Epidermal growth factor (EGF)-like repeats of human tenascin-C as ligands for EGF receptor.

Signaling through growth factor receptors controls such diverse cell functions as proliferation, migration, and differentiation. A critical question has been how the activation of these receptors is regulated. Most, if not all, of the known ligands for these receptors are soluble factors. However, as matrix components are highly tissue-specific and change during development and pathology, it has been suggested that select growth factor receptors might be stimulated by binding to matrix components. Herein, we describe a new class of ligand for the epidermal growth factor (EGF) receptor (EGFR) found within the EGF-like repeats of tenascin-C, an antiadhesive matrix component present during organogenesis, development, and wound repair. Select EGF-like repeats of tenascin-C elicited mitogenesis and EGFR autophosphorylation in an EGFR-dependent manner. Micromolar concentrations of EGF-like repeats induced EGFR autophosphorylation and activated extracellular signal-regulated, mitogen-activated protein kinase to levels comparable to those induced by subsaturating levels of known EGFR ligands. EGFR-dependent adhesion was noted when the ligands were tethered to inert beads, simulating the physiologically relevant presentation of tenascin-C as hexabrachion, and suggesting an increase in avidity similar to that seen for integrin ligands upon surface binding. Specific binding to EGFR was further established by immunofluorescence detection of EGF-like repeats bound to cells and cross-linking of EGFR with the repeats. Both of these interactions were abolished upon competition by EGF and enhanced by dimerization of the EGF-like repeat. Such low affinity behavior would be expected for a matrix-"tethered" ligand; i.e., a ligand which acts from the matrix, presented continuously to cell surface EGF receptors, because it can neither diffuse away nor be internalized and degraded. These data identify a new class of "insoluble" growth factor ligands and a novel mode of activation for growth factor receptors.

Human papillomavirus DNA replication compartments in a transient DNA replication system.

Many DNA viruses replicate their genomes at nuclear foci in infected cells. Using indirect immunofluorescence in combination with fluorescence in situ hybridization, we colocalized the human papillomavirus (HPV) replicating proteins E1 and E2 and the replicating origin-containing plasmid to nuclear foci in transiently transfected cells. The host replication protein A (RP-A) was also colocalized to these foci. These nuclear structures were identified as active sites of viral DNA synthesis by bromodeoxyuridine (BrdU) pulse-labeling. Unexpectedly, the great majority of RP-A and BrdU incorporation was found in these HPV replication domains. Furthermore, E1, E2, and RP-A were also colocalized to nuclear foci in the absence of an origin-containing plasmid. These observations suggest a spatial reorganization of the host DNA replication machinery upon HPV DNA replication or E1 and E2 expression. Alternatively, viral DNA replication might be targeted to host nuclear domains that are active during the late S phase, when such domains are limited in number. In a fraction of cells expressing E1 and E2, the promyelocytic leukemia protein, a component of nuclear domain 10 (ND10), was either partially or completely colocalized with E1 and E2. Since ND10 structures were recently hypothesized to be sites of bovine papillomavirus virion assembly, our observation suggests that HPV DNA amplification might be partially coupled to virion assembly.

Association of the human papillomavirus type 11 E1 protein with histone H1.

The E1 and E2 proteins are the only virus-encoded factors required for human papillomavirus (HPV) DNA replication. The E1 protein is a DNA helicase responsible for initiation of DNA replication at the viral origin. Its recruitment to the origin is facilitated by binding to E2, for which specific recognition elements are located at the origin. The remaining replication functions for the virus, provided by the host cell's replication machinery, may be mediated by further interactions with E1 and E2. Histone H1 was identified as an HPV type 11 (HPV-11) E1-binding protein by far-Western blotting and by microsequence analyses of a 34-kDa protein purified by E1 affinity chromatography. E1 also bound in vitro to H1 isolated under native conditions in association with intact nucleosomes. In addition, E1 and H1 were coimmunoprecipitated by an E1 antiserum from a nuclear extract prepared from cells expressing recombinant E1. Bound H1 was displaced from HPV-11 DNA by the addition of E1, suggesting that E1 can promote replication initiation and elongation by alteration of viral chromatin structure and disruption of nucleosomes at the replication fork. Furthermore, a region of the HPV-11 genome containing the origin of replication was identified which had weaker affinity for H1 than that of the remaining genome. This result suggests that the presence of a DNA structure at or near the HPV origin facilitates initiation of DNA replication by exclusion of H1. These results are similar to those of studies of simian virus 40 DNA replication, in which a large T antigen-H1 interaction and an H1-resistant region at the origin of DNA replication have also been demonstrated.