Interaction of Transforming Growth Factor .alpha. with the Epidermal Growth Factor Receptor: Binding Kinetics and Differential Mobility within the Bound TGF-.alpha.

Hoyt, David; Harkins, Richard N.; Debanne, Maria T.; O’Connor-McCourt, Maureen D.; Sykes, Brian D.

doi:10.1021/bi00255a009

Cited by 29 publications

(29 citation statements)

References 69 publications

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“…Expression and Purification of TGF-␣-TGF-␣ and the EGFR-ED were prepared and purified according to previous methods (26).…”

Section: Methodsmentioning

confidence: 99%

“…During the course of the NOESY experiment, the bound ligand magnetization is transferred to the excess free ligand through chemical exchange, and thus, the NMR information characterizing the bound structure is observed via the sharp resonances of the free ligand. At pH 6.0, TGF-␣ is in fast exchange with the EGFR-ED and has an off-rate of Ն300 s Ϫ1 (26), and therefore, this system has conditions amenable to study by TR-NOESY. The complete assignment of proton NMR resonances of TGF-␣ at pH 6.0 has been previously reported (26) and was utilized in order to assign the cross-peaks for a series of TR-NOESY spectra of TGF-␣ free and in the presence of 0, 2.4, 4.9, and 6.5% EGFR-ED.…”

Section: Two-dimensionalmentioning

confidence: 99%

“…At pH 6.0, TGF-␣ is in fast exchange with the EGFR-ED and has an off-rate of Ն300 s Ϫ1 (26), and therefore, this system has conditions amenable to study by TR-NOESY. The complete assignment of proton NMR resonances of TGF-␣ at pH 6.0 has been previously reported (26) and was utilized in order to assign the cross-peaks for a series of TR-NOESY spectra of TGF-␣ free and in the presence of 0, 2.4, 4.9, and 6.5% EGFR-ED. From the 150-ms NOESY data, of a total of 544 cross-peaks in the free ligand, 419 were assigned unambiguously.…”

Section: Two-dimensionalmentioning

confidence: 99%

“…Hoyt et al (26) have recently demonstrated, through a study of 1 H NMR transverse and longitudinal relaxation rates for the methyl resonances of TGF-␣ in the free state and in association with the EGFR-ED, that the C-terminal residues undergo a dramatic decrease in flexibility upon binding, while the N terminus maintains a degree of mobility similar in both bound and free forms of the ligand. The mid-portion of the molecule underwent a moderate decrease in flexibility relative to the uncomplexed polypeptide.…”

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confidence: 99%

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NMR Study of the Transforming Growth Factor-α (TGF-α)-Epidermal Growth Factor Receptor Complex

McInnes

Hoyt

Harkins³

et al. 1996

Journal of Biological Chemistry

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The study of human transforming growth factor-␣ (TGF-␣) in complex with the epidermal growth factor (EGF) receptor extracellular domain has been undertaken in order to generate information on the interactions of these molecules. Analysis of 1 H NMR transferred nuclear Overhauser enhancement data for titration of the ligand with the receptor has yielded specific data on the residues of the growth factor involved in contact with the larger protein. Significant increases and decreases in nuclear Overhauser enhancement cross-peak intensity occur upon complexation, and interpretation of these changes indicates that residues of the A-and C-loops of TGF-␣ form the major binding interface, while the B-loop provides a structural scaffold for this site. Human TGF-␣ 1 is a 50-amino acid polypeptide with 40% sequence homology to epidermal growth factor (1, 2). In addition, the structural similarity of the two molecules results in their ability to compete for binding to the EGF receptor (3-5). Complexation of TGF-␣ with this receptor is believed to mediate a variety of biological effects, including embryonic development of certain tissues and wound healing (6, 7); however, the major sphere of interest of this protein lies in its role in the transformation and maintenance of various malignant tumors (8, 9).The structural features of the homologous growth factors that contain three disulfides and hence three loops (A, B, and C) have been determined by NMR (10 -13) and include a triplestranded anti-parallel ␤-sheet comprising the N-terminal region, a smaller anti-parallel double hairpin in the C terminus of the molecule, and a helical segment in the A-loop in some structures. Previous studies undertaken to elucidate the structurally important residues of TGF-␣ (and EGF) required for complexation have implicated residues including Phe-15, Tyr-38, . The consensus of a variety of structural studies including the use of synthetic peptide fragments (19 -21), recombinant chimeric proteins (22, 23), and anti-TGF-␣ and anti-EGF antibodies (24, 25) is that receptor binding occurs with multiple domains of TGF-␣, although conflicting results have been obtained concerning the involvement of the A-, B-, and C-loops. The multidomain binding model is consistent with the observation that, at present, it is not possible to reduce the size of the growth factor without significantly compromising its affinity for the EGF receptor. This was illustrated by deletion studies where the N-terminal residues outside the A-loop were truncated. This mutant had 3% of the binding affinity of the intact protein (20). Further data from receptor-bound TGF-␣ may lead to the structure-based design of reductant molecules through the precise identification of ligand binding determinants. Hoyt et al. (26) have recently demonstrated, through a study of 1 H NMR transverse and longitudinal relaxation rates for the methyl resonances of TGF-␣ in the free state and in association with the EGFR-ED, that the C-terminal residues undergo a dramatic decrease in flexibility upo...

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“…Expression and Purification of TGF-␣-TGF-␣ and the EGFR-ED were prepared and purified according to previous methods (26).…”

Section: Methodsmentioning

confidence: 99%

Section: Two-dimensionalmentioning

confidence: 99%

Section: Two-dimensionalmentioning

confidence: 99%

mentioning

confidence: 99%

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NMR Study of the Transforming Growth Factor-α (TGF-α)-Epidermal Growth Factor Receptor Complex

McInnes

Hoyt

Harkins³

et al. 1996

Journal of Biological Chemistry

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“…ErbB4 was immunoprecipitated with an anti-ErbB4 antibody and ErbB tyrosine phosphorylation was detected by antiphosphotyrosine immunoblotting Figure 7 Phe45 of NRG2b may interact with Leu437 and Lys438 of ErbB4. A molecular model of the interaction between Phe45 of NRG2b and ErbB4 was generated using the data from the recently published crystal structure of EGF in complex with EGFR (Ogiso et al, 2002 (Hoyt et al, 1994). Finally, scanning alanine mutagenesis of NRG1b reveals that substitution of an alanine for Tyr48 causes a greater than twofold loss of affinity for ErbB4 .…”

Section: Phe45 Regulates Nrg2b Potencymentioning

confidence: 99%

Five carboxyl-terminal residues of neuregulin2 are critical for stimulation of signaling by the ErbB4 receptor tyrosine kinase

et al. 2003

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The neuregulins (NRGs) are members of the epidermal growth factor (EGF) family of peptide growth factors. These hormones are agonists for the ErbB family of receptor tyrosine kinases, a family that includes the epidermal growth factor receptor (EGFR/ErbB1), ErbB2/ Neu/HER2, ErbB3/HER3, and ErbB4/HER4. We recently observed that the EGF family hormone NRG2b is a potent agonist for ErbB4. In contrast, NRG2a, a splicing isoform of the same gene that encodes NRG2b, is a poor ErbB4 agonist. We hypothesized that carboxyl-terminal residues of NRG2b are critical for stimulation of ErbB4 tyrosine phosphorylation and coupling to downstream signaling events. Here, we demonstrate that the substitution of a lysine residue for Phe45 in NRG2b results in reduced ligand potency. We also demonstrate that substitution of a phenylalanine for Lys45 in NRG2a results in increased ligand potency. Finally, analyses of the gain-of-function NRG2a Chg5 mutant demonstrate that Gln43, Met47, Asn49, and Phe50 regulate ligand efficacy. Thus, these data indicate that carboxyl-terminal residues of NRG2b are critical for activation of ErbB4 signaling. Moreover, these NRG2a and NRG2b mutants reveal new insights into models for ligand-induced ErbB family receptor tyrosine phosphorylation and coupling to downstream signaling events.

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Growth factor receptors: Structure, mechanism, and drug discovery

McInnes

Sykes

1997

Biopolymers

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The focus of this review is the relationship between the three‐dimensional structure of ligands of the various members of the growth factor receptor tyrosine kinase superfamily and their interaction with the cognate receptor. Particular attention is given to the transforming growth factor—α, epidermal growth factor (EGF); nerve growth factor, neurotrophin; and insulin‐like growth factor—1 (IGF‐1), insulin systems since these have been extensively studied in recent years. The three receptor types, which bind these ligands, are the epidermal growth factor receptor family (erb B receptors), the neurotrophin or Trk receptor family, and IGF‐1/insulin receptors, respectively, and represent three distinct members of the tyrosine kinase superfamily. For each of these, formation of the ligand‐receptor complex initiates the signal transduction cascade through autophosphorylation by the intracellular tyrosine kinase domain. The extracellular portion of the receptor that contains the ligand binding domain in these systems varies significantly in organization in each case. For the EGF receptor system, ligand binding induces homo‐ and heterodimerization of the receptor leading to activation of the intracellular kinase. For the Trk receptor system, homodimerization of receptors has been shown to occur, although a second receptor, p75, is also required for high affinity binding of neurotrophins and for enhanced sensitivity of tyrosine kinase activation at low ligand concentrations. The IGF‐1 and insulin receptors exist as covalent cross‐linked dimers where each monomer is composed of two subunits. The aim of this review is also to discuss the mechanism of ligand‐receptor interaction for each of these cases; however, since no structural information is yet available for the ligand‐receptor complex, the discussion will largely be centered on the molecular requirements of ligand binding. As these receptors are activated through the ligand binding site on the extracellular domain, this represents a possible target for pharmacological intervention by inhibition or stimulation of this portion of the receptor. Thus from a drug design perspective, the focus of this review is to discuss progress in the development of agonists or antagonists of the ligand for these receptors. © 1998 John Wiley & Sons, Inc. Biopoly 43: 339–366, 1997

show abstract

Interaction of Transforming Growth Factor .alpha. with the Epidermal Growth Factor Receptor: Binding Kinetics and Differential Mobility within the Bound TGF-.alpha.

Cited by 29 publications

References 69 publications

NMR Study of the Transforming Growth Factor-α (TGF-α)-Epidermal Growth Factor Receptor Complex

NMR Study of the Transforming Growth Factor-α (TGF-α)-Epidermal Growth Factor Receptor Complex

Five carboxyl-terminal residues of neuregulin2 are critical for stimulation of signaling by the ErbB4 receptor tyrosine kinase

Growth factor receptors: Structure, mechanism, and drug discovery

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