Mutation analysis of tumor DNA reveals a point mutation in exon 3 of the VHL gene that loses the ability to bind HIF-1α.

Mutational analysis of genomic tumor DNA revealed a point mutation of the VHL gene in exon 3 at position 701. (A) The position of this mutation in relation to the intron/exon (boxes) boundaries and the functional domains of pVHL; α-domain, red; and β-domain, blue. Amino acid (top) and nucleotide (bottom) sequences are shown for wild-type (wt) and mutated (mut) VHL, illustrating the exchange of amino acid 163, leucine to proline. (B) An immunoprecipitation assay in which 10 μL of each radioactive in vitro–transcribed and –translated product of HIF-1α, wild-type pVHL (wt-pVHL.HA), and mutated pVHL (mut-pVHL.HA, Leu163Pro) were coincubated. HA tag–fused C-terminal to pVHL was used to immunoprecipitate pVHL with its potential binding partner using a specific anti-HA monoclonal antibody. Crosses indicate the input of respective proteins into the reaction. Immunoprecipitates were denatured and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Visualization was achieved by autoradiography. Arrows indicate HIF-1α and both positions of pVHL, full-length (p30.HA) and the truncated protein fragment, caused by an internal translation initiation site (p19.HA). Whereas wild-type pVHL.HA coimmunoprecipitates with HIF-1α, the mutated protein L163P shows no binding to HIF-1α.