Studies of the type I cellular retinoic acid-binding protein mutants and their biological activities.
We have mutated the type I cellular retinoic acid binding protein (CRABP-I), individually at the Arg131 (into Ala) and the Tyr133 (into Phe) residues which have been predicted to make direct contact with retinoic acid (RA) based upon previous structural studies. The RA-binding affinities of these mutants are examined and their biological effects on RA induction of reporter genes are determined. The R131A mutation drastically affects its ligand-binding property, but the Y133F mutation has little effect. By using an RA-inducible reporter, it is found that the wild type CRABP-I exerts biphasic effects on RA induction of the reporter. The early (at 12 h) effect is to enhance RA induction, whereas the delayed (at 24 h) effect is to suppress RA induction. In consistence with their RA binding property, the R131A mutant loses both its early and delayed biological activities, whereas the Y133F mutant remains as effective as the wild type. It is concluded that CRABP-I over-expression exerts biphasic effects on RA-mediated gene expression, and that Arg131, but not Tyr133, is essential for a high RA-binding affinity of this protein as well as its biological activity.
Wei
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Department of Pharmacology, University of Minnesota Medical School, Minneapolis 55455, USA.
Chang
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HU
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D000077556:Alitretinoin; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter / Q000187:drug effects; D006801:Humans; D008156:Luciferases / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D018168:Receptors, Retinoic Acid / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014212:Tretinoin / Q000378:metabolism / Q000494:pharmacology
D017931:DNA Primers; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; C082927:retinoic acid binding protein I, cellular; D000077556:Alitretinoin; D014212:Tretinoin; D008156:Luciferases