Formation of the functional complexes of m2 muscarinic acetylcholine receptors with GTP-binding regulatory proteins in solution.
Sixteen different detergents were studied for solubilization of functional complexes between m2 muscarinic acetylcholine receptors (mAChR) and guanine nucleotide-binding regulatory proteins (G proteins). More than 40% of solubilized mAChR retained their GTP-dependent high affinity for agonist binding after solubilization with sucrose monolaurate, whereas all other detergents studied gave considerably lower solubilization yields or caused the loss of the high affinity for agonist binding. The preformation of mAChR-G protein complexes in membranes revealed that a large excess of G proteins did not increase the portion of high-affinity binding sites, but caused GTP- and Mg2(+)-dependent inhibition of the binding of radioactive antagonists to mAChR. The optimization of detergent concentration and other experimental conditions revealed that up to 47% of the solubilized receptors indicated the GTP-dependent high affinity for agonist binding after mixing solubilized mAChR with purified G proteins in sucrose monolaurate in the presence of Mg2+ and carbachol. These results give the first clear proof of the formation of functional complexes between mAChR and G proteins in solution and indicate that GTP-dependent high-affinity agonist binding is connected to the direct interactions between mAChR and G proteins and that other membrane components are not necessary.
Rinken
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Department of Medical and Physiological Chemistry, University of Uppsala, Sweden.
D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D003902:Detergents; D019204:GTP-Binding Proteins / Q000737:chemistry*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006325:Heart Atria; D008274:Magnesium / Q000494:pharmacology; D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists / Q000737:chemistry; D011485:Protein Binding; D011813:Quinuclidinyl Benzilate / Q000737:chemistry; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic / Q000737:chemistry*; D018411:Spodoptera; D013552:Swine
D003902:Detergents; D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic; D011813:Quinuclidinyl Benzilate; D006160:Guanosine Triphosphate; D002217:Carbachol; D019204:GTP-Binding Proteins; D008274:Magnesium