Platelet-derived growth factor-alpha receptor activation is required for human cytomegalovirus infection

Human cytomegalovirus (HCMV) is a ubiquitous human herpesvirus that can cause life-threatening disease in the fetus and the immunocompromised host. Upon attachment to the cell, the virus induces robust inflammatory, interferon- and growth-factor-like signalling. The mechanisms facilitating viral entry and gene expression are not clearly understood. Here we show that platelet-derived growth factor-alpha receptor (PDGFR-alpha) is specifically phosphorylated by both laboratory and clinical isolates of HCMV in various human cell types, resulting in activation of the phosphoinositide-3-kinase (PI(3)K) signalling pathway. Upon stimulation by HCMV, tyrosine-phosphorylated PDGFR-alpha associated with the p85 regulatory subunit of PI(3)K and induced protein kinase B (also known as Akt) phosphorylation, similar to the genuine ligand, PDGF-AA. Cells in which PDGFR-alpha was genetically deleted or functionally blocked were non-permissive to HCMV entry, viral gene expression or infectious virus production. Re-introducing human PDGFRA gene into knockout cells restored susceptibility to viral entry and essential viral gene expression. Blockade of receptor function with a humanized PDGFR-alpha blocking antibody (IMC-3G3) or targeted inhibition of its kinase activity with a small molecule (Gleevec) completely inhibited HCMV viral internalization and gene expression in human epithelial, endothelial and fibroblast cells. Viral entry in cells harbouring endogenous PDGFR-alpha was competitively inhibited by pretreatment with PDGF-AA. We further demonstrate that HCMV glycoprotein B directly interacts with PDGFR-alpha, resulting in receptor tyrosine phosphorylation, and that glycoprotein B neutralizing antibodies inhibit HCMV-induced PDGFR-alpha phosphorylation. Taken together, these data indicate that PDGFR-alpha is a critical receptor required for HCMV infection, and thus a target for novel anti-viral therapies.     

Desensitisation of cultured glial cells to epidermal growth factor by receptor down-regulation.

Epidermal growth factor (EGF), which can be purified from the mouse submaxillary gland or from pregnant human urine, is a potent multiplication-stimulating factor for several types of cultured cells, including human fibroblasts and glial cells. The molecule binds with high affinity and saturation kinetics to a cell-surface receptor, is subsequently internalised and finally degraded. The binding event is accompanied by a reduction in the number of EGF receptors. This phenomenon--'receptor down-regulation'--has been demonstrated with several hormones and may be a general principle for the modulation of binding groups on the outer cell surface. Further, it has been proposed that receptor loss acts to regulate the cellular response to the binding ligand. The present study provides direct experimental support for this hypothesis. It demonstrates that down-regulation of EGF receptors on glial cells causes desensitisation of the mitogenic response of these cells to subsequent stimulation with EGF.     

Argos inhibits epidermal growth factor receptor signalling by ligand sequestration

The epidermal growth factor receptor (EGFR) has critical functions in development and in many human cancers. During development, the spatial extent of EGFR signalling is regulated by feedback loops comprising both well-understood activators and less well-characterized inhibitors. In Drosophila melanogaster the secreted protein Argos functions as the only known extracellular inhibitor of EGFR, with clearly identified roles in multiple stages of development. Argos is only expressed when the Drosophila EGFR (DER) is activated at high levels, and downregulates further DER signalling. Although there is ample genetic evidence that Argos inhibits DER activation, the biochemical mechanism has not been established. Here we show that Argos inhibits DER signalling without interacting directly with the receptor, but instead by sequestering the DER-activating ligand Spitz. Argos binds tightly to the EGF motif of Spitz and forms a 1:1 (Spitz:Argos) complex that does not bind DER in vitro or at the cell surface. Our results provide an insight into the mechanism of Argos function, and suggest new strategies for EGFR inhibitor design.     

Computer-based characterization of epidermal growth factor precursor

The cDNA sequence of the precursor of mouse epidermal growth factor (EGFP) has recently been reported by two groups, both of whom noted the presence of repeated similar segments, each about 40 residues long. One of these repeat units overlaps with the sequence of epidermal growth factor itself. The sequence of epidermal growth factor has been reported to be similar to that of pancreatic secretory trypsin inhibitor (PSTI) and a somewhat better match has been found with part of the sequence of bovine factor X, one of the blood coagulating factors. We report here that there is an even stronger similarity between the sequences of some of the repeat units of epidermal growth factor precursor and certain segments in factor X. This sequence similarity is also apparent in comparisons with other blood coagulation factors. On the basis of these sequence comparisons we suggest a scheme for the evolution of the epidermal growth factor precursor. We have also identified certain structural features in the precursor sequence that bear on the way in which the mature epidermal growth factor is generated.     

The solution structure of human epidermal growth factor

The epidermal growth factors (EGFs) are powerful mitogens for a wide variety of cells in culture; human EGF (hEGF), known as urogastrone, also inhibits gastric acid secretion in vivo. The transforming growth factors (TGF-alpha) are related to the EGF family both in sequence and activity and EGF-like sequences are often observed in a wide range of functionally unrelated proteins. Attempts to examine the structure of EGF by diffraction methods have not yet succeeded because of difficulties with crystallization. We report here a three-dimensional structure of a biologically active derivative (residues 1-48) of the 53-residue human EGF. An analysis of high resolution 1H nuclear magnetic resonance (NMR) spectra was used together with a combination of distance geometry, restrained energy minimization and restrained molecular dynamics methods. The three-dimensional structure provides a basis for understanding the properties of EGFs and for predicting the structures of homologous sequences in other proteins.     

Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes.

The culture lifetime of epidermal cells of newborn humans is increased from 50 to 150 generations by adding to the medium epidermal growth factor, a polypeptide mitogen. EGF seems to delay senescence of the cells by maintaining them in a state further removed from terminal differentiation. This effect is revealed by a greater ability of the cells to survive subculture and initiate new colonies, but not necessarily by an increased growth rate.     

Enhancement of polyoma virus middle T antigen tyrosine phosphorylation by epidermal growth factor

Polyoma virus codes for three proteins involved in host cell transformation: the large, middle and small T antigens. Middle T antigen is a major transforming protein which is responsible for the induction of the phenotype of transformed cells and, without it, transformation does not occur (reviewed in refs 1-4). Middle T antigen alone can transform established cell lines, although large, and possibly small, T antigens are also required for the full expression of the phenotype of transformed cells in media with a low concentration of serum. A subfraction of middle T antigen is associated with a protein kinase activity which phosphorylates middle T antigen in vitro on tyrosine. There is a strong correlation between the level of this kinase activity and the degree of expression of the phenotype of transformed cells. We report here that epidermal growth factor (EGF) stimulates tyrosine phosphorylation of middle T antigen, suggesting the possibility that mitogenic growth factor(s) regulates this phosphorylation activity.     

Rapid induction of mRNAs forPC3 by partial hepatectomy and epidermal growth factor

The expression of immediate early gene plays a pivotal role in rat hepatocyte proliferation from G₀ to G₁ phases and the progression through G₁ phase of the cell cycle within several hours after 2/3 hepatectomy. We investigated the different gene expressions within 1 h after 2/3 hepatectomy by representational difference analysis. Sequence analysis indicated thatPC3 induced by NGF was a kind of immediate early gene and might be correlated with liver regeneration. Moreover, we found that 2/3 hepatectomy could induce the expressing ofPC3 mRNA by Northern blot with a peak 1–2 h after surgery. In primary cultures of rat hepatocytes, addition of EGF resulted in rapid and transient induction ofPC3 mRNA. It was first reported thatPC3 gene belongs to immediate early gene associated with liver regeneration.     

Transformation by murine and feline sarcoma viruses specifically blocks binding of epidermal growth factor to cells.

Normal cells in culture have membrane receptors for epidermal growth factor (EGF); EGF stimulates cells to divide by binding to these receptors. Cells transformed by murine and feline sarcoma viruses rapidly lose the ability to bind EGF, whereas cells transformed by the DNA tumour viruses, polyoma and SV40, or infected with non-transforming RNA tumour viruses have normal levels of functional EGF receptors. The results suggest that a product of the sarcoma virus genome specifically changes cell EGF receptors; the sarcoma gene product may, then, be functionally related to EGF.     

XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis

In eukaryotic cells, chromosomal DNA replication begins with the formation of pre-replication complexes at replication origins. Formation and maintenance of pre-replication complexes is dependent upon CDC6 (ref. 1), a protein which allows assembly of MCM2-7 proteins, which are putative replicative helicases. The functional assembly of MCM proteins into chromatin corresponds to replication licensing. Removal of these proteins from chromatin in S phase is crucial in origins firing regulation. We have identified a protein that is required for the assembly of pre-replication complexes, in a screen for maternally expressed genes in Xenopus. This factor (XCDT1) is a relative of fission yeast cdt1, a protein proposed to function in DNA replication, and is the first to be identified in vertebrates. Here we show, using Xenopus in vitro systems, that XCDT1 is required for chromosomal DNA replication. XCDT1 associates with pre-replicative chromatin in a manner dependent on ORC protein and is removed from chromatin at the time of initiation of DNA synthesis. Immunodepletion and reconstitution experiments show that XCDT1 is required to load MCM2-7 proteins onto pre-replicative chromatin. These findings indicate that XCDT1 is an essential component of the system that regulates origins firing during S phase.     

Emi1 is required for cytostatic factor arrest in vertebrate eggs

Vertebrate eggs are arrested at metaphase of meiosis II with stable cyclin B and high cyclin B/Cdc2 kinase activity. The ability of the anaphase-promoting complex/cyclosome (APC), an E3 ubiquitin ligase, to trigger cyclin B destruction and metaphase exit is blocked in eggs by the activity of cytostatic factor (CSF) (reviewed in ref. 1). CSF was defined as an activity in mature oocytes that caused mitotic arrest when injected into dividing embryos. Fertilization causes a transient increase in cytoplasmic calcium concentration leading to CSF inactivation, APC activation, cyclin B destruction and mitotic exit. The APC activator Cdc20 is required for APC activation after fertilization. We show here that the APC(cdc20) inhibitor Emi1 (ref. 6) is necessary and sufficient to inhibit the APC and to prevent mitotic exit in CSF-arrested eggs. CSF extracts immunodepleted of Emi1 degrade cyclin B, and exit from mitosis prematurely in the absence of calcium. Addition of Emi1 to these Emi1-depleted extracts blocks premature inactivation of the CSF-arrested state. Emi1 is required to arrest unfertilized eggs at metaphase of meiosis II and seems to be the long-sought mediator of CSF activity.     

A glycophospholipid anchor is required for Qa-2-mediated T cell activation

A number of lymphocyte surface proteins are anchored in the cell membrane by glycophosphatidyl inositol (known as GPI) linkages instead of hydrophobic protein domains. Treatment of mouse T lymphocytes with antibodies specific for two such proteins, Thy-1 and Ly-6, are known to induce proliferation. We have found that antibodies specific for Qa-2, a GPI-anchored class I histocompatibility antigen, can also activate mouse T cells. To determine whether the GPI-anchor is important for this pathway of cell activation, we produced transgenic mice expressing either normal GPI-anchored Qa-2, or Qa-2 molecules with a membrane-spanning protein domain derived from H-2. Our studies show that only lymphocytes from transgenic mice carrying GPI-anchored forms of Qa-2 can be activated in vitro by Qa-2-specific antibodies. We also show that transgenic mouse T cells expressing a GPI-anchored form of H-2Db can be activated by anti-H-2Db antibodies. These results strongly indicate that the GPI-anchor is critical for this pathway of T cell activation.