Monitoring Receptor Oligomerization Using Time-resolved Fluorescence Resonance Energy Transfer and Bioluminescence Resonance Energy Transfer

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254

Several G protein-coupled receptors have been shown to exist as homo-and hetero-oligomeric complexes in living cells. However, the link between ligand-induced receptor activation and its oligomerization state as well as the proportion of the total receptor population that can engage in oligomeric complexes remain open questions. Here, the closely related human MT1 and MT2 melatonin receptors (MT1R, MT2R) were used to address these issues. Bioluminescence resonance energy transfer (BRET) experiments in living HEK 293 cells revealed that these receptors form homo-and hetero-oligomers. Constitutive energy transfer was observed for all receptor combinations at physiological expression levels and could be detected in single cell BRET experiments. Inhibition of the energy transfer by dilution of the BRET partners identified MT1R and MT2R dimers as the predominant receptor species, and this oligomerization state did not change upon agonist and antagonist binding. Agonists, neutral antagonists, and inverse agonists all promoted increases in BRET values for MT2R but not for MT1R homodimers in living cells and isolated plasma membranes. This indicates that no correlation could be inferred between the receptor activation state and the dimerization state of the receptor. This also suggests that ligand-promoted BRET increases represent specific ligand-induced conformational changes of pre-existing dimers rather then increased dimerization. The observation that ligands favored the energy transfer within the hetero-oligomer from MT1R to MT2R but not in the reverse orientation, from MT2R to MT1R, supports this view.Membrane proteins such as tyrosine kinase, cytokine, or transforming growth factor receptors have been known for many years to form oligomers, and the link between their oligomerization and activity states has been well established (1). In contrast, G protein-coupled receptor (GPCR) 1 oligomerization has been documented only recently (2), and the relation between receptor activation and oligomerization is still poorly understood. Even the proportion between monomeric and oligomeric receptor species and the exact oligomerization state (dimer, trimer, tetramer, etc.) remains a matter of controversy. Currently, two models are proposed. In the first model GPCR are monomeric in their inactive state, and agonist activation induces the formation of receptor oligomers. This model is based on low basal and strong agonist-induced energy transfer signals observed in fluorescence and bioluminescence resonance energy transfer (FRET, BRET) experiments for the gonadotropin-releasing hormone (3), the somatostatin SSTR5 (4), and the SSTR5/dopamin D2R receptor oligomers (5). The second model proposes that GPCR are constitutively oligomerized and is supported by studies reporting high basal BRET or FRET signals for ␣-mating factor (6), ␤2-adrenergic (␤2AR) (7), tyrothropin-releasing hormone (8), ␦-opioid (9), type A cholecystokinin (10), and dopamine D2 receptors (11). Agonist-promoted increases in signals were observed in some of thes...

Section: Detection Of Mt1r and Mt2r In Hek 293supporting

confidence: 65%

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confidence: 80%

Section: Fig 3 Determination Of the Oligomerization State Of Mtr Hmentioning

confidence: 99%

See 1 more Smart Citation

Monitoring of Ligand-independent Dimerization and Ligand-induced Conformational Changes of Melatonin Receptors in Living Cells by Bioluminescence Resonance Energy Transfer

Ayoub¹,

Couturier²,

Lucas-Meunier³

et al. 2002

287

254

“…This shows that the coil-coiled domains in the C-terminal tails of these subunits strongly stabilize the heterodimer. We therefore conducted TR-FRET experiments as described previously (40). In this assay, a FRET signal is measured at the surface of intact COS-7 cells between a donor molecule (europium cryptate-pyridine bipyridine) linked to an anti-HA monoclonal antibody and an acceptor molecule (Alexa Fluor® 647) linked to an anti-c-Myc monoclonal antibody (Fig.…”

Section: Gb2 Increases Agonist (But Not Antagonist) Affinity Inmentioning

confidence: 99%

Molecular Determinants Involved in the Allosteric Control of Agonist Affinity in the GABAB Receptor by the GABAB2 Subunit

Liu

Maurel

Etzol

et al. 2004

The ␥-aminobutyric acid type B (GABA B ) receptor is an allosteric complex made of two subunits, GABA B1 (GB1) and GABA B2 (GB2). Both subunits are composed of an extracellular Venus flytrap domain (VFT) and a heptahelical domain (HD). GB1 binds GABA, and GB2 plays a major role in G-protein activation as well as in the high agonist affinity state of GB1. How agonist affinity in GB1 is regulated in the receptor remains unknown. Here, we demonstrate that GB2 VFT is a major molecular determinant involved in this control. We show that isolated versions of GB1 and GB2 VFTs in the absence of the HD and C-terminal tail can form hetero-oligomers as shown by time-resolved fluorescence resonance energy transfer (based on HTRF® technology). GB2 VFT and its association with GB1 VFT controlled agonist affinity in GB1 in two ways. First, GB2 VFT exerted a direct action on GB1 VFT, as it slightly increased agonist affinity in isolated GB1 VFT. Second and most importantly, GB2 VFT prevented inhibitory interaction between the two main domains (VFT and HD) of GB1. According to this model, we propose that GB1 HD prevents the possible natural closure of GB1 VFT. In contrast, GB2 VFT facilitates this closure. Finally, such inhibitory contacts between HD and VFT in GB1 could be similar to those important to maintain the inactive state of the receptor.

“…Heterodimerization of opioid receptors and ␦ results in novel ligand binding and function, proving that GPCR oligomerization has physiologic relevance (12). More recently, energy transfer studies, using either fluorescence (FRET) or bioluminescence (BRET), have also shown the proximity of monomers of many members of the rhodopsin family of GPCRs, including ␤ 2 -adrenergic receptor (13), somatostatin receptors (14), opioid receptors (15), and the C5a receptor (57).…”

mentioning

confidence: 99%

C5a Receptor Oligomerization

Klco

Lassere

Baranski

2003

108

G protein-coupled receptors (GPCRs), stimulated by hormones and sensory stimuli, act as molecular switches to relay activation to heterotrimeric G proteins. Recent studies suggest that GPCRs form dimeric or oligomeric structures, a phenomenon that has long been established for growth factor receptors. The elucidation of the domains of GPCRs that mediate receptor association is of critical importance for understanding the function of GPCR oligomers. Using a disulfide-trapping strategy to probe the intermolecular contact surfaces, we demonstrate cross-linking of C5a receptors in membranes prepared from both human neutrophils and stably transfected mammalian cells that is mediated by a cysteine in the second intracellular loop. To explore other surfaces that might be involved in the oligomerization of C5a receptors, we constructed receptors with individual cysteines in other intracellular regions. C5a receptors with a cysteine in the first intracellular loop or the carboxyl terminus displayed the fastest kinetics of dimer formation, whereas an intracellular loop 3 cysteine displayed minimal cross-linking. Since the rate of disulfide trapping reflects the proximity of sulfhydryl groups, assuming similar accessibility and flexibility, these results imply a symmetric dimer interface that may involve either transmembrane helices 1 and 2 or helix 4. However, neither model can account for the ability of the native cysteine in the second intracellular loop to mediate efficient crosslinking. Based on these observations, we propose that C5a receptors form higher order oligomers (i.e. tetramers) or clusters in the membrane.G protein-coupled receptors (GPCRs) 1 are an evolutionarily conserved family of transmembrane receptors that are characterized by seven-transmembrane helixes connected by intracellular loops (ICs) and extracellular loops (1, 2). GPCRs mediate a multitude of physiologic responses and serve as common pharmacological targets; more than half of all pharmacological agents that are currently prescribed target GPCRs (3). Receptor activation is accomplished by a remarkably diverse group of ligands to catalyze the exchange of GTP for GDP on the ␣ subunit of heterotrimeric G proteins. This exchange results in the dissociation of the ␣-GTP subunit from the ␤␥ dimer. Both complexes can then activate downstream targets, such as effector enzymes and ion channels (4, 5).Despite numerous structure-function studies of many different GPCRs, a fundamental question remains: Do GPCRs function as monomers or as oligomers? For the ϳ25 members of class C receptors, the answer is clear. These receptors form dimers through specialized structures, such as disulfidebonded extracellular domains (metabotropic glutamate receptors) (6, 7) or coiled-coil interactions of the carboxyl-terminal tails (GABA B receptors) (8). For the more than 500 members of the rhodopsin family of GPCRs and the 60 members of the secretin family of GPCRs, studies now demonstrate that a rapidly increasing number of these GPCRs form oligomers (9, 10). Early work...