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Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists
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Functional analyses of an LXXLL motif in nuclear receptor corepressor (N-CoR)

Kristina Loinder1, Mats Söderström

  • 1Department of Biomedicine and Surgery, Division of Cell Biology, Linköping University, S-58185 Linköping, Sweden.

The Journal of Steroid Biochemistry and Molecular Biology
|September 1, 2004
PubMed

Insights

The nuclear receptor corepressor (N-CoR) contains an LXXLL motif that mediates its interaction with nuclear receptors like retinoic acid receptor alpha (RARalpha) and thyroid hormone receptor beta (TRbeta). This motif is crucial for transcriptional repression in various cellular processes.

Area of Science:

  • Molecular Biology
  • Gene Regulation
  • Cellular Differentiation

Background:

  • Transcriptional repression is vital for cell differentiation, organogenesis, and oncogenesis.
  • Nuclear receptor corepressor (N-CoR) and SMRT are key repressors of ligand-dependent transcription factors.
  • Nuclear receptor coactivators often utilize LXXLL motifs for ligand-dependent interactions.

Purpose of the Study:

  • To investigate the functional significance of the LXXLL motif in N-CoR.
  • To determine if the LXXLL motif in N-CoR mediates interactions with nuclear receptors.

Main Methods:

  • Utilized glutathione S-transferase (GST) pull-down assays to test in vitro interactions.
  • Employed a mammalian one-hybrid system and bacterial two-hybrid assays for in vivo binding studies.
  • Performed site-directed mutagenesis (LDNLL to LDNAA) to assess interaction specificity.

Main Results:

  • A 57-amino acid portion of N-CoR containing the LDNLL sequence (N-CoR(LDNLL)) interacted with retinoic acid receptor alpha (RARalpha) and thyroid hormone receptor beta (TRbeta) in vitro.
  • N-CoR(LDNLL) demonstrated binding to RARalpha both in vitro and in vivo.
  • Mutation of the LDNLL motif abolished the interaction, confirming its specificity.

Conclusions:

  • The LDNLL motif within N-CoR plays a functional role in mediating interactions with nuclear receptors.
  • This motif is essential for the interaction between N-CoR and nuclear receptors such as RARalpha and TRbeta.
  • Findings highlight the importance of the LXXLL motif in N-CoR's corepressor function.

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