Conformational Changes Of The Transmembrane Domain During The Activation Of A Calcium-Sensing Receptor (CaSR) Dimer

G protein-coupled receptors(GPCR)are key cell-surface proteins which mediate physiological responses to a large array of stimuli.These receptors are classified into 4groups based on the sequence similarity of their common seven transmembrane domains(7TM).The calcium-sensing receptor(CaSR)is one member of class C GPCR,CaSR functions as a homodimer and each monomer consists of four domains,a large Venus flytrap domain(VFT),a cysteine-rich domain(CRD),seven transmembrane domains,and C-terminal intracellular domain(ICD).Previous structural and functional data suggest CaSR dimer undergoes large-scale conformational changes.Agonist binding at the VFT leads to a compaction of the inter-subunit dimer interface and brings the CRD into close proximity,enabling the transmission of the signals from the extracellular VFT to the 7TM and finally activating the intracellular G protein.Despite of the known structure of the active and inactive CaSR homodimer extracellular domain,little is known about 7TM structure,the receptor conformational change during the activation is still elusive.In this study,we used cysteine cross-linking CuP involved and combination of FRET with SNAP-tag labeling technology to identify the transmembrane domain interface in CaSR dimer.After CuP treatment,we observed a decrease of Ca²⁺induced FRET change suggesting an inhibition of rearrangement of the ECDs dimer induced by the agonist,which can be weakened by pre-incubation with agonist before CuP treatment for mutants in TM4 and TM5.In contrast,we observed a low FRET signal without agonist for mutants in TM6 presenting active state in ECDs,which can also be strengthened by pre-incubation with agonist before CuP treatment.What's more,we found the ability of positive allosteric modulator to enhance the agonist efficacies was abolished when locking the TM4 interface,while locking the TM6 interface suppressed negative allosteric modulators effect.Above all,we proposed that TM4 and TM5 form the inactive interface while the TM6 is involved in the active interface.We further examined the cross-linking of CaSR dimer with a cysteine substitution in TM4-6 and confirmed that TM4-TM5 is the dimer interface of the inactive state.Finally,we investigated the relationship between the conformational changes of 7TM and receptor function by locking the dimer interface.We demonstrated that the activation induced by agonist is significantly inhibited by locking TM4 or TM5 interface.In contrast,we observed a robust constitutive activity by locking TM6 interface which can be increased by pre-incubation with agonist before CuP treatment.These results further demonstrated the CaSR dimer 7TM rearrangement during activation,from the inactive interface consisting of TM4/5 to the active interface of TM6.In conclusion,we revealed CaSR dimer 7TM conformational changes during activation for the first time through ECDs movement,protein cross-linking and receptor function using cysteine cross-linking,and confirmed the orientation of TM6 is essential for the activation process which provides a basis for natural pathogenic mutants located on TM6.