Affinity labelling of steroid hormone receptors

Abstract

Affinity labelling techniques have proved indispensable for the study of reversible biological recognition systems, since they conserve ligand-receptor interaction by covalent linkage. Using photo- and electrophilic labelling, it has become possible to unequivocally identify steroid hormone receptors and their proteolytic degradation products and it is simple to establish receptor peptide maps even in crude receptor preparations. The isolation of receptor proteins has been greatly simplified, as their integrity can be analyzed at any step of a purification protocol by SDS-PAGE analysis after crosslinking. Moreover, affinity-labelled receptors can be purified under denaturing conditions, e.g., in high-resolving preparative SDS-PAGE, and the material obtained can be efficiently used to generate anti-receptor antibodies. Peptide mapping after crosslinking of related receptors has been used to assess the degree of structural homology between different forms of steroid hormone receptors and receptors of different species. Peptide sequence analysis of purified crosslinked receptor protein and anti-receptor antibodies have provided the basis for cloning corresponding genes. Techniques have been established to demonstrate--via crosslinking--that the cloned DNA sequences correspond to the receptor gene binding the correct ligand. The analytical and preparative crosslinking methods developed for steroid receptors are potentially important for the study of any system in which signal transduction proceeds via the reversible interaction between biological macromolecules.