Characterization, localization, and functional analyses of VHL gene products

Abstract

von Hippel-Lindau (VHL) disease is an autosomal dominantly inherited family cancer syndrome that predisposes affected individuals to a variety of tumor types. Inactivation of the VHL gene has been demonstrated in tumors from patients with VHL disease and in the corresponding sporadic forms of these cancers.;To analyze the native products of the VHL gene, we produced monoclonal antibodies (mAbs). Two native products of the VHL gene, with apparent molecular masses of 24 and 18 kilodaltons (kDa), were identified. Peptide mapping of the native 18 kDa product suggested that this protein was produced by internal translation from the second AUG in the VHL open reading Came. Internal initiation was confirmed by an in vitro translation assay. The native 18 kDa VHL product was the more abundant VHL product in nearly all cell lines examined. Importantly, the 18 kDa product demonstrated elongin binding and mediated tumor suppression in nude mice. Furthermore, expression of this smaller VHL product led to down-regulation of vascular endothelial growth factor (VEGF) mRNA and glucose transporter GLUT1 protein.;Subcellular localization of native VHL in renal cell lines was investigated by indirect immunofluorescence using a panel of VHL-specific antibodies. Native VHL was observed in a cytoplasmic, perinuclear immunostaining pattern. Colocalization of VHL and markers for the endoplasmic reticulum (ER) was demonstrated by confocal microscopy. Deletion analysis, using chimeric VHL-green fluorescent protein (Grp) constructs, demonstrated that a 64 amino acid region of VHL (residues 114--177) was necessary and sufficient for ER localization.;A possible role of VHL in ubiquitination was investigated. Using MG132, which blocks proteasomal degradation, a ubiquitinated form of VHL-GFP product was detected. Again, VHL amino acids 114--177 were necessary and sufficient for ubiquitination. In subcellular fractionation experiments, both native VHL products were predominantly cytosolic and VHL amino acids 114--177 were also shown to be necessary for cytosolic localization. Therefore, both ubiquitination of VHL-GFP products and ER/cytosolic localization of VHL proteins were dependent on VHL amino acids 114--177, providing evidence of a relationship between these cellular phenomena. Based on these observations, we propose that key VHL biological activities occur at the cytosolic face of the ER.