Domains regulating transcriptional activity of the inducible orphan receptor NGFI-B

Abstract

NGFI-B is an early response gene which encodes a protein that has strong homology with nuclear receptors in the DNA binding domain and in carboxyl-terminal domains responsible for ligand binding and regulation of transcriptional activity. Previously, we have demonstrated that NGFI-B is transcriptionally active in cells grown in vitro in the absence of exogenously added ligand. However, the ligand for NGFI-B does not appear to be a component of cell culture medium, as NGFI-B remained active when expressed in cells grown in medium lacking phenol red, serum, essential vitamins, or essential amino acids. To define the transactivation domains, a mutational analysis was conducted which revealed that a serine/threonine-rich area of 18 amino acids within the amino terminus, termed TAB-1, is an important transcriptional activation domain. The mutation of two adjacent serine and threonine residues within TAB-1 significantly decreased transactivation by NGFI-B. An examination of the role of the carboxyl terminus in regulating NGFI-B transcriptional activity revealed that, in accordance with other nuclear receptors, mutants lacking portions of the carboxyl terminus had greatly decreased activity. The similarity with other receptors was further supported by studies with the mutant B delta 414-597 which encodes a fully active, truncated receptor analogous to a hormone-independent, constitutively active glucocorticoid receptor truncation mutant. This NGFI-B truncation mutant had activity similar to wild type NGFI-B in a number of mammalian cell lines; however, in contrast, it was 8-fold more active than the wild type receptor in the Drosophila S2 cell line, suggesting that insect cells either lack the NGFI-B ligand or obligatory accessory factors.