Native Epidermal Growth Factor Research Articles

The Agonistic Activity of the Human Epidermal Growth Factor is Reduced by the D46G Substitution.

Resistance to anti-tumor agents targeting the epidermal growth factor receptor (EGFR) reduces treatment response and requires the development of novel EGFR antagonists. Mutant epidermal growth factor (EGF) forms with reduced agonistic activity could be promising agents in cancer treatment. EGF D46G affinity to EGFR domain III was assessed with affinity chromatography. EGF D46G acute toxicity in Af albino mice at 320 and 3200 μg/kg subcutaneous doses was evaluated. EGF D46G activity in human epidermoid carcinoma cells at 10 ng/mL concentration in serum-free medium and in subcutaneous Ehrlich ascites carcinoma mice model at 320 μg/kg dose was studied. The D46G substitution decreases the thermal stability of EGF complexes with EGFR domain III by decreasing the ability of the C-terminus to be released from the intermolecular β- sheet. However, with remaining binding sites for EGFR domain I, EGF D46G effectively competes with other EGF-like growth factors for binding to EGFR and does not demonstrate toxic effects in mice. EGF D46G inhibits the proliferation of human epidermoid carcinoma cells compared to native EGF. A single subcutaneous administration of EGF D46G along with Ehrlich carcinoma cells injection inhibits the proliferation of these cells and delays tumor formation for up to seven days. EGF D46G can be defined as a partial EGFR agonist as this mutant form demonstrates reduced agonistic activity compared to native EGF. The study emphasizes the role of the EGF C-terminus in establishing interactions with EGFR domain III, which are necessary for EGFR activation and subsequent proliferation of cells.

Equilibrium Between Dimeric and Monomeric Forms of Human Epidermal Growth Factor is Shifted Towards Dimers in a Solution

An interplay between monomeric and dimeric forms of human epidermal growth factor (EGF) affecting its interaction with EGF receptor (EGFR) is poorly understood. While EGF dimeric structure was resolved at pH 8.1, the possibility of EGF dimerization under physiological conditions is still unclear. This study aimed to describe the oligomeric state of EGF in a solution at physiological pH value. With centrifugal ultrafiltration followed by blue native gel electrophoresis, we showed that synthetic human EGF in a solution at a concentration of 0.1mg/ml exists mainly in the dimeric form at pH 7.4 and temperature of 37°C, although a small fraction of its monomers was also observed. Based on bioinformatics predictions, we introduced the D46G substitution to examine if EGF C-terminal part is directly involved in the intermolecular interface formation of the observed dimers. We found a reduced ability of the resulting EGF D46G dimers to dissociate at temperatures up to 50°C. The D46G substitution also increased the intermolecular antiparallel β-structure content within the EGF peptide in a solution according to the CD spectra analysis that was confirmed by HATR-FTIR results. Additionally, the energy transfer between Tyr and Trp residues was detected by fluorescence spectroscopy for the EGF D46G mutant, but not for the native EGF. This allowed us to suggest the elongation and rearrangement of the intermolecular β-structure that leads to the observed stabilization of EGF D46G dimers. The results imply EGF dimerization under physiological pH value and temperature and the involvement of EGF C-terminal part in this process.

Virtual screening of Osimertinib and Dacomitinib Analogues with potential activity on EGFR (T790M and l858R Mutations) for non-small cell lung cancer treatment


 
 
 Introduction: Tyrosine kinase inhibitors (TKIs) drugs act on epidermal growth factor (EGFR) receptors to treat Non-small cell lung cancer (NSCLC). However, mutations on EGFR receptors T790M and L858R allow just a global response rate (GRR) of 80% with Osimertinib, while Erlotinib and Gefitinib only 10%.
 Objective: To identify promising molecules analogues to tyrosine kinase inhibitor (TKIs) drugs with the potential capacity to bind the native and mutated EGFR receptor (T790M and L858R) to avoid mutational resistance in NSCLC.
 Methods: Virtual screening by molecular docking between analogues of Osimertinib (DB09330) and Dacomitinib (DB11963) drugs retrieved of DRUGBANK database and receptors of native EGFR and mutated on L585R and T790M obtained of Protein Data Bank was performed, using PyRx software. Finally, toxicological prediction was made using GUSAR.
 Results: Analogues studied, DB03878, DB04739, DB07280 and DB06876 achieved significant affinity (-9,1 y -8,3 Kcal/mol) on mutated T790M EGFR compared with osimertinib (-7,6 Kcal/mol). Similarly, DB08091, DB08730, DB07220 and DB06920 achieved significant affinity (between 9.4 and -8.9 Kcal/mol) on L858R EGFR mutated compared with dacomitinib (-7.0 Kcal/mol). Overall, there were predominance of Van der Waals forces and links π-alkyl. Also, two analogues were safe with category IV according to predictions, DB08730 and DB03878.
 Conclusions: Eight TKI analogues showed superior binding energy over EGFR compared to reference drugs. According to toxicological predictions only 2 analogues were selected as promising TKI-type safe candidates for the treatment of resistant NSCLC.
 
 

Hyaluronate-Epidermal Growth Factor Conjugate for Skin Wound Healing and Regeneration.

Epidermal growth factor (EGF) has been recognized as an excellent wound healing agent due to its therapeutic function stimulating skin cell growth, proliferation and differentiation. However, the transdermal delivery of EGF poses a significant challenge due to its short half-life and lack of efficient formulation. Here, to improve the transdermal delivery efficiency, EGF was conjugated to hyaluronate (HA), which was formulated into a patch-type film for skin wound healing. HA-EGF conjugate was synthesized by coupling reaction between aldehyde-modified HA and N-terminal amine group of EGF to minimize the loss of biological activities. The HA-EGF conjugates exhibited similar biological activities with native EGF as confirmed by ELISA and proliferation tests using murine and human fibroblasts. For the efficient topical delivery, HA-EGF conjugates were incorporated into a matrix film of high molecular weight HA. Two-photon microscopy clearly visualized more efficient transdermal delivery of HA-EGF conjugates to both normal skin and peripheral tissues around the wound area rather than that of EGF. Optical imaging and ELISA after in vivo transdermal delivery showed that the conjugation of EGF to HA retarded its degradation and extended its residence time in the wound area. Furthermore, in vivo transdermal delivery of HA-EGF conjugate in the patch-type HA film resulted in significantly improved regeneration of skin tissues even into hypodermis.

Collagen-Binding Human Epidermal Growth Factor Promotes Cellularization of Collagen Scaffolds

Previous studies have demonstrated that epidermal growth factor (EGF) can promote tissue regeneration effectively. The aim of the present study was to combine a collagen-binding domain (CBD) with native EGF (NAT-EGF) to form a fusion protein, aiming at that it could bind to collagen scaffolds specifically. This approach could restrict the diffusion of EGF despite of extracellular fluids and limit the release of EGF to surrounding tissues. Thus, the retained CBD-EGF would render a continual function during the tissue regeneration. In our study, in vitro bioactivity assay showed that there was no significant difference between CBD-EGF and NAT-EGF to stimulate human fibroblasts to proliferate. In the collagen-binding assay, CBD-EGF exhibited a specific binding ability to collagen compared to NAT-EGF. Finally, in vivo experiment demonstrated that CBD-EGF-loaded collagen membranes uniformly possess a high capability to promote cellularization of scaffolds as compared with that of NAT-EGF. All results aforementioned suggested that collagen loaded with CBD-EGF is a functional biomaterial with potential clinical applications.

Cell-Targeted Self-Assembled DNA Nanostructures

We present two strategies for attaching self-assembled DNA arrays to the surfaces of cells. Our first approach uses biotin-streptavidin interactions to bind DNA architectures to biotinylated cells. The second approach takes advantage of specific antibody-cell surface interactions, conjugated arrays and the subsequent binding to native epidermal growth factor receptors expressed on cancer cells. DNA array-cell surface interactions were visualized by fluorescence, confocal microscopy, and scanning electron microscopy. This novel application of DNA nanoarrays provides strategies to specifically label cell surfaces with micrometer-sized patches, bind cells onto a designed functionalized DNA scaffold, engineer cell/cell networks into microtissues, and deliver materials to cell surfaces.

A chimeric epidermal growth factor with fibrin affinity promotes repair of injured keratinocyte sheets

The aim of the present study is to create a novel chimeric protein of epidermal growth factor (EGF) with fibrin affinity and demonstrate its potential for repairing injured tissues by immobilization to fibrin. The chimeric protein (FBD–EGF) was produced by the fusion of the fibronectin fibrin-binding domain (FBD) to EGF. It showed dose-dependent binding to fibrin and its binding was stable for at least 7 days, while native EGF showed little affinity. FBD–EGF promoted the growth of fibroblasts and keratinocytes in the fibrin-bound state as well as in the soluble state. Its activity was further studied in a keratinocyte culture system in which fibrin was exposed upon injury of cell sheets. Fibrin-bound FBD–EGF promoted growth of the sheets over the injured area at a significantly faster rate (approximately eightfold) than native EGF ( p < 0.01). Wounds 2 mm wide were closed in 7–9 days. This repair process was inhibited by anti-EGF. Keratinocytes proliferated more extensively in the leading edges of sheets contacting fibrin with FBD–EGF, approximately 1.7-fold more than in the adjacent regions. These results imply that the stable binding of chimeric EGF to fibrin is effective for the repair of injured keratinocyte sheets, suggesting a potential use in tissue engineering.

The presence of carboxypeptidase-M in tumour cells signifies epidermal growth factor receptor expression in lung adenocarcinomas

Carboxypeptidase-M (CPM) is a membrane-bound peptidase that metabolizes peptides, and is present in pneumocytes. CPM hydrolyses the C-terminal arginine of epidermal growth factor (EGF) resulting in des-Arg53-EGF which binds to the EGF receptor (EGFR) with an equal or greater affinity than native EGF. Therefore, this study focused on the possible presence of CPM in human lung adenocarcinomas (ADC) and evaluated the relationship between CPM and EGFR by assessing the impact of expressions on patient clinical outcome. This is a retrospective study of 110 patients who underwent resection of the primary tumour (92) or metastatic tissues (18) for treatment or diagnosis. Immunohistochemistry (IHC) for CPM and EGFR was made in serial sections using standard methods. This study demonstrates for the first time that 23.6% of ADCs express carboxypeptidase-M (26/110), mainly in membrane-bound forms. The amounts and the extent of CPM within tumours vary from low levels to obviously overexpressed forms. The immunohistochemical positivity (+) for CPM in ADCs negatively correlated with disease survival. In addition, 80% of CPM+ adenocarcinomas (21/26) showed a coexpression with EGFR suggesting a high prevalence for coexistence. The follow up data indicated a significantly shorter 5-year survival time for patients with CPM+-EGFR+ (double-positive) tumours compared to those harbouring neoplasias negative for both proteins (9.5 vs. 60.4% survivals, P < 0.001). The fact that CPM+ ADCs often co-express with EGFR suggests a functional-regulatory link between these proteins which might have therapeutical consequences. The present novel data could lead to improved IHC tests in lung adenocarcinomas for EGFR expression.

Rational design of an EGF-IL18 fusion protein: Implication for developing tumor therapeutics

Interleukin-18 (IL-18) is a proinflammatory cytokine. This protein has a role in regulating immune responses and exhibits significant anti-tumor activities. Epidermal growth factor (EGF) is an important growth factor that plays a central role in the regulation of cell cycle and differentiation. It was proposed that a targeted delivery of IL-18 by generation of IL-18-EGF fusion protein might decrease adverse effects and result in enhancing cytotoxic and antitumor activities. In the present study, a fusion protein, consisting of EGFR binding domain fused to human IL-18 mature peptide via a linker peptide of (Gly 4Ser) 3, was constructed and expressed in the insect cell line Sf9 using Bac-to-Bac baculovirus expression system. We showed that the purified recombinant fusion protein induced similar levels of IFN-γ to that of native IL-18 protein in human PBMC in the presence of ConA. Furthermore, EGF receptor competitive test in human epithelial cancer A431 cell line showed that EGF-IL18 fusion protein can specifically bind with EGFR by competing with native EGF protein. These suggest that this rationally designed protein can be further developed as novel tumor therapeutics.

Structural Analysis of an Epidermal Growth Factor/Transforming Growth Factor-α Chimera with Unique ErbB Binding Specificity

Various chimeras of the ErbB1-specific ligands epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha) display an enlarged repertoire as activators of ErbB2.ErbB3 heterodimers. Mutational analysis indicated that particularly residues in the N terminus and B-loop region of these ligands are involved in the broadened receptor specificity. In order to understand the receptor specificity of T1E, a chimeric ligand constructed by the introduction of the linear N-terminal region of TGFalpha into EGF, we determined in this study the solution structure and dynamics of T1E by multidimensional NMR analysis. Subsequently, we studied the structural characteristics of T1E binding to both ErbB1 and ErbB3 by superposition modeling of its structure on the known crystal structures of ErbB3 and liganded ErbB1 complexes. The results show that the overall structure of T1E in solution is very similar to that of native EGF and TGFalpha but that its N terminus shows an extended structure that is appropriately positioned to form a triple beta-sheet with the large antiparallel beta-sheet in the B-loop region. This conformational effect of the N terminus together with the large overall flexibility of T1E, as determined by 15N NMR relaxation analysis, may be a facilitative property for its broad receptor specificity. The structural superposition models indicate that hydrophobic and electrostatic interactions of the N terminus and B-loop of T1E are particularly important for its binding to ErbB3.

N-terminal site-specific mono-PEGylation of epidermal growth factor.

N-terminal site-specific mono-PEGylation of recombinant human epidermal growth factor (EGF) was accomplished using polyethyleneglycol (PEG) derivatives (Mw = 2000 and 5000) through a reactive terminal aldehyde group. The site-specific PEG conjugation was conducted ata slightly acidic pH condition (pH 5.5). The mono-PEGylation was targeted to an alpha-amine group at the N-terminal end of EGF to minimize reduction of biologic activity. Tryptic digestion mapping and MALDI-TOF MS techniques were applied to show the occurrence of mono-PEGylation at the N-terminus of EGF. The site-specific mono-PEGylated EGF, when compared with native EGF, fully retained its in vitro biologic activities such a cell proliferation and intracellular signal transduction. This revealed that although a synthetic polymer of a PEG was covalently conjugated to EGF, the internalized complex of PEGylated EGF-receptor within cells did not hamper the intracellular signal transduction events. The PEGylated EGF also exhibited a prolonged circulation in blood stream in vivo and markedly enhanced physical stability whe incubated with tissue homogenate. N-terminally mono-PEGylated EGF shows increased physical stability while retaining its biologic activity.

The disulfide structure of denatured epidermal growth factor: preparation of scrambled disulfide isomers.

The conformational stability of human epidermal growth factor (EGF) and the structure of denatured EGF were investigated using the technique of disulfide scrambling. Under denaturing conditions and in the presence of a thiol catalyst, the native EGF denatures by shuffling its three native disulfide bonds and converts to a mixture of scrambled isomers. Analysis by HPLC reveals that the denatured EGF is composed of about 10 fractions of scrambled isomers. The heterogeneity varies under different denaturing conditions, with the heat-denatured samples exhibiting the highest degree of heterogeneity. The disulfide structures of eight major scrambled isomers of EGF were determined. The most predominant isomer adopts the bead-form structure with disulfide bonds bridged by three pairs of neighboring cysteines: Cys6-Cys14, Cys20-Cys31, and Cys33-Cys42. The denaturation curve of EGF is determined by the relative yield of the scrambled and native species of EGF. EGF is a highly stable molecule and can be effectively denatured only by guanidine chloride at a concentration of greater than 4-5 M. At 8 M urea, less than 16% of the native EGF was denatured. The unusual conformational stability of EGF was compared with that of eight different disulfide proteins that were similarly characterized by the method of disulfide scrambling.

Europium-Labeled Epidermal Growth Factor and Neurotensin: Novel Probes for Receptor-Binding Studies

We investigated the possibility of labeling two biologically active peptides, epidermal growth factor (EGF) and neurotensin (NT), with europium (Eu)-diethylenetriaminepentaacetic acid. More specifically, we tested them as probes in studying receptor binding using time-resolved fluorescence of Eu3+. The relatively simple synthesis yields ligands with acceptable binding characteristics similar to isotopically labeled derivatives. The binding affinity (Kd) of labeled Eu-EGF to human A431 epidermal carcinoid cells was 3.6 ± 1.2 nM, similar to the reported Kd values of EGF, whereas the Kd of Eu-NT to human HT29 colon cancer cells (7.4 ± 0.5 nM) or to Chinese hamster ovary (CHO) cells transfected with the high-affinity NT receptor (CHO-NT1) were about 10-fold higher than the Kd values of NT. The bioactivity of the Eu-labeled EGF as determined by stimulation of cultured murine D1 hematopoietic cell proliferation was nearly the same as that obtained with native EGF. The maximal stimulation of Ca2+ influx with NT and Eu-NT in CHO-NT1 cells was similar, but the respective K0.5 values were 20 pM and 1 nM, corresponding to differences in the binding affinities previously described. The results of these studies indicate that Eu labeling of peptide hormones and growth factor molecules ranging from 103 to 105 Da can be conveniently accomplished. Importantly, the Eu-labeled products are stable for approximately 2 years and are completely safe for laboratory use compared to the biohazardous radioligands. Thus, Eu-labeled peptides present an attractive alternative for commonly used radiolabeled ligands in biological studies in general and in receptor assays in particular.

Rapid Microscale Enzymic Semisynthesis of Epidermal Growth Factor (EGF) Analogues

Epidermal Growth Factor (EGF) is a small growth factor containing 53 amino acid residues capable of stimulating the proliferation of both mesenchymal and epithelial cells. Comparison of the amino acid sequences of EGF from several species, and related proteins that can bind to the EGF receptor (e.g. TGFf, VGF, heparin-binding EGF, and betacellulin), suggests that Leu47, which is highly conserved, is important for biological function. Additionally, we have shown previously, using a combination of trypsin and carboxypeptidase Y digestion of native murine EGF, that removal of Leu47 results in more than 100-fold decrease in both receptor binding and mitogenic activity. We now describe a micromethod for the rapid generation of C-terminally modified EGFs to investigate further the role of C-terminal residues in determining functional activity. These analogues have been generated by digesting native murine EGF with trypsin, purifying the biologically inactive, but structurally intact, EGF1-45 core by micropreparative RP-HPLC, and then reversing the action of trypsin to couple synthetic peptides (e.g. DL, DI, DF, EL, DLLW) onto the C-terminus of the EGF1-45 core. This enzymic semisynthesis method allows multiple derivatives to be generated rapidly from microgram quantities of EGF1-45 in sufficient quantities for sensitive biological and physicochemical analysis. We have validated the method by regenerating EGF1-47 from EGF1-45 with equivalent mitogenic and receptor binding activity to EGF1-47 generated from wild type EGF by digestion with trypsin and carboxypeptidase Y. We have also investigated the effect of substituting alternative normal or non-physiological amino acids (e.g. allo-Ile) for Asp46, Leu47 or Arg48. Even small changes in these C-terminal residues reduce the mitogenic potency of the analogue.

Epidermal growth factor receptor glycosylation is required for ganglioside GM3 binding and GM3-mediated suppression [correction of suppresion] of activation.

Gangliosides are able to bind to the epidermal growth factor receptor and inhibit its activation, but the mechanism of this inhibition is unknown. To address the role of receptor carbohydrates in facilitating interaction with gangliosides, we examined the ability of GM3 to bind the deglycosylated receptor and inhibit its autophosphorylation. Flow cytometry studies demonstrated that deglycosylation of the receptor did not affect its ability to be transported to the cell membrane. In contrast with the native (fully glycosylated) receptor, GM3 did not coimmunoprecipitate with the deglycosylated receptor. Using a novel colorimetric bead binding assay, GM3 was shown to bind well to the immunoprecipitated native receptor but not at all to the deglycosylated receptor. Finally, the addition of GM3 to cells with deglycosylated epidermal growth factor receptors did not result in significant further inhibition of autophosphorylation of the receptor, despite a 10-fold decrease in phosphorylation of the native epidermal growth factor receptor by 200 microM GM3. These studies suggest that ganglioside affects epidermal growth factor receptor activity through a direct interaction that requires receptor glycosylation, and contribute to our understanding of the role of gangliosides in cell membrane function.

Design, expression, and renaturation of a lesion-targeted recombinant epidermal growth factor-von Willebrand factor fusion protein: efficacy in an animal model of experimental colitis.

In the present study, the mature epidermal growth factor (EGF) protein was engineered to incorporate a high affinity collagen-binding domain (CBD) derived from co-agulation von Willebrand factor, to specifically target EGF to colonic lesions. The fusion protein was expressed in an E. coli bacterial expression system, purified by metal chelate chromatography, and renatured by oxidative refolding into a soluble biologically active growth factor. The EGF-CBD fusion protein bound tightly to collagen matrices under conditions in which native non-targeted EGF was washed away. In biologic assays, the EGF-CBD fusion protein stimulated NIH3T3 cell proliferation with near wild-type biological activity. In vivo binding studies showed that the collagen-targeted EGF, but not the non-targeted EGF, accumulated at areas of exposed collagen on the luminal surface of the inflamed colon. Finally, a single colonic instillation of the collagen-targeted EGF-induced a more rapid regeneration of intestinal crypts 24 h after treatment (no. of crypts = 89.2+/-8.1) compared to the non-targeted EGF (no. of crypts = 52.2+/-29.8; p=0.027), and the PBS control (no. of crypts = 24. 0+/-22.9; p=0.001). Taken together, these findings indicate that intracolonic delivery of collagen-targeted EGF represents a potentially effective therapeutic strategy for acute or chronic inflammatory bowel disease.

Regulation of EGF signaling by cell polarity in MDCK kidney epithelial cells.

Although the presence of a dominant basolateral sorting signal ensures that the majority of newly synthesized epidermal growth factor (EGF) receptors are delivered directly to the basolateral surface in polarized epithelial cells, a fraction of the receptors are also delivered to the apical surface. Similar to most basolateral membrane proteins, the EGF receptor has an additional signal(s) that selectively targets molecules lacking a dominant basolateral signal to the apical surface. Although the physiological relevance of signal hierarchy is not known, alternative targeting may occur in different epithelial cell types or during development. The goal of this study, therefore, was to determine the effect of membrane domain location on EGF receptor function, focusing on EGF-induced MAP kinase signaling and DNA synthesis. Whereas ligand responsiveness was restricted to the basolateral domain in Madin-Darby canine kidney (MDCK) cells expressing a normal complement of receptors, apical ligand was effective if apical receptor density was increased by overexpression of an exogenous wild-type human gene. Unexpectedly, cells expressing apically localized, cytoplasmically truncated receptors, which behave as dominant negative mutations in other cell types, were also responsive to apical EGF. The cytoplasmically truncated molecules appear to have at least two effects: first, to increase the local concentration of ligand at the apical cell surface; and second, to facilitate activation of the relatively few native EGF receptors normally located at the apical surface. These results indicate that cell context is a critical determinant of receptor mutant protein phenotype.

Stability and distribution of orally administered epidermal growth factor in neonatal pigs

Stability and distribution of orally administered epidermal growth factor (EGF) were examined in newborn and 5-day-old pigs. Forty-five minutes after oral administration of iodine-125 labeled EGF, 60 and 50% of the radioactivity administered were recovered from the internal organs in newborn and 5-day-old pigs, respectively. In both age groups, over 95% of the recovered radioactivity was found in the gastrointestinal tract, of which 78–86% was found in the luminal contents with the remaining found in the gastrointestinal wall. Within the gastrointestinal tract, 65–71% of radioactivity was found in the stomach, 27–30% in the proximal and mid small intestine and 3–4% was found in the distal part of the small intestine. There were no significant differences in the overall distribution of orally administered radioactivity between two age groups. Based on liquid chromatography and trichloroacetic acid precipitation, a substantial amount of EGF recovered from the luminal contents (63–86%) and the gastrointestinal wall (42–81%) remained “intact”. The receptor binding ability of the EGF recovered from the gastric contents was 96–102% comparable to the native EGF tracer. The receptor binding ability remained high (40–58%) in the proximal small intestinal lumen and it decreased to 15% in the distal small intestinal lumen in newborn pigs. In 5-day-old pigs, EGF recovered from the small intestinal contents had 5 to 24% receptor binding ability when compared with native EGF tracer. The receptor binding ability of the EGF recovered from all other organs was below 5% with an exception of the gastric wall, from which recovered EGF retained 9 to 26% receptor binding ability. These results indicate that most of orally ingested EGF remained in the gastrointestinal tract in neonatal pigs 45 min after oral ingestion, and significant amount of the ingested EGF remained biologically active. It suggests that milkborne EGF can survive in the gastrointestinal tract and may play a role in regulating gut development in neonatal animals.

A Gly → Ser Change Causes Defective Folding in Vitro of Calcium-binding Epidermal Growth Factor-like Domains from Factor IX and Fibrillin-1

The calcium-binding epidermal growth factor-like (cbEGF) domain is a common motif found in extracellular proteins. A mutation that changes a highly conserved Gly residue to Ser in this domain has been identified both in the factor IX (FIX) and fibrillin-1 genes, where it is associated with relatively mild variants of hemophilia B and Marfan syndrome, respectively. We have investigated the structural consequences in vitro of this amino acid change when introduced into single cbEGF domains from human FIX (G60S) and human fibrillin-1 (G1127S), and a covalently linked pair of cbEGF domains from fibrillin-1. High pressure liquid chromatography analysis, mass spectrometry, and 1H NMR analysis demonstrate that wild-type cbEGF domains purified in the reduced form and refolded in vitro adopt the native fold. In contrast, the Gly --> Ser change causes defective folding of FIX and fibrillin-1 cbEGF domains. However, in the case of the factor IX mutant domain, a Ca2+-dependent change in conformation, identified by NMR in a proportion of the refolded material, suggests that some material refolds to a native-like structure. This is consistent with enzyme-linked immunosorbent assay analysis of FIX G60S from a hemophilia B patient Oxford d2, which demonstrates that the mutant protein is partially recognized by a monoclonal antibody specific for this region of FIX. NMR analysis of a covalently linked pair of fibrillin cbEGF domains demonstrates that the C-terminal domain adopts the native epidermal growth factor fold, despite the fact that the adjacent mutant domain is misfolded. The implications of these results for disease pathogenesis are discussed.

Epidermal growth factor regulates adenylate cyclase activity via Gs and Gi1-2 proteins in pancreatic acinar membranes

In the present study, Western-blot and radioreceptor analyses have revealed the presence of the epidermal growth factor (EGF) receptor in pancreatic acinar membranes. Isolated pancreatic acinar membranes, which allow access of functional antibodies to individual components of the signal transduction cascade, were used to examine EGF-induced regulation of adenylate cyclase activity. Forskolin, vasoactive intestinal peptide (VIP) and to a smaller extent EGF increased cAMP production in pancreatic acinar membranes. Preincubation of the membranes with anti-GS alpha antibody abolished EGF- and VIP-induced cAMP production, but had no effect on forskolin-induced cAMP accumulation. In the presence of either VIP or forskolin, EGF inhibited the VIP- and forskolin-induced cAMP production with an IC50 of 5 nM. Anti-G alpha i1-2 protein antibody, but not anti-G alpha i3 antibody, increased basal cAMP production, indicating that Gi proteins exert an inhibitory influence on basal adenylate cyclase activity. Anti-G alpha i1-2 antibody, but not anti-G alpha i3 antibody, abolished the inhibitory effect of EGF on the forskolin- and VIP-induced cAMP accumulation. A peptide corresponding to the juxtamembrane region in the cytosolic domain of the rat EGF receptor increased cAMP production in pancreatic acinar membranes in an anti-G alpha s antibody-sensitive fashion, whereas the EGF receptor peptide did not mimic the inhibitory effect of the native EGF receptor. The tyrosine kinase inhibitors genistein and pp60v-src (137-157) inhibited both the stimulatory and the inhibitory effects of EGF on cAMP production. Thus the data of the present study show that EGF regulates adenylate cyclase via activation of Gs and Gi proteins by a tyrosine phosphorylation-dependent mechanism in pancreatic acinar membranes. This leads to stimulation of basal and inhibition of forskolin- and VIP-induced adenylate cyclase activity respectively.