Core LXXLL Motif Sequences in CREB-binding Protein, SRC1, and RIP140 Define Affinity and Selectivity for Steroid and Retinoid Receptors

David M Heery,Susan Hoare,Sagair Hussain,Malcolm G Parker,Hilary Sheppard

doi:10.1074/jbc.m009404200

Abstract

An alpha-helical motif containing the sequence LXXLL is required for the ligand-dependent binding of transcriptional co-activators to nuclear receptors. By using a peptide inhibition assay, we have defined the minimal "core" LXXLL motif as an 8-amino acid sequence spanning positions -2 to +6 relative to the primary conserved leucine residue. In yeast two-hybrid assays, core LXXLL motif sequences derived from steroid receptor co-activator (SRC1), the 140-kDa receptor interacting protein (RIP140), and CREB-binding protein (CBP) displayed differences in selectivity and affinity for nuclear receptor ligand binding domains. Although core LXXLL motifs from SRC1 and RIP140 mediated strong interactions with steroid and retinoid receptors, three LXXLL motifs present in the global co-activator CBP were found to have very weak affinity for these proteins. Core motifs with high affinity for steroid and retinoid receptors were generally found to contain a hydrophobic residue at position -1 relative to the first conserved leucine and a nonhydrophobic residue at position +2. Our results indicate that variant residues in LXXLL core motifs influence the affinity and selectivity of co-activators for nuclear receptors.

Highlights

Ligand-dependent gene expression mediated by nuclear receptors involves the recruitment of transcriptional co-activators to the ligand binding domain (LBD).1 The LBD is structurally conserved among the NR family and consists of between 10 and 12 ␣-helices folded in a globular domain [1]
Consistent with this, it has been previously observed that peptides of 13 and 14 amino acids in length encompassing motif 2 of GRIP1 strongly compete the interaction of the GRIP1 nuclear receptor interaction domain (NID) with the TR␤ LBD, whereas the hexapeptide LLRYLL did not [6]
The crystal structure of the LBD of TR␤ in complex with a synthetic peptide corresponding to motif 2 of GRIP1 showed that the peptide adopts an amphipathic ␣-helix conformation of nearly 3 turns from Lys-688 to Gln-695

Summary

Introduction

Ligand-dependent gene expression mediated by nuclear receptors involves the recruitment of transcriptional co-activators to the ligand binding domain (LBD). The LBD is structurally conserved among the NR family and consists of between 10 and 12 ␣-helices folded in a globular domain [1]. The 160-kDa co-activators represented by SRC1, TIF2, and p/CIP each have a nuclear receptor interaction domain (NID) containing three LXXLL motifs These sequences and the spacing between them are highly conserved, and we and others [3, 5, 7, 16, 17] have shown that they mediate high affinity binding to NRs. In addition, the SRC1a isoform has an additional LXXLL motif at its C terminus [16]. By comparing the interactions of LXXLL core motifs derived from three different coactivators with a panel of NR LBDs, we have identified elements of the LXXLL core sequence that influence the affinity and selectivity of co-activators for NRs

Methods

Results

Conclusion