Introduction

Biological Background

G protein-coupled receptors (GPCRs) can be broken up into four classes: A, B, C, and F. ^[5]. Metabotropic glutamate receptor 5 (mGLu₅) is a Class C GPCR that couples with the G_q pathway ^[6].

Structure

Overall Stucture

mGlu₅ is seen as a homodimer in vivo, with each subunit comprising three domains; extracellular, trans-membrane and cysteine-rich. The structures shown here are centered on the trans-membrane domain, comprised of seven α-helices all roughly parallel to one another ^[4]. Also displayed are Intracellular Loop (ICL) 1 forming a short α-helix, ICL3 and Extracellular Loops (ECL) 1 and 3, which lack secondary structure, and ECL2, which interacts with trans-membrane (TM) helices 1,2 and 3 and ECL 1^[4].

Key Interactions

A number of intramolecular interactions within the trans-membrane domain stabilize the inactive conformation of mGlu₅. The first of these interactions is an ionic interaction, termed the , between Lysine 665 of TM3 and Glutamate 770 of TM6. Evidence for the importance of this interaction came through a kinetic study of mutant proteins where both residues were separately mutated to alanine resulting in constitutive activity of the GPCR and its coupled pathway^[4].A second critical interaction that stabilizes the inactive conformer is a between Serine 614 of ICL1 and Arginine 668 of TM3. Similarly, when Serine 614 is mutated to alanine high levels of activity are seen in the mutant GPCR^[4]. A between Cystine 644 of TM3 and Cystine 733 of ECL2 is critical at anchoring ECL2 and is highly conserved across Class C GPCR’s^[4]. The ECL2 position, in combination with the helical bundle of the trans-membrane domain, create a that restricts entrance to the allosteric binding site within the seven trans-membrane α-helices. This restricted entrance has no effect on the natural ligand, glutamate, as it binds to the extracellular domain, but dictates potential drug targets that act through allosteric modulation^[4].

Comparison with Class A and B GPCRs

The structure of the trans-membrane domain of mGlu₅ was compared to rhodopsin, a class A GPCR, and CRF₁R, a class B GPCR. The superimposition of all three structures demonstrated the greatest divergence occurs across the top half of the trans-membrane bundle, owning to the different extracellular domains that accompany each GPCR class. Furthermore, mGlu₅ TM7 is shifted inwards 5 Angstrom compared to the class A GPCR and TM5 is shifted inwards 6 Angstrom compared to both GPCRs, narrowing the allosteric binding entrance to a greater extent ^[4].

Clinical Relevance

Role in Diseases

mGlu₅ is located mainly post-synaptically and is in high abundance in the nucleus accumbens, caudate nucleus, striatum, hippocampus and cerebellar cortex ^[7].These areas of the brain are highly involved in cognition, motivation and emotion, essential neural functions for everyday life. Diseases and other mental deficiencies arise from either an over activation of the GPCR, which over activates its coupled signaling pathway, or from under activation of both. Negative allosteric modulators (NAMs) work to decrease protein activity and are being studied as treatments for fragile X-syndrome, depression, anxiety and dyskinesia. Conversely Positive allosteric modulators work to increase protein activity and are being studied for the treatment of schizophrenia and cognitive disorders^[8].

Interactions with a Negative Allosteric Modulator

The structure of mGlu₅ bound to the NAM Mavoglurant demonstrates how protein activity is decreased through drug interactions. binds within the core of the seven trans-membrane α-helices, forming multiple interactions with the protein that further stabilize the inactive conformation. The bicyclic ring system of the drug is surrounded by a pocket of mainly hydrophobic residues including Val 806, Met 802, Phe 788, Trp 785, Leu 744, Ile 651, Pro 655 and Asn 747^[4] (Figure 1). The carbamate tail of mavoglurant forms a hydrogen bond through its carbonyl oxygen to the amide side-chain of Asparagine 747 of TM4 (Figure 2). A hydroxyl group similarly forms hydrogen bonds to the protein, specifically to two serine residues (S805 and S809) of TM7 which form a hydrogen bonding network to other residues through their main chain atoms and a coordinated water molecule (omitted for clarity) (Figure 3). The interactions between Mavoglurant andmGlu₅ involved TM helices that were not previously stabilized by any strong interactions, introducing a new level of stability that favors the inactive conformation of the protein and hence decrease activity^[4].

Figure 1.Hydrophobic Pocket Surrounding Mavoglurant

Figure 3. Further Hydrogen Bonding between protein and Mavoglurant