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 1 (Estrogen Related Receptor -1)

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Abstract
PNRC (proline-rich nuclear receptor coregulatory protein) was identified using bovine SF1 (steroido- genic factor 1) as the bait in a yeast two-hybrid screening of a human mammary gland cDNA ex- pression library. PNRC is unique in that it has a molecular mass of 35 kDa, significantly smaller than most of the coregulatory proteins reported so far, and it is proline-rich. PNRC's nuclear localiza- tion was demonstrated by immunofluorescence and Western blot analyses. In the yeast two-hybrid assays, PNRC interacted with the orphan recep- tors SF1 and ERRa1 in a ligand-independent man- ner. PNRC was also found to interact with the li- gand-binding domains of all the nuclear receptors tested including estrogen receptor (ER), androgen receptor (AR), glucocorticoid receptor (GR), pro- gesterone receptor (PR), thyroid hormone receptor (TR), retinoic acid receptor (RAR), and retinoid X receptor (RXR) in a ligand-dependent manner. Functional AF2 domain is required for nuclear re- ceptors to bind to PNRC. Furthermore, in vitro glu- tathione-S-transferase pull-down assay was per- formed to demonstrate a direct contact between PNRC and nuclear receptors such as SF1. Coim- munoprecipitation 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 activation mediated by SF1, ERR1 (estrogen related receptor a-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 278-300, was shown to be critical and sufficient for the interaction with nuclear receptors. This region is proline rich and contains a SH3-binding motif, S-D-P-P-S-P-S. Results from the mutagenesis study demonstrated that the two conserved proline (P) residues in this motif are crucial for PNRC to interact with the nuclear receptors. The exact 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 pro- teins. (Molecular Endocrinology 14: 986-998, 2000)

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