Structure and function of the Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. Species difference in molecular properties of the receptors from mouse and rat hepatic cytosols.

M S Denison,A B Okey,L M Vella

doi:10.1016/s0021-9258(17)35611-9

Abstract

Molecular properties of cytosolic Ah receptors from livers of Sprague-Dawley rats and C57BL/6N mice were assessed by velocity sedimentation on sucrose gradients and by gel permeation chromatography on Sephacryl S-300. Analyses were done under conditions of both moderate ionic strength (presence of 0.1 M KCl) and high ionic strength (0.4 M KCl). [3H] 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was used as the radioligand. In conditions of moderate ionic strength the receptor from Sprague-Dawley rat liver sedimented at 8.8 +/- 0.05 S, had a Stokes radius of 7.0 +/- 0.21 nm, and an apparent relative molecular mass (Mr) of 257,000 +/- 7,700. In conditions of high ionic strength the Ah receptor from rat hepatic cytosol dissociated to a [3H]TCDD-binding subunit which sedimented at 5.6 +/- 0.58 S, had a Stokes radius of 5.2 +/- 0.24 nm, and an apparent Mr of 121,000 +/- 5,600. The Ah receptor from liver of C57BL/6N mice, in moderate ionic strength conditions, sedimented at 9.4 +/- 0.54 S, had a Stokes radius of 7.1 +/- 0.12 nm, and an apparent Mr of 277,000 +/- 4,800. Whereas the Ah receptor from rat liver readily dissociated into a [3H]TCDD-binding subunit during brief exposure to 0.4 M KCl, the mouse Ah receptor resisted dissociation. When exposed to 0.4 M KCl for 2 h, the mouse Ah receptor remained at the same molecular size that it had exhibited in moderate ionic strength conditions. Prolonged exposure (16 h) to 0.4 M KCl prior to analysis partially converted the mouse Ah receptor into a smaller [3H]TCDD-binding subunit which sedimented at 4.9 +/- 0.07 S, had a Stokes radius of 5.2 +/- 0.19 nm, and an apparent Mr of 105,000 +/- 3,800. The potency of seven different Ah receptor agonists in competing with [3H]TCDD for specific receptor sites was slightly different in mouse cytosol than in rat cytosol. By criteria of size, response to high ionic strength environments, and ligand binding preferences the mouse and rat Ah receptors appear to be similar but not identical molecular species.

Highlights

From the Division of ClinicalPharmacology, Department of Pediatrics, Research Institute, TheHospital for Sick Children and the Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada M5G 1x8
In conditions of moderate ionic strength the receptor from Sprague-Dawley rat liver sedimented at 8.8 f 0.05 S, had a Stokes radius of 7.0 f 0.21 nm, and an apparent relativemolecular mass(M,)
In conditionsof high ionicstrength the Ah receptor from rat hepatic cytosol dissociatedto a [3H]TCDD-bindingsubunit which sedimentedat 5.6 f 0.58 S, had a Stokes radius of 5.2 f 0.24 nm, and an atoma cell line has been characterized which appears to produce an unknown factor that stimulates responses normally mediated via the Ah receptor (9)

Summary

AND DISCUSSION

Supernatant (cytosol) was stored frozen in liquid nitrogen until use. Protein concentration was determined by the method of Bradford (22). Repurification of [3H]TCDDsubstantially reduced the amount of radioactivity eluting in the void volume, but the area under the specific binding peak was similar with commercial [3H]TCDDto that with the repurified [3H]TCDD.The main effect of radioactive contaminants in the commercial [3H]TCDDwas to increase the background radioactivity in Sephacryl S-300 profiles. We incubated rat blood serum with [3H]TCDD, analyzed the labeled serum0.n Sephacryl S-300 to determine the extent of [3H]TCDDbinding in serum and to determinewhich areas of the chromatogram might contain serum components that bind [3H]TCDD. For this experiment the serum was diluted to a concentration of 6.1 mgof protein/ml to make the serum protein concentration comparable to the concentration of protein used in experiments on hepatic cytosols. The concentration of these components in blood serum, does not seem sufficiently high to totally account for the [3H]TCDD-bindingpeaks that elute in the void volume whernat hepatic cytosolis analyzednor to totally

FRACTION NUMBER

Physicochemical PropertiesofAh Receptor

Vo Th Fr ABI SA Ov CyC

By direct application to sucrose gradient

Hepatic cytosol from

PhysicPorcohpeemrticeasl ofA h Receptor