An unusual form of the association binding kinetics of N-[3H]methylscopolamine to the split muscarinic M2trunk/M2tail receptor.
The muscarinic M(2) 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 M(2trunk), while the other, which contained the last two transmembrane regions and the C-terminal part of the third cytoplasmic loop, was named M(2tail). 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 M(2) receptor. Conversely, N-[(3)H]methylscopolamine ([(3)H]NMS) association binding experiments showed a substantial difference between the wild-type M(2) and the split M(2trunk)/M(2tail) receptors. The progression of the association binding kinetic of the M(2trunk)/M(2tail) receptor was strictly dependent upon the amount of the fragment DNA transfected. When the amount of transfected DNA was 4 microg/plate and the B(max) of [(3)H]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 [(3)H]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 [(3)H]NMS association binding to the muscarinic M(2trunk)/M(2tail) receptor is attributable to a less stable interaction between the trunk and the tail fragments of the split receptor.
Novi
Francesca
F
Department of Neurosciences, University of Pisa, Via Roma 55, 56126 Pisa, Italy.
Scarselli
Marco
M
Corsini
Giovanni U
GU
Maggio
Roberto
R
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000187:drug effects; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D005703:Gallamine Triethiodide / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007202:Indicators and Reagents; D007700:Kinetics; D018727:Muscarinic Antagonists; D019832:N-Methylscopolamine / Q000378:metabolism*; D018733:Nicotinic Antagonists / Q000494:pharmacology; D010276:Parasympatholytics / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011813:Quinuclidinyl Benzilate; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic / Q000235:genetics / Q000378:metabolism*
D007202:Indicators and Reagents; C474254:M(2trunk) M(2tail) receptor; D018727:Muscarinic Antagonists; D018733:Nicotinic Antagonists; D010276:Parasympatholytics; D010446:Peptide Fragments; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D011813:Quinuclidinyl Benzilate; D000262:Adenylyl Cyclases; D005703:Gallamine Triethiodide; D019832:N-Methylscopolamine