Comparison of in vitro binding properties of a series of dopamine antagonists and agonists for cloned human dopamine D_2S and D_2L receptors and for D₂ receptors in rat striatal and mesolimbic tissues, using [¹²⁵I] 2′-iodospiperone

Abstract

We investigated the ligand binding properties in vitro of two splice variants of the cloned human dopamine D₂ receptor (the 443 and 414 amino acids long forms called D_2L and D_2S, respectively), expressed in 293 human kidney cells, in comparison with those of the dopamine D₂ receptors in rat striatum, nucleus accumbens and tuberculum olfactorium. The new radioligand, [¹²⁵I]2′-iodospiperone, showed a similar high binding affinity (K_D:0.056–0.122 nM) for cloned human D_2S and D_2L receptors and for the D₂ receptors in the three rat brain areas. Binding affinities of 25 dopamine antagonists and of 10 dopamine agonists belonging to different chemical classes were measured. The IC₅₀ values of the antagonists were virtually identical in the five preparations: spiperone was the most potent compound (pIC₅₀ ≲ 9.9), remoxipride the least potent one (pIC₅₀ ≲ 5.7). The agonists showed similar IC₅₀ values for the cloned human D_2S and D_2L receptors but their affinity for rat brain D₂ receptors was 2- to 5-fold higher. Dopamine showed shallow inhibition curves, the high affinity binding was 10-fold lower for the cloned human D₂ receptors than for the rat brain D₂ receptors. Addition of stable guanosine-5′-triphosphate (GTP) analogues shifted the D₂ receptors in the rat brain tissues to the “low” affinity state, the low affinity binding of dopamine was equal to the affinity for the cloned human receptor. None of the dopamine antagonists or agonists could differentiate between the two splice forms of the cloned human D₂ receptors or between the D₂ receptors in rat striatal and mesolimbic tissues. The lower apparent affinity of some agonists and of dopamine in the absence of stable GTP analogues suggests a less appropriate receptor G-protein coupling for the cloned human D₂ receptors expressed in the 293 human kidney cells. Unexpectedly, guanosine-5′-O-(3-thiotriphosphate) (GTP-γ-S) reduced the [¹²⁵I]2′-iodospiperone binding to the D₂ receptors by 20–35% in the rat brain tissues and the cloned human D_2L receptor, and by 75% to the cloned human D_2S receptor. The inhibition in the last case could be prevented partly by submicromolar concentrations of dopamine. The GTP-γ-S effect is suggested to be due to reduction of disulphide bonds in the receptor. Recent molecular modelling studies indicated an important role of the disulphide bridge between Cys¹⁰⁷ at the start of transmembrane domain three and Cys¹⁸² in the third extracellular loop, for the binding of dopamine to the D₂ receptor.