Seven Transmembrane Receptor Core Signaling Complexes Are Assembled Prior to Plasma Membrane Trafficking

Abstract

Much is known about ␤ 2 -adrenergic receptor trafficking and internalization following prolonged agonist stimulation. However, less is known about outward trafficking of the ␤ 2 -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␤␥ complexes initially associate in the endoplasmic reticulum, whereas G␣ 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.

Intracellular activation of vasopressin V2 receptor mutants in nephrogenic diabetes insipidus by nonpeptide agonists

“…Recent observations that the ␤2-adrenergic receptor assembles with G␣s and adenylate cyclase in the ER (19,20) and that the naturally cell permeable agonist estrogen induces signaling from the ER-localized GPR30 (21,22) indicate that nonpeptide agonists may activate the V2R mutants to generate cAMP while located in the ER. However, considering the expression of some V2R mutants beyond the ER-Golgi complex, this remains to be established.…”

Rescue of Calcium-sensing Receptor Mutants by Allosteric Modulators Reveals a Conformational Checkpoint in Receptor Biogenesis

“…Rather, we infer that either mutation or drugs favor certain receptor conformations, and that this leads to either distinct protein-protein interactions or formation of nascent signaling complexes that may shield CaR from dorfin-mediated ubiquitination (1) or assist in forward trafficking of the receptor. Recent evidence suggests that assembly of G proteincoupled receptor signaling complexes may occur in the ER (40), and such complexes may function in some signaling capacity (39). Whether CaR transduces signals within intracellular compartments remains to be determined.…”

Regulation of the Human Ether-a-go-go-related Gene (hERG) Channel by Rab4 Protein through Neural Precursor Cell-expressed Developmentally Down-regulated Protein 4-2 (Nedd4-2)

“…It has been shown that Rab1 and Rab2 regulate endoplasmic retic-* This work was supported by the Canadian Institutes of Health Research ulum-to-Golgi transport (18,19); Rab4 is localized at the early sorting endosome and is responsible for rapid/direct recycling from early endosomes to the cell surface (20,21); Rab5 regulates the fusion between endocytic vesicles and early endosomes (22,23); Rab7 has primarily been implicated in the transport from early to late endosomes and plays an essential role in the maintenance of the perinuclear lysosome compartment (17,22,24); and Rab11 is involved in mediating slow recycling from endosomes to the plasma membrane (25).…”