Pharmacological and biochemical characterization of complexes of muscarinic acetylcholine receptor and guanine nucleotide-binding protein.
Complexes of agonist-bound muscarinic acetylcholine receptor (mAChR) and guanine nucleotide-binding protein (G protein) were solubilized and isolated from rat heart. Heart membranes were incubated with mAChR agonists or antagonists, solubilized using digitonin and cholate, and subjected to chromatography over wheat germ agglutinin-Affi-Gel. Eluted fractions were precipitated using a cardiac-selective anti-mAChR antibody (Luetje, C. W., Brumwell, C., Norman, M. G., Peterson, G. L., Schimerlik, M. I., and Nathanson, N. M. (1987) Biochemistry 26, 6892-6898). Using samples obtained from membranes initially incubated with carbachol (10 nM, 100 nM, or 1 mM), G alpha immunoreactivity was detected on Western blots probed using antibodies with specificity for G alpha subunits. The G alpha immunoreactivity was not detected when atropine alone (10 nM or 1 microM) or when excess atropine (1 microM) plus carbachol (100 nM) was used during the membrane preincubation. G beta immunoreactivity, when detectable on Western blots, was present in substoichiometric amounts relative to that of G alpha. The G alpha immunoreactivity was not present if GTP was included during incubation of membranes with agonist and following membrane solubilization. Further results indicate that although agonist binding to receptors is rapidly reversed by GTP or GDP (t1/2 less than 10 min), the mAChR-G protein complex is reversed more slowly or not at all. It was also shown that at high agonist concentrations, the cardiac mAChR interacts with both Go and Gi-like proteins. Together, these results demonstrate the utility of an immunoaffinity approach to the purification and biochemical study of receptor-G protein interactions.
Matesic
D F
DF
Department of Physiology and Biophysics, Hahnemann University, Philadelphia, Pennsylvania 19102.
Manning
D R
DR
Wolfe
B B
BB
Luthin
G R
GR
D000818:Animals; D000906:Antibodies; D000936:Antigen-Antibody Complex; D001285:Atropine / Q000378:metabolism; D015153:Blotting, Western; D002217:Carbachol / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D009206:Myocardium / Q000378:metabolism*; D010095:Oxotremorine / Q000378:metabolism; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D051381:Rats; D011976:Receptors, Muscarinic / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility
D000906:Antibodies; D000936:Antigen-Antibody Complex; D046911:Macromolecular Substances; D011976:Receptors, Muscarinic; D010095:Oxotremorine; D011813:Quinuclidinyl Benzilate; D001285:Atropine; D002217:Carbachol; D019204:GTP-Binding Proteins