hTGF Alpha Research Articles

Variable expression of human transgenes in SHN mice.

The mouse mammary tumour virus/human transforming growth factor alpha (MMTV/hTGF alpha) gene or the mouse whey acidic protein/human growth hormone (mWAP/hGH) gene was introduced to a high mammary tumour strain of SHN virgin females by mating with males bearing each gene. We maintained transgenic mice by backcrossing males with the hTGF alpha transgene or high serum hGH levels (> 50 ng/ml) to SHN virgins in the subsequent generations. Expression of the transgenes was examined at each generation. At the first and the second generations of backcrossing (N1 and N2), females showed hTGF alpha mRNA in the mammary glands associated with a marked outgrowth of the glands. Both females and males had high hGH levels in the circulation, but these characteristics disappeared completely after the third generation (N3). All findings indicate that SHN mice are specific in the resistance to the expression of some human transgenes.

A Single Amino Acid Exchange, Arg-45 to Ala, Generates an Epidermal Growth Factor (EGF) Mutant with High Affinity for the Chicken EGF Receptor

The finding that human epidermal growth factor (hEGF) and human transforming growth factor (hTGF) alpha bind with similar affinity to the human EGF receptor but differ in their affinity for the chicken EGF receptor was used as a model system to study ligand-receptor interaction of EGF receptor agonists. We previously constructed domain-exchange mutants of hEGF and hTGF alpha and found that the region COOH-terminal of the sixth cysteine residue in hTGF alpha is important for high affinity binding to the chicken EGF receptor (Kramer, R. H., Lenferink, A. E. G., Lammerts van Bueren-Koornneef, I., van der Meer, A., van de Poll, M. L. M., and van Zoelen, E. J. J. (1994) J. Biol. Chem. 269, 8708-8711). To analyze this domain in more detail, we now constructed four additional chimeras in which either the region between the sixth cysteine residue and the highly conserved Leu-47 was exchanged or the region COOH-terminal of Leu-47. A mutant in which the latter region in hEGF was replaced by hTGF alpha (designated E6ET) showed intermediate binding affinity, whereas replacement of the former region in hEGF by hTGF alpha was sufficient to generate a mutant (designated E6TE) with a similar high affinity for the chicken EGF receptor as wild type hTGF alpha. Furthermore, a deletion mutant of hEGF lacking three COOH-terminal amino acids, EGF50, showed intermediate binding affinity for the chicken EGF receptor similar to E6ET, but upon additional deletions (EGF49 and EGF48), this initial gain in affinity was lost. A systematic analysis of the region between the sixth cysteine residue and Leu-47 showed that the low affinity of hEGF for the chicken EGF receptor is mainly due to the presence of Arg-45. Replacement of the positively charged Arg-45 by Ala, the corresponding amino acid in hTGF alpha, was sufficient to generate a mutant growth factor with high affinity for the chicken EGF receptor. This indicates that in hEGF Arg-45 may play an important role in receptor binding. A model is proposed in which positively charged amino acids close to or within the receptor recognition site of hEGF prohibit high affinity binding to the chicken EGF receptor due to electrostatic repulsion of positively charged amino acids in the putative ligand binding domain of the chicken EGF receptor.

Human type-alpha transforming growth factor undergoes slow conformational exchange between multiple backbone conformations as characterized by nitrogen-15 relaxation measurements.

Human type-alpha transforming growth factor (hTGF alpha) is a small mitogenic protein containing 50 amino acids and three disulfide bonds. It has both sequence and structural homology with epidermal growth factor (EGF). While the three-dimensional structures of hTGF alpha and other EGF-like proteins have been studied extensively, relatively little is known about conformational dynamics of these molecules. In this paper we describe nuclear relaxation measurements which probe the molecular dynamics of hTGF alpha in aqueous solution at neutral pH. In order to characterize conformational dynamics of hTGF alpha on both the fast (i.e., sub-nanosecond) and intermediate nitrogen-15 chemical-exchange (i.e., microsecond) time scales, we measured nitrogen-15 relaxation parameters at pH 7.1 +/- 0.1 and a temperature of 30 +/- 0.5 degrees C. Measurements of nitrogen-15 longitudinal (R1) and transverse (R2) relaxation rates, and 1H-15N heteronuclear NOE effects, were then interpreted using an extended Lipari-Szabo analysis [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559; Clore, G. M., Szabo, A., Bax, A., Kay, L. E., Driscoll, P. C., & Gronenborn, A. M. (1990) J. Am. Chem. Soc. 112, 4989-4991] to provide estimates of the locations and amplitudes of fast internal motions and the locations of nitrogen-15 chemical-exchange line broadening. These results demonstrate that, under conditions of pH and temperature at which it is tightly bound by the EGF receptor, hTGF alpha is a highly dynamic molecule. Indeed, some 40% of the backbone amide groups of hTGF alpha, including many at the interface between the two subdomains, exhibit significant nitrogen-15 chemical-exchange line broadening indicative of interconversions between multiple protein conformations on the microsecond time scale. The distribution of these sites on the three-dimensional protein structure suggests that these dynamic fluctuations are due to (i) partial unfolding of the core beta-sheet, (ii) hinge-bending motions between the N- and C-terminal subdomains, and/or (iii) disulfide bond isomerization in the solution structure of hTGF alpha at neutral pH.

Contribution of the Transforming Growth Factor α B-loop β-Sheet to Binding and Activation of the Epidermal Growth Factor Receptor

We have exploited the differences in binding affinities of the chicken epidermal growth factor (EGF) receptor for EGF and transforming growth factor alpha (TGF alpha) to study the role of the B-loop beta-sheet of these ligands in receptor recognition and activation. Although EGF and TGF alpha share similar secondary and tertiary structures imposed by three highly conserved intramolecular disulfide bonds, they have only 30-40% overall sequence identity. The B-loop beta-sheet is the major structural element in EGF and TGF alpha, but sequence similarity in this region is low. To investigate its role in receptor binding, we constructed two chimeric growth factors (mEGF/hTGF alpha 21-30 and mEGF/hTGF alpha 21-32) composed of the murine EGF (mEGF) amino acid sequence with residues 21-30 of the B-loop beta-sheet replaced by the equivalent residues of human TGF alpha (hTGF alpha); in chimera mEGF/hTGF alpha 21-32, asparagine 32, which lies at the boundary of the amino and carboxyl domains of mEGF, was also replaced by its hTGF alpha counterpart (valine). In initial studies using unpurified medium, it was found that the recombinant growth factors exhibited differing mitogenic potencies (mEGF/hTGF alpha 21-32 > mEGF/hTGF alpha 21-30 > mEGF) when assayed on chicken fibroblasts, even though they were equivalent in mitogenesis assays using cells expressing the human EGF receptor. After purification, mEGF/hTGF alpha 21-32 was found to be 50 times more potent than mEGF in the chick fibroblast mitogenesis assay and exhibited a 10-fold increase in relative affinity for the chicken EGF receptor; both growth factors still exhibited equivalent mitogenic and receptor binding activity when tested on cells expressing human EGF receptors. We conclude that the B-loop beta-sheet of hTGF alpha is an important determinant of EGF receptor binding affinity and biological activity.

Digestion of h-EGF, h-TGF alpha, and BPC-15 in human gastric juice

This chapter discusses vidarabine. Vidarabine is a purine nucleoside, which does not occur naturally. Vidarabine has subsequently been obtained from fermentation cultures of Streptomyces antibioticus, tested and developed by Warner-Lambert/Parke-Davis Company. Vidarabine is an odorless, white crystalline solid. It melts in the range of 262˚ and 270˚C. The infrared spectrum of vidarabine presented in the chapter was taken as a 0.5% dispersion of vidarabine in potassium bromide using a Perkin-Elmer Model 621 infrared spectrophotometer. The nuclear magnetic resonance (NMR) spectrum of vidarabine was obtained with a Varian Model 60 MC spectrometer. Deuterated dimethylsulfoxide was used as the solvent with tetramethylsilane as an internal standard. Vidarabine is an amphoteric compound, which can act as both a weak acid and a weak base. Vidarabine has been synthesized by two different methods. The first involves a cleavage reaction of 9-(2,3-anhydro-β-Dlyxofuranosyl) adenine, which is derived from 9-β-Dxylofuranosyladenine. The second is a condensation of benzoyladenine with 2,3,5-tri-O-benzyl-β-D-arabinosylchloride.

Solution structure of human type-alpha transforming growth factor determined by heteronuclear NMR spectroscopy and refined by energy minimization with restraints.

Human type-alpha transforming growth factor (hTGF alpha) is a small mitogenic protein containing 50 amino acids and 3 disulfide bonds. Homo- and heteronuclear NMR spectra were used to determine nearly complete sequence-specific 1H and 15N resonance assignments for hTGF alpha under three conditions: pH 6.5 and a temperature of 10 degrees C, pH 6.5 and a temperature of 30 degrees C, and pH 3.5 and a temperature of 30 degrees C. The 15N-enriched samples of hTGF alpha allowed determination of many 3J(HN-H alpha) vicinal coupling constants. Solution structures of human type-alpha transforming growth factor (hTGF alpha) at pH 6.5 and a temperature of 10 degrees C were determined from NMR data using molecular structure generation calculations and restrained energy minimization. These structures are based on 425 conformational constraints, including 357 NOE-derived upper-bound distance constraints, constraints on the ranges of 26 dihedral angles based on measurements of vicinal coupling constants, 42 upper- and lower-bound constraints associated with 6 hydrogen bonds and 3 disulfide bonds, and several stereospecific 1H resonance assignments. The overall structure is similar to that described recently for hTGF alpha by other groups [Kline et al. (1990) Biochemistry 29, 7805-7813; Harvey et al. (1991). Eur. J. Biochem. 198, 555-562], but there are differences in some structural details. The resonance frequencies, vicinal coupling constants, and NOEs form the basis for comparisons of the solution structure of hTGF alpha at neutral and acidic pH. At pH 3.5 the protein structure is partially disordered, with most of the hydrogen-bonded backbone structure still intact. The hTGF alpha structure is also compared with that of murine epidermal growth factor. Coordinates for the set of hTGF alpha structures described in this paper have been deposited in the Protein Data Bank.

Endogenous ligands for the epidermal growth factor receptor in human breast tumors do not interfere in an epidermal growth factor receptor radioligand binding assay.

Ligands for the epidermal growth factor receptor (EGFR) (e.g. EGF and transforming growth factor alpha, TGF alpha) may be included in membrane preparations from human breast tumors, either complexed or not to EGFR. Assessment of EGFR in human placental membranes (HPM) after preincubation with human EGF (hEGF) or human TGF alpha (hTGF alpha) indicated that the presence of these ligands in a membrane preparations did not affect the apparent number of binding sites, but only resulted in an increased apparent dissociation constant (Kd). To obviate the effect of endogenously bound EGFR ligands in a radioligand binding assay, an acid treatment procedure has been used recently. In the present study we show that acid treatment of mammary tumor membranes does not result in increased EGFR levels but does result in an EGFR enriched membrane preparation by elimination of contaminating cytosol proteins. This however also occurred by washing the membranes with assay buffer at neutral pH. From these experiments we conclude that endogenous ligands, if present in membrane fractions from human breast tumors and occupying EGFR, do not interfere in a multipoint radioligand binding assay.

Multidomain binding of transforming growth factor .alpha. to the epidermal growth factor receptor

Solubilized epidermal growth factor receptor (EGF-R) has been used in an extension of the Geysen epitope mapping protocol in order to provide additional insight into the amino acid residues in human transforming growth factor alpha (hTGF alpha) which are critical to recognition and binding. Overlapping heptapeptides which encompassed the 50 amino acid primary sequence of hTGF alpha were synthesized on a polyethylene solid phase, and the amount of detergent-solubilized EGF-R bound to each peptide was measured using ELISA. EGF-R appeared to bind reproducibly to four heptapeptides cognate to sequences in both the N- and C-domains of hTGF alpha (residues 22-28, 28-34, 36-42, and 44-50). Visualization of these four regions on three-dimensional solution phase structures of hTGF alpha, derived from 1H NMR measurements [Kline, T.-P., Brown, F.K., Brown, S.C., Jeffs, P.W., Kopple, K.D., & Mueller, L. (1990) Biochemistry 29, 7805-7813], indicated that the peptide segments are located on a single face of the protein and suggested the presence of a potential receptor binding cavity. If peptide segments within both the N- and C-domains of hTGF alpha are involved in binding to EGF-R, then this has direct consequences for possible molecular mechanisms by which receptor activation might take place. For example, the observed conformational flexibility in the six NMR-derived hTGF alpha structures due to variations in the main-chain torsion angles of Val-33, in combination with the involvement of residues from both domains in the proposed binding cavity, may imply that receptor activation results from interdomain reorientation in the protein ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrolysis of transforming growth factor-α by cell-surface peptidases in vitro

Human transforming growth factor-alpha (h-TGF alpha), a 50-amino acid residue peptide, was incubated with some purified cell-surface peptidases and with renal microvillar membranes prepared from pig and rat. Hydrolysis was monitored by h.p.l.c. and activity by a biological assay. Prolonged incubation with relatively large amounts of endopeptidase-24.11, aminopeptidase N and peptidyl dipeptidase A (angiotensin-converting enzyme) caused no observable hydrolysis and no detectable loss of biological activity. Incubation with pig renal microvilli also failed to degrade the peptide. In contrast, rat renal microvilli readily degraded h-TGF alpha, as did endopeptidase-2, which is located in rat renal and intestinal brush borders, but is absent from pig kidneys. This enzyme degraded about 30 nmol of h-TGF alpha/h per mg of protein. The physiological significance of these results is discussed.

Sequence-specific 1H-NMR assignments and identification of two small antiparallel beta-sheets in the solution structure of recombinant human transforming growth factor alpha.

Transforming growth factor alpha (TGF alpha) is a small mitogenic protein with about 35% sequence identity with epidermal growth factor (EGF). TGF alpha-like proteins have been proposed to play a role in oncogenesis and wound healing. This report describes sequence-specific 1H-NMR resonance assignments for recombinant human TGF alpha (hTGF alpha). These assignments provide the basis for interpreting NMR data which demonstrate that the solution structure of hTGF alpha includes an antiparallel beta-sheet involving residues Gly-19 to Leu-24 and Lys-29 to Cys-34 and a second, smaller, antiparallel beta-sheet involving residues Tyr-38 and Val-39 and His-45 and Ala-46. These data, together with constraints imposed by the disulfide bonds, are combined to construct a molecular model of the polypeptide chain fold for residues Cys-8 to Ala-46. The resulting structure is similar to that of mouse and human EGF. Human TGF alpha and mouse EGF, however, differ with respect to their structural dynamics, since amide proton/deuteron exchange is much faster for hTGF alpha than for mouse EGF at pH 3.5.

TGFα stimulates growth of skin papillomas by autocrine and paracrine mechanisms but does not cause neoplastic progression

To investigate the role of transforming growth factor alpha (TGF alpha) in tumor development, we introduced the human TGF alpha (hTGF alpha) cDNA into cultured primary mouse epidermal cells or papilloma cells using a replication-defective retroviral vector and analyzed skin grafts constructed with such cells. Expression of the exogenous gene was confirmed by detection of hTGF alpha mRNA by northern RNA blot analysis, and the secreted hTGF alpha was measured by ELISA of culture supernatants. Tumor cells expressing hTGF alpha produced benign tumors (papillomas), which were 1.5- to 2-fold larger than tumors of parental cells when tested as skin grafts on nude mice. Grafts of normal cells that expressed hTGF alpha produced normal skin. When mixtures of parental tumor cells and normal mouse keratinocytes were grafted to nude mice, papilloma formation was suppressed and tumors that did form were small. Grafts of hTGF alpha-producing papilloma cells combined with either normal epidermal cells or hTGF alpha-producing epidermal cells yielded large tumors. Mixed grafts containing keratinocytes expressing hTGF alpha and parental papilloma cells also produced large tumors. While the tumor size was substantially increased by hTGF alpha expression, the tumors that developed in all groups were histologically benign and reached a stable size 4-5 wk after grafting. These results indicate that expression of hTGF alpha by either tumor cells (autocrine) or adjoining normal cells (paracrine) can stimulate tumor growth, particularly when tumor growth is suppressed by normal tissue. However, expression of this growth factor did not appear to influence tumor progression directly.