Vol. 56, Issue 4, 775-783, October 1999

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 M₁ Muscarinic Acetylcholine Receptors

Xi-Ping Huang, Frederick E. Williams, Steven M. Peseckis, and William S. Messer, Jr.

Department of Pharmacological Sciences, Diabetes and Metabolic Diseases Research Center, School of Medicine, Health Science Center, State University of New York at Stony Brook, Stony Brook, New York (X.-P.H.); and Department of Medicinal and Biological Chemistry, College of Pharmacy, The University of Toledo, Toledo, Ohio (F.E.W., S.M.P., W.S.M.)

Transmembrane domain VI of muscarinic acetylcholine receptors plays an important role in ligand binding and receptor function. A human M₁ (HM₁) mutant receptor, HM₁(S388Y, T389P), displayed significantly enhanced agonist potency, binding affinity, and G protein coupling. The mutations are located at the top of transmembrane domain VI and about two helical turns above Tyr381 and Asn382, which are important for ligand binding and receptor function. To determine the functional role of individual mutations of Ser388Tyr and Thr389Pro, we created stable A9 L cell lines expressing HM₁(S388Y) or HM₁(T389P) receptors. In phosphatidylinositol hydrolysis assays, muscarinic agonists showed greater potency at the HM₁(S388Y) and HM₁(S388Y, T389P) mutants compared with the wild-type and HM₁(T389P) receptors. Acetylcholine demonstrated 105-fold higher potency at HM₁(S388Y) receptors than at HM₁(T389P) receptors. Choline (30 µM, the concentration found in Dulbecco's modified Eagle's medium) exhibited 90% stimulation at HM₁(S388Y) receptors 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 binding affinity but rendered multiple agonist binding sites, and the high-affinity 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. The data suggest that Ser388 and Thr389 are potential targets for modulation of agonist binding and G protein coupling.