A classic model of steroid/thyroid hormone receptor activation postulates that a conformational change or "transformation" occurs upon ligand binding as a first step toward regulation of gene transcription. In order to test this model, physical studies have been carried out using purified full-length chicken thyroid hormone receptor alpha 1 (cT3R-alpha 1) expressed in Escherichia coli. Circular dichroism spectroscopic studies reveal that cT3R-alpha 1 adopts a different conformation upon specific binding to a cognate ligand triiodothyroacetic acid as well as to a thyroid hormone response element, an idealized inverted repeat AGGTCA TGACCT. These results suggest that cT3R-alpha 1 may adopt distinct conformations whether free or bound to ligand or to DNA. These states may reflect the changes in the conformation of steroid/thyroid hormone receptors in the signal transduction pathway.

A triple mutant of murine epidermal growth factor (mEGF), N1Q/H22Y/R45K-mEGF, was constructed by site-directed mutagenesis, expressed, purified, and characterized for use in an affinity cross-linking study to identify aminoacyl residues of the EGF receptor adjacent to a residue in the carboxyl-terminal domain of bound EGF thought to be important in distinguishing between EGF and transforming growth factor-␣ in their recognition by the receptor. Cyclization of Gln 1 to form pyroglutamate (pE) limited the site of cross-linking in the mutant to Lys 45, permitting identification of receptor residues that are proximal to this residue of bound EGF. The resulting N1pE/H22Y/R45K-mEGF was shown to be comparable to wild-type mEGF in receptor binding and stimulation of receptor autophosphorylation. 125ILabeled N1pE/H22Y/R45K-mEGF was reacted with the heterobifunctional cross-linking reagent sulfo-N-succinimidyl-4-(fluorosulfonyl)benzoate, and the resulting modified EGF was incubated with A431 membrane vesicles bearing EGF receptors. Incubation resulted in specific cross-linking of the labeled N1pE/H22Y/R45K-mEGF to EGF receptors. The resulting cross-linked complex was then partially purified, denatured, reduced, and carboxyamidomethylated. Digestion with endoprotease LysC resulted in a unique radiolabeled peptide that could be immunoprecipitated using antibodies to mEGF. This immunoprecipitated fragment was purified by gel electrophoresis and subjected to microsequencing. The resulting sequence was matched to that of a LysC fragment of the receptor, which begins with Thr 464 and is near the interface of receptor subdomains III and IV. Loss of signal at cycle 2 suggests that the point of attachment of cross-linked N1pE/H22Y/ R45K is Lys 465 of the receptor.Epidermal growth factor (EGF) 1 and TGF␣ mediate their biological responses by binding to the EGF receptor in target cells (reviewed in Refs. 1-3). While the three-dimensional structures of mEGF (4, 5), hEGF (6), and TGF␣ (7) are known from high field NMR studies, no three-dimensional structure is yet available for the EGF receptor. Much structural information about the receptor has been gleaned, however, by mapping onto the derived primary sequence of the receptor (8) information from chemical, immunochemical, and molecular biological studies of the receptor (reviewed in Refs. 9 and 10). In the present study, we focus on the extracellular, ligand-binding domain of the receptor, identifying residues of the receptor adjacent to the carboxyl-terminal domain of bound EGF, a region shown to be important in the discrimination of EGF and TGF␣ by the EGF receptor (11).Murine EGF has no lysyl residues (12), so the ␣-amino terminus is the only primary amino group. We exploited this aspect of the chemistry of the hormone in a study in which the N terminus of wild-type mEGF was modified with SSFSB, a heterobifunctional cross-linking reagent, and affinity crosslinked to the receptor (13). The site of cross-linking was identified by Edman degradation of a purified fragment of the rece...

We have found that the epidermal growth factor (EGF) receptor kinase can utilize the fluorescent ATP derivative, methylanthraniloyl ATP, as a substrate. On the basis of this observation, together with our previous studies that showed that 5'-(p-fluorosulfonylbenzoyl)adenosine (5'-FSBAdo) is a highly specific affinity label for the ATP site of the kinase domain of the EGF receptor, we prepared new derivatives of 5'-FSBAdo, 5'-(p-fluorosulfonyl)-2'(or 3')-(methylanthraniloyl)adenosine (FSBMantAdo), as fluorescent affinity labels for adenine nucleotide binding 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 absorbance spectroscopy, mass spectrometry, nuclear magnetic resonance spectroscopy, and fluorescence spectroscopy. Incubation of membrane vesicles 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 with AMP imidodiphosphate (AMPPNP), a hydrolysis-resistant ATP analog, prior to treatment with FSBMantAdo resulted in the protection of the receptor kinase activity' from FSBMantAdo inactivation. Steady state fluorescence spectra (with excitation at 360 nm) revealed a blue shift in the emission maximum of partially purified FSBMantAdo-labeled receptor (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 FSBMantAdo is a specific affinity label for the ATP site of the EGF receptor. FSBMantAdo may also prove useful as a fluorescent affinity label for other ATP binding sites.

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