An Unusual Form of the Association Binding Kinetics of N-[3H]Methylscopolamine to the Split Muscarinic M2trunk/M2tail Receptor

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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.