Characterization of human cellular retinoic acid-binding proteins-I and -II: ligand binding affinities and distribution in skin.
Cellular retinoic acid-binding proteins (CRABPs) are a family of proteins that specifically bind retinoic acid (RA) and have been implicated in mediating its action, although their exact function is still unknown. Two CRABPs have been identified and cloned. CRABP-I is present in many tissues and cultured cells; and CRABP-II, first detected in embryonic and neonatal skin of rats and chicks, is now recognized as the predominant form in human epidermis. Previous studies of CRABP protein expression and function could not distinguish between the two forms and perhaps for that reason have yielded conflicting results, particularly with regard to RA-binding affinity in human tissues. In the present study, we have used the FLAG technology to generate recombinant CRABP-II and developed an anion-exchange HPLC assay in order to allow an accurate discrimination of the two proteins. CRABP-II eluted first with a retention time of 6 min, and CRABP-I with a retention time of 14 min. Both CRABP-II and CRABP-I were found to be expressed in human skin, CRABP-II by fibroblasts and keratinocytes and CRABP-I by as yet unidentified cells. This divergent origin supports the hypothesis that CRABP-II and CRABP-I differentially mediate RA effects. Binding studies demonstrated that CRABP-I and CRABP-II possess two classes of RA-binding sites: one class of high-affinity binding sites with a constant of dissociation (Kd) of 1.5 nM for CRABP-I and 4.7 nM for CRABP-II and one class of low-affinity binding sites with a Kd of 69 nM for CRABP-I and 101 nM for CRABP-II. These data further elucidate the complex regulation of retinoid effects in human skin.
Sanquer
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USDA Human Nutrition Research Centre on Aging, Tufts University, Boston, Massachusetts.
Gilchrest
B A
BA
D001483:Base Sequence; D001665:Binding Sites; D005347:Fibroblasts / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D015603:Keratinocytes / Q000737:chemistry / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012867:Skin / Q000737:chemistry* / Q000378:metabolism; D014212:Tretinoin / Q000378:metabolism
D012333:RNA, Messenger; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; C082927:retinoic acid binding protein I, cellular; C082928:retinoic acid binding protein II, cellular; D014212:Tretinoin