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 auth entic rm3 receptor gene expressed about 100 functional receptor molecu les per cell, corresponding to a B-max for [H-3]-NMS of 36 +/- 9 fmol/ mg of protein in isolated membranes. Genetic fusion of the Dictyosteli um contact site A (csA) leader peptide to the amino terminus of rm3 in creased the receptor expression by about 17-fold. Remarkable, in [H-3] -NMS ligand binding experiments performed with whole cells no characte ristic saturable binding was observed and there was no significant dif ference in [H-3]-NMS binding to whole cells of rm3 and csA/rm3 transfo rmants. The recombinant rm3 receptor showed an about 10-fold higher af finity to the M3-selectdive antagonist p-F-HHSiD compared to the M2-se lective antagonist AQ-RA 741, suggesting that membranes derived from t ransgenic D. discoideum cells may be useful for the search of new subt ype specific muscarinic receptor ligands.