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) through a nine-amino-acid segment of E7 (21-29). This segment of E7 is homologous to the pRb-binding domains of the simian virus 40 large T and adenovirus E1A transforming proteins. Each of these viral transforming proteins bind to the same region of pRb. To isolate cellular proteins that interact with this viral protein-binding domain on pRb, 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 E1A14. 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-1 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.
Defeo-Jones
D
D
Department of Cancer Research, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
Huang
P S
PS
Jones
R E
RE
Haskell
K M
KM
Vuocolo
G A
GA
Hanobik
M G
MG
Huber
H E
HE
Oliff
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D005260:Female; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D010920:Placenta / Q000502:physiology; D011247:Pregnancy; D011994:Recombinant Proteins / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism*; D056485:Retinoblastoma-Binding Protein 2; D012689:Sequence Homology, Nucleic Acid; D025521:Tumor Suppressor Proteins
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; D016160:Retinoblastoma Protein; D025521:Tumor Suppressor Proteins; D004247:DNA; C070050:KDM5A protein, human; D056485:Retinoblastoma-Binding Protein 2