Expression Cloning and Characterization of PREB (Prolactin Regulatory Element Binding), a Novel WD Motif DNA-Binding Protein with a Capacity to Regulate Prolactin Promoter Activity

Abstract

Previous studies have implied that a transcription factor(s) other than Pit-1 is involved in homeostatic regulation of PRL promoter activity via Pit-1-binding elements. One such element, 1P, was employed to clone from a rat pituitary cDNA expression library a novel 417-amino acid WD protein, designated PREB (PRL regulatory element binding) protein. PREB contains two PQ-rich potential transactivation domains, but no apparent DNA-binding motif, and exhibits sequence-specific binding to site 1P, to a site nonidentical to that for Pit-1. The PREB gene (or a related gene) is conserved, as an apparently single copy, in rat, human, fly, and yeast. A single approximately 1.9-kb PREB transcript accumulates in GH3 rat pituitary cells, to levels similar to Pit-1 mRNA. PREB transcripts were detected in all human tissues examined, but the observation of tissue-specific multiple transcript patterns suggests the possibility of tissue-specific alternative splicing. RT-PCR analysis of human brain tumor RNA samples suggested region-specific expression of PREB transcripts in brain. Western and immunocytochemical analysis implied that PREB accumulates specifically in GH3 cell nuclei. Transient transfection employing PREB-negative C6 rat glial cells showed that PREB is as active as, and additive with, Pit-1 in transactivation of a PRL promoter construct, and that PREB, but not Pit-1, can mediate transcriptional activation by protein kinase A (PKA). Expression in GH3 cells of a GAL4-PREB fusion protein both strongly transactivated a 5XGAL indicator construct and yielded a further stimulation of expression of this construct by coexpressed PKA, implying that PREB can mediate both basal and PKA-stimulated transcriptional responses in pituitary cells. These observations imply that PREB will prove to play a significant transcriptional regulatory role, both in the pituitary and in other organs in which transcripts of its gene are expressed.

Prolactin Regulatory Element Binding Protein Is Involved in Hepatitis C Virus Replication by Interaction with NS4B

“…PREB is mainly located in the ER and is detected in a variety of tissues, including human liver (39). PREB is also called the mammalian guanine nucleotide exchange factor (Sec12) (39,40).…”

“…PREB is mainly located in the ER and is detected in a variety of tissues, including human liver (39). PREB is also called the mammalian guanine nucleotide exchange factor (Sec12) (39,40). Sec12 is known to promote COPII vesicle DRMs from transfected cells were treated with trypsin (25 g/ml) at 37°C for 10 min, as indicated, and were then used to determine the protein level by an immunoblotting assay using anti-PREB, anti-NS4B, and anti-NS5A antibodies.…”

The transcription factor prolactin regulatory element-binding protein mediates prolactin transcription induced by thyrotropin-releasing hormone in GH3 cells

“…An antibody targeting a portion of the PREB protein sequence, between amino acid residues 330 and 410 [3], was generated. The corresponding cDNA fragment was amplified from rat pituitary cDNA by polymerase chain reaction (PCR).…”

“…This selective expression is due to a pituitary-specific transcription factor (Pit-1) that regulates the expression of the gene encoding PRL in these cells [1,2]. The Pit-1 binding motif of PRL was used to screen a rat pituitary cell line library [3], and a cDNA encoding PRL regulatory element-binding protein (PREB) was isolated. PREB binds to and activates the PRL promoter, and it can also mediate the action of protein kinase A (PKA) [3].…”

“…The Pit-1 binding motif of PRL was used to screen a rat pituitary cell line library [3], and a cDNA encoding PRL regulatory element-binding protein (PREB) was isolated. PREB binds to and activates the PRL promoter, and it can also mediate the action of protein kinase A (PKA) [3]. The predicted amino acid sequence of PREB had three regions of high similarity with the tryptophanaspartic acid (WD)-repeat consensus sequence and two potential trans-regulatory PQ-rich regions.…”