The Structure And Function Study On Class B GPCR

G protein-coupled receptors (GPCR) are the largest family of membrane proteins and contain a common architecture of seven transmembrane-spanning helical domains. They mediate most of our physiological responses to hormones and neurotransmitters, and have great potential as therapeutic targets for a broad spectrum of diseases. So far, about 50% of drugs target on GPCR but non-peptidic GLP-IR agonists have not been designed as therapeutic targets due to the lack of the complex structure of class B GPCR and small molecule, greatly limit our knowledge of class B receptors’functions and structures related to the regulatory mechanism of many human diseases. But how natural peptide engaged with the N-terminal domains and transmembrane helices of the receptors to activate the receptors are still to be clarified.To investigate the mechanisms of the ligand specifical recognition and binding amino-terminal extracellular domain (ECD) and transmembrane domain (TMD) we identify the activity of class B GPCR TMD and full length receptors by measurment of their intracellular cAMP. We have demonstrated that class B GPCRs differ in their requirement for the ECD in receptor activation. One group of receptors, represented by CRF1R, PTH1R and PAC1R are activated without ECD, while in the other group, represented by GCGR and GLP-1R, require the ECD even in the presence of a very high concentration of native peptide or when the hormones were artificially tethered to the TMD.Furthermore, Membrane tethering of glucagon and GLP-1 can only active the full length receptors, they failed to stimulate signaling through GCGR and GLP-1R lacking respective ECDs. It confirmed that the ECD is required for signaling.In order to understand the mechanisms of the ECD requirement for activation of GCGR and GLP-1R, we carried out mutational studies of the ECD and the fused hormone peptide in the context of the GCG-5GSA-GCGR fusion protein. These mutations, W36, D63 and P86, abrogated glucagon binding and almost abolished receptor activation, both in the presence or absence of exogenously added glucagon. It shows that these residues are related to glucagon-binding. We also analyzed the effects of some corresponding GLP-1/GLP-1R mutants in the fusion protein.They had similar result that the ECD not only functions as affinity trap, but also is required for receptor activation.To study the mechanisms of type 2 diabetes for the prevention and treatment and provides an experimental basis for drug design and development, we studied that the small molecule GLP-1R agonist Boc5, S4P. WB4-24. which not only have the species selectivity by measurment of their intracellular cAMP. but also directly bind to the receptor ECD and require the ECD for signaling. In contrast, BETP, a small molecule GLP-1R modulator that does not bind to the ECD. BETP functions by forming covalent adducts with C347 in the intracellular loop 3 (ICL3) of GLP-1R. which may mimic a physiological covalent modification. These results suggest that the ECD plays a direct role in the activation of GLP-1R. It can provide us the new ideas of the mechanisms of small molecule-receptor interactions and offer new opportunities for the design of new and potent small molecules for the treatment of type 2 diabetes.GLP-1R is one of the most important antidiabetic drug targets, but it is low expression and poor stability and it is very difficult to get the crystal structure of the full length class B receptor. We studied the function of class B GPCR and express GLP-1R protein using the insert cell and mammalian cell of BacMam expression system. We can get the stabilized protein for crystallization high quality of GLP-1R crystals by gene engineering and optimization of purification conditions. Solving the structure of the full length of class B GPCR can offer a direct conformation basis for the mode of the interaction of ligand and receptor in the agonist-bound state. It can also lay the foundation of carrying out structure-based drug discovery aimed at the therapy of type 2 diabetes and has inestimable value.