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Role of Helix 8 in Dopamine Receptor Signaling.

ABSTRACT: G protein-coupled receptors (GPCRs) are membrane receptors whose agonist-induced dynamic conformational changes trigger heterotrimeric G protein activation, followed by GRK-mediated phosphorylation and arrestin-mediated desensitization. Cytosolic regions of GPCRs have been studied extensively because they are direct contact sites with G proteins, GRKs, and arrestins. Among various cytosolic regions, the role of helix 8 is least understood, although a few studies have suggested that it is involved in G protein activation, receptor localization, and/or internalization. In the present study, we investigated the role of helix 8 in dopamine receptor signaling focusing on dopamine D1 receptor (D1R) and dopamine D2 receptor (D2R). D1R couples exclusively to Gs, whereas D2R couples exclusively to Gi. Bioinformatic analysis implied that the sequences of helix 8 may affect GPCR-G protein coupling selectivity; therefore, we evaluated if swapping helix 8 between D1R and D2R changed G protein selectivity. Our results suggest that helix 8 is not involved in D1R-Gs or D2R-Gi coupling selectivity. Instead, we observed that D1R with D2R helix 8 or D1R with an increased number of hydrophobic residues in helix 8 relative to wild-type showed diminished ?-arrestin-mediated desensitization, resulting in increased Gs signaling.

SUBMITTER: Yang HS

PROVIDER: S-EPMC6824627 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Role of Helix 8 in Dopamine Receptor Signaling.

Yang Han-Sol HS Sun Ningning N Zhao Xiaodi X Kim Hee Ryung HR Park Hyun-Ju HJ Kim Kyeong-Man KM Chung Ka Young KY

Biomolecules & therapeutics 20191101 6

G protein-coupled receptors (GPCRs) are membrane receptors whose agonist-induced dynamic conformational changes trigger heterotrimeric G protein activation, followed by GRK-mediated phosphorylation and arrestin-mediated desensitization. Cytosolic regions of GPCRs have been studied extensively because they are direct contact sites with G proteins, GRKs, and arrestins. Among various cytosolic regions, the role of helix 8 is least understood, although a few studies have suggested that it is involve ...[more]

PMID: 30971061

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Project description:The C-terminal tail of G-protein-coupled receptors (GPCR) contain important regulatory sites that enable interaction with intracellular signaling effectors. Here we examine the relative contribution of the C-tail serine/threonine phosphorylation sites (Ser383-385, Ser387-Thr392) and the helix-8 palmitoylation site (Cys361) in signaling regulation downstream of the proteolytically activated GPCR, PAR2. We examined Gαq/11-coupled calcium signaling, β-arrestin-1/-2 recruitment, and MAPK activation (p44/42 phosphorylation) by wild-type and mutant receptors expressed in a CRISPR/Cas9 PAR2-knockout HEK-293 cell background with both peptide stimulation of the receptor (SLIGRL-NH2) as well as activation with its endogenous trypsin revealed a tethered ligand. We find that alanine substitution of the membrane proximal serine residues (Ser383-385Ala) had no effect on SLIGRL-NH2- or trypsin-stimulated β-arrestin recruitment. In contrast, alanine substitutions in the Ser387-Thr392 cluster resulted in a large (∼50%) decrease in β-arrestin-1/-2 recruitment triggered by the activating peptide, SLIGRL-NH2, but was without an effect on trypsin-activated β-arrestin-1/-2 recruitment. Additionally, we find that alanine substitution of the helix-8 cysteine residue (Cys361Ala) led to a large decrease in both Gαq/11 coupling and β-arrestin-1/-2 recruitment to PAR2. Furthermore, we show that Gαq/11 inhibition with YM254890, inhibited ERK phosphorylation by PAR2 agonists, while genetic deletion of β-arrestin-1/-2 by CRISPR/Cas9 enhanced MAPK activation. Knockout of β-arrestins also enhanced Gαq/11-mediated calcium signaling. In line with these findings, a C-tail serine/threonine mutant that has decreased β-arrestin recruitment also showed enhanced ERK activation. Thus, our studies point to multiple mechanisms that regulate β-arrestin interaction with PAR2 and highlight differences in regulation of tethered-ligand- and peptide-mediated activation of this receptor.

Project description:A dopamine D2 receptor mutation was recently identified in a family with a novel hyperkinetic movement disorder. Compared to the wild type D2 receptor, the novel allelic variant D2-I212F activates a Gαi1β1γ2 heterotrimer with higher potency and modestly enhanced basal activity in human embryonic kidney (HEK) 293 cells and has decreased capacity to recruit arrestin3. We now report that omitting overexpressed G protein-coupled receptor kinase-2 (GRK2) decreased the potency and efficacy of quinpirole for arrestin recruitment. The relative efficacy of quinpirole for arrestin recruitment to D2-I212F compared to D2-WT was considerably lower without overexpressed GRK2 than with added GRK2. D2-I212F exhibited higher basal activation of GαoA than Gαi1 but little or no increase in the potency of quinpirole relative to D2-WT. Other signs of D2-I212F constitutive activity for G protein-mediated signaling, in addition to basal activation of Gαi/o, were enhanced basal inhibition of forskolin-stimulated cyclic AMP accumulation that was reversed by the inverse agonists sulpiride and spiperone and a ∼4-fold increase in the apparent affinity of D2-I212F for quinpirole, determined from competition binding assays. In mouse midbrain slices, inhibition of tonic current by the inverse agonist sulpiride in dopamine neurons expressing D2-I212F was consistent with our hypothesis of enhanced constitutive activity and sensitivity to dopamine relative to D2-WT. Molecular dynamics simulations with D2 receptor models suggested that an ionic lock between the cytoplasmic ends of the third and sixth α-helices that constrains many G protein-coupled receptors in an inactive conformation spontaneously breaks in D2-I212F. Overall, these results confirm that D2-I212F is a constitutively active and signaling-biased D2 receptor mutant and also suggest that the effect of the likely pathogenic variant in a given brain region will depend on the nature of G protein and GRK expression.

Dataset Information

Role of Helix 8 in Dopamine Receptor Signaling.

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Role of Helix 8 in Dopamine Receptor Signaling.

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