5'-(P-FLUOROSULFONYLBENZOYL)-2' (OR 3')-(METHYLANTHRANILOYL)ADENOSINE, FLUORESCENT AFFINITY LABELS FOR ADENINE-NUCLEOTIDE BINDING-SITES - INTERACTION WITH THE KINASE ACTIVE-SITE OF THE RECEPTOR FOR EPIDERMAL GROWTH-FACTOR

We have found that the epidermal growth factor (EGF) receptor kinase c an utilize the fluorescent ATP derivative, methylanthraniloyl ATP, as a substrate, On the basis of this observation, together with our previ ous studies that showed that 5'-(p-fluorosulfonylbenzoyl)adenosine (5' -FSBAdo) is a highly specific affinity label for the ATP site of the k inase domain of the EGF receptor, we prepared new derivatives of 5'-FS BAdo, 5'-(p-fluorosulfonyl)-2'(or 3')-(methylanthraniloyl)adenosine (F SBMantAdo), as fluorescent affinity labels for adenine nucleotide bind ing sites, and in particular for the ATP site of the EGF receptor. The two products were purified by HPLC and were characterized by UV-Vis a bsorbance spectroscopy, mass spectrometry, nuclear magnetic resonance spectroscopy, and fluorescence spectroscopy, Incubation of membrane ve sicles containing the EGF receptor with either the 2' or 3' derivative resulted in irreversible inhibition of the receptor kinase activity, as assessed by autophosphorylation assays. Preincubation of vesicles w ith AMP imidodiphosphate (AMPPNP), a hydrolysis-resistant ATP analog, prior to treatment with FSBMantAdo resulted in the protection of the r eceptor kinase activity from FSBMantAdo inactivation, Steady state flu orescence spectra (with excitation at 360 nm) revealed a blue shift in the emission maximum of partially purified FSBMantAdo-labeled recepto r (426 nm), as compared with the emission maximum of free FSBMantAdo ( 441 nm) in aqueous solution, suggesting that the receptor-bound label is in a relatively low polarity environment. These studies show that F SBMantAdo is a specific affinity label for the ATP site of the EGF rec eptor. FSBMantAdo may also prove useful as a fluorescent affinity labe l for other ATP binding sites.