Biological function of the LxCxE-binding pocket of retinoblastoma protein

The product of the retinoblastoma gene (pRb) is a tumor suppressor protein that regulates cellular proliferation, apoptosis and differentiation of numerous tissues in mice. It contains multiple peptide-binding pockets through which it interacts with a host of cellular and viral proteins. The LxCxE-binding pocket of pRb has been highly conserved between pRb proteins from evolutionary distant species; however, the in vivo function of this binding pocket is unknown. The crystal structure of pRB bound to LxCxE peptide was used to design a single point mutation, N757F, which specifically inactivates interactions between pRB and LxCxE-containing proteins without affecting the pRB-E2F interaction. The N750F mutation (analogous to N757F in the human RB) was introduced into the mouse Rb-1 locus by homologous recombination. The RbN750F/N750F mice do not exhibit the phenotype of embryonic lethality observed in Rb-null mice. The pRb-N750F protein does not co-immunoprecipitate with E1A, demonstrating disruption of the LxCxE-binding pocket. The Rb+/- mice develop pituitary tumors with 90% penetrance through LOH. By contrast, the pRb-N750F protein retains its pituitary tumor suppression function as evidenced by the lack of pituitary tumors in RbN750F/N750F and Rb+ /N750F mice. This is consistent with the data from tissue culture experiments demonstrating that RbN750F/N750F fibroblasts do not exhibit any cell cycle defects.; The lack of embryonic lethality in RbN750F/N750F mice allowed us to study the effect of this mutation on adult tissues. The RbN750F/N750F mice have elevated levels of platelets and lymphocytes in the peripheral blood. The RbN750F/N750F females are infertile due to anovulation. These findings demonstrate for the first time that the LxCxE-binding pocket of pRb plays a role in thromobopoiesis, lymphopoiesis and ovulation. The Rb N750F/- mice are born at the frequency of 13% and die by the age of 8 months, indicating that, unlike Rb+ allele, the RbN750F allele is haploinsufficient. The RbN750F/- females are also infertile due to the lack of FSH and LH function in the ovaries.