Epidermal Growth Factor Precursor Research Articles

Calcium induces a conformational change in the ligand binding domain of the low density lipoprotein receptor

We previously have shown that LDL-R354, a truncated low density lipoprotein (LDL) receptor, is a calcium binding protein. LDL-R354 is composed of the ligand binding domain and repeat A of the EGF precursor homology domain of the full-length human LDL receptor. We also found that Ca2+ was required for the interaction between LDL-R354 and its ligand, LDL (Dirlam et al. 1996. Protein Expr. Purif.8: 489–500). In the current study, calcium-induced changes in the structure and function of LDL-R354 were examined. When calcium bound to LDL-R354, its apparent size increased, as determined by native and SDS-gel electrophoresis. The calcium-saturated form of LDL-R354 was more resistant to trypsin proteolysis than the calcium-depleted form. In the presence of calcium, the disulfide bonds in the truncated receptor were stabilized, rendering them more resistant to reduction by dithiothreitol. Calcium binding affinities were measured by monitoring increased tryptophan fluorescence intensities. LDL-R354 bound Ca2+ with high affinity (EC50 = 60 nm at pH 7.4) and specificity, as 400 μm Mg2+ did not compete for calcium binding. The affinity of LDL-R354 for calcium decreased when the pH was lowered. These results suggest that calcium induces a conformational change in the ligand binding domain of the LDL receptor and that a receptor conformer capable of binding ligand should be stabilized at physiological extracellular Ca2+ concentration and pH. Drops in pH may regulate LDL receptor function by altering the amount of calcium bound to the receptor.—Dirlam-Schatz, K. A., and A. D. Attie. Calcium induces a confirmation change in the ligand binding domain of the low density lipoprotein receptor.

Pig epidermal growth factor precursor contains segments that are highly conserved among species

The 53-aa polypeptide epidermal growth factor (EGF) is synthesized as a 1200-aa precursor. The non-EGF part of the precursor is very long compared with EGF, and can therefore be expected to have a biological role of its own. We have sequenced cDNA of the pig EGF precursor and compared a 668-aa segment with that of the human, the rat and the mouse EGF precursors, in order to identify highly conserved domains. The examined part of the precursor contains EGF itself and six so-called EGF-like modules. The overall amino acid identity among the four species is 64%. However, the amino acid identity differed from around 30% in some segments to around 70% in others. The highest amino acid identity, 71%, was observed for a 345-aa segment that contains three EGF-like modules and which is homologous to a part of the low-density lipoprotein receptor (LDL receptor). The amino acid identities are 64% for EGF itself, and 50-67% for the remaining three EGF-like modules. The segment of the LDL receptor that is homologous to a part of the EGF precursor is important for the function of the LDL receptor, and EGF-like modules seem to be involved in protein-protein interactions in a number of proteins. In conclusion, some segments of the EGF precursor are remarkably well conserved among species and it is tempting to speculate that they have a biological function.

The rat exorbital lacrimal gland as a site of synthesis of EGF-like growth factors.

The lacrimal gland produces the complex aqueous portion of tears that contains many components including electrolytes and proteins.1,2 The pH, electrolyte concentration, and protein composition of lacrimal fluids are crucial to maintain the health of the ocular surface. The proteins that lacrimal glands synthesize and secrete are very specific, but only a few of them have been characterized. These proteins are thought to be involved in the bacteriostatic action of tears. It has been shown that human3,4 and mouse5 tears contain epidermal growth factor (EGF) or an EGF-like immunoreactivity. The EGF precursor mRNA has been detected by Northern blot in moûse6 and by RT-PCR in human7 lacrimal glands. Moreover, EGF-like immunoreactivities have been detected in mouse,6 rabbit,8 and rat9,10 lacrimal tissues, but the immunoreactive proteins have not been further characterized. The EGF receptor mRNA has also been detected, by RT-PCR, in the human lacrimal gland,11 and by RT-PCR and Northern blot in the rat exorbital lacrimal gland.12 The EGF receptor protein has been identified and shown to be activated by EGF in the rat tissue.12

Novel stop mutation causing familial hypercholesterolemia in a Costa Rican family

Combined heteroduplex single-strand conformation polymorphism (HEX-SSCP) analysis of the promoter and coding region of the low density lipoprotein receptor (LDLR) gene revealed a novel C to T mutation at nucleotide position 2056 in a Costa Rican patient with heterozygous familial hypercholesterolemia (FH). This nonsense mutation, Q665X, results in a termination codon in the epidermal growth factor (EGF) precursor homology domain of the mature LDLR.

Apical enrichment of human EGF precursor in Madin-Darby canine kidney cells involves preferential basolateral ectodomain cleavage sensitive to a metalloprotease inhibitor.

EGF precursor (proEGF) is a member of the family of membrane-anchored EGF-like growth factors that bind with high affinity to the epidermal growth factor receptor (EGFR). In contrast to human transforming growth factor-alpha precursor (proTGFalpha), which is sorted basolaterally in Madin-Darby canine kidney (MDCK) cells (Dempsey, P., and R. Coffey, 1994. J. Biol. Chem. 269:16878-16889), we now demonstrate that human proEGF overexpressed in MDCK cells is found predominantly at the apical membrane domain under steady-state conditions. Nascent proEGF (185 kD) is not sorted but is delivered equally to the apical and basolateral membranes, where it is proteolytically cleaved within its ectodomain to release a soluble 170-kD EGF form into the medium. Unlike the fate of TGFalpha in MDCK cells, the soluble 170-kD EGF species accumulates in the medium, does not interact with the EGFR, and is not processed to the mature 6-kD peptide. We show that the rate of ectodomain cleavage of 185-kD proEGF is fourfold greater at the basolateral surface than at the apical surface and is sensitive to a metalloprotease inhibitor, batimastat. Batimastat dramatically inhibited the release of soluble 170-kD EGF into the apical and basal medium by 7 and 60%, respectively, and caused a concordant increase in the expression of 185-kD proEGF at the apical and basolateral cell surfaces of 150 and 280%, respectively. We propose that preferential ectodomain cleavage at the basolateral surface contributes to apical domain localization of 185-kD proEGF in MDCK cells, and this provides a novel mechanism to achieve a polarized distribution of cell surface membrane proteins under steady-state conditions. In addition, differences in disposition of EGF and TGFalpha in polarized epithelial cells offer a new conceptual framework to consider the actions of these polypeptide growth factors.

Leucine zipper, nuclear targeting and P130 interaction motifs in the epidermal growth factor precursor: The nucleocrine connection further unveiled

Leucine zipper, nuclear targeting and P130 interaction motifs in the epidermal growth factor precursor: The nucleocrine connection further unveiled

Renal epidermal growth factor precursor: proteolytic processing in an in vitro cell-free system

The enzymatic processing of the membrane-bound renal epidermal growth factor precursor (proEGF) could be an important step in the control of nephrogenic repair consecutive to kidney insult. The enzyme machinery responsible for that processing was examined in a cell-free system consisting of renal membranes isolated from kidney homogenates by differential centrifugation, and incubated in vitro. After a 24-h incubation at 37°C, 6–14% of membrane-bound proEGF was processed and soluble products with EGF immunoreactivity were released. As revealed by HPLC and Western blotting analysis, the products of proEGF proteolysis consisted of 6 kDa EGF (the molecular weight of mature EGF) and two polypeptides with molecular weights around 45 kDa. Interestingly the 45 kDa EGF forms, like the 6 kDa EGF, exhibited mitogenic activity toward growth-arrested NRK-52E renal cell line. The kinetic study of proEGF degradation gave data consistent with the 45 kDa product(s) being processing intermediate(s) between proEGF and 6 kDa EGF. The enzymatic activity responsible for proEGF nicking was inhibited by divalent heavy metal ions (Cu 2+ or Zn 2+) and several protease inhibitors (aprotinin, PMSF, leupeptin, soybean trypsin inhibitor), suggesting that proEGF is processed by kallikrein-like serine proteases present in the membrane preparations. Along with previous studies, the current observations suggest that renal kallikreins might play a role in renal tubular regeneration by promoting the release of soluble EGF in renal tissue.

Elements of Neural Adhesion Molecules and a Yeast Vacuolar Protein Sorting Receptor Are Present in a Novel Mammalian Low Density Lipoprotein Receptor Family Member

Normal cell development depends to a large part on multifunctional proteins that have evolved by recombination of proven modular elements. We now have discovered and characterized in rabbit such a multi-domain protein, and classify it as novel member of the low density lipoprotein (LDL) receptor gene family. The extracellular portion of the approximately 250-kDa membrane protein, termed LR11, contains a cluster of 11 LDL receptor ligand binding repeats, a group of 5 LDL receptor "YWTD" repeats, a large hexarepeat domain of structural elements found in neural cell adhesion molecules, and a domain with similarity to a yeast receptor for vacuolar protein sorting, VPS10. The cytoplasmic domain exhibits features typical of endocytosis-competent coated-pit receptors. The mosaic, and presumably multifunctional, receptor is expressed abundantly in brain, in particular the hippocampus, dentate gyrus, and cerebral cortex, and is present at significant levels in liver, adrenal glands, and testis. Western blotting of tissues and ligand blotting of LR11-transfected cells demonstrated that the novel protein binds apolipoprotein E-containing lipoproteins. In contrast to the LDL receptor, hepatic expression of LR11 is unaffected by hyperlipidemia. The identification of this highly conserved and superbly complex protein offers the opportunity to gain new insights into the emergence of multifunctional mosaic proteins akin to the ever expanding LDL receptor gene family.

Molecular genetics of familial hypercholesterolemia in Israel.

Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by a multitude of low density lipoprotein receptor (LDL-R) mutations. The purpose of the current investigation was to define the spectrum of mutations causing FH in Israel and determine their relative distribution among diverse origin groups. A total of 193 FH families were recruited in Israel, 54 of them through the MED PED (Make Early Diagnosis Prevent Early Death) FH program. Molecular analysis of the LDL-R using single-strand conformation polymorphism (SSCP) or denaturing gradient gel electrophoresis (DGGE) or both has been completed in 95 index cases. This analysis resulted in the identification of 15 LDL receptor mutations, including 7 novel mutations (del 197, C308G, R385W, splice junction mutation of intron 14, del 328, del 502-505, stop 10, del 165), that were present in 49 index cases (52%). The 15 mutations are mapped to three known functional domains of the receptor (7 in the LDL-binding region, 7 in the epidermal growth factor precursor homology region and 1 in the membrane-spanning region). Screening for the identified mutations in the remaining 98 index cases enabled the molecular diagnosis of 31 additional cases. It is therefore concluded that 80 out of 193 index cases (41%) harbor 1 of the 15 mutations described here. Three mutations-del197 (FH-Lithuania), D147H (FH-Sephardic), and stop660 (Lebanese allele)-were found in a total of 66 index cases (34%); these may be regarded as founder mutations in the three respective origin groups. In conclusion, in Israel molecular heterogeneity at the LDL receptor gene locus reflects the ethnic distribution of its origin groups. The results of the present investigation provide valuable diagnostic tools for a subset of the Israeli patients with FH who are at high risk for atherosclerosis and its complications.

Isoproterenol decreases LDL receptor expression in rat adipose cells: activation of cyclic AMP-dependent proteolysis

The low density lipoprotein (LDL) receptor is part of a family of proteins that mediate the uptake of lipoproteins into cells. In this paper we have demonstrated the over-expression in E. coli of a rat LDL receptor fusion protein that contains the region of the receptor sharing homology with the EGF precursor. The fusion protein was utilized to immunize rabbits and successfully generate antibodies that recognize the intact LDL receptor. These anti-LDL receptor/fusion protein antibodies were used to examine the effects of cyclic AMP on the expression of LDL receptors in isolated rat adipocytes. Incubation of adipocytes with isoproterenol caused a dose-dependent diminution in intact LDL receptors in the plasma membrane with the concomitant appearance of smaller immunoreactive proteins. Pulse-chase experiments demonstrated that isoproterenol rapidly shortened the initial half-life of intact, immunoprecipitable LDL receptors in the plasma membrane. The effects of isoproterenol on LDL receptor expression were mimicked by forskolin, by an analog of cyclic AMP, and by ACTH. In contrast, incubation with propranolol blocked the effects of isoproterenol on LDL receptor expression. While antioxidants and several different protease inhibitors had no effects, N-acetyl-leucine-leucine-methionine (ALLM) was able to prevent the isoproterenol-induced effects on LDL receptors. Thus, it appears that agents acting via cyclic AMP cause a rapid decrease in LDL receptors in the plasma membranes of isolated adipose cells due to the apparent stimulation of an ALLM-sensitive protease that degrades the LDL receptor. These results suggest a novel mechanism for the posttranscriptional regulation of LDL receptor expression in adipocytes.

GPI-anchored diphtheria toxin receptor allows membrane translocation of the toxin without detectable ion channel activity.

We have investigated the role of the transmembrane and cytoplasmic domains of the diphtheria toxin (DT) receptor [heparin-binding epidermal growth factor (HB-EGF) precursor] in the intoxication pathway. Two mutants were constructed in which these domains were replaced by either a 37 amino acid sequence signalling membrane attachment via a glycosylphosphatidylinositol (GPI) anchor (DTR-GPI) or by the transmembrane and cytoplasmic domains of the human EGF receptor (DTR-EGFR). Similar amounts of DTA fragment were translocated through the plasma membrane of NIH 3T3 cells transfected with the wild-type receptor (DTR), DTR-GPI and DTR-EGFR, but translocation was about six times less efficient in the case of DTR-GPI and DTR-EGFR when taking into account the number of receptors expressed. Interestingly, DT-induced 22Na+ influx was weak in DTR-EGFR cells and not detectable in DTR-GPI cells. Whole cell patch-clamp analysis showed the DT at low pH induced depolarization and decreased input resistance in DTR cells (and to a lesser extent also in DTR-EGFR cells) but not in DTR-GPI cells. These results suggest that the transmembrane and cytoplasmic part of the receptor might be involved in channel activity and that translocation of the A fragment is independent of toxin-induced cation channel activity.

The LDL receptor and LRP are receptors for beta VLDL on pigeon monocyte-derived macrophages.

Receptors for the lipoprotein, beta very low density lipoprotein (beta VLDL), have been identified through the binding of beta VLDL-gold conjugates on two ligand-induced regions of pigeon monocyte-derived macrophages. These regions were microvilli/retraction fibers and membrane ruffles. The present study investigated the location and identity of beta VLDL receptors using an antiserum directed against the epidermal growth factor (EGF) precursor region of the human low density lipoprotein (LDL) receptor. The anti-receptor serum recognized two membrane proteins from pigeon monocyte-derived macrophages, a 116 kDa (LDL receptor) protein and a 600 kDa (low density lipoprotein receptor-related protein; LRP) protein. Ligand blot analysis demonstrated that pigeon beta VLDL bound to both the LDL receptor and LRP. Immuno-gold electron microscopy using the anti-receptor serum resulted in immunoglobulin localization on the same two ligand-induced regions, microvilli/retraction fibers and membrane ruffles, to which the ligand had bound. Furthermore, simultaneous immunogold localization of the lipoprotein receptor antigens and beta VLDL-gold (ligand) binding substantiated co-localization of the receptor antigens and beta VLDL on the ligand-induced regions. Cross-competition studies with the anti-receptor serum and beta VLDL-gold conjugate documented that increasing concentration of the anti-receptor serum resulted in 70% inhibition of beta VLDL-gold conjugate binding. These data suggest that pigeon monocyte-derived macrophages utilize both the LDL receptor and LRP as receptors for pigeon beta VLDL.

Epidermal Growth Factor Precursor Is Present in a Variety of Human Renal Cyst Fluids

Progressive human renal cystic diseases are characterized by proliferation of the epithelial cells lining the cyst. The kidney synthesizes epidermal growth factor and its presence in renal cyst fluid might contribute to renal epithelial cell proliferation. We screened autosomal dominant polycystic kidney disease, acquired renal cystic disease, the von Hippel-Lindau syndrome, multilocular cystic nephroma, multicystic dysplastic kidney disease and simple cyst fluids for the presence of epidermal growth factor by radioreceptor assay, specific radioimmunoassay, immunoprecipitation and immunoblotting. Multiple epidermal growth factor immunoreactive species of approximately 180 kD. and lower molecular weights were present in almost all cyst fluids examined, suggesting endogenous synthesis and limited proteolysis of epidermal growth factor precursor protein in cyst fluid. Tamm-Horsfall protein was detected by enzyme-linked immunosorbent assay in most cysts (for example 55 of 59 autosomal dominant polycystic kidney disease samples). The majority of simple and autosomal dominant polycystic kidney disease renal cysts contained high sodium ion concentration, epidermal growth factor precursor protein and Tamm-Horsfall protein, characteristic of the early thick ascending limb. Rather than the mere presence of epidermal growth factor in renal cyst fluids, increased sensitivity to epidermal growth factor or other mitogens present in renal cyst fluid may be pathogenic in progressive renal cystic disease.

Epidermal Growth Factor Precursor Is Present in a Variety of Human Renal Cyst Fluids

Progressive human renal cystic diseases are characterized by proliferation of the epithelial cells lining the cyst. The kidney synthesizes epidermal growth factor and its presence in renal cyst fluid might contribute to renal epithelial cell proliferation. We screened autosomal dominant polycystic kidney disease, acquired renal cystic disease, the von Hippel-Lindau syndrome, multilocular cystic nephroma, multicystic dysplastic kidney disease and simple cyst fluids for the presence of epidermal growth factor by radioreceptor assay, specific radioimmunoassay, immunoprecipitation and immunoblotting. Multiple epidermal growth factor immunoreactive species of approximately 180 kD. and lower molecular weights were present in almost all cyst fluids examined, suggesting endogenous synthesis and limited proteolysis of epidermal growth factor precursor protein in cyst fluid. Tamm-Horsfall protein was detected by enzyme-linked immunosorbent assay in most cysts (for example 55 of 59 autosomal dominant polycystic kidney disease samples). The majority of simple and autosomal dominant polycystic kidney disease renal cysts contained high sodium ion concentration, epidermal growth factor precursor protein and Tamm-Horsfall protein, characteristic of the early thick ascending limb. Rather than the mere presence of epidermal growth factor in renal cyst fluids, increased sensitivity to epidermal growth factor or other mitogens present in renal cyst fluid may be pathogenic in progressive renal cystic disease.

Triiodothyronine stimulates renal epidermal growth factor expression in adult rat

To define the role that thyroid hormones play in regulation of renal epidermal growth factor (EGF) production, we characterized the effect of triiodothyronine (T3) administration on renal EGF expression in adult rats. The action of T3 to regulate EGF production was examined under one condition in which renal EGF expression is known to be diminished, posthypophysectomy, and in pituitary-intact rats. Levels of mature EGF, EGF precursor, and EGF mRNA, reduced in kidneys of hypophysectomized rats compared with pituitary-intact animals, increased significantly following the administration of T3 to hypophysectomized rats. Thus replacement of thyroid hormone alone was sufficient to enhance renal EGF expression. Induction of a hyperthyroid state in normal rats by injection of T3 for 4 days increased levels of extractable immunoreactive mature EGF, EGF precursor present in renal membranes, and EGF mRNA measured in kidneys. Levels of EGF in circulation were undetectable under all experimental conditions. We conclude that T3 enhances the renal synthesis of EGF in both hypopituitary and pituitary-intact rats. Enhancement of renal EGF expression is one mechanism that must be considered to explain the actions of thyroid hormones on kidney.

Three novel mutations in the EGF precursor homology domain of the low-density lipoprotein receptor gene in Northern Irish patients with familial hypercholesterolemia

Three novel mutations in the EGF precursor homology domain of the low-density lipoprotein receptor gene in Northern Irish patients with familial hypercholesterolemia

Detection of a kallikrein in the mouse lactating mammary gland: a possible processing enzyme for the epidermal growth factor precursor.

Kallikreins are a multigene subfamily of serine proteases that may have a role in processing precursors of polypeptide hormones and growth factors. The epidermal growth factor (EGF) immunoreactivity in mouse milk is derived from the membrane-bound EGF precursor located on the lumenal border of the alveolar cells in the mammary gland. Release of EGF into the milk requires the hydrolysis of the EGF precursor at Arg-X cleavage sites. We report the presence of a candidate EGF precursor-processing enzyme in the lactating mouse mammary gland. Kallikrein transcripts in the mouse lactating mammary gland were detected by primer-directed enzyme amplification of complementary DNA (cDNA). Primers to selected conserved regions of the kallikrein cDNA resulted in an amplified product of the predicted size (573 basepairs). Sequence analysis of the product over three nonconserved regions identified mGK-6 (mouse renal kallikrein) as the primary kallikrein in BALB/c mouse lactating mammary gland. Transcription products for the EGF-binding protein (mGK-9), mGK-1, MGK-3, and mGK-4 were not detected by enzyme amplification with specific primers corresponding to these kallikrein cDNAs. Positive immunohistochemical staining of the apical membrane of mammary alveolar cells was detected with a polyclonal antiserum to mouse kallikrein. Incubation of cell membranes isolated from lactating mammary glands released soluble EGF-immunoreactive material. Aprotinin partially inhibited the release of this material, whereas other protease inhibitors, such as leupeptin, benzamidine, and limabean trypsin inhibitor, had no detectable effect. These results support the hypothesis that the release of EGF-immunoreactive material into the milk is in part dependent upon a kallikrein enzyme (mGK-6) in the BALB/c mouse lactating mammary gland.

Detection of a kallikrein in the mouse lactating mammary gland: a possible processing enzyme for the epidermal growth factor precursor

Detection of a kallikrein in the mouse lactating mammary gland: a possible processing enzyme for the epidermal growth factor precursor

The cytoplasmic domain of the diphtheria toxin receptor (HB-EGF precursor) is not required for receptor-mediated endocytosis.

Diphtheria toxin is believed to enter toxin-sensitive mammalian cells by receptor-mediated endocytosis employing the transmembrane cell surface precursor of heparin-binding epidermal growth factor-like growth factor as a receptor. To investigate the contribution of the receptor's cytoplasmic domain to the toxin internalization process, we constructed stable cell lines that express receptor molecules containing cytoplasmic domain mutations. Our results indicate that Tyr192 and surrounding amino acid residues are important for high toxin sensitivity. Cells expressing mutant receptors are less sensitive to toxin and have fewer toxin-specific binding sites but internalize toxin at rates similar to those of cells expressing the intact receptor. This rate of internalization is much slower (1-2%/min) than that of classical endocytic receptors (10-50%/min). Our results are consistent with a model in which the cytoplasmic domain of the toxin receptor lacks a signal for rapid internalization. We suggest that toxin-receptor complexes, nevertheless, are internalized by receptor-mediated endocytosis by entrapment in clathrin-coated pits as part of bulk phase turnover of cell surface proteins. Although the rate is slow, successful intoxication occurs because a single internalized enzymatically-active toxin molecule is sufficient to inhibit protein synthesis in the cell.

Partial duplication of the EGF precursor homology domain of the LDL-receptor protein causing familial hypercholesterolemia (FH-Salerno).

A novel mutation of low density lipoprotein (LDL)-receptor gene was found in an Italian family hypercholesterolemia (FH) patient during a screening of 300 FH patients. The proband as well as her daughter were found to be heterozygotes for the mutation. Binding, internalization, and degradation of 125I-labeled LDL by the proband's fibroblasts were reduced to approximately 50% compared to values found in control cells. DNA analysis by Southern blotting showed that the mutant allele was characterized by an insertion of about 10 kb, which resulted from a duplication of exons 9-14 of the LDL-receptor gene. In addition, Northern blot analysis of the proband's RNA showed, besides the normal sized LDL-receptor mRNA (5.3 kb), an additional mRNA of about 6.2 kb. The junction between exon 14 and the duplicated exon 9 was amplified by polymerase chain reaction (PCR) from the cDNA. The sequence of the amplified fragment showed that exon 14 joined the duplicated exon 9 without changing the reading frame. The derived amino acid sequence indicated that the mutated receptor protein had a partial duplication of the EGF precursor homology domain. Ligand and immunoblotting revealed that proband's fibroblasts contained one-half of the normal amount of LDL-receptor protein (molecular mass 130 kDa) and an abnormally large receptor of approximately 160 kDa. The amount of this abnormal receptor as detected by two monoclonal antibodies (10A2 and 4B3) was found to be approximately 30% that of the normal LDL-receptor present in the same cells.(ABSTRACT TRUNCATED AT 250 WORDS)