title: Single molecule analysis of functionally asymmetric G protein-coupled receptor (GPCR) oligomers reveals diverse spatial and structural assemblies.
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 (LHR(B-)) and a signaling-deficient (LHR(S-)) 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 LHR(B-) to LHR(S-) 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.
authors.affiliation: From the Institute of Reproductive and Developmental Biology, Department of Surgery and Cancer, Imperial College London, Du Cane Road, London W12 0NN, United Kingdom.
authors.forename: Kim C
authors.initials: KC
authors.lastname: Jonas
authors.affiliation: the Computational Structural Biology Lab, Department of Life Sciences, University of Modena and Reggio Emilia, via Campi 183-41100 Modena, Italy, and.
authors.forename: Francesca
authors.initials: F
authors.lastname: Fanelli
authors.affiliation: From the Institute of Reproductive and Developmental Biology, Department of Surgery and Cancer, Imperial College London, Du Cane Road, London W12 0NN, United Kingdom, the Institute for Biomedicine, Department of Physiology, University of Turku, 20520 Turku, Finland ilpo.huhtaniemi@imperial.ac.uk.
authors.forename: Ilpo T
authors.initials: IT
authors.lastname: Huhtaniemi
authors.affiliation: From the Institute of Reproductive and Developmental Biology, Department of Surgery and Cancer, Imperial College London, Du Cane Road, London W12 0NN, United Kingdom, a.hanyaloglu@imperial.ac.uk.
authors.forename: Aylin C
authors.initials: AC
authors.lastname: Hanyaloglu
mesh_terms: D005456:Fluorescent Dyes / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D011974:Receptors, LH / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
chemical_list: D005456:Fluorescent Dyes; D011974:Receptors, LH
keywords: Bioluminescence Resonance Energy Transfer (BRET); Cell Signaling; Dimer; G Protein; G protein-Coupled Receptor (GPCR); Oligomer; Structural Biology; Super-resolution Imaging