3. J. T. Stull, M. H. Nunnally, C. H. Michnoff, in The Enzymes: Control by Phosphorylation, P. D. Boyer and E. G. Krebs, Eds. (Academic Press, New York, 1986), vol 17, p. 113. 4. J. M. Anderson, H. Charbonneau, H. P. Jones, R. 0. McCann, M. J. Cormier, Biochemisiiy 19,3113 (1980). 5. R. Ranjeva, G. Refeno, A. M. Boudet, D. Marme, Proc. Nad. Acad. Sci. U. S.A. 80, 5222 (1983). 6. A. C. Harmon, C. Putnam-Evans, M. J. Cormier, Plant Physiol. 83, 830 (1987). 7. C. Putnam-Evans, A. C. Harmon, M. J. Cormier, Biochemistry 29, 2488 (1990). 8. The lambda bacteriophage cDNA library was ob- tained from Clonetech, Palo Alto, CA. 9. J. F. Harper, L. Manney, N. D. DeWitt, M. H. Yoo, M. R. SussmanJ. Biol. Chem. 265, 13601 (1990). 10. P. P. Mueller and A. G. Hinnebush, Cell 45, 201 (1986). 11. Y. Nishizuka, Nature 334, 661 (1988). 12. J. Harper, unpublished cDNA sequence from soy- bean CDPK clone pSK2, Arabidopsis cDNA clone pAkl, and genomic clone pgAKl. 13. S. K. Hanks, A. M. Quinn, T. Hunter, Science 241, 42 (1988). 14. M. K. Bennett and M. B. Kennedy, Proc. Nad. Acad. Sci. U.SA. 84, 1794 (1987). 15. T. J. Lukas, D. B. Iverson, M. Schleicher, D. M. Watterson, Plant Physiol. 75, 788 (.1984). 16. R M. Tufty and R. H. Kretsinger, Science 187, 167 (1975). 17. D. M. Roberts, T. J. Lukas, D. M. Watterson, Crit. Rev. Plant Sci. 4, 311 (1986). 18. K. Suzuki, Trends Biochem. Sci. 12, 103 (1987). 19. G. Hardie, Nature 335, 592 (1988). 20. C. Putnam-Evans, A. C. Harmon, B. A. Palevitz, M. Fechheimer, M. J. Cormier, Cell Motil. Cytoskeleton 12, 12 (1989). 21. G. E. Schaller, A. C. Harmon, M. R. Sussman, in preparation. 22. W-7 is (N-(6-aminohexyl)-5-chloro-1-napthalene sulfonamide). 23. H. Charbonneau et aL, Biochemistry 24, 6762 (1985). 24. M. A. Frohman, M. K. Dush, G. R. Martin, Proc. Natl. Acad. Sci. U. SA. 85, 8998 (1988). 25. We thank S. Kumar and L. Manney for technical assistance with purification of peptides and cDNA sequencing, respectively, M. J. Cormier for fostering the early work on CDPK, R. Amasino for soybean RNA, and K. Walsh for advice and helpful discus- sions. Supported by grants from the USDA (M.R.S. and A.C.H.), DOE (M.R.S.), and NSF (A.C.H.) and a grant to K. Walsh from NIH (GM15731). 13 November 1990; accepted 7 March 1991 Cloning of a Factor Required for Activity of the Ah (Dioxin) Receptor EMILY C. HOFFMAN,* HERmINio REYES,* FONG-FONG CHU,t FRED SANDER,,t LINDA H. CONLEY, BARBARA A. BROOKS, OLIVER HANKINSON§ The aryl hydrocarbon (Ah) receptor binds various environmental pollutants, such as polycyclic aromatic hydrocarbons, heterocyclic amines, and polychlorinated aromatic compounds (dioxins, dibenzofurans, and biphenyls), and mediates the carcinogenic effects of these agents. The complementary DNA and part of the gene for an 87-kilodalton human protein that is necessary for Ah receptor function have been cloned. The protein is not the ligand-binding subunit of the receptor but is a factor that is required for the ligand-binding subunit to translocate from the cytosol to the nucleus after binding ligand. The requirement for this factor distinguishes the Ah receptor from the glucocorticoid receptor, to which the Ah receptor has been presumed to be similar. Two portions of the 87-kilodalton protein share sequence similarities with two Drosophila proteins, Per and Sim. Another segment of the protein shows conformity to the consensus sequence for the basic helix-loop-helix motif found in proteins that bind DNA as homodimers or heterodimers. and P450IA2 are important in the metab- olism of polycyclic aromatic hydrocarbons (found in cigarette smoke and smog) and certain heterocyclic amines (found in cooked meat) to carcinogenic intermedi- ates (3). The pathological effects of the polychlorinated aromatic compounds also depend on the action of the Ah receptor, but the mechanism of pathogenesis is un- known (4). The Ah receptor is a soluble protein com- plex of -280 kD. The -95-kD ligand- binding subunit, which has not been cloned, and the 90-kD heat shock protein (Hsp90) are both components of this complex (5). After cells are treated with ligand, receptor molecules become tightly bound in the nu- cleus. However, the location of the receptor before ligand binding has not been fully resolved (6). After conventional subcellular fractionation, the unoccupied receptor is found in the cytosol. Operationally, there- fore, ligand treatment leads to "nuclear translocation" of the receptor. The Ah re- ceptor resembles the steroid hormone recep- tors (7), and this resemblance has led to the suggestion that the ligand-binding subunit of the Ah receptor may be a member of the steroid receptor superfamily. The mouse hepatoma cell line Hepa-1 shows P4501A1 inducibility. Mutants of Hepa-1 cells defective in induction have been isolated, and those mutants that are recessive have been assigned to four comple- mentation groups (8, 9). Mutations in groups B. C, and D affect functioning of the Ah receptor (9, 10). In the group C mu- tants, the receptor is present in normal amounts but does not translocate to the nucleus after binding ligand. We now de- scribe the isolation of part of the human C gene [termed the Ah receptor nuclear trans- locator gene (arnt)] and the isolation and T HE AH RECEPTOR IS DETECTABLE IN many tissues and organs. The best understood activity of the receptor concerns its role in the induction of cy- tochrome P4SOIAL. Complexes between the Ah receptor and ligand bind to specific DNA sequences upstream of the P450IA1 gene, termed xenobiotic responsive ele- ments (XREs), and stimulate transcription of the gene (1, 2). The receptor also medi- Department of Pathology and Laboratory of Biomedical and Environmental Sciences, University of California, Los Angeles, CA 90024. *The contribution of the first two authors should be considered equal. tPresent adess: Department of Medical Oncology, City of Hope Medical Center, Duarte, CA 91090. tPresent address: Exogene, Monrovia, CA 91016. STo whom correspondence should be addressed. 954 ates induction of cytochrome P450IA2 and several other enzymes of xenobiotic metab- olism. The enzymatic activities of P450IA1 (aryl hydrocarbon hydroxylase, or- AHH) Fig. 1. DNA blot analysis of transfectants. Ge- nomic DNA (10 gg) was digested with Pst I, subjected to agarose gel electrophoresis, and transferred to nitrocellulose. Hybridization was performed in 50% formamide, 5 x SSC (standard saline citrate), 20 mM sodium phosphate (pH 6.5), 10% dextran sulfate, 1x Denhardt's solu- tion, and denatured salmon sperm DNA (250 ,ug/ml) at 42°C. After hybridization, the filter was washed in 0.1 x SSC (standard saline citrate) plus 0.1% SDS at 65°C. The probe was the Bam HI, Alu-containing fragment recloned from pBLUR8 into Ml3mp8 and isolated from the latter. Ar- rows indicate bands common to all secondary transfectants. Numbers at the left margin indicate sizes in kilobases. SCIENCE, VOL. 252 kb 23.7- 9.7- 6.6- 4.3- 2.2- 2.1- _ csj c _1 _y ' ' ;: .7 . C.) ~- I- I-~ 4*- -4- -4- 0.6- on August 23, 2014 www.sciencemag.org Downloaded from on August 23, 2014 www.sciencemag.org Downloaded from on August 23, 2014 www.sciencemag.org Downloaded from on August 23, 2014 www.sciencemag.org Downloaded from on August 23, 2014 www.sciencemag.org Downloaded from