Showing papers by "Michel Bouvier published in 2000"
TL;DR: The data demonstrate that GPCR exist as functional dimers in vivo and that BRET-based assays can be used to study both constitutive and hormone-promoted selective protein–protein interactions.
Abstract: Heptahelical receptors that interact with heterotrimeric G proteins represent the largest family of proteins involved in signal transduction across biological membranes. Although these receptors generally were believed to be monomeric entities, a growing body of evidence suggests that they may form functionally relevant dimers. However, a definitive demonstration of the existence of G protein-coupled receptor (GPCR) dimers at the surface of living cells is still lacking. Here, using bioluminescence resonance energy transfer (BRET), as a protein–protein interaction assay in whole cells, we unambiguously demonstrate that the human β2-adrenergic receptor (β2AR) forms constitutive homodimers when expressed in HEK-293 cells. Receptor stimulation with the hydrophilic agonist isoproterenol led to an increase in the transfer of energy between β2AR molecules genetically fused to the BRET donor (Renilla luciferase) and acceptor (green fluorescent protein), respectively, indicating that the agonist interacts with receptor dimers at the cell surface. Inhibition of receptor internalization did not prevent agonist-promoted BRET, demonstrating that it did not result from clustering of receptors within endosomes. The notion that receptor dimers exist at the cell surface was confirmed further by the observation that BS3, a cell-impermeable cross-linking agent, increased BRET between β2AR molecules. The selectivity of the constitutive interaction was documented by demonstrating that no BRET occurred between the β2AR and two other unrelated GPCR. In contrast, the well characterized agonist-dependent interaction between the β2AR and the regulatory protein β-arrestin could be monitored by BRET. Taken together, the data demonstrate that GPCR exist as functional dimers in vivo and that BRET-based assays can be used to study both constitutive and hormone-promoted selective protein–protein interactions.
698 citations
TL;DR: It is shown that by binding to newly synthesized mutant receptors, small ligands can act as pharmacological chaperones, promoting the proper folding and maturation of receptors and their targeting to the cell surface.
Abstract: Over 150 mutations within the coding sequence of the V2 vasopressin receptor (V2R) gene are known to cause nephrogenic diabetes insipidus (NDI). A large number of these mutant receptors fail to fold properly and therefore are not routed to the cell surface. Here we show that selective, nonpeptidic V2R antagonists dramatically increase cell-surface expression and rescue the function of 8 mutant NDI-V2Rs by promoting their proper folding and maturation. A cell-impermeant V2R antagonist could not mimic these effects and was unable to block the rescue mediated by a permeant agent, indicating that the nonpeptidic antagonists act intracellularly, presumably by binding to and stabilizing partially folded mutants. In addition to opening new therapeutic avenues for NDI patients, these data demonstrate that by binding to newly synthesized mutant receptors, small ligands can act as pharmacological chaperones, promoting the proper folding and maturation of receptors and their targeting to the cell surface.
547 citations
TL;DR: The overall low efficiency of receptor maturation, less than 50% of the precursor being processed to the fully glycosylated protein, suggests that only a fraction of the synthesized receptors attain properly folded conformation that allows exit from the ER.
303 citations
TL;DR: The discovery that ligands with pharmacological selectivity (pharmacological chaperones) can rescue the proper targeting and function of misfolded proteins, including receptors, might help to develop new treatments for 'conformational diseases'.
265 citations
TL;DR: A growing body of evidence indicates that GPCRs could exist and be active as oligomeric complexes, and their existence as homo- or heterodimers could have important implications for the development and screening of new drugs.
Abstract: In contrast to other families of cell surface receptors for which dimerization is an integral part of the activation process, G-protein-coupled receptors (GPCRs) were thought, until recently, to function as monomeric units. However, a growing body of evidence indicates that GPCRs could exist and be active as oligomeric complexes. Because they are major pharmacological targets, their existence as homo- or heterodimers could have important implications for the development and screening of new drugs.
138 citations
TL;DR: DAE is a new imaging approach to anorectal dynamic disorders, providing a highly reliable means of diagnosing perineal insufficiency as well as rectocele, and should be substituted for previous methods since it makes it possible at the same time to assess the anal sphincters and to avoid pelvic irradiation.
Abstract: Buts de l'Etude: Le diagnostic morphologique des troubles de la statique rectale et de 1 insuffisance perineale repose actuellement sur la defecographie Nous avons developpe une nouvelle technique d echographie endoanale (EE) dynamique dont nous avons compare les resultats a la defecographie dans une etude prospective aveugle Patients et Methodes: Nous avons etudie 43 patients qui presentaient une constipation d'allure terminale, d'âge moyen 51 ans, dont 19 (44%) presentaient une incontinence urinaire d'effort L'EE etait realisee a l'aide d'une sonde lineaire de 7,5 MHz (Toshiba, Tokyo, Japon), avec un enregistrement au repos et en poussee Une manometrie anorectale et une defecographie etaient realisees, aucun des operateurs n'etant informes des differents resultats Resultats: La defecographie a diagnostique une descente perineale chez 29 patients (68 %), une rectocele chez 25 patients (58 %), et une procidence rectale interne chez 8 patients (18%) La concordance entre descente du col vesical (EE dynamique) et descente perineale (defecographie) etait de 35/43 patients (80%), entre descente du plan des releveurs (EE dynamique) et descente perineale (defecographie) etait de 40/43 patients (93 %), entre rectocele en EE dynamique et en defecographie etait de 27/43 patients (57%), entre procidence muqueuse interne en EE dynamique et en defecographie etait de 34/43 patients (80%) L'adjonction de 50 ml d'eau dans le rectum realisee chez les 15 derniers patients a permis de faire passer la sensibilite de l'EE dynamique pour le diagnostic de rectocele de 36 a 86% et la precision diagnostique de 57 a 87%, Conclusions: L'EE dynamique semble etre un examen fiable pour deceler une insuffisance perineale mais egalement pour diagnostiquer une rectocele ou une procidence rectale interne L'EE dynamique pourrait remplacer la defecographie dans la mesure ou elle permet en plus une evaluation de l'etat sphincterien anal et evite une irradiation perineale
65 citations
TL;DR: This work monitors the agonist-promoted interaction between the β2-adrenoceptor–luciferase fusion and the cytosolic protein arrestin-3–GFP (green fluorescent protein) and indicates that BRET can be used to study dynamic interactions involving proteins that are located in different cellular compartments under basal conditions but that transiently come into contact on activation of one of the partners.
6 citations
TL;DR: In this article, the cascade d'activation des recepteurs couples aux proteines G (RCPG) semblait etre lineaire and n'impliquer que les proteine G heterotrimeriques.
Abstract: Jusqu'a recemment, la cascade d'activation des recepteurs couples aux proteines G (RCPG) semblait etre lineaire et n'impliquer que les proteines G heterotrimeriques. De nouveaux partenaires ainsi que de nouvelles voies de signalisation sont aujourd'hui identifies. Ainsi, les kinases specifiques aux RCPG et les arrestines, impliquees dans leur decouplage des proteines G, semblent aussi activer d'autres voies de signalisations en servant de proteines adaptatrices ou effectrices. L'interaction directe entre les RCPG et des petites proteines G (Rho, ARF), des canaux ioniques, ou encore des proteines d'echafaudages via des motifs specifiques (SH2, SH3), suggere aussi l'existence de voies de signalisation alternatives. Enfin, ces recepteurs peuvent exister sous forme homo- ou heterodimerique et des proteines chaperons peuvent moduler l'expression et la specificite de certains de leurs ligands.
1 citations