scholarly journals Single-molecule imaging reveals distinct effects of ligands on CCR5 dynamics depending on its dimerization status

2021 ◽  
Author(s):  
Fanny Momboisse ◽  
Giacomo Nardi ◽  
Philippe Colin ◽  
Melanie Hery ◽  
Nelia Cordeiro ◽  
...  
Keyword(s):  
Cell Surface ◽  
Single Molecule ◽  
Opposite Effect ◽  
Receptor Activation ◽  
Agonist Stimulation ◽  
Ligand Free ◽  
Human Immunodeficiency Viruses ◽  
Cc Chemokine Receptor 5 ◽  
G Protein Coupled ◽  
Hiv 1

G protein-coupled receptors (GPCR) are present at the cell surface in different conformational and oligomeric states. However, how these states impact GPCRs biological function and therapeutic targeting remains incompletely known. Here, we investigated this issue in living cells for the CC chemokine receptor 5 (CCR5), a major receptor in inflammation and the principal entry co-receptor for Human Immunodeficiency Viruses (HIV-1). We used TIRF microscopy and an original statistical method to track and classify the motion of different receptors subpopulations. We showed a diversity of ligand-free forms of CCR5 at the cell surface constituted of various oligomeric states and exhibiting transient Brownian and restricted motions. These forms were stabilized differently by distinct ligands. In particular, agonist stimulation restricted the mobility of CCR5 and led to its clustering, a feature depending on b-arrestin, while inverse agonist stimulation exhibited the opposite effect. These results suggest a link between receptor activation and immobilization. Applied to HIV-1 envelope glycoproteins gp120, our quantitative analysis revealed agonist-like properties of gp120s. Distinct gp120s influenced CCR5 dynamics differently, suggesting that they stabilize different CCR5 conformations. Then, using a dimerization-compromized mutant, we showed that dimerization (i) impacts CCR5 precoupling to G proteins, (ii) is a pre-requisite for the immobilization and clustering of receptors upon activation, and (iii) regulates receptor endocytosis, thereby impacting the fate of activated receptors. This study demonstrates that tracking the dynamic behavior of a GPCR is an efficient way to link GPCR conformations to their functions, therefore improving the development of drugs targeting specific receptor conformations.

scholarly journals Trispecific broadly neutralizing HIV antibodies mediate potent SHIV protection in macaques

Science ◽  
2017 ◽  
Vol 358 (6359) ◽  
pp. 85-90 ◽  
Author(s):  
Ling Xu ◽  
Amarendra Pegu ◽  
Ercole Rao ◽  
Nicole Doria-Rose ◽  
Jochen Beninga ◽  
...  
Keyword(s):  
Single Molecule ◽  
Neutralizing Antibodies ◽  
Broadly Neutralizing Antibodies ◽  
Aids Vaccine ◽  
Single Protein ◽  
Hiv Antibodies ◽  
Cd4 Binding Site ◽  
Human Immunodeficiency Viruses ◽  
Glycan Site ◽  
Hiv 1

The development of an effective AIDS vaccine has been challenging because of viral genetic diversity and the difficulty of generating broadly neutralizing antibodies (bnAbs). We engineered trispecific antibodies (Abs) that allow a single molecule to interact with three independent HIV-1 envelope determinants: the CD4 binding site, the membrane-proximal external region (MPER), and the V1V2 glycan site. Trispecific Abs exhibited higher potency and breadth than any previously described single bnAb, showed pharmacokinetics similar to those of human bnAbs, and conferred complete immunity against a mixture of simian-human immunodeficiency viruses (SHIVs) in nonhuman primates, in contrast to single bnAbs. Trispecific Abs thus constitute a platform to engage multiple therapeutic targets through a single protein, and they may be applicable for treatment of diverse diseases, including infections, cancer, and autoimmunity.

Multiple nonfunctional alleles of CCR5 are frequent in various human populations

Blood ◽  
2000 ◽  
Vol 96 (5) ◽  
pp. 1638-1645 ◽  
Author(s):  
Cédric Blanpain ◽  
Benhur Lee ◽  
Marie Tackoen ◽  
Bridget Puffer ◽  
Alain Boom ◽  
...  
Keyword(s):  
Cell Surface ◽  
Receptor Activation ◽  
Human Populations ◽  
Type I ◽  
Binding Affinities ◽  
Functional Responses ◽  
Functional Consequences ◽  
Relative Inability ◽  
Specific Alteration ◽  
Hiv 1

CCR5 is the major coreceptor for macrophage-tropic strains of the human immunodeficiency virus type I (HIV-1). Homozygotes for a 32-base pair (bp) deletion in the coding sequence of the receptor (CCR5Δ32) were found to be highly resistant to viral infection, and CCR5 became, therefore, one of the paradigms illustrating the influence of genetic variability onto individual susceptibility to infectious and other diseases. We investigated the functional consequences of 16 other natural CCR5 mutations described in various human populations. We found that 10 of these variants are efficiently expressed at the cell surface, bind [125I]-MIP-1β with affinities similar to wtCCR5, respond functionally to chemokines, and act as HIV-1 coreceptors. In addition to Δ32, six mutations were characterized by major alterations in their functional response to chemokines, as a consequence of intracellular trapping and poor expression at the cell surface (C101X, FS299), general or specific alteration of ligand binding affinities (C20S, C178R, A29S), or relative inability to mediate receptor activation (L55Q). A29S displayed an unusual pharmacological profile, binding and responding to MCP-2 similarly to wtCCR5, but exhibiting severely impaired binding and functional responses to MIP-1α, MIP-1β, and RANTES. In addition to Δ32, only C101X was totally unable to mediate entry of HIV-1. The fact that nonfunctional CCR5 alleles are relatively frequent in various human populations reinforces the hypothesis of a selective pressure favoring these alleles.

scholarly journals Cell-surface residence of sphingosine 1-phosphate receptor 1 on lymphocytes determines lymphocyte egress kinetics

2010 ◽  
Vol 207 (7) ◽  
pp. 1475-1483 ◽  
Author(s):  
Shobha Thangada ◽  
Kamal M. Khanna ◽  
Victoria A. Blaho ◽  
Myat Lin Oo ◽  
Dong-Soon Im ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
Agonist Stimulation ◽  
Primary Determinant ◽  
Sphingosine 1 Phosphate ◽  
Lymphocyte Egress ◽  
G Protein Coupled ◽  
Kinetics In Vivo ◽  
Knockin Mouse

The sphingosine 1-phosphate receptor 1 (S1P1) promotes lymphocyte egress from lymphoid organs. Previous work showed that agonist-induced internalization of this G protein–coupled receptor correlates with inhibition of lymphocyte egress and results in lymphopenia. However, it is unclear if S1P1 internalization is necessary for this effect. We characterize a knockin mouse (S1p1rS5A/S5A) in which the C-terminal serine-rich S1P1 motif, which is important for S1P1 internalization but dispensable for S1P1 signaling, is mutated. T cells expressing the mutant S1P1 showed delayed S1P1 internalization and defective desensitization after agonist stimulation. Mutant mice exhibited significantly delayed lymphopenia after S1P1 agonist administration or disruption of the vascular S1P gradient. Adoptive transfer experiments demonstrated that mutant S1P1 expression in lymphocytes, rather than endothelial cells, facilitated this delay in lymphopenia. Thus, cell-surface residency of S1P1 on T cells is a primary determinant of lymphocyte egress kinetics in vivo.

scholarly journals The Nef protein of the macrophage tropic HIV-1 strain AD8 counteracts human Bst-2/tetherin

2020 ◽  
Author(s):  
Sebastian Giese ◽  
Scott P. Lawrence ◽  
Michela Mazzon ◽  
Bernadien M. Nijmeijer ◽  
Mark Marsh
Keyword(s):  
Cell Surface ◽  
Virus Release ◽  
Viral Budding ◽  
Primary Monocyte ◽  
Human Tetherin ◽  
Human Immunodeficiency Viruses ◽  
Infected Cells ◽  
Translation Initiation Codon ◽  
Hiv 1 ◽  
Tetherin Antagonism

AbstractBst-2/tetherin inhibits the release of numerous enveloped viruses by physically attaching nascent particles to infected cells during the process of viral budding from the cell surface. Tetherin also restricts human immunodeficiency viruses (HIV), and pandemic main (M) group HIV-1s are thought to exclusively rely on their Vpu proteins to overcome tetherin-mediated restriction of virus release. However, at least one M group HIV-1 strain, the macrophage-tropic primary AD8 isolate, is unable to express vpu due to a mutation in its translation initiation codon. Here, using primary monocyte-derived macrophages (MDMs), we show that AD8 was able to use its Nef protein to compensate for the absence of Vpu and restore virus release to wild type levels. We demonstrate that HIV-1 AD8 Nef reduces endogenous tetherin levels from the cell surface, physically separating it from the site of viral budding and thus preventing HIV retention. Mechanistically, AD8 Nef enhances l-tetherin internalisation, leading to perinuclear accumulation of the restriction factor. Finally, we show that Nef proteins from other HIV strains also display varying degrees of tetherin antagonism. Overall, this is the first report showing that M group HIV-1s can use an accessory protein other than Vpu to antagonise human tetherin.

scholarly journals Heterogeneity in VEGF Receptor-2 mobility and organization on the endothelial cell surface leads to diverse activation models by VEGF

2019 ◽  
Author(s):  
Bruno da Rocha-Azevedo ◽  
Sungsoo Lee ◽  
Aparajita Dasgupta ◽  
Anthony R. Vega ◽  
Luciana R. de Oliveira ◽  
...  
Keyword(s):  
Cell Surface ◽  
Single Molecule ◽  
Receptor Activation ◽  
Cell Surface Receptor ◽  
Vegf Receptor ◽  
Surface Receptors ◽  
Endothelial Cell Surface ◽  
Heterogeneous Nature ◽  
Surface Receptor ◽  
Complex Relationships

SummaryThe nanoscale organization of cell surface receptors plays an important role in signaling. We determined this organization and its relation to receptor activation for VEGF Receptor-2 (VEGFR-2), a critical receptor tyrosine kinase in endothelial cells (ECs), by combining live-cell single-molecule imaging of endogenous VEGFR-2 with rigorous computational analysis. We found that surface VEGFR-2 can be mobile or immobile/confined, and monomeric or non-monomeric, with a complex interplay between the two. The mobility and interaction heterogeneity of VEGFR-2 in the basal state led to heterogeneity in the sequence of steps leading to VEGFR-2 activation by VEGF. Specifically, we found that VEGF can bind to both monomeric and non-monomeric VEGFR-2, and, when binding to monomeric VEGFR-2, promotes dimer formation but only for immobile/confined receptors. Overall, our study highlights the dynamic and heterogeneous nature of cell surface receptor organization and its complex relationships with receptor activation and signaling.

scholarly journals Title: Single-molecule diffusion-based estimation of ligand effects on G protein-coupled receptors

2017 ◽  
Author(s):  
Masataka Yanagawa ◽  
Michio Hiroshima ◽  
Yuichi Togashi ◽  
Mitsuhiro Abe ◽  
Takahiro Yamashita ◽  
...  
Keyword(s):  
Diffusion Coefficient ◽  
Cell Surface ◽  
G Protein ◽  
Single Molecule ◽  
G Protein Coupled Receptors ◽  
Single Molecule Imaging ◽  
Imaging Analysis ◽  
Coated Pits ◽  
Ligand Effects ◽  
G Protein Coupled

AbstractG protein-coupled receptors (GPCRs) are major drug targets and have high potential for drug discovery. The development of a method for measuring the activities of GPCRs is essential for pharmacology and drug screening. However, it is difficult to measure the effects of a drug by monitoring the receptor on the cell surface, and changes in the concentrations of downstream signaling molecules, which depend on signaling pathway selectivity of the receptor, are used as an index of the receptor activity. Here, we show that single-molecule imaging analysis provides an alternative method for assessing ligand effects on GPCR. We monitored the dynamics of the diffusion of metabotropic glutamate receptor 3 (mGluR3), a class C GPCR, under various ligand conditions by using total internal reflection fluorescence microscopy (TIRFM). The single-molecule tracking analysis demonstrates that changes in the average diffusion coefficient of mGluR3 quantitatively reflect the ligand-dependent activity. Then, we reveal that the diffusion of receptor molecules is altered by the common physiological events associated with GPCRs, including G protein binding or accumulation in clathrin-coated pits, by inhibition experiments and dual-color single-molecule imaging analysis. We also confirm the generality of agonist-induced diffusion change in class A and B GPCRs, demonstrating that the diffusion coefficient is a good index for estimating the ligand effects on many GPCRs regardless of the phylogenetic groups, chemical properties of the ligands, and G protein-coupling selectivity.One Sentence Summary: Single-molecule imaging for evaluating ligand effects on GPCRs by monitoring the diffusion dynamics on the cell surface.

scholarly journals Lipid Raft-dependent Glucagon-like Peptide-2 Receptor Trafficking Occurs Independently of Agonist-induced Desensitization

2004 ◽  
Vol 15 (8) ◽  
pp. 3673-3687 ◽  
Author(s):  
Jennifer L. Estall ◽  
Bernardo Yusta ◽  
Daniel J. Drucker
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Lipid Raft ◽  
Receptor Activation ◽  
Receptor Internalization ◽  
Cell Surface Receptor ◽  
G Protein Coupled Receptor ◽  
Endosomal Acidification ◽  
Glucagon Like Peptide ◽  
G Protein Coupled

The intestinotrophic and cytoprotective actions of glucagon-like peptide-2 (GLP-2) are mediated by the GLP-2 receptor (GLP-2R), a member of the class II glucagon-secretin G protein-coupled receptor superfamily. Although native GLP-2 exhibits a short circulating half-life, long-acting degradation-resistant GLP-2 analogues are being evaluated for therapeutic use in human subjects. Accordingly, we examined the mechanisms regulating signaling, internalization, and trafficking of the GLP-2R to identify determinants of receptor activation and desensitization. Heterologous cells expressing the transfected rat or human GLP-2R exhibited a rapid, dose-dependent, and prolonged desensitization of the GLP-2–stimulated cAMP response and a sustained GLP-2–induced decrease in levels of cell surface receptor. Surprisingly, inhibitors of clathrin-dependent endocytosis failed to significantly decrease GLP-2R internalization, whereas cholesterol sequestration inhibited ligand-induced receptor internalization and potentiated homologous desensitization. The hGLP-2R localized to both Triton X-100–soluble and –insoluble (lipid raft) cellular fractions and colocalized transiently with the lipid raft marker caveolin-1. Although GLP-2R endocytosis was dependent on lipid raft integrity, the receptor transiently associated with green fluorescent protein tagged-early endosome antigen 1–positive vesicles and inhibitors of endosomal acidification attenuated the reappearance of the GLP-2R on the cell surface. Our data demonstrate that GLP-2R desensitization and raft-dependent trafficking represent distinct and independent cellular mechanisms and provide new evidence implicating the importance of a clathrin- and dynamin-independent, lipid raft-dependent pathway for homologous G protein-coupled receptor internalization.

scholarly journals Revealing the Stoichiometry of G Protein-Coupled Receptors (GPCRs) at the Cell Surface using Single Molecule Imaging

2013 ◽  
Vol 104 (2) ◽  
pp. 525a ◽  
Author(s):  
Laura Weimann ◽  
Steven F. Lee ◽  
James H. Felce ◽  
Simon J. Davis ◽  
David Klenerman
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Single Molecule ◽  
G Protein Coupled Receptors ◽  
Single Molecule Imaging ◽  
G Protein Coupled

scholarly journals The Role of G-Protein-Coupled Receptor Activation by Conformational Selection as Revealed by Single-Molecule Fluorescence

2017 ◽  
Vol 112 (3) ◽  
pp. 327a-328a
Author(s):  
Dennis D. Fernandes ◽  
Libin Ye ◽  
Yuchong Li ◽  
Zhenfu Zhang ◽  
Gregory-Neal Gomes ◽  
...  
Keyword(s):  
G Protein ◽  
Single Molecule ◽  
Receptor Activation ◽  
G Protein Coupled Receptor ◽  
Single Molecule Fluorescence ◽  
Conformational Selection ◽  
G Protein Coupled

Multiple nonfunctional alleles of CCR5 are frequent in various human populations

Blood ◽  
2000 ◽  
Vol 96 (5) ◽  
pp. 1638-1645 ◽  
Author(s):  
Cédric Blanpain ◽  
Benhur Lee ◽  
Marie Tackoen ◽  
Bridget Puffer ◽  
Alain Boom ◽  
...  
Keyword(s):  
Cell Surface ◽  
Receptor Activation ◽  
Human Populations ◽  
Type I ◽  
Binding Affinities ◽  
Functional Responses ◽  
Functional Consequences ◽  
Relative Inability ◽  
Specific Alteration ◽  
Hiv 1

Abstract CCR5 is the major coreceptor for macrophage-tropic strains of the human immunodeficiency virus type I (HIV-1). Homozygotes for a 32-base pair (bp) deletion in the coding sequence of the receptor (CCR5Δ32) were found to be highly resistant to viral infection, and CCR5 became, therefore, one of the paradigms illustrating the influence of genetic variability onto individual susceptibility to infectious and other diseases. We investigated the functional consequences of 16 other natural CCR5 mutations described in various human populations. We found that 10 of these variants are efficiently expressed at the cell surface, bind [125I]-MIP-1β with affinities similar to wtCCR5, respond functionally to chemokines, and act as HIV-1 coreceptors. In addition to Δ32, six mutations were characterized by major alterations in their functional response to chemokines, as a consequence of intracellular trapping and poor expression at the cell surface (C101X, FS299), general or specific alteration of ligand binding affinities (C20S, C178R, A29S), or relative inability to mediate receptor activation (L55Q). A29S displayed an unusual pharmacological profile, binding and responding to MCP-2 similarly to wtCCR5, but exhibiting severely impaired binding and functional responses to MIP-1α, MIP-1β, and RANTES. In addition to Δ32, only C101X was totally unable to mediate entry of HIV-1. The fact that nonfunctional CCR5 alleles are relatively frequent in various human populations reinforces the hypothesis of a selective pressure favoring these alleles.

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