Different Classes of Coactivators Recognize Distinct but Overlapping Binding Sites on the Estrogen Receptor Ligand Binding Domain

Frank C.S Eng,Annie Barsalou,Naotake Akutsu,Isabelle Mercier,Christina Zechel,Sylvie Mader,John H White

doi:10.1074/jbc.273.43.28371

Abstract

We have analyzed interaction of coactivators with the wild-type estrogen receptor alpha (ER), HEG0, and a mutant, L536P-HEG0, which is constitutively active in several transiently transfected cells and a HeLa line that stably propagates an estrogen-sensitive reporter gene. Different classes of coactivators do not recognize the ER ligand binding domain (LBD) in the same manner. Steroid receptor coactivator-1 (SRC-1), amplified in breast cancer-1 (AIB-1), transcriptional intermediary factor-1 (TIF-1), transcriptional intermediary factor-2 (TIF-2), and receptor interacting protein 140 (RIP140) interacted with HEG0 and L536P-HEG0 in the presence of estradiol, but generally not in the presence of anti-estrogens. However, ICI164,384 stimulated some interaction of RIP140 with LBDs. SRC-1, AIB-1, and RIP140 interacted constitutively with the L536P ER, whereas TIF-1 and TIF-2 interacted only weakly in the absence of hormone. Reciprocal competition for binding to the ER LBD was observed between different classes of coactivators. Moreover, coexpression of RIP140 blocked enhanced transactivation by HEG0 observed in the presence of TIF-2, suggesting that RIP140 may play a negative role in ER signaling. We conclude that constitutive activity of L536P-HEG0 is manifested to similar degrees in different cell types and likely arises from constitutive coactivator binding; different classes of coactivators recognize distinct but overlapping binding sites on the ER LBD. Finally, the observation that L536P-HEG0 interacted constitutively with AIB-1, a coactivator that has been implicated in ER signaling in breast and ovarian cancer, suggests that similar mutations in the ER may contribute to hormone-independent proliferation of breast and ovarian cells.

Highlights

The estrogen receptor (ER)1 is a member of the family of nuclear receptors [1,2,3,4,5,6]
Different classes of coactivators have been identified, including Steroid receptor coactivator-1 (SRC-1) and related proteins such as pCIP/amplified in breast cancer-1 (AIB-1)/RAC3 [12,13,14,15,16], transcriptional intermediary factor-2 (TIF-2)/GRIP-1 [17, 18], receptor interacting protein 140 (RIP140) [19], transcriptional intermediary factor-1 (TIF-1)␣ and -␤ [20, 21], all of which apparently interact with receptors through signature motifs containing the core LXXLL [14, 21, 22]
Given the evidence that different coactivators may function by distinct mechanisms, it is possible that recruitment of several coactivators to a target gene would stimulate preinitiation complex formation through multiple pathways

Summary

Introduction

The estrogen receptor (ER) is a member of the family of nuclear receptors [1,2,3,4,5,6]. Similar to other nuclear receptors, the ER is a ligand-activated regulator of transcription that functions through stimulating formation of transcriptional preinitiation complexes. The ER stimulates the assembly of these components through interaction with factors collectively known as coactivators that interact with the receptor ligand binding domain (LBD) in the presence of hormone [11]. This interaction requires the AF-2 activating function, located at the extreme C terminus of the LBD. Active templates in transient transfection assays may not require extensive chromatin remodeling and, all of the components essential for preinitiation complex formation on endogenous genes. Using ligand binding domains from the wild-type receptor and a constitutively active

Methods

Results

Conclusion