G Protein Coupling and Ligand Selectivity of the D_2Land D₃Dopamine Receptors

Abstract

The human dopamine D 2L receptor couples promiscuously to multiple members of the G␣ i/o subfamily. Despite the high homology of the D 2L and D 3 receptors, the G protein coupling specificity of the human D 3 receptor is less clearly characterized. The primary aim of this study, then, was the parallel characterization of the G protein coupling specificity of the D 2L and D 3 receptors. By using both receptor-G protein fusion proteins and stable cell lines in which pertussis toxin-resistant mutants of individual G␣ i -family G proteins were expressed in an inducible fashion, we demonstrated highly selective coupling of the D 3 receptor to G␣ o1 . Furthermore, by using the fusion proteins to ensure identical stoichiometry of receptor to G protein for each pairing, a range of ligands displayed higher potency and, for partial agonists, higher efficacy at the D 3 receptor when coupled to G␣ o1 compared with the D 2L receptor. The second aim of this study was to investigate the molecular basis of the above differential G protein coupling specificity. The importance of a 12-amino acid sequence from the C-terminal end of the third intracellular loop of the D 2L receptor in providing promiscuity in G protein coupling was demonstrated using a chimeric D 3 /D 2 receptor in which the equivalent region of the D 3 receptor was exchanged for this sequence. This chimera displayed D 3 -like affinity for [ 3 H]spiperone and potency for agonists but gained D 2 -like ability to couple to each of G␣ i1-3 as well as G␣ o1 .In recent studies, we have shown that the long isoform of the human dopamine D 2 receptor (D 2L receptor) couples efficiently and promiscuously to all four G␣ i/o subtypes . This confirmed previous work by other groups, although the relative order of preference remains controversial (Watts et al., 1998;Jiang et al., 2001;Gazi et al., 2003;Senogles et al., 2004). Although the D 2 receptor is considered a key target for antipsychotic drugs, there has been considerable interest in the contribution that the dopamine D 3 receptor may provide (Gurevich et al., 1997;Joyce, 2001;Joyce and Millan, 2005;Sokoloff et al., 2006). Human dopamine D 2 and D 3 receptors have an overall amino acid similarity of 52%, which increases to 78% if only transmembrane regions are considered (Sokoloff et al., 1990). As a result, D 2 and D 3 receptors exhibit a similar pharmacological profile (Ahlgren-Beckendorf and Levant, 2004). However, signal transduction by the D 3 receptor is less clearly understood than that of the D 2 receptor. Studies to elucidate cellular responses to D 3 receptor activation are hampered by the coexpression of D 2 and D 3 receptors in brain tissue and the lack of definitively selective pharmacological tools. It follows, then, that the majority of data have been obtained by expression of the receptors in heterologous systems. Initial reports indicated that the D 3 receptor, when expressed in a variety of cell systems, did not exhibit a decrease in affinity for agonists in the presence of guanine nucleotides a...

Discovery and Characterization of a G Protein–Biased Agonist That Inhibits β-Arrestin Recruitment to the D2 Dopamine Receptor

“…Although the mechanisms underlying functional selectivity are not known, a leading hypothesis is that GPCRs can adopt multiple functionally "active" conformational states that are either stabilized or induced by these selective ligands (Kobilka and Deupi, 2007;Wess et al, 2008). Although relatively few biased ligands have been described for the D2R Mottola et al, 2002;Gay et al, 2004;Lane et al, 2007Lane et al, , 2008, existing examples strongly support the concept of the D2R being able to adopt multiple signalingbiased confirmations. Recently, a structural basis for functional selectivity of several GPCRs has been proposed (Liu et al, 2012;Dror et al, 2013;Kruse et al, 2013;Wacker et al, 2013;Abdul-Ridha et al, 2014) suggesting that rational design of functionally selective compounds may be possible.…”

Discovery of a Novel Class of Negative Allosteric Modulator of the Dopamine D2 Receptor Through Fragmentation of a Bitopic Ligand

“…15 The D 3 R has been shown to selectively couple to the Gα o1 G protein whereas the D 2 R can couple to all members of the Gα i/o G protein family. 24 Accordingly, we assessed the action of 3 at the D 2 R (Figure 2f) compared to the D 3 R (Figure 2g) in an assay measuring activation of Gα o1 G protein using a bioluminescence resonance energy transfer (BRET) biosensor. 25 One would also expect that extension of the purely allosteric pharmacophore to include structural features of the orthosteric moiety, incorporating a suitable linker moiety in between, would result in the generation of ligands that displayed competitive behaviour with an orthosteric ligand.…”

Functional Homomers and Heteromers of Dopamine D2L and D3 Receptors Co-exist at the Cell Surface

“…Although the D 2L receptor interacts effectively with each member of the pertussis toxinsensitive G␣ i family of G proteins (29), the D 3 receptor appears to be more selective, showing greatest activation of G␣ o (30,33). We therefore generated fusion proteins containing either the D 2L or D 3 receptors linked to a pertussis toxin-insensitive, C351I, a variant of G␣ o that both receptors still activate (29,30). To produce the first set of inactive fusions, modifications were made at the G protein level where a further G204A mutation was introduced into C351I-G␣ o .…”

“…To do so we employed the potential for functional complementation following co-expression of pairs of nonequivalent and nonfunctional fusions between dopamine receptors and a G protein they are usually able to activate. Although the D 2L receptor interacts effectively with each member of the pertussis toxinsensitive G␣ i family of G proteins (29), the D 3 receptor appears to be more selective, showing greatest activation of G␣ o (30,33). We therefore generated fusion proteins containing either the D 2L or D 3 receptors linked to a pertussis toxin-insensitive, C351I, a variant of G␣ o that both receptors still activate (29,30).…”

“…This prevents effective GDP-GTP exchange in response to receptor occupancy and activation by agonists. G204A,C351I-G␣ o was then linked to either the wild type D 2L or D 3 receptors (29,30). For the second set, mutations were introduced into the receptors such that a pair of hydrophobic residues located at equivalent positions in the 2nd intracellular loop of each of the D 2L and D 3 receptors was converted to acidic residues.…”