Single Molecule Analysis of Functionally Asymmetric G Protein-coupled Receptor (GPCR) Oligomers Reveals Diverse Spatial and Structural Assemblies

Formation of G protein-coupled receptors (GPCRs) into dimers and higher order oligomers represents a key mechanism in pleiotropic signaling, yet how individual protomers function within oligomers remains poorly understood. We present a super-resolution imaging approach, resolving single GPCR molecules to ∼8 nm resolution in functional asymmetric dimers and oligomers using dual-color photoactivatable dyes and localization microscopy (PD-PALM). PD-PALM of two functionally defined mutant luteinizing hormone receptors (LHRs), a ligand-binding deficient receptor (LHRB−) and a signaling-deficient (LHRS−) receptor, which only function via intermolecular cooperation, favored oligomeric over dimeric formation. PD-PALM imaging of trimers and tetramers revealed specific spatial organizations of individual protomers in complexes where the ratiometric composition of LHRB− to LHRS− modulated ligand-induced signal sensitivity. Structural modeling of asymmetric LHR oligomers strongly aligned with PD-PALM-imaged spatial arrangements, identifying multiple possible helix interfaces mediating inter-protomer associations. Our findings reveal that diverse spatial and structural assemblies mediating GPCR oligomerization may acutely fine-tune the cellular signaling profile. Background: GPCRs form complex oligomers whose role in signaling is poorly understood. Results: Super-resolution imaging of functionally asymmetric oligomers reveals diverse functional and structural organizations and the ability to alter signal responses. Conclusion: GPCR oligomers may fine-tune receptor signaling by altering the functional role of individual protomers. Significance: Distinct oligomers could be exploited pharmacologically to improve efficacy, selectivity, and/or specificity.