CLONING OF CDNAS FOR CELLULAR PROTEINS THAT BIND TO THE RETINOBLASTOMA GENE-PRODUCT

THE E7 transforming protein of human papilloma virus-16 binds to the retinoblastoma gene product (pRb) 1,2 through a nine-amino-acid segment of E7 (21-29) 3-5. This segment of E7 is homologous to the pRb-binding domains of the simian virus 40 large T and adenovirus E1A transforming proteins 6-9. Each of these viral transforming proteins bind to the same region of pRb 10-11. To isolate cellular proteins that interact with this viral protein-binding domain on pRb 12,13, we used recombinant pRb to screen a human complementary DNA expression library. Two cDNAs were isolated that encode retinoblastoma binding proteins (RBP-1 and RBP-2). We report here that these RBP genes exist in separate loci and produce discrete messenger RNAs. The predicted amino-acid sequence of these genes showed no homology to known proteins, but both RBPs contain the pRb binding motif conserved between E7, large T and E1A 14. In vitro expression of the RBP cDNAs yielded proteins that specifically bound to pRb. Recombinant E7 protein, the E7 21-29 peptide and the homologous RBP-L peptide inhibited RBP-pRb binding. Mutations introduced into the putative pRb-binding segment in RBP-1 impaired its binding activity. These studies indicate that the cellular RBP-1, RBP-2 and viral E7 proteins interact with pRb through similar domains.