Photoaffinity labeling the beta-adrenergic receptor with an iodoazido derivative of norepinephrine |
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Authors: | J F Resek A E Ruoho |
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Institution: | Department of Pharmacology, University of Wisconsin Medical School, Madison 53706. |
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Abstract: | 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. |
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