Binding of Hormone to Tissue: The First Step in Polypeptide Hormone Action

Abstract

Polypeptide hormones produce a wide range of metabolic and morphologic responses in specific target tissues.1 Clearly the initial interaction of hormone with sensitive tissue must be binding of the hormone to some cellular constituent. To delineate this interaction we have studied the effects of thyroid-stimulating hormone (TSH) on thyroid slices and of insulin on skeletal muscle. Tissues that were exposed to hormone and washed thoroughly in hormone-free medium showed a persistent hormone effect. However, when the washed tissues were subsequently exposed to antibody or to trypsin, this effect was obliterated. Thus, the persistent hormone effect was due to the continued presence of relatively intact hormone at a readily accessible tissue site. .05 M phosphate, pH 7.4, at 500 v at 40 on Whatman 3MM chromatography paper, over 95% of the radioactivity of each of the labeled proteins migrated appropriately as a single peak. Substantially less than 1% of the radioactivity was iodide. At least 80% of the TSH-I131 was bound by TSH antibody. Labeled proteins were stored at -20°and used within 72 hr of preparation.Antibodies were produced in a burro by four intramuscular injections of 6 mg of bovine TSH (4 IU/mg) in complete Freund's adjuvant (Difco). Antibody was prepared from citrated plasma by defibrination with calcium and thrombin and precipitation at 230 with ammonium sulfate at 40% saturation. The guinea pig anti-insulin serum (Suburban Laboratories) was similarly purified. Hereafter antiserum refers to the purified gamma globulin. The anti-TSH serum at a final dilution of 1: 5 neutralized bovine TSH at 50 mU/ml but had little effect on human TSH. The anti-insulin serum at a 1:20 dilution neutralized insulin at 10 mU/ml. Neutralization indicates that a solution of hormone and antibody, allowed to mix for 30 min, failed to stimulate glucose-1-C14 oxidation in thyroid or glucose-i-C14 incorporation into rat diaphragms. An aliquot of burro anti-TSH serum was treated with papain (Worthington) at pH 6 for 5 hr.5 Papain was inactivated by iodoacetamide, and Porter fraction III was precipitated by dialysis in water and removed by centrifugation. Simultaneous with the preparation of each antiserum, serum from an unimmunized animal was treated identically and served as control throughout. All sera were dialyzed against Krebs-Ringer bicarbonate before use.The buffer for in vitro experiments was Krebs-Ringer bicarbonate pH 7.4 containing bovine fraction V (fr. V, Armour) at 5 mg/ml. Routinely, dog thyroid slices weighing 15-30 mg were preincubated with bovine TSH (4 IU/mg) in 2-5 ml of buffer and washed three times by agitation for 1 min in 10 ml of hormone-free buffer at 10. The slices were then blotted, weighed, and incubated at 370 in 1 ml of buffer containing 1 mg of glucose and 0.5 ,uc of glucose-i-C14. The C1402 was collected in hyamine.6 Data are expressed as cpm/mg of thyroid slice per hr of incubation. The thyroid of a single normal dog was used for each experiment.The effect of insulin on glucose utilization in the r...

Insulin Receptors in the Liver: Specific Binding of [ 125 I]Insulin to the Plasma Membrane and Its Relation to Insulin Bioactivity

“…The first step in the action of polypeptide hormones appears to be rapid, reversible interaction between hormone in the extracellular medium and specific recognition sites on the plasma membrane of target cells (1)(2)(3). In previous studies (3), using [1251 ]ACTH, we have demonstrated directly the binding of ACTH to its biologically important receptors.…”

Colchicine-Binding Protein and the Secretion of Thyroid Hormone

“…Oll the other hand, the bulk of the circulating organic iodine exists in the form of iodoamino acids derived hydrolytically from the above iodinated proteins. Although other pathways of hormone secretion have not been ruled out, the present concept for activation of thyroid secretion by thyroid-stimulating hormone (TSH) ~ involves the following steps (a) T S H combines with a membrane receptor and activates the membrane-bound adenylate cyclase (2,3). (b) The increased intra-1 Abbrevzations used include: cyclic AI',IP, adenosine 3~,5 r cyclic monophosphate; dibutyryl cyclic AlXlP, N~-2-O-dibutyryl-adenosine 3 t, 5 r cyclic monophosphate; SP),IG, 0.25 ~ sucrose, 10 m~ Na phosphate, pFI 7.0, 10 ima l~lgC12, and 0.1 rmt GTP solution; ~P2~IO, the same buffer without sucrose, TSH, thyroid-stimulating hormone.…”

Insulin Receptors in Human Circulating Cells and Fibroblasts

“…Using biologically-active iodinated hormones and various in vitro preparations of target tissue, we and others have studied directly the binding of polypeptide hormones to specific receptors on target cells (1)(2)(3)(4)(5)(6)(7)(8). These techniques have not been applied to the study of clinical disorders in man largely because it has not been feasible to obtain sufficient quantities of these tissues under appropriate physiological conditions.…”