IDENTIFICATION OF DISCRETE STRUCTURAL DOMAINS IN THE RETINOBLASTOMA PROTEIN - AMINO-TERMINAL DOMAIN IS REQUIRED FOR ITS OLIGOMERIZATION

To characterize the protein product of the retinoblastoma tumor suppre ssor gene biochemically, a recombinant human protein was produced in a n Escherichia coli expression system. The full-length protein, p110RB, and an amino-terminal truncated form, p56RB, were expressed and purif ied to near homogeneity by conventional chromatographic procedures. To probe, the structural organization of the retinoblastoma protein the purified proteins were subjected to partial proteolysis by trypsin, ch ymotrypsin, and subtilisin. Four discrete structural domains were reve aled in p110RB by this method. Two of these structural domains, found in both p56RB and p110RB, were mapped to the carboxyl-terminal half of the protein and corresponded to the SV40 large T binding domains defi ned previously by genetic methods. In addition two distinct domains in the amino-terminal half of the protein were also defined. A potential role for these newly defined amino-terminal domains was uncovered upo n analysis of the purified proteins by nondenaturing polyacrylamide ge l electrophoresis. p110RB revealed multiple bands by this method, sugg esting the formation of oligomeric structures by the protein, while th is property was not observed for p56RB. Electron microscopy of p110RB revealed linearly extended, macromolecular structures, further support ing the formation of homologous higher order structures by the full-le ngth retinoblastoma protein. Analysis of the interactions between reti noblastoma protein molecules using the yeast two-hybrid system confirm ed that the retinoblastoma protein could self-associate and that this association was mediated by interactions between the amino- and carbox yl-terminal ends of the protein. These observations suggest that the r etinoblastoma protein contains multiple structural domains with the am ino-terminal domains being required for oligomerization of the full-le ngth protein.