Abstract
The G protein-coupled ghrelin receptor GHSR1a is a potential pharmacological target for treating obesity and addiction because of the critical role ghrelin plays in energy homeostasis and dopamine-dependent reward. GHSR1a enhances growth hormone release, appetite, and dopamine signaling through G(q/11), G(i/o), and G(12/13) as well as β-arrestin-based scaffolds. However, the contribution of individual G protein and β-arrestin pathways to the diverse physiological responses mediated by ghrelin remains unknown. To characterize whether a signaling bias occurs for GHSR1a, we investigated ghrelin signaling in a number of cell-based assays, including Ca(2+) mobilization, serum response factor response element, stress fiber formation, ERK1/2 phosphorylation, and β-arrestin translocation, utilizing intracellular second loop and C-tail mutants of GHSR1a. We observed that GHSR1a and β-arrestin rapidly form metastable plasma membrane complexes following exposure to an agonist, but replacement of the GHSR1a C-tail by the tail of the vasopressin 2 receptor greatly stabilizes them, producing complexes observable on the plasma membrane and also in endocytic vesicles. Mutations of the contiguous conserved amino acids Pro-148 and Leu-149 in the GHSR1a intracellular second loop generate receptors with a strong bias to G protein and β-arrestin, respectively, supporting a role for conformation-dependent signaling bias in the wild-type receptor. Our results demonstrate more balance in GHSR1a-mediated ERK signaling from G proteins and β-arrestin but uncover an important role for β-arrestin in RhoA activation and stress fiber formation. These findings suggest an avenue for modulating drug abuse-associated changes in synaptic plasticity via GHSR1a and indicate the development of GHSR1a-biased ligands as a promising strategy for selectively targeting downstream signaling events.
Highlights
The G protein coupled receptor GHSR1a mediates feeding and addictive behaviors
-Arrestin-2 Interaction with the GHSR1a in HEK293 Cells— A specific cluster of phosphorylated serine/threonine residues in the C terminus of class B G protein-coupled receptor (GPCR) enables them to form stable complexes with -arrestin during endocytosis [37]. These complexes are readily apparent by confocal microscopy because they distinctly label the membranes of early endosomes but are absent from their interior [38, 39], and we previously identified the location of these clusters for representative GPCRs by C-tail mutagenesis [37, 39]
L585 was more potent than ghrelin in stimulating Ca2ϩ, with EC50 values of 2.7 Ϯ 1.2 nM and 93.5 Ϯ 11.6 nM, respectively (Fig. 4A). This order of potency is consistent with reports on similar small molecule agonists of the ghrelin receptor [4], but it is the reverse of what we found for the -arrestin translocation assay
Summary
The G protein coupled receptor GHSR1a mediates feeding and addictive behaviors. Results: Mutagenesis of the second intracellular loop of GHSR1a generates biased receptors, favoring distinct signaling events. Our results demonstrate more balance in GHSR1a-mediated ERK signaling from G proteins and -arrestin but uncover an important role for -arrestin in RhoA activation and stress fiber formation These findings suggest an avenue for modulating drug abuse-associated changes in synaptic plasticity via GHSR1a and indicate the development of GHSR1a-biased ligands as a promising strategy for selectively targeting downstream signaling events. We report that GHSR1a forms transient complexes with -arrestin-2 on the cell membrane that can be stabilized by the addition of phosphate acceptor sites in the receptor tail or destabilized by substitution of a conserved proline residue in the receptor ICL2 This interaction with -arrestin is essential for the GHSR1a-mediated induction of RhoA signaling and actin remodeling, whereas GHSR1a-mediated ERK1/2 phosphorylation involves both -arrestin and G protein components. Most significantly, mutating amino acid residues for the interaction with -arrestin or G proteins in ICL2 generated signaling-biased receptors, potentially allowing future studies to determine their individual contribution to various in vivo physiological processes mediated by ghrelin
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