Abstract
G protein-coupled receptors (GPCRs) constitute the most abundant protein family in mammalian genome. Indeed, GPCRs are the target for about 30% of the drugs on the market. In human particularly, there are 22 genes encoding class C GPCRs, which consists of GABAb receptor, calcium sensing receptor, retinoic acid-inducible receptors (orphan), taste receptors, metabotropic glutamate receptors, and a few additional orphan receptors. Metabotropic glutamate receptors (mGluRs) are involved in controlling synaptic transmission, and involved in various CNS disorders including pain, Parkinson's disease, schizophrenia etc... They are naturally homodimers, and each protomer consists of a heptahelical transmembrane domain linked to a bilobate Venus flytrap domain (VFT) by a Cystein-rich domain. Understanding the conformational changes of mGluRs is essential to decipher the allosteric transition associated with their activation. Crystallographic studies suggested that in the inactive and active states the second lobes of the VFTs are distant and close, respectively. However certain ambiguities and discrepancies about these two states have been observed by X-ray crystallography. For the first time in GPCR activation mechanism studies, we proposed structural dynamics investigations by single-molecule FRET (sm-FRET) in solution. Our results show that the isolated mGluR VFTs oscillate between the resting and active states in a time range of 50-100μs (Olofsson et al. Nat. Comm. 2014). Following the success of the powerful sm-FRET methodology on isolated VFTs, we employ here Multi-parameter Fluorescence Detection (MFD) and Pulsed Interleaved Excitation (PIE) to study the full-length receptors in order to gain additional insights into mGluR activation. Our current results confirm the structural dynamics obtained by the mGluRs VFT, and suggest a stabilizing role of the transmembrane domain. Further studies on allosteric modulation and G protein effect are on-going.
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