Pharmacological and structural integrity of muscarinic M2 acetylcholine receptors produced in Sf9 insect cells.
Muscarinic acetylcholine receptors (human m2 subtype), expressed in Sf9 cells, using the baculovirus system, were purified and found to display the expected ligand binding properties, whether membrane-bound or affinity-purified. The purified recombinant receptors were specifically photolabelled with p-N,N-[3H]dimethylamino and p-N,N-[3H]dibutylamino benzene diazonium derivatives. Electrophoretic patterns for covalent radioactive incorporation of the probes were essentially similar to those for [3H]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 reflect structural integrity of the protein as substantial proteolytic fragmentation was detected at a prolonged infection time, i.e., at the highest level of expression. Selection of overexpression conditions, as illustrated here for muscarinic receptors, thus requires not only pharmacological controls, but also analysis of the covalently labelled protein under strongly dissociating conditions.
Weill
C
C
Laboratoire de Chimie Bio-Organique, U.R.A. 1386 C.N.R.S., Faculté de Pharmacie, Université Louis Pasteur, Illkirch, France.
Autelitano
F
F
Guenet
C
C
Heitz
F
F
Goeldner
M
M
Ilien
B
B
D000818:Animals; D016367:Baculoviridae; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007313:Insecta / Q000821:virology; D018721:Muscarinic Agonists / Q000378:metabolism / Q000494:pharmacology*; D018727:Muscarinic Antagonists / Q000378:metabolism / Q000494:pharmacology*; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic / Q000096:biosynthesis / Q000187:drug effects* / Q000235:genetics; D011994:Recombinant Proteins / Q000187:drug effects
D018076:DNA, Complementary; D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins