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 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 β-arrestin (βarr). 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, GHSR ^L149P (L149P), we establish a unique role of ICL2 conformations 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 into non-lipid raft PM microdomains, (iii) enhanced agonist-directed endocytosis, and (iv) dramatic signaling bias towards βarr1/2 over Gα _q , Gα _i/o , and Gα _12/13 in a cholesterol-dependent manner. Using a combination of pharmacological and genetic tools, we demonstrate that βarr1/2 recruitment to L149P requires G protein-coupled receptor kinase-2/3 (GRK2/3)-mediated phosphorylation, but it does not utilize protein kinase C (PKC), Gβγ-dependent GRK2/3 translocation, or Gα _i/o , supporting a G protein-independent mechanism. Lastly, we found that βarr1/2 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 GPCR ^ICL2 -directed drugs.