Local GPCR density tips the balance of μ-opioid receptor trafficking

The extent to which local GPCR surface density governs engagement of downstream signaling and trafficking pathways remains unclear. Using single-particle tracking of the μ-opioid receptor (MOR), we show that receptor density differentially regulates G protein signaling and GRK2/3-β-arrestin-dependent receptor trafficking. At low surface density, MORs activate G proteins but fail to enter clathrin-coated structures despite the presence of endogenous GRK2/3 and β-arrestin. Increasing MOR density, co-expressing other class A GPCRs, or elevating GRK2 or β-arrestin abundance rescues agonist-induced MOR trafficking. In contrast, the class B GPCR V2R blocks MOR trafficking at both low and high MOR densities. These results support a model in which increasing class A GPCR density, despite worsening effector-to-receptor stoichiometry, promotes trafficking by forming an affinity matrix that enables reversible GRK2/3 and β-arrestin interactions to be productively used by neighboring receptors in a density-dependent manner, whereas class B GPCRs sequester β-arrestin and block trafficking.