The molecular basis of the interaction between the proline-rich SH3-binding motif of PNRC and estrogen receptor alpha.

PNRC and PNRC2 are members of a new family of nuclear receptor coactivators. We systematically determined the molecular basis and the structure/function relationship for the PNRC-ERalpha interaction. PNRC was found to interact with ERalpha mainly through its C-terminus region, amino acids 270-327, and an SH3-binding motif within this region was shown to be essential for PNRC to interact with and function as coactivator of ERalpha. The importance of the flanking sequences of SH3-binding motif in the interaction between PNRC and ERalpha was also investigated. The PNRC-interacting domain(s) on ERalpha was also mapped. PNRC was found to interact with both AF1 and LBD of ERalpha, and to function as a coactivator for both AF1 and AF2 transactivation functions. The interaction of ERalpha mutants, I358R, K362A, V376R, L539R and E542K, with PNRC/PNRC2 was further investigated. ERalpha/HBD/V376R could bind to PNRC or PNRC2, with similar affinity as wild-type ERalpha/HBD, and the transactivation activity of ERalpha/V376R was enhanced 5-fold by PNRC. Since GRIP1, a well-characterized coactivator, was found not to be able to enhance the transactivation function of this mutant, our results indicate that the PNRC-ERalpha interaction interface is not exactly identical to that of GRIP1-ERalpha interaction.