Desensitization of human muscarinic acetylcholine receptor m2 subtypes is caused by their sequestration/internalization.
Desensitization of human muscarinic acetylcholine receptor m2 subtypes (hm2 receptors) stably expressed in chinese hamster ovary cells was measured as decreases in the carbamylcholine-stimulated [35S]GTPgammaS binding activity in membrane preparations after pre-treatment of cells with carbamylcholine. The extent of carbamylcholine-stimulated [35S]GTPgammaS binding activity was found to decrease to 64% following pretreatment of cells with 10 microM carbamylcholine for 30 min, and under the same conditions 51-59% of hm2 receptors were sequestered/internalized as assessed by decreases in the [3H]N-methylscopolamine binding activity on the cell surface. A similar reduction in the carbamylcholine-stimulated [35S]GTPgammaS binding activity was observed by pretreatment of cells with 5 nM propylbenzylylcholine mustard, which irreversibly bound to and inactivated 58% of the hm2 receptors. When the cells were pretreated with 10 microM carbamylcholine in the presence of 0.32 M sucrose, which is known to inhibit clathrin-mediated endocytosis, no sequestration/internalization of hm2 receptors was observed, and the extent of carbamylcholine-stimulated [35S]GTPgammaS binding activity did not change. These results indicate that desensitization of hm2 receptors may be caused by reduction of receptor number on the cell surface through sequestration/internalization rather than by loss of the function of receptors.
Tsuga
H
H
Department of Neurochemistry, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, 113-0033, Japan.
Kameyama
K
K
Haga
T
T
D000818:Animals; D016466:CHO Cells; D002217:Carbachol / Q000494:pharmacology*; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic / Q000145:classification / Q000187:drug effects / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D051552:beta-Adrenergic Receptor Kinases
D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D011813:Quinuclidinyl Benzilate; D002217:Carbachol; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases