Photoaffinity labeling the beta-adrenergic receptor with an iodoazido derivative of norepinephrine.
An iodinated photosensitive derivative of norepinephine, N-(p-azido-m-iodophenethylamidoisobutyl)-norepinephrine (NAIN), has been synthesized and characterized. NAIN stimulated adenylate cyclase activity in guinea pig lung membranes in a manner similar to (-)-isoproterenol and was inhibited by (-)-alprenolol. NAIN was shown to compete with [125I]iodocyanobenzylpindolol for the beta-adrenergic receptor in guinea pig lung membranes with an affinity which was dependent on the presence of guanyl nucleotides. Carrier-free radioiodinated NAIN ([125I]NAIN) was used at 2 nM to photoaffinity label the beta-adrenergic receptor in guinea pig lung membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of (-)-alprenolol (1 microM) protectable [125I]NAIN labeling showed the same molecular mass polypeptide (65 kDa) that was specifically derivatized with the antagonist photolabel [125I]iodoazidobenzylpindolol. Specific labeling of the beta-adrenergic receptor with [125I]NAIN was dependent on the presence of MgCl2 and the absence of guanyl nucleotide. Guanosine-5'-O-(3-thiotriphosphate (100 microM) abolished specific labeling by [125I]NAIN. N-Ethylmaleimide (2 mM) in the presence of [125I]NAIN protected against the magnesium and guanyl nucleotide effect. These data show that NAIN is an agonist photolabel for the beta-adrenergic receptor.
Resek
J F
JF
Department of Pharmacology, University of Wisconsin Medical School, Madison 53706.
Ruoho
A E
AE
D000262:Adenylyl Cyclases / Q000378:metabolism; D000345:Affinity Labels / Q000138:chemical synthesis*; D000526:Alprenolol / Q000494:pharmacology; D000818:Animals; D001386:Azides / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006168:Guinea Pigs; D007202:Indicators and Reagents; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D008168:Lung / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D009638:Norepinephrine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D011943:Receptors, Adrenergic, beta / Q000187:drug effects / Q000378:metabolism*; D013873:Thionucleotides / Q000494:pharmacology
D000345:Affinity Labels; D001386:Azides; D007202:Indicators and Reagents; D011943:Receptors, Adrenergic, beta; D013873:Thionucleotides; C057279:N-(4-azido-3-iodophenethylamidoisobutyl)norepinephrine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D000526:Alprenolol; D000262:Adenylyl Cyclases; D008274:Magnesium; D007545:Isoproterenol; D009638:Norepinephrine