The Drosophila EGF receptor gene homolog: conservation of both hormone binding and kinase domains

Chicken v-erB probe was used to isolate a unique clone of Drosophila melanogaster DNA. It maps by in situ hybridization to position 57F on chromosome 2. A complete nucleotide sequence of the coding region has been obtained. The putative Drosophila EGF receptor protein is similar in overall organization to the human homolog. It shows three distinct domains: an extracellular putative EGF binding domain, a hydrophobic transmembrane region, and a cytoplasmic kinase domain. The overall amino acid homology is 41% in the extracellular domain and 55% in the kinase domain. Two cysteine-rich regions, a hallmark of the human ligand-binding domain, have also been conserved. Fusion of the coding sequences of the kinase and extracellular domains generating the receptor gene must have occurred over 800 million years ago.     

Characterization of a comparative model of the extracellular domain of the epidermal growth factor receptor

The Epidermal Growth Factor (EGF) receptor is a tyrosine kinase that mediates the biological effects of ligands such as EGF and transforming growth factor alpha. An understanding of the molecular basis of its action has been hindered by a lack of structural and mutational data on the receptor. We have constructed comparative models of the four extracellular domains of the EGF receptor that are based on the structure of the first three domains of the insulin-like growth factor-1 (IGF-1) receptor. The first and third domains of the EGF receptor, L1 and L2, are right-handed beta helices. The second and fourth domains of the EGF receptor, S1 and S2, consist of the modules held together by disulfide bonds, which, except for the first module of the S1 domain, form rod-like structures. The arrangement of the L1 and S1 domains of the model are similar to that of the first two domains of the IGF-1 receptor, whereas that of the L2 and S2 domains appear to be significantly different. Using the EGF receptor model and limited information from the literature, we have proposed a number of regions that may be involved in the functioning of the receptor. In particular, the faces containing the large beta sheets in the L1 and L2 domains have been suggested to be involved with ligand binding of EGF to its receptor.     

N-Terminal domain linkage modulates the folding properties of protein S epidermal growth factor-like modules

Protein S interacts with activated protein C to play a crucial role in blood anticoagulation, and protein S deficiency is associated with increased risk of thrombosis. Despite the large volume of functional data available for this protein, no atomic resolution structure data have yet been reported. This is due at least in part to difficulties encountered when trying to produce fragments dissected from the intact protein; however, a few successful strategies have been described. In this research we have expressed a number of constructs containing protein S epidermal growth factor-like (EGF) domains 1 and 2 in Escherichia coli and Pichia pastoris. None of the proteins produced was stably folded as assayed by solution nuclear magnetic resonance spectroscopy. We therefore constructed a series of non-native protein S EGF concatemers to investigate the role of pairwise domain linkage in domain folding. Our results demonstrate that N-terminal domain linkage can either positively or negatively impact on the refolding of an adjacent domain. Furthermore, analysis of the NMR data for EGF3-4 reveals the expected interdomain NOEs that are characteristic of an extended arrangement of calcium-binding EGF domains and a similar average [(1)H]-(15)N heteronuclear NOE value for each of the two domains. These results provide the first data in support of protein S EGF3-4 adopting the same extended domain orientation as observed for the functionally distinct proteins fibrillin-1 and the low-density lipoprotein receptor. The results also have important implications for future studies, particularly when a dissection approach is used, of tandem EGF domains from protein S and other proteins.     

Molecular dynamics simulations on human fibulin-4 mutants D203A and E126K reveal conformational changes in EGF domains potentially responsible for enhanced protease lability and impaired extracellular matrix assembly

Fibulin-4 is a 50 kDa glycoprotein of elastic fibers and plays an important role in development and function of elastic tissues. Fibulin-4 consists of a tandem array of five calcium-binding epidermal growth factor-like modules flanked by N- and C-terminal domains. Mutations in the human fibulin-4 gene EFEMP2 have been identified in patients affected with various arteriopathies including aneurysm, arterial tortuosity, or stenosis, but the molecular basis of most genotype-phenotype correlations is unknown. Here we present biochemical and computer modelling approaches designed to gain further insight into changes in structure and function of two fibulin-4 mutations (E126K and D203A), which are potentially involved in Ca²⁺ binding in the EGF2 and EGF4 domain, respectively. Using recombinantly produced fibulin-4 mutant and wild type proteins we show that both mutations introduced additional protease cleavage sites, impaired extracellular assembly into fibers, and affected binding to to fibrillin-1, latent TGF-β-binding proteins, and the lysyl oxidase LOXL2. Molecular dynamics studies indicated that the E126K and D203A mutations do not necessarily result in a direct loss of the complexed Ca²⁺ ion after 500 ns simulation time, but in significantly enhanced fluctuations within the connecting loop between EGF3 and EGF4 domains and other conformational changes. In contrast, intentionally removing Ca²⁺ from EGF4 (D203A ΔCa) predicted dramatic changes in the protein structure. These results may explain the changes in protease cleavage sites, reduced secretion and impaired extracellular assembly of the E126K and D203A fibulin-4 mutants and provide further insight into understanding the molecular basis of the associated clinical phenotypes.     

Beta-hydroxyaspartic acid in the first epidermal growth factor-like domain of protein C. Its role in Ca2+ binding and biological activity

Protein C is a vitamin K-dependent regulator of blood coagulation. It has beta-hydroxyaspartic acid in position 71 which is in the first of its two domains that are homologous to epidermal growth factor (EGF). This region has recently been demonstrated to have a Ca2+ binding site with a Kd of approximately 100 microM. Recombinant human protein C, expressed in mammalian tissue culture, had full biological activity and contained beta-hydroxyaspartic acid. Furthermore, it had a Ca2+-dependent epitope in the EGF-like domain, recognized by a monoclonal antibody. In contrast, a mutant recombinant human protein C in which beta-hydroxyaspartic acid had been replaced with glutamic acid in position 71 did not have the Ca2+-dependent epitope, and its biological activity was reduced to about 10% of normal. Fab' fragments of this antibody inhibited the anticoagulant activity of plasma-derived activated protein C, apparently by interfering with the interaction between activated protein C and its cofactor, protein S. The latter contains four tandemly arranged EGF homology domains. We propose that beta-hydroxyaspartic acid is directly involved in Ca2+ binding in protein C and in related proteins and that protein C interacts with protein S by means of its EGF homology regions.     

A chimeric EGF-R-neu proto-oncogene allows EGF to regulate neu tyrosine kinase and cell transformation.

The neu oncogene, characterized by Weinberg and colleagues, is a transforming gene found in ethylnitrosourea-induced rat neuro/glioblastomas; its human proto-oncogene homologue has been termed erbB2 or HER2 because of its close homology with the epidermal growth factor receptor (EGF-R) gene (c-erbB1). Expression of the rat neu oncogene is sufficient for transformation of mouse NIH 3T3 fibroblasts in culture and for the development of mammary carcinomas in transgenic mice, but the neu proto-oncogene has not been associated with cell transformation. We constructed a vector for expression of a chimeric cDNA and hybrid protein consisting of the EGF-R extracellular, transmembrane and protein kinase C-substrate domains linked to the intracellular tyrosine kinase and carboxyl terminal domain of the rat neu cDNA. Upon transfection with the construct, NIH 3T3 cells gave rise to EGF-R antigen-positive cell clones with varying amounts of specific EGF binding. Immunofluorescence and immunoprecipitation using neu- and EGF-receptor specific antibodies demonstrated a correctly oriented and positioned chimeric EGF-R-neu protein of the expected apparent mol. wt on the surface of these cells. EGF or TGF alpha induced tyrosine phosphorylation of the chimeric receptor protein, stimulated DNA synthesis of EGF-R-neu expressing cells and led to a transformed cell morphology and growth in soft agar. In contrast, the neu proto-oncogene did not show kinase activity or transforming properties when expressed at similar levels in NIH 3T3 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solution structure of the Ca2+-Binding EGF3-4 pair from vitamin K-dependent protein S: identification of an unusual fold in EGF3

Vitamin K-dependent protein S is a cofactor of activated protein C, a serine protease that regulates blood coagulation. Deficiency of protein S can cause venous thrombosis. Protein S has four EGF domains in tandem; domains 2-4 bind calcium with high affinity whereas domains 1-2 mediate interaction with activated protein C. We have now solved the solution structure of the EGF3-4 fragment of protein S. The linker between the two domains is similar to what has been observed in other calcium-binding EGF domains where it provides an extended conformation. Interestingly, a disagreement between NOE and RDC data revealed a conformational heterogeneity within EGF3 due to a hinge-like motion around Glu186 in the Cys-Glu-Cys sequence, the only point in the domain where flexibility is allowed. The dominant, bent conformation of EGF3 in the pair has no precedent among calcium-binding EGF domains. It is characterized by a change in the psi angle of Glu186 from 160 degrees +/- 40 degrees , as seen in ten other EGF domains, to approximately 0 degrees +/- 15 degrees . NOESY data suggest that Tyr193, a residue not conserved in other calcium-binding EGF domains (except in the homologue Gas6), induces the unique fold of EGF3. However, SAXS data, obtained on EGF1-4 and EGF2-4, showed a dominant, extended conformation in these fragments. This may be due to a counterproductive domain-domain interaction between EGF2 and EGF4 if EGF3 is in a bent conformation. We speculate that the ability of EGF3 to adopt different conformations may be of functional significance in protein-protein interactions involving protein S.     

The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling.

A cDNA clone encoding a novel, widely expressed protein (called growth factor receptor-bound protein 2 or GRB2) containing one src homology 2 (SH2) domain and two SH3 domains was isolated. Immunoblotting experiments indicate that GRB2 associates with tyrosine-phosphorylated epidermal growth factor receptors (EGFRs) and platelet-derived growth factor receptors (PDGFRs) via its SH2 domain. Interestingly, GRB2 exhibits striking structural and functional homology to the C. elegans protein sem-5. It has been shown that sem-5 and two other genes called let-23 (EGFR like) and let-60 (ras like) lie along the same signal transduction pathway controlling C. elegans vulval induction. To examine whether GRB2 is also a component of ras signaling in mammalian cells, microinjection studies were performed. While injection of GRB2 or H-ras proteins alone into quiescent rat fibroblasts did not have mitogenic effect, microinjection of GRB2 together with H-ras protein stimulated DNA synthesis. These results suggest that GRB2/sem-5 plays a crucial role in a highly conserved mechanism for growth factor control of ras signaling.     

A potent DNA synthesis inhibitor expressed by the immortal cell line SUSM-1

We have previously reported the production of DNA synthesis inhibitor proteins by both quiescent and senescent human diploid fibroblasts. Young, proliferating fibroblasts do not produce such inhibitors, but are capable of responding to either the quiescent or senescent cell DNA synthesis inhibitors. Recently, we have analyzed the immortal cell line SUSM-1 (derived from normal liver fibroblasts following exposure to carcinogen) for inhibitory activity. We have found that SUSM-1 cells produce a factor capable of inhibiting DNA synthesis in young fibroblasts. Crude extracts prepared from SUSM-1 cells inhibit DNA synthesis in a dose-dependent manner at concentrations 10-fold lower than those of either senescent or quiescent fibroblast cell extracts. SUSM-1 cells are incapable of responding to the inhibitor they produce, as are three other immortal human cell lines tested. One immortal cell line, HeLa, does respond to the SUSM-1 inhibitor, though to a lesser degree than observed with normal young fibroblasts. One hypothesis is that the DNA synthesis inhibitor protein(s) of senescent cells plays a role in determining the finite in vitro life span of normal cells. The results reported here suggest that SUSM-1 cells may have escaped senescence through loss of a receptor or cofactor for the inhibitor protein(s).     

Identification and characterization of interactions between the vertebrate polycomb-group protein BMI1 and human homologs of polyhomeotic.

In Drosophila melanogaster, the Polycomb-group (PcG) genes have been identified as repressors of gene expression. They are part of a cellular memory system that is responsible for the stable transmission of gene activity to progeny cells. PcG proteins form a large multimeric, chromatin-associated protein complex, but the identity of its components is largely unknown. Here, we identify two human proteins, HPH1 and HPH2, that are associated with the vertebrate PcG protein BMI1. HPH1 and HPH2 coimmunoprecipitate and cofractionate with each other and with BMI1. They also colocalize with BMI1 in interphase nuclei of U-2 OS human osteosarcoma and SW480 human colorectal adenocarcinoma cells. HPH1 and HPH2 have little sequence homology with each other, except in two highly conserved domains, designated homology domains I and II. They share these homology domains I and II with the Drosophila PcG protein Polyhomeotic (Ph), and we, therefore, have named the novel proteins HPH1 and HPH2. HPH1, HPH2, and BMI1 show distinct, although overlapping expression patterns in different tissues and cell lines. Two-hybrid analysis shows that homology domain II of HPH1 interacts with both homology domains I and II of HPH2. In contrast, homology domain I of HPH1 interacts only with homology domain II of HPH2, but not with homology domain I of HPH2. Furthermore, BMI1 does not interact with the individual homology domains. Instead, both intact homology domains I and II need to be present for interactions with BMI1. These data demonstrate the involvement of homology domains I and II in protein-protein interactions and indicate that HPH1 and HPH2 are able to heterodimerize.     

1H, 13C and 15N resonance assignments for the fibrillin-1 EGF2-EGF3-hybrid1-cbEGF1 four-domain fragment

Fibrillins are large extracellular glycoproteins that form the principal component of microfibrils. These perform a vital structural function in the extracellular matrix of many tissues. Fibrillins have also been implicated in mediating a number of protein–protein interactions, some of which may be significant in regulating growth factors such as transforming growth factor β. Here we present the backbone and side-chain ¹H, ¹³C and ¹⁵N assignments for a 19 kDa protein fragment derived from the N-terminus of human fibrillin-1, encompassing four domains in total. These domains include the second and third epidermal growth factor-like (EGF) domains, the first hybrid domain (hyb1), and the first calcium-binding EGF domain of fibrillin-1. This region of fibrillin-1 is of particular interest as the hyb1 domain has been suggested to play a role in microfibril assembly, as well as several other protein–protein interactions.     

Contribution of the CR Domain to P-Selectin Lectin Domain Allostery by Regulating the Orientation of the EGF Domain

The allostery of P-selectin has been studied extensively with a focus on the Lec and EGF domains, whereas the contribution of the CR domain remains unclear. Here, molecular dynamics simulations (MDS) combined with homology modeling were preformed to investigate the impact of the CR domain on P-selectin allostery. The results indicated that the CR domain plays a role in the allosteric dynamics of P-selectin in two ways. First, the CR1 domain tends to stabilize the low affinity of P-selectin during the equilibration processes with the transition inhibition from the S1 to S1’ state by restraining the extension of the bent EGF orientation, or with the relaxation acceleration of the S2 state by promoting the bending of the extended EGF orientation. Second, the existence of CR domain increases intramolecular extension prior to complex separation, increasing the time available for the allosteric shift during forced dissociation with a prolonged bond duration. These findings further our understanding of the structure-function relationship of P-selectin with the enriched micro-structural bases of the CR domain.     

Selective Loss of CDC2 and CDK2 Induction by Tumor Necrosis Factor-α in Senescent Human Diploid Fibroblasts

Normal human diploid fibroblasts undergo a finite number of doublings in culture. This process of senescence is accompanied by a loss in the ability to respond to proliferative stimuli and is therefore distinct from the quiescent state induced by nutrient deprivation. We have studied changes in gene expression induced in these cells following exposure to the cytokine, tumor necrosis factor-α (TNF). We observed that TNF induced CDC2 and CDK2 expression in early-passage quiescent WI-38 fibroblasts. However, as cells approached senescence, their ability to induce CDC2 and CDK2, as well as stimulate DNA synthesis in response to TNF, progressively declined, with minimal to absent induction in senescent cells. This occurred despite the TNF-dependent induction of such proliferation-independent genes as manganese superoxide dismutase and interleukin-6 in senescent and quiescent cells. Serum was similarly unable to induce CDC2 or CDK2 expression in senescent cells. These results demonstrate that senescent cells are selectively deficient in TNF-mediated induction of CDC2 and CDK2, genes crucial to DNA synthesis and mitosis.     

Molecular interactions between the protein products of the neurogenic loci Notch and Delta, two EGF-homologous genes in Drosophila

Genetic analyses have raised the possibility of interactions between the gene products of the neurogenic loci Notch and Delta, each of which encodes a transmembrane protein with EGF homology. To examine the possibility of intermolecular association between the products of these two genes, we studied the effects of their expression on aggregation in Drosophila S2 cells. We find that Notch-expressing cells form mixed aggregates specifically with cells that express Delta and that this process is calcium dependent. In addition, we show that Notch and Delta can associate within the membrane of a single cell, and further, that they form detergent-soluble intermolecular complexes. Our analyses suggest that Notch and Delta proteins interact at the cell surface via their extracellular domains.