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

Abstract

An ␣-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.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). A central hydrophobic pocket accommodates the cognate ligand, which upon binding induces a conformational change in the LBD, exposing a co-activator docking site on the LBD surface. Sequence conservation, mutational analyses, and crystal structures have indicated that the co-activator docking site is made up of residues from helices 3, 5, and 12, which form a hydrophobic channel conserved among the NR family members. The integrity of this surface is essential for co-activator binding and as a consequence the transactivation function (AF-2) of the LBD (reviewed in Ref.2).We have previously shown that a short ␣-helical sequence, the LXXLL motif, is necessary and sufficient for mediating the interaction of the co-activators RIP140, SRC1 and CBP/p300 with NRs (3). LXXLL motifs have also been shown to mediate binding of other co-activators including TIF1 (4), TIF2/GRIP1 (5, 6), p300/CBP-interacting protein/ACTR (7, 8), TRAP220/ DRIP205 (9 -11), PPAR␥ co-activator-1 (12), and ASC-2/ RAP250/NRC (13-15) to NRs. The number and sequence of LXXLL motifs varies considerably among the co-activators and is likely to account for observed differences in binding of coactivators to selected NRs or classes of NRs. 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 addi...

An Extended LXXLL Motif Sequence Determines the Nuclear Receptor Binding Specificity of TRAP220

“…For example, TIP60 has been reported to bind the Class I receptors, but displayed little interaction with VDR, TR, or RXR (22). The p160s interact with a wide range of NRs, although individual LXXLL motifs derived from these proteins display differential binding to NRs (14,38). In addition, we have shown that LXXLL sequences derived from CBP and RIP140 show selectivity that is at least partly determined by the LXXLL core sequences (14).…”

“…The constructs AAD-AR-LBD-(625-919), AAD-PR-LBD-(633-933) (44), AAD-ER␣-LBD-(282-595) (4), AAD-RAR␣-LBD-(200 -462), and AAD-RXR␣-LBD-(230 -467) (14) have been described previously. The constructs AAD-PPAR␥-LBD-(173-475) and AAD-TR␤-(1-456) were generated by cloning PCR fragments into the modified pASV3.…”

“…The NID and Core LXXLL Motifs of TRAP220 Exhibit Distinct NR Binding Specificities-It has been shown previously that 8 -10 amino acid sequences encompassing the signature motif LXXLL are sufficient to bind to liganded NR LBDs, and this has been referred to as the core LXXLL motif (4,14). To explore the nature of the specificity exhibited by the TRAP220 NID in more detail, we assessed the ability of LXXLL core motifs derived from SRC1 and TRAP220 to bind NRs.…”

“…This channel accommodates the LXXLL ␣-helix, which is held in place by hydrophobic interactions and a charged clamp involving two amino acids (lysine and glutamate) that are conserved throughout the NR family (12,13). The minimal sequence that can bind the AF2 surface (the LXXLL core motif) is contained within 8 amino acids (Ϫ1 to ϩ7) (14).…”

“…The p160s interact with a wide range of NRs, although individual LXXLL motifs derived from these proteins display differential binding to NRs (14,38). In addition, we have shown that LXXLL sequences derived from CBP and RIP140 show selectivity that is at least partly determined by the LXXLL core sequences (14). Similarly, LXXLL motifs (or variants such as LXXIL and FXXLL) from other cofactors, such as PERC (31), NRIF3 (32), and NSD1 (33) have been reported to display selectivity in their interactions with NRs.…”