The influence of G protein subtype on agonist action at D2 dopamine receptors
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 D2S/αo (preparation 1) and D2S/αi2 (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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2012, Progress in Neuro-Psychopharmacology and Biological PsychiatryCitation Excerpt :While both receptor variants interact with Gα0, Gαi1, Gαi2 and Gαi3 proteins, the long isoform shows preferential coupling to the Gα0 and Gαi2 protein subtypes (Cordeaux et al., 2001; Gazi et al., 1999; Guiramand et al., 1995). Indeed, some compounds including dopamine and (+)-3-PPP appeared to be more potent when the D2L receptor was co-expressed in Sf9 cells with Gα0 protein, as compared to combination with Gαi protein subtypes (Cordeaux et al., 2001), an effect that was similarly observed when fusioning the D2L receptor with Gα0 protein compared to Gαi subtypes (Lane et al., 2007; Nickolls and Strange, 2004). It is noteworthy that a similar difference in the potency of dopamine was observed in this previous report when examining the coupling to either Gα0 and Gαi protein subtypes or in the present study when examining guanylyl nucleotide exchange in NMDG or NaCl containing buffers.
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2010, Pharmacology and TherapeuticsCitation Excerpt :This possibility merits systematic clinical investigation: for example, in a comparative study of piribedil vs pramipexole or ropinirole. Dopamine D2 and D3 receptors couple to multiple intracellular cascades and certain drugs differentially favour recruitment of one pathway over another (Gazi et al., 2003; Gay et al., 2004; Neve et al., 2004; Nickolls and Strange, 2004; Heusler et al., 2007). This phenomenon is seen at many classes of GPCR and is called “conformational selection”, “agonist-directed trafficking” or “biased agonism” (Galandrin et al., 2007; Mailman, 2007).
Differential agonist and inverse agonist profile of antipsychotics at D<inf>2L</inf> receptors coupled to GIRK potassium channels
2007, NeuropharmacologyCitation Excerpt :The reasons for such a behaviour are unclear, but differential coupling to distinct G protein pools (with some of them being more readily activated than others) after receptor activation might be an explanation. Interestingly, there is evidence for “agonist-directed trafficking of receptor response” or “functional selectivity” at the D2 receptor (Nickolls and Strange, 2004; Gay et al., 2004). Importantly, evidence for pathway-specific agonism has been reported for aripiprazole in several studies (Lawler et al., 1999; Shapiro et al., 2003; Urban et al., in press; Jordan et al., 2005).
Generation of Gαi knock-out HEK293 cells illuminates Gαi-coupling diversity of GPCRs
2023, Communications Biology
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