Morley D. Hollenberg

Membrane Receptors and Hormone Action

Publisher Summary Studies of receptor binding generally employ highly radioactive ligand derivatives; however, knowledge of the biological activity of the radioactive derivative is essential for a complete interpretation of the binding data. This chapter describes the relation between biological activity and binding, physicochemical detection and assay of receptors, solubilization of membrane receptors, provides examples of membrane-localized receptors, affinity techniques, polyvalent hormone derivatives, and receptor studies, and theoretical mechanisms of hormone–receptor binding and action. The biological response toward many neurotransmitters and hormones, such as insulin, acetylcholine, and epinephrine is rapid both in onset and in the termination of action on removal of these agents; therefore, it may be anticipated that the initial hormone–receptor interaction will also be reversible. The chapter shows that progress has been made toward understanding the way by which hormones, via interactions with cell–surface receptors, modulate overall cellular function. This interaction is only the first in a presumably complicated series of protein–protein interactions within the plane of the cell membrane; the mechanism, whereby the ultimate signal is transmitted to the internal cellular milieu is undoubtedly an equally intricate process. The chapter is considered to be useful in delineating the challenge that the elucidation of these processes presents and in providing a basis and incentive for more discoveries in this field.

Epidermal growth factor: Characteristics of specific binding in membranes from liver, placenta, and other target tissues☆

Abstract Epidermal growth factor, a 6,400-dalton polypeptide from the mouse submaxillary gland, binds specifically to cells and membranes derived from a variety of human, rat, mouse, and bovine tissues. Liver, placenta, skin, cornea, and cultured chondrocytes, Hela cells, and Chang liver cells bind large amounts of epidermal growth factor, whereas fat cells, resting and lectin-stimulated human peripheral lymphocytes, mouse thymocytes, cultured rat hepatoma cells, and mammary cells from virgin and pregnant mice bind little or no epidermal growth factor. The binding site for epidermal growth factor is distinct from receptors for other anabolic peptides such as insulin, nerve growth factor, and growth hormone. The binding of epidermal growth factor is rapid and reversible. The rate constant of association is approximately 10 6 mole −1 sec −1 , the rate constant of dissociation is about 6 × 10 −4 sec −1 , and the apparent equilibrium dissociation constant is about 10 −9 m . Trypsin at low concentrations (50–200 μg/ml) destroys the receptor site for epidermal growth factor. The binding of epidermal growth factor by membranes is not accompanied by appreciable degradation of the peptide present in the medium or of that bound to the membranes. Use can be made of the high affinity and specificity of membranes for epidermal growth factor to measure by a competitive binding assay as little as 200 pg of EGF per ml (3 × 10 −11 m ).

Binding of insuling and other hormones to non-receptor materials: Saturability, specificity and apparent “negative cooperativity”

Summary Studies of interactions of 125 I-insulin with non-tissue materials (talc, silica, protein-agarose derivatives, glass test tubes) are presented as examples of non- receptor (“nonspecific”) interactions which share, at least superficially, the criteria (saturability, specificity, high affinity and reversibility) commonly attributed to “specific” hormone-receptor interactions. Such “specific” nonreceptor interactions, which can potentially complicate interpretations of receptor binding studies, also exhibit characteristics analogous to those observed in liver membranes where the data have been interpreted (3) to indicate “negative cooperativity” between receptors. Similar data are presented here for insulin and epidermal growth factor binding to placenta membranes, and the effects of porcine insulin are compared to those of guinea pig insulin, a derivative which dimerizes very poorly. The “negative cooperativity” observed in the nonreceptor systems is most likely attributable to insulin-insulin interactions. Wherever ligand (e.g., hormone) self-association or isomerization occurs, the binding data (e.g. Scatchard plot) may yield nonlinear relationships which can falsely be interpreted as indicating cooperative interactions between receptors, or the presence of “additional groups” of binding sites.

Epidermal Growth Factor-Urogastrone, a Polypeptide Acquiring Hormonal Status*

Publisher Summary This chapter discusses the characteristics of epidermal growth factor—urogastrone, a polypeptide acquiring hormonal status. The discovery, isolation, and complete sequence determination of two related polypeptides, one from the mouse and the other from man, that are both highly potent stimulators of cellular proliferation and inhibitors of gastric acid secretion is described. The structure of human urogastrone is determined by Cohen who isolated two polypeptides from human urine concentrates, both of which are observed to interact with the same membrane receptor and one of which is demonstrated to be an active mitogen both in vivo and in vitro . The physicochemical data obtained by Cohen and colleagues suggest that the polypeptides isolated are the same as the isolated urogastrones. Human urine provides the richest convenient source of human epidermal growth factor-urogastrone (hEGF-URO). By radioreceptor assay, it can be estimated that random urine samples from male donors contain, on average, about 9.5 nmol/liter. The isolation requires the initial preparation from urine of either tannic acid-acetone or benzoic acid-acetone powder of urinary proteins, followed by multiple gel filtration and ion exchange steps.

Receptors for insulin and epidermal growth factor: relation to synthesis of DNA in cultured rabbit lens epithelium.

Abstract Epidermal growth factor (EG factor) and insulin stimulate the incorporation of thymidine into contact-inhibited rabbit lens epithelial cells in culture. The maximal stimulation observed with EG factor is greater than with insulin. Half-maximal stimulation by EG factor is observed at 6 × 10 −10 m and for insulin at 1 × 10 −9 m . [ 125 I]-labeled EG factor (2000 Ci/mmol, about 1 g atom of iodine per mol) is equipotent with native EG factor in stimulating DNA synthesis. Both insulin and EG factor bind to distinct high-affinity sites in intact lens cell monolayers; half-maximal binding is observed at about 10 −9 m for both polypeptides. A maximum of approximately 8 × 10 4 insulin molecules and 4 × 10 4 EG factor molecules are bound per cell. These observations indicate that cultured rabbit lens cells possess receptors for insulin and EG factor by biological and physicochemical criteria and raise the possibility that both peptides may play a role in lens growth and development.

Hormone Receptor Complexes and Their Modulation of Membrane Function

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.

Adult Hemoglobin Synthesis by Reticulocytes from the Human Fetus at Midtrimester

The synthesis of adult-type hemoglobin was measured in small samples of peripheral blood cells from 9- to 18-week human fetuses. Hemoglobin indistinguishable from hemoglobin A was identified by ion-exchange chromatography, electrophoresis at pH 8.6, tryptic peptide analysis, and the insensitivity of its synthesis to the action of O-methylthreonine. Synthesis of hemoglobin A accounted for 8 to 14 percent of total hemoglobin synthesis and was demonstrated in as little as 10 microliters of fetal blood. These studies provide sensitive methods for the detection of β chain types in hemoglobin synthesized by the human fetus at midtrimester. If methods to obtain small quantities of fetal blood at midtrimester become available, these techniques should be applicable to the antenatal detection of sickle cell anemia and related hemoglobinopathies.

Age-related parallel decline in beta-adrenergic receptors, adenylate cyclase and phosphodiesterase activity in rat erythrocyte membranes

Abstract The activities of three components of the cyclic AMP system were compared in erythrocyte ghost membranes prepared from the blood of rats at various ages from 1.5 to 15 months. The apparent number of β-adrenergic receptor sites, adenylate cyclase activity and cyclic AMP phosphodiesterase activity all declined about 50% in the membranes from the older animals (>5 months) as compared to the 1.5 month ones. The soluble erythrocyte phosphodiesterase also declined with age, but the decline did not parallel that of the membrane-associated activity. In contrast, there was no age-related change in the number of β-adrenergic receptors in membranes from the brains of the same animals. In erythrocyte ghosts, both the ratio of isoproterenol-stimulated adenylate cyclase activity to basal activity and the ratio of sodium fluoride-stimulated activity to basal were constant with age. Neither the dissociation constant for the β-adrenergic receptor nor the Michaelis constant for the phosphodiesterase changed as a function of age. Together with other data in the literature, these results suggest a close functional association of the components of the cyclic AMP system in the mature erythrocyte membrane, and support a physiological role for the cyclic AMP mediated β-adrenergic effects in the red blood cell.

Independent variation of β-adrenergic receptor binding and catecholamine-stimulated adenylate cyclase activity in rat erythrocytes

Abstract Isoproterenol-stimulated adenylate cyclase activity in membrane preparations of reticulocyte-rich (90%) erythrocytes from phenylhydrazine-treated rats is 9 times greater than in untreated animals (1% reticulocytes); basal and fluoride-stimulated activities are also enhanced 2 and 4-fold respectively. In contrast, the number of β-adrenergic receptor sites detected by the binding of 125 I-hydroxybenzylpindolol ( 125 I-HYP) is increased only 40% in these same preparations. The dissociation constant (K D ) of 125 I-HYP and the IC 50 of (-)-isoproterenol for receptor binding sites are unchanged, as is the EC 50 of (-)-isoproterenol for activation of adenylate cyclase. The disproportionately large increase in the activity of the isoproterenol-sensitive adenylate cyclase, compared with the small increase in the number of 125 I-HYP binding sites indicates that the functions of catecholamine recognition and consequent adenylate cyclase response can vary independently and suggest that the receptor and the cyclase may be autonomous molecular entities.

Characterization of the particulate and soluble acceptor for transcobalamin II from human placenta and rabbit liver

We describe in both human placenta and rabbit liver membranes specific acceptors which bind the human transcobalamin II-vitamin B-12 (cobalamin) complex with an affinity of 2.3 . 10(9) (placenta) and 6.7 . 10(9) (liver) M-1 and which bind the rabbit transcobalamin II-cobalamin complex with an affinity of 1.1 . 10(9) (placenta) and 1.9 . 10(9) (liver) M-1, respectively. The binding requires Ca2+ and is sensitive to both 1 M NaCl and acid pH. A new ligand binding assay, based on the ability of the acceptor, but not transcobalamin II, to bind to concanavalin A, is described and is used to characterize the solubilized acceptors. The solubilized acceptors bind human transcobalamin II-cobalamin with high affinity (about 2-9 . 10(9) M-1) but do not bind free cobalamin; unsaturated transcobalamin II is bound with an affinity approximately one-third of that for transcobalamin II saturated with cobalamin. On gel filtration, the human acceptor saturated with transcobalamin II-cobalamin exhibits a Stokes radius of 6.7 nm, whereas the free acceptor has a Stokes radius of 5.1 nm. The rabbit liver acceptor either unsaturated or saturated with transcobalamin II-cobalamin exhibits a Stokes radius of 5.7 nm. Both acceptors bind to lectins such as concanavalin A, wheat germ agglutinin and phytohemagglutinin, indicating their glycoprotein nature, and both acceptors can be purified approximately 30-fold by affinity chromatography on wheat germ agglutinin-Sepharose columns. The concanavalin A assay, combined with lectin-Sepharose and transcobalamin II-cobalamin-Sepharose affinity chromatography will provide for the isolation and study of pure acceptors from a variety of tissue sources.