Introduction
Metabotropic glutamate receptors are found in the central nervous system and play a critical role in modulating cell excitability and synaptic transmission[1]. Glutamate is the main neurotransmitter in the brain and activates 8 different types of metabotropic glutamate receptors[2]. Metabotropic Glutamate Receptor 2(mGlu2) is a member of the Class C GPCRFamily and can further be classified into the Group II subgroup of metabotropic receptors. Since mGlu2 is a part of the Class C GPCR family, it undergoes small conformational changes to the transmembrane domain (TMD) to move from the inactive to the fully active structure[1]. Functionality of mGlu2 will be dependent on the concentration of glutamate. Higher concentrations of glutamate will promote stronger signal transduction from the extracellular domain to the transmembrane domain.
mGlu2 plays vital roles in memory formation, pain management, and addiction, which makes it an important drug target for Parkinson’s Disease[3], Schizophrenia (blue link), Cocaine Addiction[4], and many other neurological conditions.
Structure
Overall Structure
Cryo-EM studies of mGlu2 have yielded adequate structures that have acted as maps to aid in producing a better structural understanding of the inactive and active states of mGlu2[1]. The overall structure of the mGlu2 is composed of 3 main parts: a ligand binding Venus FlyTrap Domain(VFT), followed by a Cysteine Rich Domain linker to the Transmembrane Domain that contains 7 alpha helices (7TM) on both the that aid in the binding of the G-Protein. Class C CPCRs such as mGlu2, are activated by their ability to form dimers. MGlu2 is a homodimer which is imperative to the receptor’s ability to relay signals induced by glutamate from the extracellular domain(ECD) to its transmembrane domain(TMD). The homodimer of mGlu2 contains an alpha chain and a beta chain. Occupation of both ECDs with the agonist, glutamate, is necessary for a fully active mGlu2[5]. However, only one chain in the dimer is responsible for activation of the G-protein, this suggests an asymmetrical signal transduction mechanism for mGlu2[1].
Inactive State
A few hallmarks of the inactive structure of mGlu2 are the Venus FlyTrap Domain in the open conformation, well separated Cysteine-Rich Domains, and distinct orientation of the 7 Transmembrane Domains (7TM). Perhaps the most critical component of the inactive form is the asymmetric TM3-TM4 interface formed by both of the 7 alpha helices in the alpha and beta chains in the transmembrane domain. The transmembrane domain is mediated mainly by helix IV on the alpha chain and helix lll on the beta chain of the dimer through hydrophobic interactions. These hydrophobic interactions between both transmembrane helices stabilize inactive conformation of mGlu2[1].
Intermediate Form
Although there are no Cryo-EM images of the intermediate form, it is still a very important state that mGlu2 goes through. The agonist binding site is formed by both lobes of the Venus FlyTrap Domain. The receptor will remain in this inactive state if there are insufficient concentrations of glutamate available[5]. Since glutamate is the main excitatory neurotransmitter in the central nervous system, its ability to bind is extremely important, especially for cell excitability.
PAM and NAM Bound Form
A positive allosteric modulator (PAM) or a negative allosteric modulator (NAM) can bind to mGlu2. PAM binds to the receptor, induces conformational changes, which help promote greater affinity for G protein binding. PAM binds in a binding pocket that is created by alpha helices III, V, VI, VII in the transmembrane domain. Upon binding of PAM, it interacts with helix VI, including residues W773, F776, L777, and F780. Due to spatial hindrance, helix VI is shifted downward, causing conformational changes. NAM, however, reduces the affinity for G protein binding. NAM binds to the same binding pocket as PAM and also interacts with residue W773. Due to the structure of NAM, it occupies the binding site a little deeper than PAM. This causes NAM to push on the side chain of W773 towards helix VII[1]. PAM and NAM induce different conformational changes, which result in different outcomes.
Figure 3:This is PAM located in its binding pocket. PAM, JNJ-40411813, is shown in magenta and colored by atom. The image shows four labelled alpha helices (III, V, VI, and VII) that create the binding pocket in the 7TM region of mGlu2 for PAM to bind within. The binding of PAM promotes the function of the mGLu2.
Active State
Upon binding of the PAM, helix VI is shifted downward in the transmembrane domain. This downward shift induces a reorientation of the transmembrane domain from its original TM3-TM4 asymmetric dimer interface in the inactive form to now a TM6-TM6 asymmetric dimer interface. The downward shift of helix VI is crucial for the receptor’s transformation from the inactive to the active form for 2 main reasons: (1) reorientation breaks key interactions in the transmembrane domain that stabilize the inactive form (2) positions intracellular loops of the helices in the transmembrane domain to assist in the binding and recognitions of the G-Protein.
G-Protein Recognition
Reorientation positions helix lll on either the alpha or beta chain because both have the ability to bind to the G-protein but only one chain is required for full receptor activation. The intracellular region of helix lll mainly contributes to the interactions with the alpha subunit of the G-protein. Intracellular Loop 2 plays a key role in G-protein coupling as well by building polar interaction networks through its ionic interactions with the alpha subunit of the G-protein. Lastly, mGlu2 residue E666 forms a salt bridge with an alpha N residue (R32) on the alpha subunit which further destabilizes the inactive conformation[1].
G-protein Binding
The PAM induced downward shift of helix IV coupled with the reorientation of the transmembrane domain to a TM6-TM6 asymmetric interface, opens up a cleft on the intracellular surface of the receptor. This cleft allows a “hook-like” region, that is composed of the last 4 residues of the alpha subunit of the G-protein, to move in adjacent to helix IV in the transmembrane domain. One very important residue in this interaction is C351on the hook that participates in hydrophobic interactions with Intracellular loop 2 and helix IV. It is due to these interactions that the C-terminal region of the alpha subunit of the G-protein binds in the shallow groove formed by intracellular loops 2 and 3 and residues on helices lll and lV[1].The receptor is now fully active with the dimer coupled only to one G-protein, the Venus FlyTrap Domain in the closed conformation resulting in a tighter form, and the transmembrane domain helices reoriented on both the alpha and beta chains to form an asymmetric dimer interface.
Clinical Relevance
