Study Of The Mechanism By Which Ligands Activate Histamine Receptor H₁R And H₂R

Histamine receptors are members of the class A G protein-coupled receptor superfamily,including four subtypes:histamine type 1 receptor（H₁R）,histamine type 2receptor（H₂R）,histamine type 3 receptor（H₃R）,and histamine 4 receptor（H₄R）.Histamine receptors play pivot roles in numerous physiological and pathological conditions,and have shown potential in the treatment of allergic inflammation,gastric acid-related ulcer.H₁R is an effective target for anti-allergic treatment.For more than 70years,antihistamines targeting H₁R have been the best therapy for allergic diseases.The crystal structure of the inactive H₁R receptor bound by the inverse agonist doxepin has been solved.However,the exact mechanism by which ligand activate receptor is still unclear,which poses a major challenge for the development of novel antihistamines.Recently,the cryo-EM structure of the H₁R/G_qcomplex bound to histamine has been resolved by our group.Basing on the structure of the H₁R receptor,we conducted an in-depth study on the mechanism of ligand induced activation of H₁R,the mechanism of H₁R inhibition by inverse agonists,as well as the mechanism of H₂R activation.In order to verify the contribution of the target amino acid residues on the activation/inactivation of the receptor,and further explore the specific mechanism of receptor activation,we used the molecular docking to model ligand-receptor interaction;we also use mutagenesis study on the key sites to verify the strucurtral observation and the insight mechamism,and to characterize the activity of the downstream signaling pathway by the dual luciferase reporter assay system.The mutatgenesis study shows that histamine binds to the receptor through the formation of hydrogen bonds with polar amino acid residues such as D107,T112,N198,Y431,and through the hydrophobic interaction through non-polar amino acids such as W158,W428 and F435.We also proposed the H₁R"squash to activate and expand to deactivate"model by verifying the mutation of key residues on helix 3 and helix 6.Histamine activates the receptor by interacting with D107 on transmembrane helix 3and Y431 on transmembrane helix 6,squashing the binding pocket on the extracellular side,opening the intracellular cavity and recruiting G_qprotein.On the contrary,antihistamines（inverse agonists）use their bulky groups to push helix 6 and helix 3 apart,expand the ligand binding pocket and squeeze the intracellular cavity,thereby prevent the recruitment of G_qprotein.In the mutagenesis study on the coupling mechanism of H₁R/G_q,we found that R125 on helix 3,N464 and Y468 on helix 7 may directly participate in the coupling of H₁R/G_q.In addition,we also conducted mutation studies on the mechanism of inverse agonists inhibiting H₁R and the activation mechanism of H₂R,and the key sites of the ligand binding pocket have not been screened.The results of this study will help to further analyze the ligand selectivity and signal transduction mechanism of H₁R and even the histamine receptor family,and provide new clues for the design of new antihistamine drugs.