title: Seven transmembrane receptor core signaling complexes are assembled prior to plasma membrane trafficking.
abstract: Much is known about beta2-adrenergic receptor trafficking and internalization following prolonged agonist stimulation. However, less is known about outward trafficking of the beta2-adrenergic receptor to the plasma membrane or the role that trafficking might play in the assembly of receptor signaling complexes, important for targeting, specificity, and rapidity of subsequent signaling events. Here, by using a combination of bioluminescence resonance energy transfer, bimolecular fluorescence complementation, and confocal microscopy, we evaluated the steps in the formation of the core receptor-G protein heterotrimer complex. By using dominant negative Rab and Sar GTPase constructs, we demonstrate that receptor dimers and receptor-G betagamma complexes initially associate in the endoplasmic reticulum, whereas G alpha subunits are added to the complex during endoplasmic reticulum-Golgi transit. We also observed that G protein heterotrimers adopt different trafficking itineraries when expressed alone or with stoichiometric co-expression with receptor. Furthermore, deliberate mistargeting of specific components of these complexes leads to diversion of other members from their normal subcellular localization, confirming the role of these early interactions in targeting and formation of specific signaling complexes.
authors.affiliation: Department of Pharmacology and Therapeutics, McGill University, Montréal, Quebec H3G 1Y6, Canada.
authors.forename: Denis J
authors.initials: DJ
authors.lastname: Dupré
authors.forename: Mélanie
authors.initials: M
authors.lastname: Robitaille
authors.forename: Nathalie
authors.initials: N
authors.lastname: Ethier
authors.forename: Louis R
authors.initials: LR
authors.lastname: Villeneuve
authors.forename: Aida M
authors.initials: AM
authors.lastname: Mamarbachi
authors.forename: Terence E
authors.initials: TE
authors.lastname: Hébert
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D019281:Dimerization; D004721:Endoplasmic Reticulum / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits / Q000235:genetics / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000235:genetics / Q000378:metabolism*; D005799:Genes, Dominant; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D007425:Intracellular Membranes / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D011869:Radioligand Assay; D051381:Rats; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D015398:Signal Transduction; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D018343:Receptors, Adrenergic, beta-2; D043562:Receptors, G-Protein-Coupled; C450520:seven-transmembrane G-protein-coupled receptor; C493083:SAR1A protein, human; D020559:Monomeric GTP-Binding Proteins; D020691:rab GTP-Binding Proteins