Differential modulation of agonist potency and receptor coupling by mutations of Ser388Tyr and Thr389Pro at the junction of transmembrane domain VI and the third extracellular loop of human M-1 muscarinic acetylcholine receptors

Transmembrane domain VI of muscarinic acetylcholine receptors plays an impo rtant role in ligand binding and receptor function. A human M-1 (HM1) mutan t receptor, HM1 (S388Y, T389P), displayed significantly enhanced agonist po tency, binding affinity, and G protein coupling. The mutations are located at the top of transmembrane domain VI and about two helical turns above Tyr 381 and Asn382, which are important for ligand binding and receptor functio n. To determine the functional role of individual mutations of Ser388Tyr an d Thr389Pro, we created stable A9 L cell lines expressing HM1 (S388Y) or HM 1 (T389P) receptors. In phosphatidylinositol hydrolysis assays, muscarinic agonists showed greater potency at the HM1 (S388Y) and HM1 (S388Y, T389P) m utants compared with the wild-type and HM1 (T389P) receptors. Acetylcholine dem onstrated 105- fold higher potency at HM1 (S388Y) receptors than at HM 1 (T389P) receptors. Choline (30 mu M, the concentration found in Dulbecco' s modified Eagle's medium) exhibited 90% stimulation at HM1 (S388Y) recepto rs but was inactive at HM1 (T389P) receptors. In ligand binding experiments , mutation of Ser388Tyr resulted in significantly increased agonist binding affinity. In contrast, mutation of Thr389Pro did not change agonist bindin g affinity but rendered multiple agonist binding sites, and the high-affini ty binding was sensitive to GTP analogs. These results demonstrate that the Ser388Tyr mutation is responsible for enhanced agonist potency and binding affinity, whereas the Thr389Pro mutation alters G protein interactions. Th e data suggest that Ser388 and Thr389 are potential targets for modulation of agonist binding and G protein coupling.