Expression of the rat muscarinic receptor gene m3 in Dictyostelium discoideum.
We functionally expressed the rat muscarinic m3 receptor (rm3) in the cellular slime mold Dictyostelium discoideum under the control of the homologous discoidin I gamma promoter. Cells transfected with the authentic rm3 receptor gene expressed about 100 functional receptor molecules per cell, corresponding to a Bmax for [3H]-NMS of 36 +/- 9 fmol/mg of protein in isolated membranes. Genetic fusion of the Dictyostelium contact site A (csA) leader peptide to the amino terminus of rm3 increased the receptor expression by about 17-fold. Remarkable, in [3H]-NMS ligand binding experiments performed with whole cells no characteristic saturable binding was observed and there was no significant difference in [3H]-NMS binding to whole cells of rm3 and csA/rm3 transformants. The recombinant rm3 receptor showed an about 10-fold higher affinity to the M3-selective antagonist p-F-HHSiD compared to the M2-selective antagonist AQ-RA 741, suggesting that membranes derived from transgenic D. discoideum cells may be useful for the search of new subtype-specific muscarinic receptor ligands.
Voith
G
G
Institute for Pharmaceutical Biology, Johann Wolfgang Goethe-University, Frankfurt/Main, Germany.
Kramm
H
H
Zündorf
I
I
Winkler
T
T
Dingermann
T
T
D000818:Animals; D015152:Blotting, Northern; D004023:Dictyostelium / Q000378:metabolism*; D005822:Genetic Vectors; D007700:Kinetics; D008024:Ligands; D009154:Mutation; D051381:Rats; D011976:Receptors, Muscarinic / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism
D008024:Ligands; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins