Association with Ets-1 Causes Ligand- and AF2-Independent Activation of Nuclear Receptors

Abstract

The vitamin D receptor (VDR) normally functions as a ligand-dependent transcriptional activator. Here we show that, in the presence of Ets-1, VDR stimulates the prolactin promoter in a ligand-independent manner, behaving as a constitutive activator. Mutations in the AF2 domain abolish vitamin D-dependent transactivation but do not affect constitutive activation by Ets-1. Therefore, in contrast with the actions of vitamin D, activation by Ets-1 is independent of the AF2 domain. Ets-1 also conferred a ligand-independent activation to the estrogen receptor and to peroxisome proliferator-activated receptor ␣. In addition, Ets-1 cooperated with the unliganded receptors to stimulate the activity of reporter constructs containing consensus response elements fused to the thymidine kinase promoter. There is a direct interaction of the receptors with Ets-1 which requires the DNA binding domains of both proteins. Interaction with Ets-1 induces a conformational change in VDR which can be detected by an increased resistance to proteolytic digestion. Furthermore, a retinoid X receptor-VDR heterodimer in which both receptors lack the core C-terminal AF2 domain can recruit coactivators in the presence, but not in the absence, of Ets-1. This suggests that Ets-1 induces a conformational change in the receptor which creates an active interaction surface with coactivators even in the AF2-defective mutants. These results demonstrate the existence of a novel mechanism, alternative to ligand binding, which can convert an unliganded receptor from an inactive state into a competent transcriptional activator.Nuclear receptors normally act as ligand-inducible transcriptional factors by binding as homodimers or as heterodimers with the retinoid X receptor (RXR) to hormone response elements (HREs) in target genes (15). Transcriptional regulation by nuclear receptors is achieved through autonomous activation functions (AFs): a constitutive N-terminal AF1 and a C-terminal ligand-dependent AF2. Ligand binding causes a conformational change in the receptors that allows recruitment of CREB binding protein (CBP)-and p160-related coactivator proteins with histone acetylase activity (26). The multisubunit coactivator complex TRAC-DRIP also binds the nuclear receptors in a ligand-and AF2-dependent manner and may interact directly with the basic transcriptional machinery (9, 21). However, recent evidence has shown that several receptors may also be activated in a ligand-independent manner. A variety of agents including growth factors and cyclic AMP activate the receptors, presumably by stimulation of cellular protein kinases which cause receptor phosphorylation (3,12,22,24,31). This activation appears to involve a ligand-and AF2-independent recruitment of p160 coactivators (8, 27). Furthermore, the estrogen receptor (ER) can be also stimulated in a ligand-independent manner by association with cyclin D1 (35). Cyclin D1 interacts with p160 coactivators (36) and also with the acetylase p/CAF (CBP-associated factor) (18) and can recruit the coactivators...

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