Abstract
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.
Footnotes
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Send reprint requests to: William S. Messer, Jr., Ph.D., Department of Medicinal and Biological Chemistry, College of Pharmacy, The University of Toledo, 2801 W. Bancroft St., Toledo, OH 43606-3390. E-mail:wmesser{at}uoft02.utoledo.edu.
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This work was supported by Grants NS01493, NS31173, and NS35127. Portions of this work have appeared in abstract and poster form and were presented at the 8th International Symposium on Subtypes of Muscarinic Receptors, Danvers, MA, August 1998, and at the 28th annual meeting of the Society for Neuroscience, Los Angeles, CA, November 1998. This work is part of the Ph.D. dissertation of X.-P.H., Department of Medicinal and Biological Chemistry, College of Pharmacy, The University of Toledo.
- Abbreviations:
- ACh
- acetylcholine
- mAChR
- muscarinic acetylcholine receptor
- CCh
- carbachol
- Gpp(NH)p
- guanosine-5′-(β,γ-imido)triphosphate
- PI
- phosphatidylinositol
- HM1
- human muscarinic acetylcholine receptor subtype 1
- NMS
- N-methylscopolamine
- Oxo
- oxotremorine
- Oxo-M
- oxotremorine-M
- PLC
- phospholipase C
- (R)-QNB
- (R)-3-quinuclidinyl benzilate
- Ne3
- N-terminal region of the third extracellular loop
- TM
- transmembrane domain
- R-SAT
- receptor selection and amplification technology
- WT
- wild-type
- Received January 26, 1999.
- Accepted June 21, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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