| G protein-coupled receptors (GPCRs) mediate physiological responses to various ligands, such as hormones, neurotransmitters and sensory stimuli. We synthesized both active and affinity tagged photo-affinity probes specific to GABAB receptor. Such activity-based probes enabled us to first lock GABAB receptor in an inactive state and then stimulate the receptor with a positive allosteric modulator, thereby permitting monitoring of the dynamic events associated with GABAB receptor activation. We found that activation of GABAB receptor triggered a dynamic assembly and disassembly of a protein complex, including GABAB receptor, IGF-1R and its downstream effectors. Notably, both Gai and Gβ subunits were pre-assembled with the inactive GABAB receptor and disassembled from the receptor complex upon activation. Our results identify a critical role for activity-specified dynamic protein interactions between GABAB receptor and downstream signaling proteins.The extracellular domain of GABAB1subunit (GABAB1-VFT) contains the Ligand binding pocket (LBP) and was an excellent therapeutic target. For the further researches of the GABAB1-VFT domain structure, the recombinant GABAB1-VFT was cloned, expressed and purified in large scale. The functional fraction of GABAB1-VFT was isolated by the ligand affinity chromatography. This fraction has the high activity of ligand binding.In order to study the process of GABAB receptor ligand bind, we designed a platform constructed by ESI-MS, Circular Dichroism Spectroscopy and Fluorescence Spectroscopy which can monitor the Secondary structure and the Tertiary structure changes as well as the ligands binding or dissociation process. With this platform, we analyzed the Mb conformational changes during its ligand binding or dissociation process in different conditions, and demonstrated the different mechanism of the heme releasing induced by acid and organic solvents. |
