PHARMACOLOGICAL AND STRUCTURAL INTEGRITY OF MUSCARINIC M-2 ACETYLCHOLINE-RECEPTORS PRODUCED IN SF9 INSECT CELLS

Muscarinic acetylcholine receptors (human m2 subtype), expressed in Sf 9 cells, using the baculovirus system, were purified and found to disp lay the expected ligand binding properties, whether membrane-bound or affinity-purified. The purified recombinant receptors were specificall y photolabelled with p-N,N-[H-3]dimethylamino and p-N,N-[H-3]dibutylam ino benzene diazonium derivatives. Electrophoretic patterns for covale nt radioactive incorporation of the probes were essentially similar to those for [H-3]propylbenzilylcholine mustard-labelled receptor sites but were dependent on the infection time of Sf9 cells. Pharmacological properties of the recombinant receptors being unaltered did not refle ct structural integrity of the protein as substantial proteolytic frag mentation was detected at a prolonged infection time, i.e., at the hig hest level of expression. Selection of overexpression conditions, as i llustrated here for muscarinic receptors, thus requires not only pharm acological controls, but also analysis of the covalently labelled prot ein under strongly dissociating conditions. (C) 1997 Elsevier Science B.V.