Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET)

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 ␤2-adrenergic receptor (␤ 2AR) 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 ␤2AR 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 ␤2AR molecules. The selectivity of the constitutive interaction was documented by demonstrating that no BRET occurred between the ␤2AR and two other unrelated GPCR. In contrast, the well characterized agonist-dependent interaction between the ␤2AR and the regulatory protein ␤-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. G protein-coupled receptors (GPCR) represent the single largest family of transmembrane receptors involved in cell signaling. Until recently, they were believed, unlike most other membrane receptors, to function as monomeric entities that interact with G proteins once stabilized in their active conformation by agonist binding. However, a growing body of functional and biochemical evidence suggests that they may exist as homo-or heterodimers. The functional evidence is based largely on positive and negative effects that dominant receptor mutants have on wild-type receptor function and on the observation that coexpression of two defective receptors can restore activity (1-6). More recently, coexpression of the type-2b ␥-aminobutyric acid receptor GABAb-R2 was found to be essential for the cell surface expression and the function of the GABAb-R1 subtype (7-9), suggesting that heterodimerization between the two receptor molecules is required for function. Biochemically, coimmunoprecipitation of receptors bearing different epitope tags was used to support the notion that GPCR homo-(10 -13) and heterodimers (9, 14) can form. However, the cons...

Cell-surface targeting of α2-adrenergic receptors — Inhibition by a transport deficient mutant through dimerization

“…Therefore, dimers are unable to export from the ER. Dimerization has been well described for a variety of G protein-coupled receptors [9][10][11][12][13][14][15][34][35][36][37][38][39][40][41]. However, whether α 2B -AR is capable of forming homodimers has not been reported.…”

Carboxypeptidase M and Kinin B1 Receptors Interact to Facilitate Efficient B1 Signaling from B2 Agonists

“…Cross-linking of B1R and CPM-Cross-linking was carried out on whole cells using BS 3 similar to procedures described previously (29). Briefly, cells were scraped into PBS and centrifuged for 10 min at 1000 ϫ g. The cell pellets were resuspended in PBS containing 2 mM BS 3 and rotated for 2 h at 4°C.…”

Des dimères et des oligomères de récepteurs couplés aux protéines G, oui mais pourquoi ?

“…Parmi ces technologies, les techniques de transfert d'énergie par résonance BRET et FRET (pour bioluminescence ou fluorescence resonance energy transfer) se sont avérées cruciales, car elles révèlent la proximité spatiale de deux protéines [22,23]. En effet, le transfert d'énergie entre deux entités fluorescentes ne se produit que pour des distances inférieures à 100 Å. Dans le cas des RCPG, une telle distance entre deux récepteurs correspond à une interaction directe.…”