Abstract
The muscarinic M2 receptor was split at the third cytoplasmic loop into two fragments: the one containing the first five transmembrane regions and the N-terminal part of the third cytoplasmic loop was named M2trunk, while the other, which contained the last two transmembrane regions and the C-terminal part of the third cytoplasmic loop, was named M2tail. As seen in many other G protein-coupled receptors, when these two fragments were transfected together in COS-7 cells they rescued the pharmacological profile and the functional activity of the wild-type M2 receptor. Conversely, N-[3H]methylscopolamine ([3H]NMS) association binding experiments showed a substantial difference between the wild-type M2 and the split M2trunk/M2tail receptors. The progression of the association binding kinetic of the M2trunk/M2tail receptor was strictly dependent upon the amount of the fragment DNA transfected. When the amount of transfected DNA was 4 μg/plate and theBmax of [3H]NMS at equilibrium was around 200 fmol/mg protein the form of the association was that of classical saturation, but when the amount of transfected DNA was lower the [3H]NMS association reached a maximum binding point and then declined to a lower equilibrium binding level. The form of the association was temperature-dependent: as the temperature was lowered, the maximum binding point tended to be higher. We suggest that this peculiar form of the [3H]NMS association binding to the muscarinic M2trunk/M2tail receptor is attributable to a less stable interaction between the trunk and the tail fragments of the split receptor.
Footnotes
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This work was supported by a grant from Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MURST).
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DOI: 10.1124/jpet.102.045393
- Abbreviations:
- [3H]NMS
- N-[3H]methylscopolamine
- GPCR
- G protein-coupled receptor
- [3H]QNB
- [3H]quinuclidinylbenzilate
- GTPγS
- guanosine 5′-O-(3-thio)triphosphate
- PMSF
- phenylmethylsulfonyl fluoride
- Received October 9, 2002.
- Accepted January 14, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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