Effect of dithiothreitol on the beta-adrenergic receptor of S49 wild type and cyc- lymphoma cells: decreased affinity of the ligand-receptor interaction.
Dithiothreitol (DTT) treatment of WT or cyc- lymphoma membranes resulted in the simultaneous loss of epinephrine-stimulated adenylate cyclase activity and beta-adrenergic antagonist binding. The treatment produced no significant decrease in NaF-stimulated activity and only a partial loss of the PGE1 stimulation. Epinephrine partially protected against the loss of epinephrine-stimulated cyclase activity. The decrease in beta-adrenergic stimulation of adenylate cyclase was characterized by over a 100-fold increase in Kact for epinephrine stimulation of adenylate cyclase (10 mM DTT for 30 min) with no effect on the Vmax. We have previously shown that two polypeptides (Mr = 55,000 and 65,000 daltons) are specifically labeled by [125I]iodoazidobenzylpindolol (IABP) in WT and cyc-. The IABP photolabeling of the 55,000 dalton beta-receptor polypeptide was preferentially reduced by 1.0 mM DTT, whereas 10 mM DTT eliminated the photolabeling of both polypeptides. The effects of DTT were not due to either scavenging of the nitrene or reduction of the azide. A reduction in epinephrine stimulation of adenylate cyclase was also observed after treatment of intact WT, and the effects were readily reversed in the cells by washout of the DTT. The DTT effects on membranes were not reversed by washout. Our results demonstrate that the oxidized state of the lymphoma beta-receptor is necessary for maximum sensitivity to agonist stimulation of adenylate cyclase and that low concentrations of reducing agent selectively decrease specific photo-labeling of the 55,000 dalton beta-receptor polypeptide.
D000262:Adenylyl Cyclases / Q000378:metabolism; D000345:Affinity Labels; D001386:Azides; D002478:Cells, Cultured; D004229:Dithiothreitol / Q000494:pharmacology*; D004837:Epinephrine / Q000494:pharmacology; D008223:Lymphoma / Q000201:enzymology / Q000378:metabolism*; D010869:Pindolol / Q000031:analogs & derivatives; D011943:Receptors, Adrenergic, beta / Q000187:drug effects*
D000345:Affinity Labels; D001386:Azides; D011943:Receptors, Adrenergic, beta; C029987:1-(indol-4-yloxy)-3-(1-(4-azido-3-iodophenyl)-2-isobutylamine)-2-propanol; D010869:Pindolol; D000262:Adenylyl Cyclases; D004229:Dithiothreitol; D004837:Epinephrine