Evolutionary Analysis For The VFTM Of Class C GPCRs And Research On The Probe Targeting GB1-VFTM

Class C G protein-coupled receptors (GPCRs) represent a distinct group among the large family of GPCRs, which structurally posses a large extracellular domain including a Venus flytrap module (VFTM). The VFTMs of class C GPCRs are responsible for the ligands recognizing and binding, and share sequence similarity with bacterial periplasmic amino acid binding proteins (PBPs) involved in the transport of small molecules like amino acids. Considering the structural homology and the functional divergence in the VFTMs, an extensively phylogenetic investigation based on the functional divergence analysis and the test for positive selection was conducted for five typical groups of class C GPCRs (mGluRs, CaSR, T1R, GB1 and GB2). The altered selective constraints were determined to identify the sites undergone functional divergence driven by the positive selections. In order to structurally demonstrate the change patterns during the evolutionary process, ancestral VFTMs were inferred and reconstructed with three-dimensional (3D) structures. The results indicate that functional divergences driven by positive selection are responsible for the evolutionary patterns via three continuous duplicate events of the VFTMs of class C GPCRs. The sites involved in functional divergence may provide more candidates for further researches on the relationship between structures and functions, and also shed lights on the activation mechanism of class C GPCRs.In order to label the GABA_b receptors on living cells, a trimodular activity-based fluorescent probe was designed, synthesized and characterized based on the structure of CGP64213, an antagonist of GABAb receptor. This probe can be applied to photoaffinity label the GABAb receptor transiently expressed in Chinese hamster ovary cells. Moreover, it exhibits specific binding activity at the ligand-binding pocket of GB 1 subunits and high specificity of photoaffinity labeling, which makes the probe valuable for studying the localization and function of GABAb receptors on living cells.