The Cysteine Shuffle

To test the feasibility of tagging receptor cysteines with a thiol-labeling reagent, the redox sensitivity of a radioligand receptor assay (RRA) for the 25-kDa glycoprotein hormone TSH was established. Unlike the 12 cysteines in the amino terminal extension of the related LH receptor, TSH (and FSH) receptors have only 11 extracellular cysteines, beyond the 2 involved in the conserved cystine bridge, suggesting that a free thiol may be available and influence ligand binding. Indeed, low concentrations of the thiol alkylating agent, N-ethyl maleimide (NEM), or thiol oxidizing agents promote the binding of TSH to its receptor, implying that reduced thiols hinder TSH binding. Dithiol reductants such as dithiothreitol (DTT) destroy binding and also reverse this effect of NEM, reinforcing the importance of intact cystine bridges to the maintenance of receptor structure.

The results in Figure 10.8A are from an early experiment designed to show that ligand binding to a receptor increases the sensitivity of the receptor to redox reagents — an indirect demonstration of ligand-induced conformational change in the receptor. The extent of radiolabeled-TSH binding in the control log-dose response curve of the TSH RRA is lowered when the receptor preparation is pretreated with a maximally tolerating dose of NEM (about 300 |M), to block already extant thiols. Addition of 300 |M NEM to the control curve or the pretreated receptor curve lowers binding to background levels as the receptor is destroyed. These results

control

NEM-pretreated receptor

NEM during the assay

control

NEM-pretreated receptor

NEM during the assay

10 9 8 7 -log10[TSH] (M)

conformational tagging disulphide exchange redox switch

Cys-reactive * label

Cys-reactive * label

FIGURE 10.8 Cysteine shuffling as a marker of receptor rearrangement. A, experimental and B, diagrammatic demonstration of enhanced receptor cysteine accessibility during ligand binding.47 C, various ways by which ligand-induced tagging of receptor cysteines may arise.

dimerization modulation

FIGURE 10.8 Cysteine shuffling as a marker of receptor rearrangement. A, experimental and B, diagrammatic demonstration of enhanced receptor cysteine accessibility during ligand binding.47 C, various ways by which ligand-induced tagging of receptor cysteines may arise.

demonstrate unequivocally that the receptor preparation is more sensitive to the damaging effects of NEM in the presence of ligand than in its absence. By using an NEM that incorporates a label or marker molecule, it should be possible to tag the receptor cysteines that have become newly exposed, as a direct consequence of ligand binding and receptor conformational changes.

The procedure for ligand-induced tagging of receptor cysteines is depicted in Figure 10.8B. Readily accessible cysteines are blocked by pretreatment of the receptor by NEM. The pretreated receptor is then exposed to ligand, which binds and induces a conformational change in the receptor. Any newly exposed receptor cysteines, as a consequence of ligand binding, can be tagged by a subsequent or concomitant exposure of the ligand-receptor mixture to a labeled NEM preparation.

The principle of the assay allows the order and/or duration of reagent addition and exposure to be altered according to circumstance, e.g., the second pictogram in Figure 10.8B of NEM* with bound ligand can precede the first, and is an effective way of capturing receptor thiol groups in flux between hidden and exposed. By applying this technique to other receptors in the rhodopsin family, including adrenergic, peptide, and protein hormone receptors, it became rapidly clear that all tested receptors were redox sensitive and capable of ligand-induced labeling. Even peptide ligands with (endothelin, oxytocin) and without (GnRH, angiotensin) endogenous cystine bonds conformed and their receptors were labeled. Because the procedure was applicable to intact cells as well as membranes, it was eminently suitable for HTS, and the Cys-screen method of ligand-induced labeling of receptor cysteines as a measure of receptor conformational change has been patented.47

Ligand binding can influence the exposure of receptor cysteines in several ways and some of the most interesting are depicted in Figure 10.8C. The cysteine shuffle can involve straightforward conformational tagging or result from disulfide exchange between ligand and receptor, a ligand-induced redox switch, or even through the modulation of multicomponent complexes (as observed for the metabotropic 7TMR family and receptor tyrosine kinases). Each of these mechanisms is capable of producing a readout in a screening scenario.

The proposition that the transition from the inactive to the active form of the receptor is dependent upon or reflected in the disposition of the cysteine residues in the receptor is attractive for two major reasons: (1) the general placement of cysteine residues in proteins means that they are ideal monitors of conformational changes in protein molecules and (2) cysteines are the most chemically reactive amino acids, which provide a convenient group for specific labeling and also a reactive species with the potential for signal transmission. Therefore, an assay that measures ligand-induced thioreactive molecular tagging of a receptor could have the potential to discriminate between agonists and antagonists in a single screen.

Regardless of whether the cysteine residues are oxidized or reduced, their relative hydrophobicity means that they are usually to be found traversing the hydrophobic-hydrophilic boundary of proteins.6 A survey of cysteine locations in protein structures reveals that the majority of cysteines reside just below the solventexposed surface of protein molecules (Scherzler, Procter, and Willey, unpublished observations).

In extracellular proteins, cysteines form S-S bonds that are conformational constrainers and also tie exterior and interior peptide chains together. Upon reduction of these bonds, a protein is no longer constrained and is free to adopt other conformations. Binding of ligand would be expected to induce a conformational change in the receptor, such that the cysteines lying at or near the surface of the molecule could become newly hidden or newly exposed. An exposed thiol group at or near the surface of the receptor can be detected readily using a multitude of marker molecules. The receptor is the pharmacological discriminator and it is highly appropriate for it to be tagged in the course of the Cys-screen. A major advantage of covalent cysteine labeling is its "permanent" nature, which allows signal detection to be measured as and when required.

The fundamental question is whether the cysteines are passively associated with or actively involved in hormone recognition and activation. Either activity may be useful in the development of a readout of hormone-receptor interaction, and it is conceivable that different cysteine residues exhibit differential activity profiles in one receptor compared with another.

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