Identification of amino acids critical for the DNA binding and dimerization properties of the human retinoic acid receptor alpha. Importance of lysine 360, lysine 365, and valine 361.
Retinoic acid receptors (RARs) and retinoid X receptors (RXRs) activate target genes by binding to retinoic acid response elements (RAREs) as heterodimeric, asymmetrical complexes, and display a high degree of cooperativity in binding to RAREs. We have examined here the effect of lysine, cysteine, arginine, histidine, and tyrosine side chain chemical modification on the DNA binding, homo- and heterodimerization properties of the full-length human retinoic acid receptor alpha (hRARalpha). Lysines are the only residues to be engaged in the dimerization with human retinoid X receptor alpha (hRXRalpha) in the absence of DNA, whereas histidines are selectively involved in the homodimerization of hRARalpha in the presence of a RARE. Arginine modification affected the DNA binding activity of each type of dimer, whereas cysteines and tyrosines were primarily involved in the homo- or heterodimerization process in the presence of the same RARE. Modified lysines, interfering with the dimerization with hRXRalpha, were identified by receptor labeling and peptide mapping. They are located in the hormone binding domain eighth heptad repeat, at positions 360 and 365. In keeping with these results, mutation of Lys360, Val361, and Lys365 diminished strongly the DNA binding activity of hRARalpha as a homodimer or a heterodimer. Our results thus provide direct evidence for the differential involvement of basic, polar, or aromatic amino acids in the DNA binding, homodimerization, and heterodimerization properties of hRARalpha. Furthermore, they demonstrate the use of distinct dimerization interfaces and identify the type of amino acids involved in these protein-protein interactions.
Rachez
C
C
CJF INSERM 92-03, Laboratoire de Biochimie Structurale, Faculté de Médecine de Lille 1, France.
Sautière
P
P
Formstecher
P
P
Lefebvre
P
P
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D018168:Receptors, Retinoic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014212:Tretinoin / Q000378:metabolism
D000596:Amino Acids; D004268:DNA-Binding Proteins; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D014212:Tretinoin