Hormone receptors,affinity labeling

Katzenellenbogen JA, Katzenellenbogen BS (1984) Affinity labeling of receptors for steroid and thyroid hormones. Academic Press, New York... 

With the availability of labeled hormones of high specific activity and the application of the principles of affinity chromatography, researchers were able to isolate cellular proteins that bind to plant hormones in vitro. Such proteins have been referred to as receptor proteins, binding proteins, or acceptor proteins. Tacit in the concept of hormone receptor proteins is the stereo-specific interaction of the hormone and the receptor protein (19). The resulting hormone-protein complex participates in growth processes that depend on new or enhanced protein synthesis. Advances in molecular biology and related sciences have enabled many researchers to study the role of receptors in the control of nuclear functions or other activities and to determine the site of primary hormonal action. 

Affinity labelling of thyroid hormones to nuclear receptors indicated the presence of an abundant 47000 Da component and a less abundant 57000 Da species [40], Micrococcal nuclease also excises the two receptor forms. It is yet not clear whether these two forms are products of different genes or if the 57 000 Da form is converted or processed to the 47 000 Da species. 

Amongst resistant variants of CEM-7 cells an additional type of receptor defect was detected which has so far not been found in other cell systems [68,69], It was called activation labile because these receptors are very unstable under conditions which normally activate receptor complexes to forms with an exposed DNA binding site. These defective receptors have recently been shown to behave normally in terms of biochemical and immunochemical properties if the hormonal ligand is covalently attached by affinity labelling and therefore is unable to dissociate upon activation [70]. 

Our present information about the hormone binding domain, although still limited, comes from a combination of biochemical and genetic studies. The amino acid which reacts with the chemical affinity label dexamethasone 21-mesylate has now been identified as cysteine in position 656 of the rat glucocorticoid receptor [109]. This cysteine is therefore located within or very close to the hormone binding site. It corresponds to cysteine 644 of the mouse receptor (Fig. 2). 

There have been a number of technological advances in recent years which are responsible for much of the progress made in our understanding of steroid receptor mechanisms. These include the ability to purify receptors, affinity labeling techniques, production of antireceptor antibodies for direct detection of receptor proteins, and isolation and characterization of hormone responsive genes as well as the genes for receptors themselves. 

Top S, El Hafa H, Vessieres A, Huche M, Vaissermann J, Jaouen G (2002) Novel estradiol derivatives labeled with Ru, W, and Co complexes. Influence on hormone receptor affinity of several organometallic groups at the 170 position. Chem Eur J 8 5241-5249... 

This small country occupies a leading position in the world of peptides. As early as 1949, publications on activation of N-blocked amino acids in the form of their reactive esters appeared from the laboratory of Robert Schwyzer (Plate 39) at CIBA in Basel. After his appointment to professor at the Eidgenossische Technische Hochschule (ETH) in Zurich, he established there, at the Institute of Molecular Biology and Biophysics, a distinguished school of peptide research. One of his students, P.W. Schiller, is professor at the University of Montreal. Another outstanding former student of Schwyzer, Alex Eberle, is a lecturer in Zurich, yet active in Basel, where he is engaged in affinity-labelling of hormone receptors. 

Biochemical characterization of thyroid hormone-binding species from various tissue sources revealed these to be proteins with high-affinity for thyroid hormones which are found predominantly in cell nuclei (14-16). Purification of the thyroid hormone receptor protein has been hampered by its low abundance in cells, but biochemical analysis and photoaffinity labelling studies indicate that two nuclear polypeptides of 57 and 47 kD specifically bind (17-20). 

Nikodem, V. M., Cheng, S.-Y., and Rail, J. E. (1980). Affinity labeling of rat liver thyroid hormone nuclear receptor. Proc. Natl. Acad. Sci. USA 77 7064-7068. 

Another approach to demonstrating intracellular penetration is to examine nuclear uptake in the cells. After incubation of neuroblast monolayers with T3 or T, the cells are disrupted and the nuclei are isolated and their hormone content measured. We have shown that the accumulation of labeled hormone in nuclei is strongly diminished in the presence of antimycin or MDC. These agents, however, do not inhibit uptake by isolated nuclei. Further evidence is presented in Fig. 5, based on the apparent affinity of nuclear receptors after whole cell incubations compared to the affinity determined with previously isolated nuclei. It can be seen that the receptor affinity for L-T3 and L-T measured in intact cells is more than 3 times that in isolated nuclei. This can be attributed to a step-up in the concentration of free hormone in the cytoplasm over the concentration in the medium, the latter value being used in the calculation of Ka in intact cells. 

The demonstration of specific hormone receptors in most systems has relied either on direct isolation and characterization of protein-hormone complexes or on analysis of the kinetics of uptake and/or dissociation of labeled hormones by cells or macromolecules. Under equilibrium conditions, uptake at speeifie receptors has been discriminated from nonspecific uptake by displaeement with nonlabeled physiologically active steroids or competitive inhibitors. Such studies have shown that steroids have a high affinity but a relatively slow rate of association with specific receptors, that the reeeptors are saturable at physiological concentrations of hormone, and that binding is competed for by other active steroids... 

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