Elsevier

Neuropharmacology

Volume 47, Issue 6, November 2004, Pages 860-872
Neuropharmacology

The influence of G protein subtype on agonist action at D2 dopamine receptors

https://doi.org/10.1016/j.neuropharm.2004.06.010Get rights and content

Abstract

In previous studies, we have shown that agonists influence the ability of D2 dopamine receptors to couple to G proteins and here we extend this work. The human D2Short dopamine receptor and a natural polymorphism of this D2Short(Ser311Cys), have been studied by co-expressing the receptors in insect cells with Gβ1γ2 and either Gαo, Gαi1, Gαi2 or Gαi3 G protein subunits. These preparations have been used to study the G protein coupling profiles of the two receptors and the influence of agonists. Receptor/G protein coupling was analysed in dopamine/[3H]spiperone competition binding experiments and through stimulation of [35S]GTPγS binding. Although the Ser311Cys polymorphism itself had no appreciable effect on the G protein coupling specificity of the D2 receptor, agonist stimulation of [35S]GTPγS binding, revealed that both dopamine and (+)-3PPP showed a clear preference for Gαo compared to the Gαi subtypes, but quinpirole did not. These results indicate that agonists are able to stabilise different receptor conformations with different abilities to couple to G proteins.

Introduction

The neurotransmitter, dopamine exerts its actions by binding to G protein-coupled receptors (GPCRs). Dopamine receptors are divided into two subclasses; the D1-like, which includes D1 and D5 receptors, and the D2-like, which includes D2, D3, and D4 receptors. In addition, the D2 receptor exists as two splice variants of the same gene, D2L and D2S, which differ by a 29 amino acid insert in the third intracellular loop of D2L (Grandy et al., 1989, Giros et al., 1989). Agonist binding to D2 receptors results in the activation of several different signalling pathways including inhibition of adenylyl cyclase (Hall and Strange, 1997) and modulation of both calcium channels (Liu et al., 1994) and potassium channels (Liu et al., 1999). The pertussis toxin-sensitive nature of the majority of these responses leads to the conclusion that these responses are mediated by G proteins in the Gi/o family.

The concept of agonist-induced receptor trafficking (Kenakin, 1995) describes the phenomenon of different agonists being able to stabilise different conformations of the same receptor which have the ability to selectively activate one G protein over another. Evidence for this phenomenon has come from a number of systems including the 5-HT2C receptor (Berg et al., 1998), the muscarinic acetylcholine receptor (Akam et al., 2001) and the β2 adrenoceptor (Wenzel-Seifert and Seifert, 2000), see also Kenakin (2002) and Hermans (2003) for reviews of the topic. Evidence suggesting agonist trafficking at the D2 dopamine receptor first came from the observation that the agonists (+)-3PPP and quinpirole exhibited a reversal of relative efficacy between D2 receptors in different locations (striatum and pituitary gland) (Meller et al., 1992). More recently, we have demonstrated that the rat D2L receptor preferentially couples to Gαo over Gαi2 (Cordeaux et al., 2001), as does the human D2S receptor (Nickolls and Strange, 2003) and that the human D2L receptor preferentially couples to Gαo over each of the three Gαi subtypes (Gazi et al., 2003). Additional reports (Watts et al., 1998, Kilts et al., 2002, Shapiro et al., 2003) also support the idea that D2 dopamine receptor agonists can have specific effects on signalling systems.

It has been proposed that polymorphisms in the gene encoding the D2 dopamine receptor could underlie the aetiology of a number of pathologies including alcoholism, substance dependence, and schizophrenia. Three polymorphic variants of the D2 receptor have been identified in the coding region, encoding the amino acid substitutions Val96Ala, Pro310Ser and Ser311Cys (D2Cys311) (Gejman et al., 1994). Previously, the D2Cys311 receptor has been reported to exhibit a decreased ability to mediate the inhibition of adenylyl cyclase (Cravchik et al., 1996). The amino acid substitution in the D2Cys311 receptor occurs in the third intracellular loop of the D2 receptor. As the third intracellular loop is an important region for determining the coupling of GPCRs to G proteins (Strader et al., 1987), the reduced ability of D2Cys311 to inhibit adenylyl cyclase could, therefore, be related to impaired coupling to G proteins.

In the present study, we have compared the coupling of D2S and D2SCys311 receptor variants to different G proteins by co-expression with the G protein subunits β1γ2 and either αo, αi1, αi2 and αi3 in insect cells. The functional coupling of the receptors to these G proteins has been investigated using [35S]GTPγS binding assays and dopamine/[3H]spiperone competition binding assays. This is the first time that the coupling specificity of the D2S receptor for different G proteins has been investigated thoroughly and the first time that the G protein coupling specificity of the D2SCys311 receptor has been investigated.

Section snippets

Materials

[Phenyl-4-3H]-spiperone (0.5–1.1 TBq mmol−1) was obtained from Amersham (Amersham, Bucks, UK) and [35S]-GTPγS (37–55 TBq mmol−1) was obtained from NEN DuPont (Stevenage, Herts, UK) All other chemicals were from Sigma (Poole, Dorset, UK). Oligonucleotides were synthesised and desalted by Invitrogen (Carlsbad, CA, USA). The baculoviruses expressing the human G protein subunits αo, αi1, αi2, αi3, β1 and γ2 were generously donated by Dr. T Kozasa (University of Illinois, Chicago, USA).

Construction of baculovirus expressing epitope tagged D2 receptors

The

Results

In order to examine the ability of D2S and D2SCys311 to activate the G protein subunits, αo, αi1, αi2 and αi3, each of the receptors were (a) expressed alone, (b) co-expressed with β1γ2 G protein subunits and (c) co-expressed with β1γ2 G protein subunits and one of the Gα subunits, to produce six different membrane preparations for each receptor variant. Data on preparations D2So (preparation 1) and D2Si2 (preparation 1) have been published previously in Nickolls and Strange (2003), but are

Discussion

We have recently analysed the ability of the rat D2L dopamine receptor (Cordeaux et al., 2001) and the human D2S dopamine receptor (Nickolls and Strange, 2003) to couple to αo and αi2 G protein subunits and the ability of the human D2L dopamine receptor to couple to αo, αi1, αi2 and αi3 G protein subtypes (Gazi et al., 2003). In this study, this work is extended to compare the coupling profile of the human D2S receptor with Go, Gi1, Gi2 and Gi3 proteins and to investigate the ability of a

Acknowledgements

We thank the University of Reading for financial support and Dr A Cravchik for the gift of the D2SCys311 plasmid.

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    Present address: Neurocrine Biosciences Inc., 10555 Science Center Drive, San Diego, CA, USA.

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