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

Abstract

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 ...

Alterations of mGluR5 and its endogenous regulators Norbin, Tamalin and Preso1 in schizophrenia: towards a model of mGluR5 dysregulation

“…In view of the fact that G-protein coupled receptor dimerization occurs soon after biosynthesis [20], it is also likely that these captive mGluR5 are in the dimer form. A growing body of evidence suggests that mGluR5 proteins are only functional in the form of disulfide-linked dimers [5,29], suggesting an increase in mGluR5 dimers represents an increase in functional mGluR5 units in the present study. However, considering reduced mGluR5 signaling (by mGluR5 knockout or pharmacological antagonism) produces schizophrenia-like behaviors, and mGluR5 PAMs have therapeutic efficacy in preclinical rodent models [see 18], we hypothesize a model of mGluR5 dysregulation in which higher protein expression and dimer units does not represent increased activity and/or function in the schizophrenia brain, but rather, alterations to mGluR5 localization (Fig 4a and b) and also signaling (Fig.…”

Fine tuning of sub-millisecond conformational dynamics controls metabotropic glutamate receptors agonist efficacy

“…Indeed, the glutamate affinity on the isolated ECDs is very similar to that determined on the full-length receptor 51,[55][56][57] . Moreover, we showed that on a an isolated 7TM domain of mGlu2R and on a full-length monomeric mGlu2R construct reconstituted in lipids nanodiscs, positive and negative allosteric modulators acted as full agonists and antagonists, whereas glutamate alone had no effect 9 . The activation of the G-protein by glutamate was recovered upon using of the full-length dimeric receptor, underlying the key role of the ECDs, as a dimer, in the activation process 9 .…”

“…Moreover, we showed that on a an isolated 7TM domain of mGlu2R and on a full-length monomeric mGlu2R construct reconstituted in lipids nanodiscs, positive and negative allosteric modulators acted as full agonists and antagonists, whereas glutamate alone had no effect 9 . The activation of the G-protein by glutamate was recovered upon using of the full-length dimeric receptor, underlying the key role of the ECDs, as a dimer, in the activation process 9 . In summary, we clearly demonstrate that the extracellular domain of the mGluR exists in a preformed equilibrium of conformational states and that the exclusive role of ligands is to modulate the transition rate between states, rather than stabilizing individual conformations at the expense of others.…”

“…It cannot be excluded that these molecules are in reality in slow exchange with the population of dimers engaged in the fast A2R exchange, even though this could not be examined in our study. Determining whether the exploration of the A S state is required for the activation of the receptor in its native environment and proper signal transduction will be the subject of a future study using full-length receptors reconstituted in lipid nanodiscs 9 , an approach successfully used in FCS studies of other membrane proteins 42,43 . Our data explain how mGluR ECDs could be crystallized in an 'active' conformation despite antagonist binding (PDB 3KS9, Structural Genomics Consortium,) and in a 'resting' conformation in the presence of an agonist 16 .…”

“…1a) whose activation mechanism depends on major conformational changes, modulated by the binding of ligands. They are obligate dimers, activated by glutamate 9 , the major excitatory neurotransmitter in the central nervous system, and they are essential to the regulation of synaptic activity 10 , representing, therefore, promising targets in drug development 11 . The eight mGluR subtypes belong to the class C GPCR family, which also includes the receptors for GABA, calcium and for the flavours sweet and umami 12,13 .…”