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Abstract
We have determined protein levels of total and individual nuclear retinoic acid (RAR-alpha, -beta, -gamma) and retinoid X (RXR-alpha, -beta, -gamma) receptors by ligand binding, Western analysis, and gel shift assays, in adult human skin, a major retinoid-responsive tissue. Total RARs and RXRs, measured by direct binding of specific ligands, were 0.24 +/- 0.01 fmol/micrograms (n = 13) and 1.26 +/- 0.08 fmol/micrograms (n = 7), respectively. These values calculated on an average per cell basis were 1790 RARs/cell and 9400 RXRs/cell. Similar results were obtained with competitive ligand binding assays. RAR-alpha, -beta, and -gamma were each specifically immunoprecipitated, and their levels determined by ligand binding assays of supernatants and Western analysis of precipitates. RAR-gamma was the most abundant, representing 87% of RAR protein. The remaining 12-14% of RAR protein was RAR-alpha. No RAR-beta was detected. Similar immunoprecipitation studies revealed that RXR-alpha represented 90% of RXR protein expressed in human skin. No RXR-beta or RXR-gamma proteins were detected by Western blot. Supershift gel retardation with antibodies to RARs detected probe-RAR-alpha and probe-RAR-gamma complexes in a 1 to 4 ratio. No probe-RAR-beta complex was detected. With antibodies to both RAR-gamma and RXR, a double supershifted complex was formed, indicating that RAR-gamma/RXR heterodimers bound to the probe. These data demonstrate 1) protein levels of RXRs are five times greater than RARs, 2) relative protein levels of RAR and RXR family members are compatible with their previously described relative mRNA levels, and 3) RXR-alpha/RAR-gamma heterodimers are the major retinoid receptors that have the potential to regulate transcription of target genes, in adult human skin.
Highlights
- We have determined protein levelosf total and indi- target gene transcription, is believed to be the predominant vidualnuclearretinoicacid
Three nuclear receptors, measuredby direct bindingof specific ligands, we0re.24 termed retinoic acid receptor-a (RAR-a),' RAR-p, and RAR-y, d 0.01 fmoUpg and 1.26 t 0.08 fm0Upg, were discovered, which boundand were activated by all-transrespectively. These values calculatedon an average per retinoic acid [14,17,18,19,20,21]. This was followed by identification of cell basis were1790 RARsfcell and9400 RKRaeell
Simi- Studies on the regulation of expression of RARs and in lar ~ ~ o p ~ cipita tstiuodines revealed that RXR-ar cells and tissues have, for the most part, examined levels and represented W o of RXR protein expressed in human location of transcripts by Northern analysis [9, 17,18,19,20,21,26,27,28]
Summary
For direct Western analysis, nuclear extracts (1ml containing 3 mg of protein) were concentrated to 0.1 ml using a Centricon 30 concentrawith isotype control IgG, or removal of RAR-a, RAR-p, or. R.AR--y by immunoprecipitation with specific monoclonal antibodies. The levels of RAR-a, RAR-p, and RAR--y were calculated tor (Amicon, Inc.). Samples (105-210 pg of protein) were analyzed by from decreased [3H]CD367binding, which resulted from spe-. The level (Novex).The monoclonal antibodies utilized were: RAR-a,Ab9dF) 9A6) [39];RAR-p, Ab8p(F)2(8P-lOE2)(40);RAR-7,Ab4$bF) (4y-7A11) [41];RXR-a!,4RX-1F6; RXR+ [54], and RXR-y, 12RX-2D3.Immunoreactive proteins on Western blots were detected by ‘‘’I-goat anti-mouse IgG and radioactivity in bands quantified by a PhosphorImager (Molecular Dynamics). Of =-a was calculated from decreased [3H]9-cis-retinoicacid binding, which resulted from RXR-a immunoprecipitation, taking intoaccount the contribution of RARs to L3H19-cis retinoic acid binding
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