Single-Molecule Analysis of G Protein-Coupled Receptor Stoichiometry: Approaches and Limitations.

How G protein-coupled receptors (GPCRs) are organized at the cell surface remains highly contentious. Single-molecule (SM) imaging is starting to inform this debate as receptor behavior can now be visualized directly, without the need for interpreting ensemble data. The limited number of SM studies of GPCRs undertaken to date have strongly suggested that dimerization is at most transient, and that most receptors are monomeric at any given time. However, even SM data has its caveats and needs to be interpreted carefully. Here, we discuss the types of SM imaging strategies used to examine GPCR stoichiometry and consider some of these caveats. We also emphasize that attempts to resolve the debate ought to rely on orthogonal approaches to measuring receptor stoichiometry

Referenced Single-Molecule Measurements Differentiate between GPCR Oligomerization States.

The extent to which Rhodopsin family G-protein-coupled receptors (GPCRs) form invariant oligomers is contentious. Recent single-molecule fluorescence imaging studies mostly argue against the existence of constitutive receptor dimers and instead suggest that GPCRs only dimerize transiently, if at all. However, whether or not even transient dimers exist is not always clear due to difficulties in unambiguously distinguishing genuine interactions from chance colocalizations, particularly with respect to short-lived events. Previous single-molecule studies have depended critically on calculations of chance colocalization rates and/or comparison with unfixed control proteins whose diffusional behavior may or may not differ from that of the test receptor. Here, we describe a single-molecule imaging assay that 1) utilizes comparisons with well-characterized control proteins, i.e., the monomer CD86 and the homodimer CD28, and 2) relies on cell fixation to limit artifacts arising from differences in the distribution and diffusion of test proteins versus these controls. The improved assay reliably reports the stoichiometry of the Glutamate-family GPCR dimer, γ-amino butyric acid receptor b2, whereas two Rhodopsin-family GPCRs, β2-adrenergic receptor and mCannR2, exhibit colocalization levels comparable to those of CD86 monomers, strengthening the case against invariant GPCR oligomerization.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.bpj.2015.09.00

Receptor Quaternary Organization Explains G Protein-Coupled Receptor Family Structure.

The organization of Rhodopsin-family G protein-coupled receptors (GPCRs) at the cell surface is controversial. Support both for and against the existence of dimers has been obtained in studies of mostly individual receptors. Here, we use a large-scale comparative study to examine the stoichiometric signatures of 60 receptors expressed by a single human cell line. Using bioluminescence resonance energy transfer- and single-molecule microscopy-based assays, we found that a relatively small fraction of Rhodopsin-family GPCRs behaved as dimers and that these receptors otherwise appear to be monomeric. Overall, the analysis predicted that fewer than 20% of ∼700 Rhodopsin-family receptors form dimers. The clustered distribution of the dimers in our sample and a striking correlation between receptor organization and GPCR family size that we also uncover each suggest that receptor stoichiometry might have profoundly influenced GPCR expansion and diversification

Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

Signal integration between activating Fc receptors and inhibitory signal regulatory protein α (SIRPα) controls macrophage phagocytosis. Here, using dual-color direct stochastic optical reconstruction microscopy, we report that Fcγ receptor I (FcγRI), FcγRII, and SIRPα are not homogeneously distributed at macrophage surfaces but are organized in discrete nanoclusters, with a mean radius of 71 ± 11 nm, 60 ± 6 nm, and 48 ± 3 nm, respectively. Nanoclusters of FcγRI, but not FcγRII, are constitutively associated with nanoclusters of SIRPα, within 62 ± 5 nm, mediated by the actin cytoskeleton. Upon Fc receptor activation, Src-family kinase signaling leads to segregation of FcγRI and SIRPα nanoclusters to be 197 ± 3 nm apart. Co-ligation of SIRPα with CD47 abrogates nanocluster segregation. If the balance of signals favors activation, FcγRI nanoclusters reorganize into periodically spaced concentric rings. Thus, a nanometer- and micron-scale reorganization of activating and inhibitory receptors occurs at the surface of human macrophages concurrent with signal integration

Distribution maps of cetacean and seabird populations in the North‐East Atlantic

1. Distribution maps of cetaceans and seabirds at basin and monthly scales are needed for conservation and marine management. These are usually created from standardized and systematic aerial and vessel surveys, with recorded animal den- sities interpolated across study areas. However, distribution maps at basin and monthly scales have previously not been possible because individual surveys have restricted spatial and temporal coverage. 2. This study develops an alternative approach consisting of: (a) collating diverse survey data to maximize spatial and temporal coverage, (b) using detection func- tions to estimate variation in the surface area covered (km2) among these surveys, standardizing measurements of effort and animal densities, and (c) developing species distribution models (SDM) that overcome issues with heterogeneous and uneven coverage. 3. 2.68 million km of survey data in the North-East Atlantic between 1980 and 2018 were collated and standardized. SDM using Generalized Linear Models and General Estimating Equations in a hurdle approach were developed. Distribution maps were then created for 12 cetacean and 12 seabird species at 10 km and monthly resolution. Qualitative and quantitative assessment indicated good model performance. 4. Synthesis and applications. This study provides the largest ever collation and standardization of diverse survey data for cetaceans and seabirds, and the most comprehensive distribution maps of these taxa in the North-East Atlantic. These distribution maps have numerous applications including the identification of im- portant areas needing protection, and the quantification of overlap between vul- nerable species and anthropogenic activities. This study demonstrates how the analysis of existing and diverse survey data can meet conservation and marine management needs.Versión del editor4,7

A survey of G protein-coupled receptor stoichiometry

G-protein coupled receptors (GPCRs) represent the largest family of transmembrane proteins in the human genome. Their biological and medical significance has driven extensive research into their structure and function, yet a number of important aspects of their behaviour remain unresolved. Arguably the most contentious debate in the field concerns whether or not the receptors form stable homo- and hetero-oligomeric interactions, and there is currently no consensus on the extent or purpose of GPCR oligomerisation. In this thesis, the ‘typical’ stoichiometry of Rhodopsin family GPCRs is investigated via the examination of more than 60 receptors natively expressed by human HEK 293T cells using bioluminescence resonance energy transfer (BRET). Assaying receptors in the cells in which they are natively expressed maximises the likelihood of authentic assembly and trafficking while simultaneously providing an unbiased cross-section of the whole GPCR family. In order to make such an investigation possible, the sensitivity of existing BRET approaches for partial homodimers had to be, and was, confirmed, and a complementary competition-based BRET method suitable for a semi high-throughput analysis was developed. Application of these assays to the HEK 293T GPCR repertoire revealed that the Rhodopsin family is very predominantly monomeric but contains a small fraction of independently evolved dimers comprising small phylogenetic clusters of receptors. The mechanism of Rhodopsin family dimerisation was in some cases found to be reliant on interactions between transmembrane helices, in contrast to other families of GPCRs, which were observed or are known to be exclusively dimeric due to interactions between their N-terminal domains. The mechanism of dimerisation in Rhodopsin family GPCRs may preclude constitutive dimerisation but allow heterodimerisation of closely related receptors, as observed for a subset of receptors using a third type of assay designed to detect heterodimers. Taken together, these observations suggest a model of GPCR evolution in which dimers either have a selective disadvantage compared to monomers, or for which dimerisation offers no apparent selective advantage. These findings suggest that receptor stoichiometry is at least partly responsible for several of the remarkable features of GPCR family structure, including the very large size of the family as a whole, the great diversity of Rhodopsin family GPCRs, and the origins of sensory receptors.</p

A survey of G protein-coupled receptor stoichiometry

G-protein coupled receptors (GPCRs) represent the largest family of transmembrane proteins in the human genome. Their biological and medical significance has driven extensive research into their structure and function, yet a number of important aspects of their behaviour remain unresolved. Arguably the most contentious debate in the field concerns whether or not the receptors form stable homo- and hetero-oligomeric interactions, and there is currently no consensus on the extent or purpose of GPCR oligomerisation. In this thesis, the ‘typical’ stoichiometry of Rhodopsin family GPCRs is investigated via the examination of more than 60 receptors natively expressed by human HEK 293T cells using bioluminescence resonance energy transfer (BRET). Assaying receptors in the cells in which they are natively expressed maximises the likelihood of authentic assembly and trafficking while simultaneously providing an unbiased cross-section of the whole GPCR family. In order to make such an investigation possible, the sensitivity of existing BRET approaches for partial homodimers had to be, and was, confirmed, and a complementary competition-based BRET method suitable for a semi high-throughput analysis was developed. Application of these assays to the HEK 293T GPCR repertoire revealed that the Rhodopsin family is very predominantly monomeric but contains a small fraction of independently evolved dimers comprising small phylogenetic clusters of receptors. The mechanism of Rhodopsin family dimerisation was in some cases found to be reliant on interactions between transmembrane helices, in contrast to other families of GPCRs, which were observed or are known to be exclusively dimeric due to interactions between their N-terminal domains. The mechanism of dimerisation in Rhodopsin family GPCRs may preclude constitutive dimerisation but allow heterodimerisation of closely related receptors, as observed for a subset of receptors using a third type of assay designed to detect heterodimers. Taken together, these observations suggest a model of GPCR evolution in which dimers either have a selective disadvantage compared to monomers, or for which dimerisation offers no apparent selective advantage. These findings suggest that receptor stoichiometry is at least partly responsible for several of the remarkable features of GPCR family structure, including the very large size of the family as a whole, the great diversity of Rhodopsin family GPCRs, and the origins of sensory receptors.This thesis is not currently available on ORA