Locking the Dimeric GABA B G-Protein-Coupled Receptor in Its Active State

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The eight metabotropic glutamate receptors (mGluRs) are key modulators of synaptic transmission and are considered promising targets for the treatment of various brain disorders. Whereas glutamate acts at a large extracellular domain, allosteric modulators have been identified that bind to the seven transmembrane domain (7TM) of these dimeric G-protein-coupled receptors (GPCRs). We show here that the dimeric organization of mGluRs is required for the modulation of active and inactive states of the 7TM by agonists, but is not necessary for G-protein activation. Monomeric mGlu2, either as an isolated 7TM or in full-length, purified and reconstituted into nanodiscs, couples to G proteins upon direct activation by a positive allosteric modulator. However, only a reconstituted full-length dimeric mGlu2 activates G protein upon glutamate binding, suggesting that dimerization is required for glutamate induced activation. These data show that, even for such well characterized GPCR dimers like mGluR2, a single 7TM is sufficient for G-protein coupling. Despite this observation, the necessity of dimeric architecture for signaling induced by the endogenous ligand glutamate confirms that the central core of signaling complex is dimeric.M etabotropic glutamate receptors (mGluRs) play key roles in the modulation of both excitatory and inhibitory synapses in the brain. These eight G-protein-coupled receptors (GPCRs) represent major targets for pharmaceutical companies in search of new treatments for a variety of neurological and psychiatric disorders (1-3). These receptors are part of the class C GPCR family that also includes the GABA B , calcium sensing, and sweet and umami taste receptors, which are all major targets for drug development (4).The structural complexity of class C GPCRs, compared with rhodopsin-like class A GPCRs, offers multiple possibilities in designing molecules that modulate their activity. Not only are mGluRs strict constitutive dimers (5, 6), but each protomer is composed of several domains (7,8). Agonists bind in a bilobate venus fly-trap domain (VFT) (9), which is linked through a cysteine-rich domain (CRD) to the heptahelical transmembrane domain (7TM) that is responsible for G-protein activation (7). The 7TM is the target of a number of synthetic compounds acting either as negative or positive allosteric modulators (NAMs and PAMs, respectively). Given their ability to finely tune endogenous signaling, such compounds present exciting opportunities for drug development (10).The functional mechanism of such a complex machine remains to be characterized, although some critical steps have been well documented. Previous studies have shown that receptor activation results from the closure of the VFT upon agonist binding (9,(11)(12)(13)(14)(15). This conformational change in the extracellular domain is coupled to a conformational change in the intracellular side of at least one 7TM that is responsible for G-protein coupling (16)(17)(18)(19). The mechanism for allosteric communication between the VFT and 7TM ...

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confidence: 99%

Distinct roles of metabotropic glutamate receptor dimerization in agonist activation and G-protein coupling

Moustaine

Granier

Doumazane

et al. 2012

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133

“…Structural and mutagenesis studies indicated that the closure of the VFT resulting from agonist binding in the cleft represents a key step in receptor activation (12)(13)(14)(15)(16). It is assumed that this VFT conformational change is associated with a reorientation of the two VFTs in these dimeric receptors (15,16), leading to a relative movement of the two 7TMs and the activation of one of them (17).…”

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confidence: 99%

Interdomain movements in metabotropic glutamate receptor activation

Huang

Cao

Jiang

et al. 2011

Many cell surface receptors are multimeric proteins, composed of several structural domains, some involved in ligand recognition, whereas others are responsible for signal transduction. In most cases, the mechanism of how ligand interaction in the extracellular domains leads to the activation of effector domains remains largely unknown. Here we examined how the extracellular ligand binding to the venus flytrap (VFT) domains of the dimeric metabotropic glutamate receptors activate the seven transmembrane (7TM) domains responsible for G protein activation. These two domains are interconnected by a cysteine-rich domain (CRD). We show that any of the four disulfide bridges of the CRD are required for the allosteric coupling between the VFT and the 7TM domains. More importantly, we show that a specific association of the two CRDs corresponds to the active state of the receptor. Indeed, a specific crosslinking of the CRDs with intersubunit disulfide bridges leads to fully constitutively active receptors, no longer activated by agonists nor by allosteric modulators. These data demonstrate that intersubunit movement at the level of the CRDs represents a key step in metabotropic glutamate receptor activation.transmembrane signaling | G protein-coupled receptor | allosteric modulation M ost cell surface receptors are multimeric complexes of which each subunit is produced through the association of different domains throughout evolution (1-5). The activation of such receptor complexes is a result of coordinated conformational changes or movement of these different domains. Although an increasing amount of 3D crystal structures are becoming available, there is still limited information available on the structural basis of interdomain communication.Class C G protein-coupled receptors (GPCRs) represent key examples of such receptor complexes (6, 7). These receptors are obligatory dimers, either homo-or heterodimers, made by the association of two domains over evolutionary time; an extracellular bilobate venus flytrap (VFT) domain associated with a G protein activating 7 transmembrane (7TM) domain (Fig. 1A) (8). The VFTs are evolved from certain types of bacterial periplasmic binding proteins, especially those of the leucine-isoleucinevaline binding protein family involved in the transport of amino acids, sugars, or ions. Not surprisingly, class C GPCRs are activated by amino acids, i.e., the receptors for the two major neurotransmitters, the eight metabotropic glutamate receptors (mGluRs), and the GABA B receptor; sugar (the sweet taste receptors); or ions [the calcium-sensing receptor (CaSR)]. Accordingly, class C GPCRs represent exciting new targets for drug development for both the pharmaceutical and food industries, as illustrated by the number of drugs targeting these receptors already on the market (the GABA B receptor agonist baclofen, umami compounds, and various sweeteners such as aspartame, and cinacalcet, a positive allosteric modulator of the CaSR), and those in clinical trials (9-11).The precise mechanisms of how...

“…Recently, activation of some class A (rhodopsin-like) GPCRs has been monitored in real-time by using a fluorescence resonance energy transfer (FRET)-based approach (6). The FRET responses observed upon agonist binding were well described by monoexponential fits whose time constants were faster than previously expected: approximately 40 ms for the ␣ 2A -adrenergic receptor (7), 70 ms for the A 2A -adenosine receptor (8), and 60 ms for the ␤ 1 -adrenergic receptor (9), although not as fast as for light receptor rhodopsin (10) and ligand-gated ion channels (11).A distinctive feature of class C GPCRs is a very large extracellular domain (12), comprising a noteworthy Venus Flytrap module (VFTM), a bilobate structure in the cleft of which the agonist binds and induces its closure (13,14). Compared with other classes, the overall conformational change leading to activation of class C GPCRs appears more complex since it is preceded by the closure of the VFTM (12), an additional step that may delay activation.…”

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confidence: 99%

Optical measurement of mGluR1 conformational changes reveals fast activation, slow deactivation, and sensitization

Marcaggi

Mutoh

Dimitrov

et al. 2009

Metabotropic glutamate receptor (mGluR) activation has been extensively studied under steady-state conditions. However, at central synapses, mGluRs are exposed to brief submillisecond glutamate transients and may not reach steady-state. The lack of information on the kinetics of mGluR activation impairs accurate predictions of their operation during synaptic transmission. Here, we report experiments designed to investigate mGluR kinetics in real-time. We inserted either CFP or YFP into the second intracellular loop of mGluR1␤. When these constructs were coexpressed in PC12 cells, glutamate application induced a conformational change that could be monitored, using fluorescence resonance energy transfer (FRET), with an EC50 of 7.5 M. The FRET response was mimicked by the agonist DHPG, abolished by the competitive antagonist MCPG, and partially inhibited by mGluR1-selective allosteric modulators. These results suggest that the FRET response reports active conformations of mGluR1 dimers. The solution exchange at the cell membrane was optimized for voltageclamped cells by recording the current induced by co-application of 30 mM potassium. When glutamate was applied at increasing concentrations up to 2 mM, the activation time course decreased to a minimum of approximately 10 ms, whereas the deactivation time course remained constant (ϳ50 ms). During long-lasting applications, no desensitization was observed. In contrast, we observed a robust sensitization of the FRET response that developed over approximately 400 ms. Activation, deactivation, and sensitization time courses and amplitudes were used to derive a kinetic scheme and rate constants, from which we inferred the EC50 and frequency dependence of mGluR1 activation under non-steady-state conditions, as occurs during synaptic transmission.he amino acid L-glutamate, the main neurotransmitter in the CNS, is referred to as a fast neurotransmitter because it mediates fast excitatory neurotransmission by activating ionotropic glutamate receptors (i.e., NMDA, AMPA, and kainate receptors). It also targets metabotropic glutamate receptors (mGluRs, 8 types) that belong to the class C G protein-coupled receptor (GPCR) subfamily. As opposed to ionotropic glutamate receptors, mGluRs mediate slower effects of glutamate, downstream of G protein activation (1). In addition, mGluRs have often been considered to play a role as extrasynaptic receptors because they are not located within the synaptic cleft (2), and their affinity for glutamate may be high enough (3) to detect glutamate concentration in extrasynaptic locations. Their extrasynaptic localization appears consistent with the slow effects of their activation because glutamate transients are expected to be less sudden at extra-and perisynaptic sites than opposite to the release site. However, even at extrasynaptic locations, glutamate transients evoked by synaptically released glutamate occur within milliseconds, as shown by recording of glutamate transporter-mediated currents in glial cells that wrap around synapses (4, 5...