Structural And Functional Studies Of Class B G Protein-Coupled Receptors

Class B G protein-coupled receptors(GPCRs)belong to the secretin family containing 15 members targeted by the hormone peptides.These class B GPCRs comprise an important family of drug-targeted receptors and their dysfunction can cause different diseases such as diabetes,cancer,neurodegeneration,osteoporosis and cardiovascular diseases.Despite much efforts in the study of class B GPCR,its activated structure and the detailed activation mechanism of full length class B GPCR remain largely unknown,preventing further understanding of the receptor’s interaction with the hormone peptide and the induction of downstream signaling.In this thesis,I provided new insights into this topic organized in a total of four chapters.The first chapter,introduced and reviewed existing biochemical and structural studies of class B GPCR,and summarized the major significance of my work and the main scientific issue addressed.In the second chapter,I focused on the specific class B GPCR-glucagon-like peptide-1 receptor(GLP-1R)and reported the mechanistic basis of the ECD of GLP-1R required for activation by mutagenesis,peptide fusion,peptide deletion,and co-expressed approach [1].We demonstrated that the ECD of GLP-1R could activate the receptor TMD in trans when exposed to the intact peptide hormone,demonstrating that the existing interaction between ECD and TMD when GLP-1R was activated by the hormone peptide,and further specific mutations in extracellular loops(EC1,EC2,EC3)could abolish this ECD trans-activation [1],suggesting extracellular loops were the major interaction interfaces.These findings report a new activation mechanism in which the ECD of activated GLP-1R interacting with TMD conflicting to the currently accepted two-domain mechanism for class B GPCR activation.This interesting discovery then raised discussion about conformation changes between ECD and TMD when the receptors were activated by the hormone peptide[1].In the third chapter,using a combination of structural modeling and mutagenesis studies on GCGR,we studied the mechanism of how the class B GPCRs governed a conformational switch between the active and inactive states [2].We identified a hydrophobic lock in GCGR comprising M338 and F345 within IC3 loop and TM6,L329 and L333 at TM5 which stabilized TM6 in the inactive conformation [2].Disruptions of this hydrophobic lock by single mutation,especially F345 or M338 mutation,led to constitutive G-protein and arrestin signalling [2].However,this hydrophobic lock was only specific to GCGR [2].Furthermore,we discovered a polar core comprising conserved residues H177 from TM2,E245 from TM3,T351 from TM6 and Y400 from TM7 and mutations that disrupted this polar core led to constitutive GCGR activity [2].Based on these data,we proposed a mechanistic model of GCGR activation,in which the TM6 was held in inactive conformation by the conserved polar core and the hydrophobic lock [2].Mutations that disrupted these inhibitory elements allowed TM6 to swing outwards to adopt an active TM6 conformation[2].Importantly,the residues forming the polar core were 100% conserved in class B GPCRs and mutations in the corresponding polar core of parathyroid hormone 1 receptor(PTH1R),pituitary adenylate cyclase-activating polypeptide type 1 receptor(PAC1R),vasoactive intestinal peptide receptor 1(VIP1R)and corticotropin-releasing factor receptor type 1(CRFR1)also induced constitutive G-protein signalling,suggesting that the rearrangement of the polar core was a conserved mechanism for class B GPCR activation[2].Together,this conclusion extends our mechanistic understanding about class B GPCR activation and lays the solid foundation for designing the non-peptide small molecular agonist.In the fourth chapter,I focused on parathyroid hormone 1 receptor(PTH1R) and efforts towards the structure of activated receptor using crystallography.With the poor yield and behavior of full length PTH1 R,I chose to work on the TMD of PTH1 R in the first place.In order to obtain PTH1 R activated by the hormone peptide,I fused the peptide at the N termini of TMD directly and used crosslinking and new detergent to improve the behavior and thermostability.I used mammalian system to express this fusion protein and further improved the protein’ behavior by optimizing the expression constructs,purification and crystallographic condition.Finally,I obtained well-behaved activated protein and small but promising crystal hits.In this part,my work prepared for next crystallization and hits screening for PTH1 R,whose structure could help drug design for osteoporosis diseases.