Immunological identification and functional quantitation of retinoic acid and retinoid X receptor proteins in human skin.
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.
Fisher
G J
GJ
Department of Dermatology, University of Michigan, Ann Arbor 48109.
Talwar
H S
HS
Xiao
J H
JH
Datta
S C
SC
Reddy
A P
AP
Gaub
M P
MP
Rochette-Egly
C
C
Chambon
P
P
Voorhees
J J
JJ
D001483:Base Sequence; D001667:Binding, Competitive; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D011233:Precipitin Tests; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D012867:Skin / Q000378:metabolism*; D014157:Transcription Factors; D014212:Tretinoin / Q000378:metabolism*
D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D018160:Receptors, Cytoplasmic and Nuclear; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D014157:Transcription Factors; D014212:Tretinoin