Encoding the β-Arrestin Trafficking Fate of Ghrelin Receptor GHSR1a: C-Tail-Independent Molecular Determinants in GPCRs

Abstract

G-protein-coupled receptors (GPCRs) can bias signaling through distinct biochemical pathways that originate from G-protein/receptor and β-arrestin/receptor complexes. Receptor conformations supporting β-arrestin engagement depend on multiple receptor determinants. Using ghrelin receptor GHR1a, we demonstrate by bioluminescence resonance energy transfer and fluorescence microscopy a critical role for its second intracellular loop 2 (ICL2) domain in stabilizing β-arrestin/ GHSR1a core interactions and determining receptor trafficking fate. We validate our findings in ICL2 gain-and loss-of-function experiments assessing β-arrestin and ubiquitin-dependent internalization of the CC chemokine receptor, CCR1. Like all CC and CXC subfamily chemokine receptors, CCR1 lacks a critical proline residue found in the ICL2 consensus domain of rhodopsinfamily GPCRs. Our study indicates that ICL2, C-tail determinants, and the orthosteric binding pocket that regulates β-arrestin/ receptor complex stability are sufficient to encode a broad repertoire of the trafficking fates observed for rhodopsin-family GPCRs, suggesting they provide the essential elements for regulating a large fraction of β-arrestin signaling bias.

Biased signaling: A viable strategy to drug ghrelin receptors for the treatment of obesity

“…More precisely, substitution of the proline 148 (P148A) residue generated a GHSR1a mutant that had reduced β-arrestin affinity and was biased toward the Gα q/11 signaling. However, mutating the neighboring leucine 149 (L149G) residue selectively abolished signaling to the Gα q/11 pathway, generating a complete biased GHSR1a mutant toward the β-arrestin signaling pathway [86,87]. These findings together with spectroscopic [88] and crystal structure data [89] suggest that G protein and β-arrestin cannot simultaneously engage ICL2 residues, which supports a role for conformation-dependent signaling bias at the ghrelin receptor and makes GHSR1a an effective prototype for decoding biased agonism at GPCRs.…”

“…Besides introducing mutations that could favor activation of a specific G protein signaling over another, functionally biased ghrelin receptors that could distinguish between G protein and arrestin-mediated signaling have also been described [86,87]. A recent study demonstrated that introducing a single point mutation at the second intracellular loop (ICL2) of the ghrelin receptor was enough to produce GHSR1a mutants with opposite signaling-biased phenotypes [86]. More precisely, substitution of the proline 148 (P148A) residue generated a GHSR1a mutant that had reduced β-arrestin affinity and was biased toward the Gα q/11 signaling.…”

“…Mutagenesis experiments demonstrated that phosphorylation sites at the GHSR1a C-terminal tail and the ICL2 core components act in a concerted fashion to regulate GHSR1a/β-arrestin-2 interaction kinetics. While the C-terminal tail was crucial for an initial β-arrestin recruitment, the proximal segment of ICL2 was necessary to maintain a stable and sustained β-arrestin-2 interaction with the ghrelin receptor [86]. Thus, ICL2 and the C-terminal tail determinants orchestrate β-arrestin recruitment and β-arrestin/receptor complex stability, and their interplay could be sufficient to encode a broad repertoire of GHSR1a trafficking fates [86], confirming their key role in regulating a large fraction of the β-arrestin signaling bias.…”

“…While the C-terminal tail was crucial for an initial β-arrestin recruitment, the proximal segment of ICL2 was necessary to maintain a stable and sustained β-arrestin-2 interaction with the ghrelin receptor [86]. Thus, ICL2 and the C-terminal tail determinants orchestrate β-arrestin recruitment and β-arrestin/receptor complex stability, and their interplay could be sufficient to encode a broad repertoire of GHSR1a trafficking fates [86], confirming their key role in regulating a large fraction of the β-arrestin signaling bias. Consequently, identifying the different factors that could affect ICL2 and C-terminal tail functions in regulating β-arrestin interactions and affecting the receptor fate determination should help improving ghrelin receptor expression at the cell surface and facilitate the development of more efficacious therapeutics.…”

MRAP2 inhibits β-arrestin recruitment to the ghrelin receptor by preventing GHSR1a phosphorylation

“…GHSR1a primarily couples to Gα q/11 , thus stimulating the production of intracellular inositol triphosphate (IP) 3. Like other GPCRs, agonist stimulation results in phosphorylation of GHSR1a by kinases, including GPCR kinase 2 (GRK2) and PKC ( 15 ), and β-arrestin recruitment. Notably, GHSR1a contains several phosphorylation sites within the C-terminal tail, some of which have been shown to be important for β-arrestin recruitment ( 16 ).…”