Isolation and characterization of mouse GRIP1, a novel transcriptional coactivator of steroid receptors

Steroid receptors are a group of conditional transcription factors which require transcriptional coactivators to mediate their stimulation of transcription initiation. To search for the coactivators for the glucocorticoid receptor (GR), we used the yeast two-hybrid system to identify a novel mouse protein GRIP1 (Glucocorticoid Receptor-Interacting Protein), which contains 1462-amino acids, and shares partial sequence homology with SRC-1, a recently isolated transcriptional coactivator for nuclear receptors. In the yeast two-hybrid system and in vitro, GRIP1 interacted with the HBDs of all the steroid receptors in the presence of agonists, but not in the absence of agonists or in the presence of antagonists RU486 and ZK299, although both antagonists caused GR to dissociate from hsp90. GRIP1 can also interact with the HBDs of VDR, TR{dollar}alpha{dollar}, RAR{dollar}alpha,{dollar} and RXR{dollar}alpha{dollar}, but with different degrees of hormone dependency. Point mutations in the AF-2 transactivation domain of the estrogen receptor HBD which eliminated the transactivation activity of ER also prevented interaction with GRIP1; a GR HBD mutant GA11, which showed increased transactivation activity, exhibited enhanced ability to interact with GRIP1. When fused to the DNA binding domain (DBD) of a heterologous protein, GRIP1 exhibited transactivation activity both in yeast and mammalian cells. More interestingly, while a steroid receptor HBD fused with a Gal4 DBD could not, by itself, activate reporter gene controlled by a Gal4 enhancer site in yeast, co-expression of this fusion protein with GRIP1 strongly activated the reporter gene in a hormone dependent manner. GRIP1 also enhanced the hormone dependent transactivation activity of intact GR and estrogen receptor in yeast with a reporter gene controlled by their cognate enhancer elements. Further tests indicated that while GRIP1 can function as a coactivator of the GR HBD (containing AF-2 transactivation domain), it did not interact with the GR N-terminal domain (containing AF-1 transactivation domain) and had no effect on its transactivation activity in yeast either. These results demonstrated directly that AF-1 and AF-2 domains accomplish their transactivation activities through different mechanisms.