GATA DNA-binding protein expressed in mouse I-10 Leydig testicular tumor cells

A nuclear extract of the mouse I-10 Leydig tumor cell line was analyzed by gel mobility shift assay with a combination of antibodies for various mamma lian GATA proteins. Antibodies for GATA-4 caused a supershift of the DNA-pr otein complex, which is formed through GATA-4 binding to an oligonucleotide with a typical GATA motif, while ones for GATA-1, GATA-2, GATA-3, and GATA -6 did not. These results indicated that I-10 cells express GATA-4 protein. Western blotting analysis of cellular proteins also demonstrated the prese nce of GATA-4 protein, the size of which corresponds to that of the rat ort hologous protein transiently expressed in Cos-l cells. A significant level of GATA-4 expression in I-10 cells would be advantageous for studying the r oles of this protein, especially in view of gonadal function. We further ex amined the binding site preference of GATA-4 expressed in I-10 cells. GATA- 4 showed broad sequence specificity similar to GATA-6, the order of binding core site preference being GATA > GATT > GATC, and adenine was favored on both sides of the core for strong binding. Thus the conserved zinc finger d omain of GATA proteins is suggested to contribute to the binding sequence p reference. GATA-4 expressed in I-10 cells was not susceptible to proteolysi s coupled with cAMP signaling, (C) 2001 Academic Press.