Encoding The Beta-Arrestin Trafficking Fate Of Ghrelin Receptor Ghsr1a: C-Tail-Independent Molecular Determinants In Gpcrs

G-protein-coupled receptors (GPCRs) can bias signaling through distinct biochemical pathways that originate from G-protein/receptor and beta-arrestin/receptor complexes. Receptor conformations supporting beta-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 beta-arrestin/GHSR1a core interactions and determining receptor trafficking fate. We validate our findings in ICL2 gain- and loss-of-function experiments assessing beta-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 rhodopsin-family GPCRs. Our study indicates that ICL2, C-tail determinants, and the orthosteric binding pocket that regulates beta-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 beta-arrestin signaling bias.