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Metabotropic Glutamate Receptor

spinqualitylabelsall models Metabotropic Glutamate Receptor PDB:7epa Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Introduction Within the central nervous system (CNS), various membrane receptors exist to detect extracellular signaling molecules and communicate this information intracellularly. Found in eukaryotes and known for its seven transmembrane helices, G-protein coupled receptors (GPCRs) are one type of membrane bound receptors with conserved intracellular signaling via a heterotrimeric G-protein. The metabotropic glutamate receptor (mGlu), a Class C GPCR, is a receptor utilized in glutamate signaling– which is essential in synaptic plasticity as well as the development and repair of the CNS. These receptors are specifically found in the pre- and postsynaptic neurons of the CNS. Eight different mGlu subtypes exist which are divided into three groups (I, II, III). While each mGlu has a slightly different function and location, the structures of the different mGlu subtypes are very similar (Table 1) . For each group, binding of the neurotransmitter glutamate to the mGlu introduces a conformational change that can activate a G-protein. Table 1. Classification of mGlu Subtypes Group mGlu Type Location Function I mGlu1 mGlu5 Postsynaptic neurons Activates calcium channels Excites adenylyl cyclase II mGlu2 mGlu3 Presynaptic neurons Activates potassium channels Inhibits calcium channels Inhibits adenylyl cyclase III mGlu4 mGlu6 mGlu7 mGlu8 Presynaptic neurons Activates potassium channels Inhibits calcium channels Inhibits adenylyl cyclase Figure 1. Structure of glutamate under physiological conditions (pH 7.4). To activate the mGlu transformation, glutamate acts as the protein's main agonist. Glutamate is an acidic, polar amino acid (Figure 1). This agonist binds to the extracellular portion of the glutamate receptor causing the transmembrane spanning region of the homodimer to change conformationally. This change allows for the mGlu to bind to a G-protein. This binding allows for the activation of a G-protein which initiates a signaling cascade within the cell that can ultimately lead to a difference in the synapse’s excitability. In mGlu, the binding affinity of glutamate is also controlled by the binding of either a positive (PAM) or negative (NAM) allosteric modulator to a binding site within the seven TMD. Structural Highlights mGlu receptors are dimeric proteins consisting of an alpha and beta chain. While a heterodimer of different mGlu subtypes can form, only homodimeric receptors can become active. Both the alpha and beta chains are comprised of three domains: the venus fly trap (VFT), cysteine rich domain (CRD), and the transmembrane domain (TMD). Figure 2. Proper orientation of mGlu about the cell membrane. Domains VFT: The extracellular location in which the two glutamate agonists bind is known as the VFT. This domain includes a disulfide bond between C121 of the alpha and beta chains . This disulfide bond is shifted down in the open conformation and undergoes a disulfide bond upward movement upon glutamate binding which stabilizes the active site. Glutamate binds within the binding pocket via hydrogen bonding, specifically hydrogen bonding, with R57, S143, S145, T168, and K377 of the VFT. This binding initiates a closed VFT conformation. CRD: The portion of the protomer that connects the VFT with the TMD is known as the CRD. Many disulfide bonds are located in this region between cysteines. As the connecting segment of the protein, it is critical in transmitting the conformational change caused by the binding of glutamate in the VFT to the TMD. The change resulting from the binding of glutamate brings the cysteine-rich domains of the alpha and beta chain together to alter the configuration of the seven TMD helices through its interaction with the VFT extracellular loop 2 (ECL2) . This ECL2 conformational change is mediated through interactions with amino acids at the apex of the CRD (e.g. I674, P676, and P753) . TMD: The TMD consists of seven transmembrane helices that are responsible for G-protein interactions and are able to transmit the signal from ligand binding across a membrane. In the inactive form, the asymmetric conformation of the helices is mediated by the hydrophobicity of helix 3 and 4 . This allows for a TM3-TM4 interface to form between the monomers (Figure 3A). Along with the interaction of the CRD with the ECL2 of the TMD, an allosteric modulator must bind within the transmembrane helices to allow for the conformation of the helices to be altered. This conformation allows for an active dimer interface along helix 6 of both protomers (Figure 3B). The stabilization of this conformation also enables G-protein coupling with ICL2, ICL3, TM Helix 3 and the C terminus (Figure 5). Figure 3. A) The inactive transmembrane helices conformation. B) The active transmembrane helices conformation. Conformational Changes 1. In its resting state, mGlu is in an inactive homodimeric form (Figure 3-1). In this conformation, the receptor is considered open with an inter-lobe angle of 44°. The structure has two free glutamate binding sites in the VFT, the CRDs are separated, and the TMD is not interacting with a G-protein. 2. In the intermediate activation state, also known as the open-closed conformation, one glutamate is bound in one binding pocket of VFT (Figure 3-2). This single glutamate bound state is still considered inactive as the receptor has not changed the conformations in the CRD and thus the TMD. With the same asymmetric transmembrane helices formation, a TM3-TM4 interface is still present and mGlu cannot interact with a G-protein. Figure 4. Illustration of mGlu's conformational change process. 3. A second glutamate then binds to the other binding pocket of the VFT (Figure 3-3). Mediated by L639, F643, N735, W773, and F776, a positive allosteric modulator (PAM) also binds within the seven TMD helices of the alpha chain . This closed conformation of the VFT now has an inter-lobe angle of 25° is considered to be in the active conformation. The binding of these ligands allows the CRDs to compact and come together. This conformational transformation causes the TMD to form a separate, active asymmetric conformation with a TM6-TM6 interface between the chains (Figure 3). Figure 5. The interaction between an active mGlu (magenta/lime/purple/crimson) and a G-protein (orange). Hydrogen bonds are shown through black dashes 4. The crossover of the helices from the alpha and beta chains allows for intracellular loop 2 (ICL2) and the C-terminus to be properly ordered to interact with a single G-protein (Figure 3-4/5). While hydrogen bonding is present between the C-terminus and alpha helix 5 of the G-protein, this mGlu/G-protein coupling is primarily driven by the hydrophobic interactions in the interface with the ɑ5 helix of the G-protein (Figure 5). This coupling can only occur in the presence of a PAM as the pocket in which the coupling occurs would be completely closed in its absence. 5. Upon binding, the G-protein can become active through the receptor catalyzed reaction of GDP to GTP on the alpha subunit of the G-protein (Figure 3-4/5). Depending on the type of mGlu present, this activation causes different signaling cascades to occur within the cell . These cascades are necessary for cellular function as they can play primary roles in regulating metabolic molecules, ion channels, transporter molecules, and several other parts of the cell; if these proteins are mutated, various diseases can occur. Clinical Relevance Glutamate is a major neurotransmitter in the brain and is known for its role in memory, synaptic plasticity, and neuronal development. Thus, mGlus are located throughout the CNS and play key roles in various neurodegenerative diseases such as Alzheimer's Disease (AD), Huntington's Disease (HD), Parkinson’s Disease (PD), and Amyotrophic Lateral Sclerosis (ALS). Also, due to the key role of glutamate in the brain, mGlus are also being studied for their role in psychiatric disorders like anxiety and depression. In many of these diseases, the overexpression of glutamate can lead to the overstimulation of mGlus and excitotoxicity . However, an exception to this is the under stimulation of mGlus which can result in schizophrenia. Research is ongoing, but studies have proven that agonists, antagonists, PAMs, and NAMs of mGlus are potential treatments for these diseases. 3D Structures 7epa, mGlu Inactive 7mtr, mGlu Active 7epb, mGlu Active 7mts, mGlu Active G-Protein Bound

Student Contributors

• Courtney Vennekotter
• Cade Chezem