Homologous mutations near the junction of the sixth transmembrane domain and the third extracellular loop lead to constitutive activity and enhanced agonist affinity at all muscarinic receptor subtypes

Previous studies have found that a mutation near the junction of the sixth transmembrane domain (TM6) and the third extracellular loop of the M-5 muscarinic receptor leads to constitutive activation and enhanced agonist affinity for the mutated receptor. These results were consistent with. the extended ternary complex model, which predicts a correlation between agonist affinity and constitutive activity. We have introduced. the homologous mutation into all five subtypes of the highly conserved muscarinic receptor family; SerThr --> TyrPro was introduced into M-1 and M-5, and AsnThr --> TyrPro was introduced into M-2, M-3, and M-4. In binding assays, these mutations produced increases in affinities toward acetylcholine and carbachol that ranged from 5-fold at the M-2 receptor to 15- to 20-fold at M-1, M-3, and M-4, to 40-fold at M-5. In functional assays, all five mutant receptors exhibited constitutive activity, at levels ranging between 30 and 80% of the maximal response elicited by carbachol. In every case, the muscarinic antagonist atropine Inhibited this constitutive activity with high affinity. Thus, despite differences in effector coupling and in wild-type sequence at the mutation site, all five subtypes were activated by this mutation at the top of TM6. Previous studies of the M-5 subtype have indicated that TM6 is a ligand-dependent switch that sets the activation state of the receptor. Based on the results of the present study, it is possible that TM6 represents a general switch for the activation of the muscarinic receptor family.