The RING finger binding protein is a nuclear membrane protein that interacts with RUSH transcription factors



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Texas Tech University


Molecular regulation of Uteroglobin gene expression by progesterone and prolactin is mediated by RUSH transcription factors. The RUSH family of proteins, which includes rabbit RUSH-la and P and the human, mouse and plant homologs of RUSH lα, are SWI/SNF related chromatin remodeling proteins. These proteins have a novel C3HC4 RING finger, at their -COOH terminus that has been implicated in mediating protein-protein interactions. When this motif was identified in RUSH proteins, it was used to screen an expression library to isolate cDNAs for proteins that complex with it. A single phage clone (~1.6kb insert) was identified. Sequence analysis of this RING Finger Binding Protein (RFBP) clone, revealed a partial cDNA that lacked an initiator codon but contained a stop codon. RACE PCR was then used to extend the 5' and 3' ends of the cDNA. The predicted amino acid sequence from the composite cDNA sequence (4286-bp) is that of a putative Type IV P-type ATPase. P-type ATPases are membrane transporters that use the energy of ATP hydrolysis to transport substrate across the membrane. Genomic cloning and ClustalW alignment indicate that RFBP is an atypical P-type ATPase that has only seven of eight core regions and nine of ten transmembrane domains typical of this family of proteins. Core region D that contains transmembrane domain four is absent from this protein. Western blot analysis, coupled with immunoelectron microscopy data, indicates that RFBP is present in the inner nuclear membrane. Coimmunoprecipitation and GST pulldown experiments showed a direct interaction between RUSH and RFBP. The RUSH binding site lies within aa 612-804 of the RFBP protein.

Competitive quantitative RT-PCR indicates that RFBP is ubiquitous in its expression, with the expression pattern correlating with that of RUSH in these same tissues. In addition, expression of RFBP is hormonally regulated in the endometrium, suggesting that RFBP function and expression may be closely linked with the function of the RUSH proteins in regulating gene expression in the reproductive system. Current studies provide important information about RFBP as a RUSH binding interaction between the proteins to shed light on the mechanism of hormone regulation of uteroglobin gene expression.