Steroid binding to hepatoma tissue culture cell glucocorticoid receptors involves at least two sulfhydryl groups.

N R Miller,S S Simons

doi:10.1016/s0021-9258(18)68167-0

Abstract

The presence of a thiol in the steroid binding cavity of glucocorticoid receptors has recently been proved by our affinity labeling of Cys-656 in the steroid binding domain of rat receptors (Simons, S. S., Jr., Pumphrey, J. G., Rudikoff, S., and Eisen, H. J. (1987) J. Biol. Chem. 262, 9676-9680). Studies with the sterically small, thiol-specific reagent methyl methanethiolsulfonate (MMTS) now reveal the involvement of at least two sulfhydryl groups in steroid binding. While the dose-response curves for [3H]dexamethasone binding versus thiol reagent are normally sigmoidal, an unusual bimodal curve is obtained with MMTS in which dexamethasone binding is eliminated at low, but maintained at intermediate, MMTS concentrations. This bimodal dose-response curve demands the involvement of two (or more) thiol groups. Those receptors pretreated with intermediate concentrations of MMTS retain approximately 70% of the initial binding capacity and one-fifth the affinity for dexamethasone. Solutions of this low affinity form of receptor contain essentially no accessible -SH groups, and all of the usual covalent labeling by dexamethasone 21-mesylate of various proteins, including the receptor, is blocked. The facts, that this low affinity form of the receptor is not affected by added iodoacetamide, cannot be produced from the nonsteroid binding form of receptor simply by adding more MMTS, and displays different kinetics of formation than does the nonsteroid binding form of receptor all argue that reaction of the receptor with intermediate and low MMTS, concentrations occurs via different pathways. Nevertheless, the effects of both concentrations of MMTS on the receptor are fully reversible with added dithiothreitol. The kinetics of inhibition of [3H]dexamethasone binding at low MMTS concentrations are independent of receptor concentration, indicating an intramolecular reaction. Collectively these data suggest a model of steroid binding involving two thiols, one of which appears to be Cys-656. Low concentrations of MMTS induce the formation of an intramolecular disulfide, which prevents steroid binding, while the intermediate MMTS concentrations convert both thiols directly to mixed disulfides, and steroid binding persists. Thus, reduced thiols do not appear to be required for steroid binding if the steric bulk of the oxidized thiols is small.

Highlights

The presence of a thiol in the steroid binding cavity do not appear tobe required for steroid bindinigf the of glucocorticoid receptors has recently been proved steric bulkof the oxidized thiols is small
A rather large ing versus thiol reagent are normally sigmoidal, an (-250 amino acids) steroid binding domain for all steroid unusual bimodal curve is obtainedwith methyl methanethiolsulfonate (MMTS) in receptors has been defined [1,2,3,4] and one cysteine inthe which dexamethasone binding is eliminateadt low, but steroid binding site of glucocorticoid receptors has recently maintained at intermediate, MMTS concentrations. been identified [5]
The kinetics of tive) DNA sequences is about two-fold less than that of the inhibition of [SH]dexamethasonebinding at low MMTS receptor-agonist complexes [9], but the relative affinity of concentrations are independent of receptor concentra- each type of complex for specific

Summary

Introduction

The presence of a thiol in the steroid binding cavity do not appear tobe required for steroid bindinigf the of glucocorticoid receptors has recently been proved steric bulkof the oxidized thiols is small. This bimodal dose-response curve demands thein- lysine [6,7,8] and arginine [6] may be involved in volvement of two (ormore) thiol groups. MMTS retain -70% of the initial binding capacity andwould be helpful in explaining the different classes of one-fifth the affinity for dexamethasone Solutions of this low affinity form of receptor contain essentially no accessible -SH groups, and allof the usualcovalent labeling by dexamethasone 21-mesylate of various proteins, includingthe receptor, is blocked. All of the information that is required for the expression of agonist versus antagonist activity is necessarily contained in the structure of the steroid, which somehowgives rise to receptor-steroid complexes with different biological and biochemical properties. Collec- cocorticoid regulatory elements) uersus nonspecific DNA setively these data suggest a model of steroid binding quences is the same [11].Despite the nearly identical activainvolving two thiols, one of which appears to be Cys-

Methods

Results

Conclusion