title: Single-molecule resolution of G protein-coupled receptor (GPCR) complexes.
abstract: The organization of G protein-coupled receptors (GPCRs) into dimers and higher-order oligomers has provided a potential mechanistic system in defining complex GPCR responses. Despite being studied for nearly 20 years it has, and still is, been an area of controversy. Although technology has developed to quantitatively measure these associations in real time, identify the structural interfaces and even systems to understand the physiological significance of di/oligomerization, key questions remain outstanding including the role of each individual complex from the monomer to the higher-order oligomer, in their native system. Recently, single-molecule microscopy approaches have provided the tools to directly visualize individual GPCRs in dimers and oligomers, though as with any technological development each have their advantages and limitations. This chapter will describe these recent developments in single-molecule fluorescent microscopy, focusing on our recent application of super-resolution imaging of the GPCR for the luteinizing hormone/chorionic gonadotropin to quantify GPCR monomers and formation of protomers in to dimers and distinct oligomeric forms. We present our approach, considerations, strategy, and challenges to visualize this receptor beyond the light diffraction limit via photoactivated localization microscopy with photoactivatable dyes. The addition of super-resolution approaches to the GPCR "nano-tool kit" will pave the way for novel avenues to answer outstanding questions regarding the existence and significance of these complexes to GPCR signaling.
authors.affiliation: Institute of Reproductive and Developmental Biology, Imperial College London, London, UK; Institute of Medical and Biomedical Education, St George's University of London, London, UK.
authors.forename: Kim C
authors.initials: KC
authors.lastname: Jonas
authors.affiliation: Institute of Reproductive and Developmental Biology, Imperial College London, London, UK.
authors.forename: Ilpo
authors.initials: I
authors.lastname: Huhtaniemi
authors.affiliation: Institute of Reproductive and Developmental Biology, Imperial College London, London, UK.
authors.forename: Aylin C
authors.initials: AC
authors.lastname: Hanyaloglu
mesh_terms: D006801:Humans; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D055503:Protein Multimerization; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism* / Q000648:ultrastructure; D015398:Signal Transduction; D059010:Single-Cell Analysis
chemical_list: D043562:Receptors, G-Protein-Coupled
keywords: Dimerization; G protein-coupled receptors; Oligomerization; Signaling; Single molecule; Super-resolution imaging