Interaction of the D_2short dopamine receptor with G proteins: analysis of receptor/G protein selectivity

Abstract

The human D_2short (D_2S) dopamine receptor has been expressed together with the G proteins Gi2 and Go in insect cells using the baculovirus system. Levels of receptor were determined using [${}^3\text{H}$]spiperone binding. Levels of G protein heterotrimer were determined using quantitative Western blot and using [${}^{35}\text{S}$]GTPγS saturation binding experiments. Levels of the receptor and G protein and the receptor/G protein ratio were similar in the two preparations. Stimulation of [${}^{35}\text{S}$]GTPγS binding by a range of agonists occurred with higher relative efficacy and in some cases higher potency in the preparation expressing Go, indicating that interaction of the D_2S receptor is more efficient with this G protein. The effects of various G protein-selective agents on 10,11-dihydroxy-N-n-propylnorapomorphine ([${}^3\text{H}$]NPA) binding were used to examine the receptor/G protein complex in the two preparations. Suramin inhibited [${}^3\text{H}$]NPA binding with slightly higher potency in the Gi2 preparation, whereas GppNHp inhibited [${}^3\text{H}$]NPA binding with greater potency (∼6-fold) in the Go preparation. This may imply that the G protein is more readily activated in the D_2S/Go preparation. [${}^3\text{H}$]Spiperone binding occurred with an increased B_max in the presence of suramin in the Go preparation but not in the Gi2 preparation, suggesting a higher affinity interaction between the free receptor and this G protein. It is concluded that the higher efficiency activation of Go by the D_2S receptor may be a function of higher affinity receptor/G protein interaction as well as a greater ability to activate the G protein.

Introduction

The interaction of the receptor and G protein is a critical event in the mechanism of agonist action at G protein coupled-receptors (GPCRs). Agonist activation of GPCRs is often described using the ternary complex or extended ternary complex models [1], [2]. In the extended ternary complex model, the receptor is proposed to exist in two forms, the ground state R and a partially activated state R^∗. R^∗ is able to couple more efficiently to the G protein to form the fully active species R^∗G. Agonists (A) stabilise the R^∗ and R^∗G forms of the receptor leading to the formation of the ternary complex AR^∗G. In the activated forms of the receptor (R^∗G and AR^∗G), the exchange of GTP for GDP occurs on the α subunit and once GTP is bound, both the α and the βγ subunits are released to alter the activity of effectors. The specific determinants of agonist efficacy and potency are, however, still unknown.

Some receptors have been shown to interact with multiple G proteins [3]. It has been proposed that, when a receptor interacts with more than one G protein, different agonists may have the ability to induce differentially activated forms of a receptor which have the ability to selectively activate one G protein over another. This concept has been termed agonist-induced receptor trafficking [4]. Evidence for this phenomenon has been demonstrated in a number of systems, including the 5-HT_2C receptor [5] and the Drosophila octopamine/tyramine receptor [6]. The introduction of compounds that disrupt the interaction of receptors and G proteins [3] may allow for selective disruption of signalling cascades.

Receptors for the neurotransmitter, dopamine, constitute a family of GPCRs that can be divided into D₁-like (D₁, D₅) and D₂-like (D₂, D₃, D₄) receptor subtypes [7]. The D₂ dopamine receptor is an important site of action for the antipsychotic and anti-Parkinsonian drugs and has been shown to interact with different members of the Gi/o protein family to influence different signalling events. Signalling of the D₂ receptor through Gi2 leads to the inhibition of adenylyl cyclase [8], [9], whereas signalling through Go leads to the inhibition of calcium channels [10]. A clear definition of the selectivity of the D₂ receptor for one G protein over another has, however, not yet emerged. The D₂ dopamine receptor also exists in short and long isoforms (D_2S and D_2long (D_2L)) generated by alternative splicing [11], [12]. There are also indications that the two isoforms may have different functions [13] and couple differentially to G proteins [14], [15], but the precise selectivity has not been defined.

We studied the interaction of rat D_2L with the G proteins Go and Gi2 by expression in insect cells using the baculovirus system and showed a stronger interaction between the D_2L receptor and Go [16]. The baculovirus expression system is an ideal system for the study of interaction of D₂ receptors and G proteins as it does not contain endogenous dopamine receptors and there is minimal interaction between the receptor and the endogenous G proteins of the insect cells [17], [18]. In both our previous [16] and the present study, we determined the receptor/G protein ratio in the membranes following infection as this may affect the potency and efficacy of agonists and inverse agonists [19], [20], [21].

In the present study, the human dopamine D_2S receptor has been co-expressed with G proteins Go and Gi2 in insect cells in order to examine the specificity of receptor/G protein interaction for this isoform. The potency and efficacy of a range of dopaminergic agonists has been measured using the stimulation of [${}^{35}\text{S}$]GTPγS binding. Our data suggest that the human dopamine D_2S receptor couples preferentially to the G protein Go over the G protein Gi2.