receptor. These differences in maximal response were also seen when the inhibitory effects of dopamine on adenylyl cyclase via the three receptor subtypes were compared. 5 The stimulation of [³⁵S]-GTPγS binding by dopamine in membranes from recombinant cells therefore provides an excellent system for studying the molecular nature of agonism and the receptor/G-protein interactions for these receptors.