Radioreceptor assay for epidermal growth factor.

An established cell line of human lung fibroblasts with a high number of surface receptorsfor mouse epidermal growth factor (mEGF) was used to develop a simple and highly sensitive radioreceptor assay for EGF. ¹²⁵I-Labeled mEGF competed mole for mole with unlabeled mEGF for specific receptors. Optimal range for discriminating EGF concentrations in body fluids and tissue extracts by a competitive binding assay was between 5 and 100 ng/ml. Interassay correlation of variation was 8.47% and the recovery of highly purified mEGF added to serum and urine samples was greater than 95%. Human serum and amniotic fluids contained about 24 and 4 ng/ml, respectively, of mEGF equivalents. Concentrations of mEGF in mouse urine and serum were highly variable and were 2- to 10-fold greater than that previously detected by radioimmune assay. Hypophysectomy nearly abolished submaxillary mEGF content in both male and female mice, but testosterone treatment of hypophysectomized animals restored normal concentrations of mEGF to the glands. mEGF added to culture medium disappeared with time as a function of the number of cellular EGF receptors indicating cellular degradation of the growth factor. The radioreceptor assay for EGF is based on the close biologic relationship between the cell receptor site and the native hormone and should prove to be a useful complementary tool to characterize the physiological role of EGF.     

Identification of epidermal growth factor in mouse abdominal effusion

Epidermal growth factor (mEGF)-like immunoreactive material(s) was identified in mouse abdominal effusion (approximately 2.1 ng/mg protein) by our enzyme immunoassay (EIA) for mEGF. This material(s) and mEGF from the submaxillary glands of male mice were virtually equivalent with respect to the molecular weight and the antigenicity. Also, on isoelectric focusing analysis, the mEGF-like material(s) identified in abdominal effusion gave a major peak at pH 4.2 and a minor one at pH 4.5. These results demonstrate that the mEGF-like material(s) found in abdominal effusion is a molecule identical to mouse submaxillary gland EGF. Further we found that sialoadenectomy did not cause a marked decrease in the level of mEGF in abdominal effusion, suggesting that the source of mEGF found in abdominal effusion is other than the submaxillary glands.     

Inhibition of iodine-125-labeled human follitropin binding to testicular receptor by epidermal growth factor and synthetic peptides

Two tetrapeptide sequence homologies between mouse epidermal growth factor precursor (mEGFP) and human follitropin (FSH) were revealed by a computer program that identifies identical residues among polypeptide sequences. The two tetrapeptides, Lys-Thr-Cys-Thr (KTCT) and Thr-Arg-Asp-Leu (TRDL), are present in the hormone-specific beta subunit of FSH from all species studied. These tetrapeptides are not present in the alpha subunit, which is common to all pituitary glycoprotein hormones. Both tetrapeptides are also found in mEGFP, and one tetrapeptide, TRDL, is located within the 53-residue form of mEGF purified from mouse submaxillary glands. Computer-generated hydropathy profiles predicted that both tetrapeptides are located in hydrophilic portions of the FSH beta subunit and that TRDL is in a hydrophilic portion of commercially available mEGF. Therefore, the tetrapeptides might be accessible to receptor binding sites for FSH. We report that mEGF inhibits binding of 125I-labeled human FSH to receptors in testis by 50% (I50) at a concentration of 1.8 X 10(-5) M. No binding inhibition was observed by GnRH or arginine-vasopressin at 10(-4) M, neither of which contain the tetrapeptide sequences. FSH beta subunit, which contains both tetrapeptides, also inhibited binding (I50 = 9 X 10(-8) M) of 125I-labeled human FSH to testis receptor. Thus, it appears that FSH beta subunit and mEGF are capable of inhibiting binding of FSH to testicular FSH receptors, presumably through interactions that include the homologous tetrapeptides. This presumption was supported by the observation that the synthetic tetrapeptides (KTCT or TRDL) were also active in inhibiting binding of 125I-labeled human FSH to testis receptor.     

Structure-function studies of murine epidermal growth factor: expression and site-directed mutagenesis of epidermal growth factor gene

Wild-type murine epidermal growth factor (mEGF) and mutants with Leu47 replaced by serine and valine, respectively, have been produced by recombinant DNA methodology. A synthetic gene for mEGF was fused to the coding sequence for the signal peptide of the outer membrane protein A (ompA) of Escherichia coli in the secretion vector pIN-III-ompA3, and the recombinant plasmid was used to transform E. coli. Upon induction of gene expression, mEGF and the mutants was expressed and secreted into the periplasmic space. Purification of the wild-type Leu47-mEGF and the mutants was carried out by reversed-phase and anion-exchange high-performance liquid chromatography (HPLC). Amino acid analysis and Western blot analysis further confirmed the identities of the proteins. Specific activities for wild-type and mutant proteins were measured in both mEGF receptor binding and autophosphorylation assays. The recombinant mEGF has specific activities identical with that of mEGF purified from mouse submaxillary glands, while both mutants have reduced specific activities in both bioassays. The data demonstrate the importance of the highly conserved Leu47 residue in mEGF for full biological activity.     

Rat epidermal growth factor: complete amino acid sequence. Homology with the corresponding murine and human proteins; isolation of a form truncated at both ends with full in vitro biological activity

Epidermal growth factor (EGF) isolated from the submaxillary gland of the rat (rEGF) is missing the COOH-terminal five residues present in both mouse and human EGF. rEGF competes for the binding of 125I-labelled mEGF to human carcinoma cells with the same affinity as mEGF. rEGF and mEGF have identical mitogenic activities on mouse 3T3 fibroblasts, thus the C-terminal region of the sequence is not necessary for the in vitro activity of EGF. Using reversed-phase high-performance liquid chromatography, four molecular forms of EGF have been extracted from rat submaxillary glands. These forms represent rEGF, rEGF(2-48), rEGF(3-48) and rEGF(4-48); all forms appear to be equipotent in both the receptor binding and mitogenic assays. The isoelectric points of these rEGFs are in the range of pH 5.1 to 5.2. The primary structure of rEGF was determined from approximately 10 micrograms protein by sequence analysis of the intact molecule and fragments obtained from the reduced and alkylated protein by chemical cleavage with CNBr and enzymic cleavage with chymotrypsin and a proline-specific endopeptidase. Subnanomole amounts of generated peptides were purified to homogeneity by reversed-phase microbore high-performance liquid chromatography and analysed by automated Edman degradation in a gas-phase sequencer. There are 48 amino acid residues in the complete polypeptide chain which lacks alanine, phenylalanine, lysine and tryptophan. The amino acid sequence of rat epidermal growth factor is: Asn-Ser-Asn-Thr-Gly-Cys-Pro-Pro-Ser-Tyr-Asp-Gly-Tyr-Cys-Leu-Asn- Gly-Gly-Val-Cys-Met-Tyr-Val-Glu-Ser-Val-Asp-Arg-Tyr-Val-Cys-Asn-Cys -Val-Ile-Gly-Tyr-Ile-Gly-Glu-Arg-Cys-Gln-His-Arg-Asp-Leu-Arg. The calculated relative molecular mass from the sequence analysis is 5377.     

Regulation of gonadotropin receptors and gonadotropin responses in a clonal strain of Leydig tumor cells by epidermal growth factor

The MA-10 line is a clonal strain of Leydig tumor cells that has receptors for human choriogonadotropin (hCG) and mouse epidermal growth factor (mEGF). These cells respond to hCG, cholera toxin, and 8-Br-adenosine 3':5'-monophosphate with increased steroid production. It is reported herein that exposure of the MA-10 cells to mEGF results in a substantial (80 to 90%) reduction in the number of hCG receptors per cell. The loss of hCG receptors is accompanied by a corresponding reduction in the ability of hCG to stimulate steroidogenesis. The steroidogenic responses to cholera toxin and 8-Br-adenosine 3':5'-monophosphate, however, are not affected. Other results presented show that mEGF is not a mitogen for these cells.     

Epidermal growth factor stimulation of prostaglandin E2 biosynthesis in amnion cells. Induction of prostaglandin H2 synthase

Amnion is believed to be a tissue of signal importance, anatomically and functionally, in the maintenance of pregnancy and during the initiation of parturition. Epidermal growth factor (EGF)-like agents cause a striking increase in the secretion of prostaglandin E2 (PGE2) in human amnion cells but only if arachidonic acid is present in the culture medium. To investigate the regulation of arachidonic acid metabolism by EGF-like agents in amnion, we used mEGF and human amnion cells in primary monolayer culture as a model system. The amount of PGE2 secreted into the culture medium was quantified by radioimmunoassay and the rate of conversion of [14C]arachidonic acid to [14C]PGE2 (PGH2 synthase activity) in cell sonicates was determined under optimal in vitro conditions. Treatment of amnion cells with mEGF led to a marked increase in the rate of production of PGE2. The specific activity of PGH2 synthase (viz. the combined activities of prostaglandin endoperoxide (PGH2) synthase and PGH2-PGE isomerase) was increased by 2-5-fold in cells treated with mEGF. Treatment of amnion cells with mEGF for 4 h did not affect the specific activities of phospholipase A2 or phosphatidylinositol-specific phospholipase C. By immunoisolation of newly synthesized, [35S]methionine-labeled PGH2 synthase, we found that mEGF stimulated de novo synthesis of the enzyme. Thus, mEGF acts in human amnion cells in primary monolayer culture to increase the rate of PGE2 biosynthesis by a mechanism that involves induction of PGH2 synthase; the manifestation of EGF action on PGE2 biosynthesis is dependent on the presence of nonesterified arachidonic acid.     

On the mechanisms involved in the regulation of the cell-surface receptors for human choriogonadotropin and mouse epidermal growth factor in cultured Leydig tumor cells

The MA-10 cells are a clonal strain of mouse Leydig tumor cells that have receptors for human choriogonadotropin (hCG) and mouse epidermal growth factor (mEGF). Exposure of the cells to hCG results in a reduction in the number of surface hCG receptors, and little or no change in the number of surface mEGF receptors. On the other hand, exposure of the cells to mEGF results in a reduction in the number of both surface mEGF receptors and surface hCG receptors. In order to study these phenomena, we assumed that the number of surface receptors is determined by the rate at which receptors appear at the surface and by the rate of receptor internalization. When these rates were measured, we found that hCG and mEGF reduce their respective surface receptors by increasing the rate of receptor internalization, and that mEGF reduces the surface hCG receptors by decreasing the rate of appearance of the receptor.     

Anti-phosphotyrosine immunoprecipitation of phosphatidylinositol 3' kinase activity in different cell types after exposure to epidermal growth factor.

In previous studies from this laboratory, it was shown that mouse epidermal growth factor (mEGF) or insulin increased the labeling of phosphaditylinositol-3,4-bisphosphate (PI-3,4-P2) in MA-10 cells prelabeled with different radioactive precursors (Pignataro, O.P., and Ascoli, M. (1990) J. Biol. Chem. 265, 1718-1723 and Mol. Endocrinol. (1990) 4, 758-765). In order to further characterize this phenomenon we sought to determine if we could use anti-phosphotyrosine antibodies to immunoprecipitate a phosphatidylinositol (PI) kinase activity from MA-10 cells treated with mEGF or insulin. Our data indicate that this is indeed the case, and that the PI kinase precipitated is a PI-3' kinase. A second cell type, A431 cells, in which we were unable to detect an increase in PI-3,4-P2 labeling when stimulated with mEGF or insulin, was also studied. It was found that, as in MA-10 cells, A431 cells also contain an immunoprecipitable PI-3' kinase activity that is increased in response to mEGF or insulin.     

Effects of dextranation on the pharmacokinetics of short peptides. A PET study on mEGF

The effects of dextranation on the biodistribution of mouse epidermal growth factor (mEGF, 6 kDa) were assessed. By reductive amination, mEGF was coupled to 13 and 46 kDa dextran. The two dextranated conjugates and free mEGF were labeled with the positron-emitting nuclide (76)Br (T(1/2) = 16 h). After intravenous administration to Sprague Dawley rats, the radioactivity biodistribution was evaluated by positron emission tomography (PET) and by measurements of dissected tissues. The dextranation prolonged the retention time in blood, especially when the dextran chain was long. [(76)Br]mEGF-dextran conjugates were shown to have significantly, more than 5 times, lower kidney accumulation than the nonconjugated [(76)Br]mEGF. In conclusion, dextranation affects the biodistribution of mEGF in vivo giving a prolonged circulation time, a decreased uptake in kidney, and an increased spleen accumulation.     

Epidermal growth factor activates steroid biosynthesis in cultured Leydig tumor cells without affecting the levels of cAMP and potentiates the activation of steroid biosynthesis by choriogonadotropin and cAMP

The studies presented herein were designed to investigate the effects of mouse epidermal growth factor (mEGF) on steroid biosynthesis in a clonal strain of cultured murine Leydig tumor cells (designated MA-10). We show that in short-term incubations (up to 8 h), mEGF activates steroid biosynthesis without affecting cAMP levels. The maximal activation of steroid biosynthesis by mEGF (about 10-fold) is, however, much lower than the maximal activation detected with human choriogonadotropin (hCG) or cAMP analogues (about 1000-fold). We also show that mEGF has two (opposing) effects on the activation of steroidogenesis by hCG. Initially, it transiently attenuates the increase in intracellular cAMP and steroid biosynthesis provoked by submaximal concentrations of hCG. At later times, however, it potentiates the stimulatory effects of submaximal concentrations of hCG on steroid biosynthesis in a synergistic fashion. Last, we show that mEGF and submaximal concentrations of cAMP analogues also activate steroidogenesis in a synergistic fashion and that the degree of synergism attained with cAMP analogues plus mEGF is much higher than that attained with hCG plus mEGF. Taken together, our results show that mEGF (i) activates steroidogenesis without affecting cAMP levels and (ii) modulates the activation of steroidogenesis by the cAMP second messenger system.     

Conformational changes in rodent and human alpha-fetoprotein: influence of fatty acids

Binding, spectral and immunological studies were performed to demonstrate the conformational changes in rodent and human alpha-fetoprotein (AFP) induced by a free fatty acid environment. Scatchard analysis of estradiol (E2) binding to purified rat AFP indicated that unsaturated fatty acids changed the number of binding E2 sites and the apparent E2 equilibrium dissociation constant which varied non-linearly with docosahexaenoic acid concentration. UV spectral analysis of rodent and human AFPs showed that the absorbance minimum of AFP incubated with unsaturated fatty acid (L-AFP) was red-shifted, broadened and less pronounced than that of purified native AFP (N-AFP). Immunochemical studies with specific polyclonal antibodies to purified rodent and human AFPs (N-AFP antibodies) showed that these proteins lost immunoreactivity after incubation with unsaturated fatty acid. N-AFP antibodies recognized fewer epitopes on L-AFP than on N-AFP, whatever the species. Specific anti-rat L-AFP antibodies were used to demonstrate specific epitopes on rat L-AFP. Rat L-AFP antibodies did not recognize rat N-AFP. Saturated fatty acids were without effect on the binding, spectral and immunological properties of rodent and human AFPs. RIA or ELISA values for human AFP from fetal serum, hepatoma serum, and cord serum, were reduced 80, 50 and 5%, respectively, by unsaturated fatty acids. This decrease correlated with the relative percentage of polyunsaturated fatty acid in each biological fluid. Such results indicate that an unsaturated fatty acid environment induces conformational changes in AFP which may modulate the endocrine and immune functions of this protein.     

Stimulation of the hypothalamic-pituitary-adrenal axis by epidermal growth factor

The effects of mouse epidermal growth factor (mEGF) on the hypothalamic-pituitary-adrenocortical axis were studied in vivo in conscious male rats and in vitro with cultured anterior pituitary cells. Both intravenous (i.v.) and intracerebroventricular (i.c.v.) injections of mEGF (5-20 ng: 8.3-33.3 pmol) produced significant, dose-related increases in plasma ACTH and corticosterone concentrations. The potency of mEGF is 1/20-1/50 of that of rat corticotropin-releasing factor (rCRF), and pretreatment with 150 micrograms alpha-helical CRF (9-41) completely abolished the effects of the two peptides. mEGF in concentrations ranging from 10 pM to 10 nM did not significantly affect ACTH release from dispersed anterior pituitary cells. It also failed to alter ACTH secretion in response to rCRF. These results indicate that mEGF stimulates the pituitary-adrenocortical axis through a CRF-dependent mechanism.     

From rodent reagents to human therapeutics using antibody guided selection

Guided selection is a method of producing a human version of a rodent or any other non-human antibody. The process is a serial transition from rodent to human via rodent-human chimaerics, through to a panel of human antibodies with similar characteristics to those of the starting rodent antibody. The guided selection process can be undertaken using either phage display or ribosome display, and chimaeric antibodies can be made either in series or parallel, with or without the retention of the original rodent CDR3s. Guided selection has successfully been used for the generation of a number of human versions of rodent antibodies, including HUMIRA, an inhibitor of tumour necrosis factor-alpha which is approved for the treatment of moderate to severe rheumatoid arthritis in over 40 countries.     

Molecular dynamics simulations of epidermal growth factor and transforming growth factor-α structures in water

AMBER v. 4.1 force field in 1.5 ns NPT molecular dynamics simulations of murine epidermal growth factor (mEGF), human epidermal growth factor (hEGF), and human transforming growth factor-α (hTGF-α) structures with explicit TIP3P solvation were used to investigate differences in backbone stability, changes in secondary structure, interdomain flexibility, and weakly polar interactions. Backbone root mean square deviations of sections of each peptide show that the most stable regions in mEGF and hEGF are the A-, B-, and C-loops, whereas the most stable regions in hTGF-α are the A- and B-loops. The secondary structure in the B-loops of mEGF and hEGF differ significantly from the nuclear magnetic resonance (NMR) structures of mEGF and hEGF. The position and type of turns in the B-loop of mEGF and hEGF increase the interstrand distance of the antiparallel β-sheets thereby disrupting their structure. The interdomain flexibility of simulated hTGF-α structure is greater than in either mEGF or hEGF. The ϕ, ψ dihedrals of hTGF-α occupy two distinct populations of phase space corresponding to either a C or an α-helical conformation. This change in dihedral angle is stabilized by Phe¹⁵ with Arg⁴² and Phe¹⁷ with Arg⁴² N-π weakly polar interactions that are present only in hTGF-α but not in mEGF or hEGF. Proteins 33:396–407, 1998. © 1998 Wiley-Liss, Inc.     

Processing and transfer of epidermal growth factor in developing rat jejunum and ileum

Using everted sac technique we demonstrated the transfer of ¹²⁵I-mEGF across the jejunal and ileal walls of suckling, weanling and adult rats. The transfer by the suckling rat jejunum and ileum was significantly inhibited by the presence of dinitrophenol and sodium azide or by the replacement of sodium with potassium or choline. RP-HPLC analysis detected carboxy-terminal processing of ¹²⁵I-mEGF in suckling and adult rat jejunum and ileum. Suckling rat jejunum produced ¹²⁵I-des(53)mEGF and ¹²⁵I-des(49–53)mEGF, whereas ¹²⁵I-des(48–53)mEGF was detected in suckling rat ileum or adult rat jejunum and ileum. All three forms of ¹²⁵I-mEGF bound to anti-EGF antibody and EGF receptors. The receptor binding of ¹²⁵I-des(53)mEGF was higher than that of ¹²⁵I-mEGF, but those of ¹²⁵-des(49–53)mEGF and ¹²⁵I-des(48–53)mEGF were greatly diminished. Results indicate a carboxy-terminal processing of mouse EGF during uptake and transfer in the small intestine of developing and adult rats, and the resulting products showed altered receptor binding. An identical amino acid sequence of the C-terminal pentapeptide of EGF from mouse, human and possibly rat may suggest a biological significance of C-terminal processing of EGF in the small intestine.     

Effects of intradermally injected and topically applied mouse epidermal growth factor on wool growth, skin and wool follicles of merino sheep

Twice daily intradermal (ID) injections of mouse epidermal growth factor (mEGF) in sterile saline for 1-4 days into delineated areas of skin of Merino sheep produced dose-dependent changes in wool follicles and fibres, ranging from slight reduction in follicle bulb size and transient disturbance of cuticle formation on some fibres to the induction of catagen of follicles and shedding of fibres with distorted, tapered ends. Regeneration of follicles commenced by day 7. By contrast, ID injections of saline did not affect follicle activity. The epidermis became thicker and more parakeratotic after multiple injections of mEGF than after injection of saline, but was almost normal again by day 14. Persistent small increases in sebaceous gland size, additional to those induced by ID injections of saline, and delayed small increases in sweat gland size also occurred after multiple injections of mEGF. Daily topical applications of mEGF in 50% (v/v) aqueous propylene glycol 5 days each week for 4 weeks did not affect wool growth or the follicles and other skin components. The only effect observed, due to application of the aqueous propylene glycol, was an increase in the number of layers of cornified cells in the stratum corneum of the epidermis, with the cells arranged in clearly discernible stacks. The effects produced by ID injections of mEGF indicate that mEGF acts directly on the pilosebaceous and epidermal components of skin.     

Role of the 6-20 disulfide bridge in the structure and activity of epidermal growth factor

Two synthetic analogues of murine epidermal growth factor, [Abu6, 20] mEGF4-48 (where Abu denotes amino-butyric acid) and [G1, M3, K21, H40] mEGF1-48, have been investigated by NMR spectroscopy. [Abu6, 20] mEGF4-48 was designed to determine the contribution of the 6-20 disulfide bridge to the structure and function of mEGF. The overall structure of this analogue was similar to that of native mEGF, indicating that the loss of the 6-20 disulfide bridge did not affect the global fold of the molecule. Significant structural differences were observed near the N-terminus, however, with the direction of the polypeptide chain between residues four and nine being altered such that these residues were now located on the opposite face of the main beta-sheet from their position in native mEGF. Thermal denaturation experiments also showed that the structure of [Abu6, 20] mEGF4-48 was less stable than that of mEGF. Removal of this disulfide bridge resulted in a significant loss of both mitogenic activity in Balb/c 3T3 cells and receptor binding on A431 cells compared with native mEGF and mEGF4-48, implying that the structural changes in [Abu6, 20] mEGF4-48, although limited to the N-terminus, were sufficient to interfere with receptor binding. The loss of binding affinity probably arose mainly from steric interactions of the dislocated N-terminal region with part of the receptor binding surface of EGF. [G1, M3, K21, H40] mEGF1-48 was also synthesized in order to compare the synthetic polypeptide with the corresponding product of recombinant expression. Its mitogenic activity in Balb/c 3T3 cells was similar to that of native mEGF and analysis of its 1H chemical shifts suggested that its structure was also very similar to native.     

Kallikrein localization in rodent salivary glands and kidney with the immunoglobulin-enzyme bridge technique.

Kallikrein has been localized in rodent kidney and salivary glands by means of an immunoglobulin-enzyme bridge technique. In sections of kidney, anti-kallikrein antibodies bound to the apical region of certain distal tubule segments in the cortex, to reabsorption droplets of proximal convoluted tubules, and to certain duct segments in the papilla. In salivary glands of both male and female rats and mice, and apical rim of most striated duct cells of submandibular, parotid and sublingual glands and granular tubules of submandibular glands exhibited immunoreactivity. Granular intercalated duct cells in female submandibular glands also displayed immunostaining for kallikrein. Phenylephrine administration resulted in loss of immunoreactive granules from the granular convoluted tubule cells of male mouse submandibular gland. This response was paralleled by a biochemically demonstrable decrease in kallikrein-like tosylarginine methyl ester (TAME) esterase activity.     

Morphological changes in the skin and wool fibres of Merino sheep infused with mouse epidermal growth factor

Intravenous infusion of 4.5-4.7 mg of mouse epidermal growth factor (mEGF) into nine castrated male Merino sheep for 26 h resulted in complete casting of the fleeces 6-8 days later. The morphological changes which occurred in the skin were studied in skin samples taken before infusion and at intervals between 1 h and 42 days after the infusion had begun. Wool fibres from the shed fleeces were examined with the scanning electron microscope. Increased cell proliferation occurred in the epidermis and sebaceous glands, whereas the wool follicles regressed. Transient dermal haemorrhages occurred during the first 3 h of infusion. The fibre and inner root sheath in the keratogenous zone of 30-40% of the follicles were partially disrupted within the first 6 h of mEGF infusion; catagen began in all follicle bulbs within 24 h. Fibre and inner root sheath production, although markedly reduced, continued in about 60% of follicles which had partially regressed, but production ceased in the remainder in which tapered ends formed on the fibres prior to shedding. Follicles began to regenerate asynchronously 4-8 days after the beginning of infusion and completed their development during the next 3 weeks. The follicle regression and fleece casting induced by mEGF infusion, and subsequent follicle regeneration were completed more rapidly than observed previously with other depilatory agents, and, except for prolonged epidermal thickening, there was no lasting cutaneous abnormality.