Rare variant in intracellular loop-2 alters the spatial distribution and transducer coupling selectivity of the...

G protein-coupled receptors (GPCRs) are the largest class of membrane proteins and the most common pharmaceutical drug target. Prior work from our group and others supports that a conserved, proline-hydrophobic residue (ProH) motif within intracellular loop-2 (ICL2) biases GPCR-transducer coupling between canonical G proteins and β-arrestin (βarr). Here, we systematically characterize a rare missense mutation within the ProH motif of the ghrelin receptor (growth hormone secretagogue receptor [GHSR])-Leu149Pro^34.51 (L149P)-to dissect how naturalistic perturbation of ICL2 dynamics pleiotropically regulates the spatial distribution, trafficking, and transducer selectivity of a prototypical, rhodopsin-like GPCR. Molecular dynamics simulations indicate that the L149P mutation destabilizes the α-helical conformation of ICL2, thereby increasing loop flexibility proximal to the receptor-transducer coupling interface. Using a panel of bioluminescence resonance-energy transfer biosensors combined with pharmacological and genetic tools, we demonstrate that the GHSR-L149P mutation (1) enhances receptor surface expression by reducing constitutive GHSR trafficking; (2) biases transducer engagement toward βarr1/2 and Gα_i/o over Gα_q and Gα₁₃, despite partial mini-G_q coupling efficacy; (3) expands GPCR kinase (GRK) subclass utilization from GRK2/3-dependent to an additional, partial reliance on GRK5/6; and (4) promotes βarr1/2 recruitment independent of G protein-mediated second messenger kinase activation. The evolutionary conservation of the ProH motif suggests that cognate, ICL2-destabilizing variants likely confer comparable disruptions in GPCR structure/function beyond established effects on G protein coupling. Collectively, these results establish a naturalistic model to study ICL2-dependent GPCR regulation and illuminate the ProH motif as a promising drug target for biased allosteric modulators. SIGNIFICANCE STATEMENT: G protein-coupled receptors are the most common pharmaceutical target in medicine. This study exploited a rare variant in intracellular loop-2 of the ghrelin receptor to characterize a conserved, allosteric "hotspot" controlling G protein-coupled receptor expression, trafficking, and signaling. These findings highlight intracellular loop-2 as a promising target for biased allosteric modulator drug design.