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

Authors:

Jonas, K. C., Fanelli, F., Huhtaniemi, I. T., & Hanyaloglu, A. C.

Keywords:

Bioluminescence Resonance Energy Transfer (BRET), Cell Signaling, G Protein, G protein-Coupled Receptor (GPCR), Structural Biology, Dimer, Oligomer, Super-resolution Imaging

Abstract:

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.

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