PNRC: A proline-rich nuclear receptor coregulatory protein that modulates transcriptional activation of multiple nuclear receptors including orphan receptors SF1 (steroidogenic factor 1) and ERR alpha 1 (estrogen related receptor alpha-1)

PNRC (proline-rich nuclear receptor coregulatory protein) was identified us ing bovine SF1 (steroidogenic factor 1) as the bait in a yeast two-hybrid s creening of a human mammary gland cDNA expression library. PNRC is unique i n that it has a molecular mass of 35 kDa, significantly smaller than most o f the coregulatory proteins reported so far, and it is proline-rich. PNRC's nuclear localization was demonstrated by immunofluorescence and Western bl ot analyses. In the yeast two-hybrid assays, PNRC interacted with the orpha n receptors SF1 and ERR alpha 1 in a ligand-independent manner. PNRC was al so found to interact with the ligand-binding domains of all the nuclear rec eptors tested including estrogen receptor (ER), androgen receptor (AR), glu cocorticoid receptor (GR), progesterone receptor (PR), thyroid hormone rece ptor (TR), retinoic acid receptor (RAR), and retinoid X receptor (RXR) in a ligand-dependent manner. Functional AF2 domain is required for nuclear rec eptors to bind to PNRC. Furthermore, in vitro glutathione-S-transferase pul l-down assay was performed to demonstrate a direct contact between PNRC and nuclear receptors such as SF1. Coimmunoprecipitation experiment using Hela cells that express PNRC and ER was performed to confirm the interaction of PNRC and nuclear receptors in vivo in a ligand-dependent manner. PNRC was found to function as a coactivator to enhance the transcriptional activatio n mediated by SF1, ERR1 (estrogen related receptor alpha-1), PR, and TR. By examining a series of deletion mutants of PNRC using the yeast two-hybrid assay, a 23-amino acid (aa) sequence in the carboxy-terminal region, aa 273 -300, was shown to be critical and sufficient for the interaction with nucl ear receptors. This region is proline rich and contains a SH3-binding motif , S-D-(P) under bar-P-S-(P) under bar-S. Results from the mutagenesis study demonstrated that the two conserved proline ((P) under bar) residues in th is motif are crucial for PNRC to interact with the nuclear receptors. The e xact 23-amino acid sequence was also found in another protein isolated from the same yeast two-hybrid screening study. These two proteins belong to a new family of nuclear receptor coregulatory proteins.