New insights into protein‐tyrosine kinase receptor signaling complexes

Fry, Michael; Panayotou, George; Booker, Grant W.; Waterfield, Michael D.

doi:10.1002/pro.5560021102

Cited by 64 publications

(30 citation statements)

References 107 publications

(89 reference statements)

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“…The absence of an identifiable signal in cycle two, which corresponds to Lys 465 of the receptor sequence, combined with the observation that this site is not cleaved by LysC, argues strongly that Lys 465 is the point of attachment of the receptor to Lys 45 of bound N1pE/H22Y/R45K-mEGF. 2 In this, as in our previous affinity cross-linking studies (13,14), we have observed specifically bound, but not covalently cross-linked 125 I-EGF present through the WGL-agarose purification step, which under the best of circumstances takes many hours and subjects the ligandreceptor complex to enormous dilution. The integrity of the ligandreceptor complex under such conditions can, in part, be explained by the very high rates of association observed for the hormone with the receptor (19).…”

Section: Egf Receptor Residues Near the C Terminus Of Bound Egf 19657supporting

confidence: 80%

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Identification of Residues of the Epidermal Growth Factor Receptor Proximal to Residue 45 of Bound Epidermal Growth Factor

Summerfield

Hudnall

Lukas

et al. 1996

Journal of Biological Chemistry

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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...

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Section: Egf Receptor Residues Near the C Terminus Of Bound Egf 19657supporting

confidence: 80%

“…[1][2][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.…”

mentioning

confidence: 99%

Identification of Residues of the Epidermal Growth Factor Receptor Proximal to Residue 45 of Bound Epidermal Growth Factor

Summerfield

Hudnall

Lukas

et al. 1996

Journal of Biological Chemistry

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“…A recent report (35) has suggested, however, that the ability of EGF receptors mutated at Lys-721 to stimulate MAP kinase (15,41) derives from amplification of signals from a previously undetected level of endogenous WT EGFRs through heterodimerization, transphosphorylation of the kinase-negative receptors, and subsequent activation of the Grb2/Sos/Ras pathway (42). However, the observed phosphorylation of K721R was not found to result in the stimulation of either cation transport or DNA synthesis (15), suggesting that MAP kinase activation by this mutant is not sufficient to mediate such downstream events.…”

Section: Resultsmentioning

confidence: 99%

A kinase-negative epidermal growth factor receptor that retains the capacity to stimulate DNA synthesis.

Coker

Staros²,

Guyer³

1994

Proc. Natl. Acad. Sci. U.S.A.

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The residue proposed to serve as the catalytic base for phosphoryl transfer, Asp-813, ofthe human epidermal growth factor receptor (EGFR) was mutated to Ala, and the mutant receptor (D813A) was expressed in Chinese hamster ovary (CHO) cells. Partially purified D813A exhibited no detectable kinase activity in the absence or presence of EGF. A low level of EGF-stimulable phosphorylation of D813A was detectable in intact cells, apparently due to the activity of an associated Tyr kinase(s). As previously observed for kinaseinactive Lys-721 mutants, EGF binding to D813A stimulates mitogen-activated protein kinase activity. Surprisingly, and unlike results reported for Lys-721 mutants, D813A is capable of stimulating both "Rb+ uptake and DNA synthesis in response to EGF. These data suggest not only that Asp-813 is critical to the catalytic activity of the EGFR but also that differences may exist in the signaling properties of kinasenegative Lys-721 and kinase-negative Asp-813 EGFR mutants.

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“…At the end of the labeling time, medium was removed, and cells were rinsed in ice-cold medium and scraped off with a rubber policeman. After low-speed centrifugation, cells were lysed with 0.3% SDS/1% 2-mercaptoethanol/4% (vol/vol) LKB Ampholines, pH 5-7, 5-8, [3][4][5][6][7][8][9][10]. Within 30 sec, RNase (0.1 mg/ml) and DNase 1 (0.2 mg/ml) (Worthington) were added and mixed.…”

mentioning

confidence: 99%

Early changes in protein synthesis induced by basic fibroblast growth factor, nerve growth factor, and epidermal growth factor in PC12 pheochromocytoma cells.

Hondermarck

McLaughlin

Patterson

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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Nerve growth factor (NGF) and basic flbroblast growth factor (bFGF) stimulate neuronal differentiation, whereas epidermal growth factor (EGF) promotes only mitogenic responses in PC12 pheochromocytoma cells. The early changes in protein synthesis induced by bFGF, NGF, and EGF in these cells have been determined by two-dimensl PAGE of [35S~methionine-labeled proteins and computerized image analysis. The rate of synthesis ofonly 29 proteins (out of 1500 identified) was found to be modulated during the first several hours of growth factor stimulation. Individually, 12 were affected by EGF, 23 were affected by bFGF, and 20 were affected by NGF. Eight of these were regulated by all three growth factors, while 10 proteins were commonly Induced by bFGF and NGF, in accordance with the essentially identical morphological responses induced by these two factors. In addition, the effects of bFGF and NGF were about equally divided between increases and decreases in the rate of synthesis of individual proteins, whereas EGF caused sign tiy more positive (increased) responses. All proteins modulated by NGF or FGF alone were negative in their response and those induced by only EGF were positive. Of particular interest, the rate of synthesis of two proteins of 55 kDa and pl 5.45 and 5.50 was dramatically and transiently induced during the first 2 hr of bFGF and NGF treatment and was not affected by EGF. This study indicates that all three factors elicit early increases and decreases in the synthesis of a quite limited number of proteins and provides molecular evidence for the specificity of a differentiative vs. a proliferative growth factor-induced signaling pathway in these cells.

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New insights into protein‐tyrosine kinase receptor signaling complexes

Cited by 64 publications

References 107 publications

Identification of Residues of the Epidermal Growth Factor Receptor Proximal to Residue 45 of Bound Epidermal Growth Factor

Identification of Residues of the Epidermal Growth Factor Receptor Proximal to Residue 45 of Bound Epidermal Growth Factor

A kinase-negative epidermal growth factor receptor that retains the capacity to stimulate DNA synthesis.

Early changes in protein synthesis induced by basic fibroblast growth factor, nerve growth factor, and epidermal growth factor in PC12 pheochromocytoma cells.

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