Glutamate binding triggers monomerization of unliganded mGluR2 dimers
Introduction
Metabotropic glutamate receptors (mGluRs) are members of the class C G-protein coupled receptors (GPCRs) [1]. They are expressed throughout the central nervous system (CNS) [2] and are classified as class I, II, and III based on receptor structure and physiological function [2,3]. Class I is mainly postsynaptic and contains mGluR1 and mGluR5 receptors whereas Class II and III are presynaptic [2]. mGluR2 and mGluR3 are class II receptors and mGluR4, mGluR6, mGluR7, and mGluR8 are classified as class III receptors [2,4]. During signaling events, Class I receptors couple to Gq/G11 and activate phospholipase C that leads to calcium mobilization and activation of protein kinase C (PKC) [2]. On the other hand, Group II and Group III receptors predominantly couple to Gi/o proteins which inhibit adenylyl cyclase, regulate ion channels, and other downstream signaling partners [2]. mGluRs play important roles in the modulation of both excitatory and inhibitory synapses in the brain and are involved in many neurological and psychiatric disorders such as Alzheimer's disease, Parkinson's disease, anxiety, depression, schizophrenia, etc. [2,5,6].
Structurally, mGluRs contain three domains: a large extracellular domain, a heptahelical transmembrane domain, and a short intracellular C-terminal domain [7]. The extracellular domain is comprised of a ligand binding domain (LBD) and a cysteine-rich domain (CRD) [7]. LBD harbors the ligand-binding site and is the primary site for the initiation of the conformational change leading to G-protein activation [2]. The transmembrane domain contains the sites for allosteric modulation, both negative (NAMs) and positive (PAMs) [8,9]. Allosteric modulation is an important aspect of the fine-tuning of endogenous signaling related to mGluRs.
To date, mGluR5 and mGluR2 are shown to form strict dimers [[10], [11], [12]] and the dimeric organization of mGluR2 is necessary for glutamate induced activation [11]. It has been reported that glutamate binds to the dimer and induces a conformational change in the LBD domain that is associated with downstream signaling [13]. Contradicting this notion, PAMs can interact with the monomeric form of the receptor and initiate signaling [11]. However, when both glutamate and PAMs act simultaneously, receptor activation is abrogated in its dimeric state [11]. The reasons for this phenomenon are not clear. A recent study, however, has shown that mGluR1, mGluR2, mGluR3, mGluR4, mGluR5, and mGluR7 all form dimers and the dimerization propensity of mGluR3 is stronger than the dimerization propensity of mGluR2 [14], thus contradicting the fact that mGluR2 forms a strict dimer.
X-ray structures of the ligand binding domain (LBD) of mGluR2 in both the relaxed (unliganded) and active (liganded) form lack BC loop (residues Leu110-Pro133) coordinates [15]. This loop connects helices B and C in the LBD of mGluR2 protomer and is unstructured. It is speculated that BC-loop forms an intermolecular disulfide bridge at the Cys121 position and is important to hold receptors into strict dimeric form. This speculation is based on an observation that mGluR5 is a disulfide linked dimer in a previous study [10]. However, a recent study showed that L103A, L154A, and F158A mutations in ligand binding domain reduce the dimerization propensity of mGluR2 significantly suggesting that Cys121 does not form inter- disulfide bond [12]. Furthermore, mutating Cys121 to Ala121 in mGluR2 moderately affects dimerization and that the ligand binding could induce a conformational change in the mutated receptor resulting in receptor activation [12] The role of BC-loop was deciphered in a recent study. It was shown to be essential for glutamate induced conformational change and hence the activation of the mGluR2 [16]. However, the role of BC loop in the assembly of mGluR2 remains unclear.
Thus far, the question related to the functional assembly of mGluR2 has been vaguely addressed. In this study, we aim to address the assembly state of the mGluR2 in live cells. More precisely, we addressed if mGluR2 exists in a dynamic monomer-dimer equilibrium in live cells and whether glutamate binding to mGluR2 affects the equilibrium. To answer these questions, we examined the dimerization propensity of mGluR2 with and without unstructured BC loop in the absence and in the presence of saturating concentration of glutamate using Forster Resonance Energy Transfer (FRET) in live cells. We showed that mGluR2 did not form a strict dimer but rather existed in a dynamic monomer-dimer equilibrium. The deletion of the BC loop from mGluR2 moderately reduced the dimerization propensity of mGluR2. Furthermore, glutamate binding to mGluR2 induced a conformational change in the receptor that led to monomerization of mGluR2. The deletion of the unstructured BC loop from mGluR2 prevents both conformational change and monomerization of the receptor upon binding to glutamate.
Section snippets
Plasmid constructs
For this study, N-terminally HA-SNAP tagged mGluR2 construct was used as described previously [17]. To obtain the HA-SNAP-mGluR2ΔBC construct (BC loop deletion mutant), the residues from S109 to A135 were deleted from the full-length HA-SNAP-mGluR2 construct.
Protein expression and labeling
Plasmid constructs described above were transfected in AAV 293 cells grown in DMEM media supplemented with 10% fetal bovine serum (FBS) (ThermoScientific, Waltham, MA). Transient transfection was performed using an MBS mammalian
Results
To study the dimerization propensity of mGluR2 we used FRET in live cells. AAV293 cells were transfected with plasmids encoding the fusion proteins HA-SNAP-mGluR2 or HA-SNAP-mGluR2ΔBC. The tagging of mGluR2 with HA-SNAP has been shown not to alter its function [13]. The unstructured loop (BC loop) was deleted (Fig. 1A) from full-length mGluR2 to obtain mGluR2ΔBC. Cells expressing the transfected protein were reseeded in poly-d-lysine coated four-chamber glass-bottom dishes at very low densities
Discussion
Previously, it has been reported that mGluR2 forms a strict dimer [11,12], and the dimeric assembly of mGluR2 is required for glutamate induced activation [11]. Our data suggest that FRET increases as a function of protein expression until it hits the plateau (FRETmax). This is a clear indication for a protein existing in a dynamic monomer-dimer equilibrium. We also observed a glutamate induced conformational change as reported by previous investigators [13]. Additionally, we observed that the
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgment
We acknowledge Seth L. Robia from the Department of Cell and Molecular Physiology at Stritch School of Medicine for lab facilities. KP acknowledges Dr. Rajesh Upadhyay, Dr. Amit Kumar, and financial support from IITG. There was no specific funding to support this work.
References (33)
- et al.
Dimers and beyond: the functional puzzles of class C GPCRs
Pharmacol. Therapeut.
(2011) - et al.
Evolution, structure, and activation mechanism of family 3/C G-protein-coupled receptors
Pharmacol. Ther.
(2003) - et al.
The complexity of their activation mechanism opens new possibilities for the modulation of mGlu and GABAB class CG protein-coupled receptors
Neuropharmacology
(2011) - et al.
Metabotropic glutamate receptor 5 is a disulfide-linked dimer
J. Biol. Chem.
(1996) - et al.
Mechanism of assembly and cooperativity of homomeric and heteromeric metabotropic glutamate receptors
Neuron
(2016) - et al.
Defining the homo- and heterodimerization propensities of metabotropic glutamate receptors
Cell Rep.
(2020) - et al.
Unstructured loop is essential for the activation of mGluR2
Biochem. Biophys. Res. Commun.
(2020) - et al.
Photobleaching-corrected FRET efficiency imaging of live cells
Biophys. J.
(2004) - et al.
Phosphomimetic mutations increase phospholamban oligomerization and alter the structure of its regulatory complex
J. Biol. Chem.
(2008) - et al.
Phospholamban binds with differential affinity to calcium pump conformers
J. Biol. Chem.
(2011)
Newly discovered micropeptide regulators of SERCA form oligomers but bind to the pump as monomers
J. Mol. Biol.
Cerulean, Venus, and VenusY67C FRET reference standards
Biophys. J.
GPCR homo-oligomerization
Curr. Opin. Cell Biol.
Metabotropic glutamate receptors: physiology, pharmacology, and disease
Annu. Rev. Pharmacol. Toxicol.
The metabotropic glutamate receptors: structure, activation mechanism and pharmacology
Curr. Drug Targets - CNS Neurol. Disord.
Metabotropic glutamate receptors as novel targets for anxiety and stress disorders
Nat. Rev. Drug Discov.
Cited by (2)
Glutamate receptor endocytosis and signaling in neurological conditions
2023, Progress in Molecular Biology and Translational ScienceCitation Excerpt :mGluR2 confers neuroprotection against Aβ-induced toxicity (synthetic β-amyloid protein) only when activated in combination with mGluR3, elucidating their potential importance in Alzheimer's disease.72 The existence of mGluR2 in a dynamic equilibrium of monomeric and dimeric forms rather than a strict dimer has recently been demonstrated and glutamate binding induced conformational changes monomerize the receptor in the presence of an unstructured loop (BC loop formed by certain amino acids of VFD).73,74 mGluR3—mGluR3 receptors are also found in the cerebral white matter, apart from the hippocampal regions and entorhinal cortex.68
Cross-species transmission, evolution and zoonotic potential of coronaviruses
2023, Frontiers in Cellular and Infection Microbiology
- 1
These authors contributed equally.