Metabotropic glutamate receptors belong to family C GPCR, which has common features with a large extracellular domain (ECD) functioning as an endogenous agonist binding region, cystein rich (CR) domain, seven time transmembrane domain, (7-TM) and C-terminal. The receptor functions as a dimer linked by a disulfate bridge through C140 onto the ECD (53). An extracellular domain consists of two lobes which form a venus flytrap-like structure, which has a 3-dimensional structure very similar to bacterial leucine/isoleucine/valine-binding protein (LIVBP). Thus, the first homologous model was generated by O’Hara’s group using LIVBP as a template. In this model structure, sever- al key residues (T188, D208, Y236 and D318) for agonist binding, which were highly conserved in different species, were determined (54). In 2000, Kunishima N et al. re- ported several ECD structures, including two apo forms and one holo form with L-Glu and potential Mg2+, using X-ray crystallography (47). Two years later, two additional forms bound with L-Glu, Gd3+, and (s)-MCPG were reported. In L-Glu bound structures, the L-Glu binding pocket was sketched out confirming the model study on this receptor (55). These structures revealed 3 different kinds of conformations upon the bound li- gands which were estimated as the activation mechanism of mGluR1α. Upon L-Glu binding, the receptor was stated to be a closed-open form, as LB1 and LB2 in the same protomer were closed even though two LB2 domains were kept open due to the charge repulsion in the interface (E238 and D242). In the presence of Gd3+, the negative
charge could be neutralized and two LB2 domains moved toward each other, forming a close-close form. Whether the LB2 domains were closed or open, the conformational change could still lead to a rearrangement of the transmembrane domain with the assis-
tance of a cysteine rich domain. The free form or antagonist bound form was known as a resting form, also called an open-open form. Similar to a free form, the receptor bound with s-MCPG or LY341495 shows as a relax state (Fig. 1-6). Although the constitutive activation was reported in mGluR1 due to the function of Homer1b binding to the C ter- minal, the activation of the receptor was ascribed to the predominance of a close form in dynamic equilibrium. This hypothesis was further improved by Tateyama et al. using the FRET technique. In brief, two intracellular loop 2 (i2) were brought closer upon the agonists stimulation (L-Glu and Ca2+), while the antagonists increased the space be- tween the loops (56).
1.2.3 mGluRs related diseases
Mebotropic glutamate receptor 1 (mGluR1) is widely expressed on the surface of post synapses in the central neuronal system. It senses glutamate following recruitment of hetero-trimeric G proteins, and the accumulation of DAG and IP3 which further mod- ulates protein kinases involved cascade response and intracellular Ca2+ mobilization, respectively. This consequently leads to nonselective inward cation current. Shortly af- ter the receptor was cloned, its versatility was revealed to respond to not only glutamate but also Ca2+ and other polyvalent ions. The activity of mGluR1α has been proved to be related to some important physiological and pathological processes, such as breast cancer, melanoma, and neuronal degenerative diseases. Down regulation of mGluR1α was detected in neurons of substantia nigra in Parkinson monkey models, suggesting mGluR1α plays an important role in the process of Parkinson disease. In addition, pre- pulse inhibition (PPI) was disrupted in mGluR1 knockout mice (57). PPI deficiency
usually appeared in patients with schizophrenia, this indicates mGluR1 is also involved in the process of schizophrenia. MGluR5 was suggested to be involved in Fragile X syndrome (58). Down regulating group I mGluRs could relieve Fragile X symptoms, while especially the antagonist of mGluR5, MPEP, can suppress the seizure pheno- types. MGluR4 were previously reported as a target to relieve pain. More recent, an original antagonist (PHCCC) of group I mGluRs was found to enhance the potency of an agonist of mGluR4 (L-AP4). Moreover, treating Parkinsonian rat model with PHCCC, the movement activity was reduced (59). This also suggests mGluR4 could be a thera- peutic target for Parkinson’s diseases. Furthermore, the agonist of mGluR2/3
(LY2140023) has shown to improve both positive and negative symptoms in patients with schizophrenia, and this drug has entered phase II clinical trial (60).
Figure 1-4 Folding and trafficking of GPCRs (61). Family C GPCRs could form ho- mo- or heterodimer after synthesized in ER with the facilitation of chaperones and other quality control system. The well folded proteins were translocated to Golgi complex for further modification or directly anchored on the cell membrane through exocytic vesicles while abnormal proteins were ubiquitinated and delivered to lysosomes for degradation.
1.2.4 Trafficking of mGluRs
The activity of mGluRs is dependent of the receptor expression on cell surface. For instance, surface expression of mGluR7 plays an important role to control the neu- ronal plasticity (62). Decrease of surface mGluR5 by exposing to cocaine lead to loss of endocannabinoid retrograde LTD (63). MGluRs, like other members of GPCRs, are folded in ER lumen with the facilitation of chaperones and quality control system. The properly folded proteins were further modified in Golgi complex, and finally reached cell membrane. The misfolded receptors are usually uquibinated and protelyzed by proteas- es. In presence of agonists, the surface receptors will be desensitized and internalized with the assistance of lipid raft and caveolin. Mutants of mGluR1 lacking of caveolin binding motif were demonstrated to attenuate mGluR1 coupled ERK-MAPK signaling pathway (64). Several members of mGluRs have been proved to interact with calmodu- lin (CaM), including mGluR1, mGluR5 and mGluR7. A PKC phosphorylation site (S901) was found in mGluR5. Phosphorylation of this site eliminated CaM binding, thus reduc- ing surface expression of mGluR5 (65). On the other hand, preventing S901 from phos- phorylation enhances mGluR5 activity (65). However, the role of CaM binding in
mGluR1 is not decided yet. MGluR7 also contains CaM binding site, which is highly conserved in mGluR4A and mGluR8 (66). Similarly, phosphorylation of mGluR7 also prevents CaM binding (67). The accumulating evidence suggests that CaM is the com- mon factor of mGluRs serving as switch of internalization of the receptors.