PEDRO CUATRECASAS, MORLEY D. HOLLENBERG, KWEN-JEN CHANG and VANN BENNETT,     Department of Pharmacology and Experimental Therapeutics and Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland

Publisher Summary

This chapter discusses the problems in the study of hormone receptors and their modulation of membrane function. Because physiological membrane receptors are present in extremely small quantity in membranes, the hormone must be labeled to very high specific activity without destroying the biological activity of the hormone. Virtually, all chemical compounds used as binding ligands exhibit some nonspecific adsorptive or binding properties to a variety of inert and non-receptor biological materials, and such binding may be of extremely high affinity and the number of such nonspecific binding sites may greatly exceed that of specific receptors, therefore, great difficulties may be encountered in detecting such specific receptors. The general approach in these studies has been to measure the interaction, binding, of a radioactively labeled hormone with intact target cells or with isolated membrane preparations derived from such cells. The binding is presumed to reflect specific receptor interactions if it demonstrates the following characteristics: (1) strict structural and steric specificity, (2) saturability that indicates a finite and limited number of binding sites, (3) tissue specificity in accord with biological target cell sensitivity, (4) high affinity in harmony with the physiological concentrations of the hormone, and (5) reversibility that is kinetically consistent with the reversal of the physiological effects observed upon removal of the hormone from the medium.

I Introduction

During the past few years there has been a large burst of interest in membrane receptors for hormones, and considerable progress has been made in the identification and study of receptors for such peptide hormones as insulin, glucagon, adrenocorticotropin, thyrotropin, angiotensin, calcitonin, growth hormone, prolactin, follicle-stimulating hormone, luteinizing hormone, chorionic gonadotropin, oxytocin, and vasopressin, as well as nonpeptide hormones such as catecholamines, prostaglandins, and acetylcholine (reviewed in Cuatrecasas, 1974a,b; Birnbaumer, 1973). The general approach in these studies has been to measure the interaction (binding) of a radioactively labeled hormone with intact target cells or with isolated membrane preparations derived from such cells. The binding is surmised to reflect “specific” receptor interactions if it demonstrates (a) strict structural and steric specificity; (b) saturability, which indicates a finite and limited number of binding sites; (c) tissue specificity in accord with biological target cell sensitivity; (d) high affinity, in harmony with the physiological concentrations of the hormone; and (e) reversibility which is kinetically consistent with the reversal of the physiological effects observed upon removal of the hormone from the medium. In addition, it is of considerable help if specific chemical or enzymic perturbations of the cells or of the hormone result in changes in the biological activity which parallel closely similar changes in binding.

It should be realized that the term “receptor” in all these studies is a general term used for convenience, and its use implies a lack of knowledge of the discrete or unique chemical structures involved in the interactions. As the history of the study of drugs has shown, the use of the term “receptor” is in effect a reflection of ignorance of the molecular locus of action of the drug; when this locus is known (e.g., whether a specific enzyme such as a kinase for cyclic AMP, or hemoglobin for a vitamin or certain drugs), we no longer refer to it as the receptor, but rather by its chemical nature. “Receptor” in the context used currently in hormonal studies is defined operationally as those molecules that specifically recognize and bind the hormone and, as a consequence of this recognition, can lead to other changes (or series of changes) which ultimately result in the biological response. This is done in analogy with classical enzyme–substrate systems, in which substrate binding and catalysis are separate and discrete but sequential processes which can be studied independently. As in the case of competitive inhibitors of enzymes, hormone analogs (e.g., as known for glucagon, ACTH, angiotensin, and oxytocin) may in certain cases bind to “receptors” but fail to trigger biological responses, thus serving as relative or absolute inhibitors depending on the quantitative comparison of their intrinsic activity relative to their binding affinity.

The present article is intended not as a review of the pertinent literature, but rather as a presentation of special selected topics and ideas which have been of interest and concern in this laboratory.

II Problems in the Study of Hormone Receptors

A number of problems and pitfalls can be encountered in the kinds of studies described above, and considerable caution must be exercised in the interpretation of data, especially when detailed mechanistic inferences are made or when extrapolating to complex in vivo biological systems or disease states. Some examples of such actual or potential problems relating to different aspects of hormone-binding studies will be presented here.