Rare variant in intracellular loop-2 of the ghrelin receptor reveals novel mechanisms of GPCR biased signaling and trafficking

G protein-coupled receptors (GPCRs) are the largest class of integral membrane proteins and the most common pharmaceutical drug target. Through allosteric coupling, GPCRs transduce extracellular stimuli into physiologically-relevant intracellular signaling cascades via G proteins and {beta}-arrestin ({beta}arr). The growth hormone secretagogue receptor (GHSR) is a rhodopsin-like, peptide hormone GPCR considered a promising target for both metabolic and neurological diseases. Here, by characterizing an ultra-rare coding variant in intracellular loop-2 (ICL2) of the GHSR, GHSRL149P (L149P), we establish a unique role of ICL2 in GPCR biased signaling, lipid modulation, and intracellular trafficking. Using an array of bioluminescence resonance energy transfer (BRET)-based assays, we show that the natural L149P mutant exhibits (i) a constitutive plasma membrane [PM] expression bias, (ii) preferential partitioning away from cholesterol-enriched PM microdomains, (iii) enhanced agonist-directed endocytosis, and (iv) dramatic signaling bias towards {beta}arr1/2 over Gq, Gi/o, and G12/13. Using a combination of pharmacological and genetic tools, we demonstrate that {beta}arr1/2 recruitment to L149P requires G protein-coupled receptor kinase-2/3 (GRK2/3)-mediated phosphorylation, but it does not utilize protein kinase C (PKC), G{beta}{gamma}-dependent GRK2/3 translocation, or Gi/o, supporting a G protein-independent mechanism. Lastly, we found that {beta}arr1/2 recruitment to L149P requires both GRK2/3 and GRK5/6, while the wild-type GHSR relies exclusively on GRK2/3, consistent with increased GRK6 pre-coupling to the L149P mutant. Collectively, our findings using a rare, natural variant reveal novel mechanisms of GPCR regulation that could be leveraged to improve personalized medicine and facilitate the rational design/discovery of GPCRICL2-directed drugs. SIGNIFICANCE STATEMENTG protein-coupled receptors (GPCRs) are the largest class of membrane proteins and the most prevalent drug target class in medicine. However, the structural and mechanistic determinants of GPCR signaling and membrane compartmentalization remains incompletely understood. Here, we characterize an ultra-rare natural variant of the growth hormone secretagogue receptor (GHSR), L149P, that uncovers an unanticipated role for intracellular loop 2 (ICL2) in receptor biased signaling, intracellular trafficking, and membrane lipid modulation. The variant exhibits a strong bias for {beta}-arrestin signaling independent of canonical G protein pathways, mediated instead by unique GPCR kinase interactions and a distinct subcellular distribution. These findings reveal previously unrecognized regulatory layers in GPCR structure/function and identify ICL2 as a promising target for designing precision pharmacotherapeutics.