Redefining the role of the endogenous XAP2 and C-terminal hsp70-interacting protein on the endogenous Ah receptors expressed in mouse and rat cell lines.
Studies using transient expression systems have implicated the XAP2 protein in the control of aryl hydrocarbon receptor (AHR) stability and subcellular location. Thus, studies were performed in cell lines that expressed endogenous rat or mouse Ah(b-1) (C57BL/6) or Ah(b-2) (C3H) AHRs with similar levels of endogenous XAP2. Unliganded rat and mouse Ah(b-2) receptor complexes associated with reduced levels of XAP2 and exhibited dynamic nucleocytoplasmic shuttling in comparison with Ah(b-1) receptors. Rat and mouse Ah(b-2) receptors also exhibited a greater magnitude of ligand-induced degradation than Ah(b-1) receptors. Small interfering RNA reduction of endogenous XAP2 by >80% had minimal impact on the level of Ah(b-2) receptors but resulted in a 25-30% reduction of Ah(b-1) receptors. XAP2 reduction resulted in increased susceptibility of the Ah(b-1) receptor to ligand-induced degradation yet produced higher levels of endogenous CYP1A1 induction. Stable expression of the Ah(b-2) receptor in the C57BL/6 background resulted in a protein with reduced association with XAP2, dynamic nucleocytoplasmic shuttling, and increased levels of ligand-induced degradation. Small interfering RNA reduction of endogenous XAP2 in a C-terminal hsp70-interacting protein knockout mouse cell line, exhibited a 25-30% reduction in the level of endogenous Ah(b-1) AHR and showed high levels of ligand-induced degradation. Thus, endogenous XAP2 exerts a negative function on a small fraction of the endogenous Ah(b-1) receptor complex but appears to have a minimal impact on endogenous rat or Ah(b-2) receptors. This implies that the analysis of the AHR-mediated signaling via rat and mouse Ah(b-2) receptors may better represent the physiology of this signal transduction pathway.
Pollenz
Richard S
RS
Department of Biology, University of South Florida, Tampa, Florida 33620, USA. pollenz@chuma1.cas.usf.edu
Dougherty
Edward J
EJ
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D019363:Cytochrome P-450 CYP1A1 / Q000096:biosynthesis*; D004790:Enzyme Induction; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D011233:Precipitin Tests; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D018336:Receptors, Aryl Hydrocarbon / Q000235:genetics / Q000378:metabolism*; D012235:Rhodamines
D005456:Fluorescent Dyes; D006360:Heat-Shock Proteins; D047908:Intracellular Signaling Peptides and Proteins; D000072317:Polychlorinated Dibenzodioxins; D011506:Proteins; D034741:RNA, Small Interfering; D018336:Receptors, Aryl Hydrocarbon; D012235:Rhodamines; C103265:aryl hydrocarbon receptor-interacting protein; D019363:Cytochrome P-450 CYP1A1