Identification of hAHRR splice variant AHRRΔ8 as the major variant expressed in human tissues. (A) Partial amino acid sequence alignment of AHRRs and AHRs from different species. The top sequence is human AHRR₇₁₅; immediately below it is the sequence of AHRRΔ8. Abbreviations: Hs, human; Mm, mouse; Rn, rat; Xl, frog; Fh, killifish; Tr, Japanese puffer fish; Dr, zebrafish; Mt, tomcod. For GenBank accession numbers, see the legend to Fig. S1 in the supplemental material. (B) hAHRR gene structure. Translated exons are shown in black boxes. The first exon and part of the second exon are untranslated. The additional exon (exon 8) is shown in gray. The position of the Pro185Ala polymorphism is marked by an arrow. (C) Survey of adult human tissues for the expression of AHRR₇₁₅ and AHRRΔ8 transcripts. A partial AHRR cDNA fragment was amplified from adult and fetal human cDNAs, using primers flanking exon 8 (shown in gray in panel B). The presence of the exon would have produced a 182-bp PCR product (AHRR₇₁₅), as opposed to the 128-bp product (AHRRΔ8) obtained in all tissues.

Human ARNT dimerizes with AHRRΔ8 but not with AHRR₇₁₅. (A) Synthesis of AHRRΔ8 and AHRR₇₁₅ by in vitro transcription and translation in the presence of [³⁵S]methionine, demonstrating that the two proteins were synthesized at similar levels. (B) In vitro-synthesized AHRRΔ8 and AHRR₇₁₅ were each incubated with radiolabeled ARNT and coimmunoprecipitated by either the hAHRR antibody (PAb-RR-80-2) or IgG. The asterisk indicates that ARNT was labeled with [³⁵S]methionine. The *hARNT lane contains an aliquot of the input labeled protein. The results shown are representative of three independent experiments (see also Fig. S4B in the supplemental material). Numbers at left are molecular masses in kilodaltons.

Effect of ARNT overexpression and increasing TCDD concentrations on the repression of AHR by AHRRΔ8. (A) COS-7 cells were transfected with human AHR (5 ng) and human ARNT (25 and 200 ng) with or without AHRRΔ8 (5 ng). The presence of excess ARNT did not rescue the repression of AHR by AHRRΔ8. The results shown are representative of three independent experiments. (B) HepG2 cells were transfected with the luciferase reporter pGudLuc6.1 with or without additional ARNT (50 ng). Cells were dosed with DMSO or increasing concentrations of TCDD, followed by luciferase assays. Cotransfection of AHRRΔ8 (100 ng) caused repression of the endogenous AHR. The extent of repression was unaffected by the presence of additional ARNT.

AHRRΔ8 does not inhibit TCDD-induced nuclear localization of AHR. COS-7 cells were transfected with 350 ng each of a mouse AHR-YFP fusion construct and human ARNT without (A) or with (B) 350 ng of the hAHRRΔ8 construct. Cells were dosed with DMSO or TCDD (1 nM) for 6 h. After being fixed in formaldehyde, the cells were mounted onto slides and viewed under a fluorescence microscope (Zeiss Imager.Z1). For each treatment group, two representative images are shown.

Specificity of repression by AHRRΔ8. (A) PXR. COS-7 cells were transfected with the human PXR construct (100 ng) and the luciferase reporter XREM-tkLuc (50 ng) with or without AHRRΔ8 (100 ng), AHR (5 ng), and/or ARNT (25 ng). Cells were dosed with clotrimazole (CTRZ) (10 μM final concentration) with or without TCDD (10 nM), followed by a luciferase assay. (B) ERα. COS-7 cells were transfected with the human ERα construct (20 ng) and the luciferase reporter 3×ERE-TATA-luc (50 ng) with or without AHRRΔ8 (50 and 100 ng) and with or without ARNT (25 ng). Cells were dosed with 17β-estradiol (E2) (10 nM final concentration), followed by a luciferase assay. (C and D) HIF. (C) HepG2 cells were transfected with increasing amounts of AHRRΔ8 (0, 50, 100, and 200 ng) and the hypoxia-responsive reporter 6×HRE-luc (10 ng). Cells were dosed with cobalt chloride (CoCl) (150 μM final concentration), followed by a luciferase assay. (D) COS-7 cells were transfected with 6×HRE-luc (10 ng), along with AHRRΔ8 (0 or 5 ng) and ARNT (0 or 200 ng). Cells were dosed with cobalt chloride (CoCl) (150 μM final concentration), followed by a luciferase assay. The results shown in each panel are representative of three or four independent experiments.

Repressor activity of hAHRR splice variants AHRR₇₁₅ and AHRRΔ8. (A) The hAHRR antibody does not recognize human AHR or AHRRs from other species. Cell lysates from COS-7 cells transiently expressing the indicated constructs were blotted and probed with antibody PAb-RR-80-2 raised against the hAHRR. (B) In transient-transfection assays in COS-7 cells, AHRR₇₁₅ and AHRRΔ8 are expressed at similar levels. Cell lysates were blotted and probed with the hAHRR antibody. Numbers at left of panels A and B are molecular masses in kilodaltons. (C) Repression of exogenously expressed AHR by AHRR₇₁₅ and AHRRΔ8 in COS-7 cells. COS-7 cells were transfected with human AHR (5 ng), human ARNT (25 ng), and AHRR₇₁₅ or AHRRΔ8 constructs (50 or 150 ng each), along with a luciferase reporter under the control of dioxin response elements (pGudLuc6.1) and a transfection control plasmid expressing Renilla luciferase (pRL-TK). Cells were dosed with DMSO or TCDD (10 nM final concentration), followed by a luciferase assay. The results shown are representative of seven independent experiments. (D and E) Repression of endogenous AHR in HepG2 (D) and MCF-7 (E) cells by AHRR₇₁₅ and AHRRΔ8. Cells were transfected with AHRR₇₁₅ or AHRRΔ8 constructs (25 and 100 ng each), along with a luciferase reporter under the control of dioxin response elements (for HepG2, pGudLuc6.1; for MCF-7, Cyp1a1-Luc) and pRL-TK. Cells were dosed with DMSO or TCDD (10 nM final concentration), followed by a luciferase assay. The results shown in panels D and E are representative of two and three independent experiments, respectively.

Repression of AHR transcriptional activation by AHRRΔ8-Ala¹⁸⁵ and AHRRΔ8-Pro¹⁸⁵ polymorphic variants of the hAHRR. (A) In transient-transfection assays in COS-7 cells, AHRRΔ8-Ala¹⁸⁵ and AHRRΔ8-Pro¹⁸⁵ are expressed at similar levels. COS-7 cells were transfected with 50, 100, or 150 ng of the hAHRR constructs. The cell lysates were blotted and probed with an antibody against the hAHRR. (B) COS-7 cells were transfected with human AHR (5 ng), human ARNT (25 ng), and AHRRΔ8-Ala¹⁸⁵ or AHRRΔ8-Pro¹⁸⁵ constructs (1, 5, 10, and 25 ng), along with pGudLuc6.1 and pRL-TK. Cells were dosed with DMSO or TCDD (10 nM final concentration), followed by a luciferase assay. (C) The data for TCDD-inducible luciferase activity in panel B were expressed as percent repression in comparison to the “AHR-plus-ARNT only” group. AHRRΔ8-Ala¹⁸⁵ and AHRRΔ8-Pro¹⁸⁵ repressed AHR-ARNT transactivation of pGudLuc6.1 to the same extent. The results shown are representative of three independent experiments.

Overexpression of AHR coactivators does not rescue repression by AHRR. (A) Determination of the minimal amount of AHRRΔ8 needed to repress AHR by 80%. COS-7 cells were transfected with 5 ng each of human AHR and ARNT constructs and increasing amounts (1, 5, 10, 25, and 50 ng) of the hAHRRΔ8 construct, along with the pGudLuc6.1 luciferase reporter and pRL-TK. Cells were dosed with DMSO or TCDD (1 nM), followed by luciferase assays. (B) COS-7 cells were transfected with 5 ng of human AHR and 25 ng of human ARNT, with and without 5 ng of hAHRRΔ8 construct and 200 ng each of the different receptor coactivators. The results shown are representative of three independent experiments.

Coimmunoprecipitation of in vitro-synthesized proteins. Proteins were synthesized by in vitro transcription and translation and incubated with radiolabeled ARNT or AHR. Protein complexes were immunoprecipitated with either the hAHRR antibody (PAb-RR-80-2) or an ARNT antibody (MA1-515). IgG was used to detect nonspecific complexes. The asterisks indicate proteins that were labeled with [³⁵S]methionine. The results shown are representative of at least three independent experiments. Additional results and a summary are provided in Fig. S4 in the supplemental material. Numbers at left are molecular masses in kilodaltons.