Hormone Binding by Protein Disulfide Isomerase, a High Capacity Hormone Reservoir of the Endoplasmic Reticulum

Todd P Primm,Hiram F Gilbert

doi:10.1074/jbc.m007670200

Abstract

Protein disulfide isomerase (PDI) is a folding assistant of the eukaryotic endoplasmic reticulum, but it also binds the hormones, estradiol, and 3,3',5-triiodo-l-thyronine (T(3)). Hormone binding could be at discrete hormone binding sites, or it could be a nonphysiological consequence of binding site(s) that are involved in the interaction PDI with its peptide and protein substrates. Equilibrium dialysis, fluorescent hydrophobic probe binding (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS)), competition binding, and enzyme activity assays reveal that the hormone binding sites are distinct from the peptide/protein binding sites. PDI has one estradiol binding site with modest affinity (2.1 +/- 0.5 microm). There are two binding sites with comparable affinity for T(3) (4.3 +/- 1.4 microm). One of these overlaps the estradiol site, whereas the other binds the hydrophobic probe, bis-ANS. Neither estradiol nor T(3) inhibit the catalytic or chaperone activity of PDI. Although the affinity of PDI for the hormones estradiol and T(3) is modest, the high local concentration of PDI in the endoplasmic reticulum (>200 microm) would drive hormone binding and result in the association of a substantial fraction (>90%) of the hormones in the cell with PDI. High capacity, low affinity hormone sites may function to buffer hormone concentration in the cell and allow tight, specific binding to the true receptor while preserving a reasonable number of hormone molecules in the very small volume of the cellular environment.

Highlights

Peptide/protein interactions are essential for effective catalysis of disulfide formation and isomerization
Hormone binding could be at discrete hormone binding sites, or it could be a nonphysiological consequence of binding site(s) that are involved in the interaction Protein disulfide isomerase (PDI) with its peptide and protein substrates
Ligand Binding—PDI binds a diverse set of ligands (Fig. 1), interacting with unfolded and folded proteins, E2, T3, peptidelike antibiotics such as bacitracin, and peptides such as oxidized insulin chain B (InB)

Summary

EXPERIMENTAL PROCEDURES

Materials—Recombinant rat PDI was expressed and purified as described previously [34]. Bis-ANS was obtained from Molecular Probes, and its concentration was determined by absorbance at 385 nm using an extinction coefficient of 16,790 cmϪ1 MϪ1 [36]. Fluorescence Measurements—Measurements were made in 50 mM HEPES-NaOH, pH 7.0, containing 10 mM DTT at 25 °C using various concentrations of PDI and bis-ANS. Initiation of the assay was by addition of ADH (final concentration, 11 ␮M dimer) to a 50 mM HEPES-NaOH buffer, pH 7.0, containing 10 mM DTT and PDI at various concentrations. Reduced, denatured RNase (final concentration, 8 ␮M) was added to PDI (1.4 ␮M) in a buffer containing 4.5 mM cCMP, 1 mM glutathione, 0.2 mM glutathione disulfide, 2 mM EDTA, 100 mM Tris-HCl, pH 8. Reduced, denatured RNase concentration was determined by absorbance at 280 nm using an extinction coefficient of 9300 cmϪ1 MϪ1. There was no detectable degradation of PDI during equilibration as detected by SDS-polyacrylamide gel electrophoresis analysis

RESULTS

Ligands that displace

DISCUSSION