Binding of aryl hydrocarbon receptor (AhR) to AhR-interacting protein. The role of hsp90.
The aryl hydrocarbon receptor (AhR) has been shown to interact with an immunophilin-like molecule known as AhR-interacting protein (AIP) and to enhance AhR function. We show here that AIP associates with AhR homologues from mouse and fish, which can bind ligands such as dioxin, but nonligand binding homologues from Caenorhabditis elegans or Drosophila do not bind to AIP. However, a minimal ligand-binding domain of the AhR is incapable of binding AIP. The binding of AIP to AhR in reticulocyte lysate shows several of the characteristics of an hsp90-dependent process, including sensitivity to geldanamycin and temperature and a requirement for ATP or nonhydrolyzable analogues. Purified AIP binds to the C terminus of hsp90, and mutation of a conserved basic residue in the tetratricopeptide repeats of AIP (K266A, analogous to K97A in protein phosphatase 5) abolishes binding to hsp90. Mutation of K266A in AIP reduces binding to AhR by 75-80%; the geldanamycin sensitivity of this complex shows that AhR stabilizes the AIP-hsp90-AhR complex. The alpha-helical C terminus of AIP, which is outside the tetratricopeptide repeat domain, is absolutely required for binding to AhR as shown by deletions of the C-terminal 5 amino acids or alanine-scanning mutagenesis, but it is not required for binding of AIP to hsp90. The data support a model where 1) AIP binds to both hsp90 and AhR; 2) hsp90 is required for AhR-AIP binding; and 3) the binding of AhR to AIP stabilizes the AIP-hsp90-AhR complex.
Bell
D R
DR
Centers for Disease Control, National Institute for Occupational Safety and Health, Health Effects Laboratory Division, Morgantown, West Virginia 26505, USA. david.bell@nottingham.ac.uk
Poland
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A
D000595:Amino Acid Sequence; D000818:Animals; D016227:Benzoquinones; D001665:Binding Sites; D002457:Cell Extracts; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D047029:Lactams, Macrocyclic; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009711:Nucleotides / Q000494:pharmacology; D011233:Precipitin Tests; D011485:Protein Binding / Q000187:drug effects; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011809:Quinones / Q000494:pharmacology; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012156:Reticulocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature; D014158:Transcription, Genetic
D016227:Benzoquinones; D002457:Cell Extracts; D018841:HSP90 Heat-Shock Proteins; D047908:Intracellular Signaling Peptides and Proteins; D047029:Lactams, Macrocyclic; D009711:Nucleotides; D011506:Proteins; D011809:Quinones; D018336:Receptors, Aryl Hydrocarbon; D011994:Recombinant Proteins; C103265:aryl hydrocarbon receptor-interacting protein; D008239:Lysine; C001277:geldanamycin