Modulation of human growth hormone binding to somatogenic and lactogenic receptors by monoclonal antibodies to human growth hormone.

The relationship between the structure of human growth hormone (hGH) and the hormone-receptor interaction was investigated by studying the effects of specific monoclonal antibodies (MAbs) to hGH on the binding of [125I]hGH to rabbit liver and mouse liver microsomes. Receptor binding assays were carried out using a constant dose (1 ng) of [125I]hGH and varying concentrations of MAbs. The assay was carried out in the presence of either excess ovine prolactin for the measurement of somatogenic (SOM) binding sites, or excess bovine growth hormone for the determination of lactogenic (LAC) binding sites. Anti-hGH MAbs were found to have a whole spectrum of effects on hGH binding, including inhibitory, non-effect and enhancing activities. Enhancement of the binding of [125I]hGH to both SOM and LAC receptors was observed in liver membranes of rabbit or mouse. The observed amplified signal of [125I]hGH binding to various receptors in the presence of MAb no. 8 may be due to conformational changes which occur following MAb binding to hGH. On the other hand, most of the other MAbs caused inhibition of [125I]hGH binding. A negative correlation exists between the cross-reaction of various MAbs with the N-terminus truncated forms of hGH (Met14-hGH or Met8Leu-hGH) and their respective KD/IC50 values enabled the evaluation of the crucial role of the N-terminus region in hGH binding to both LAC and SOM receptors. MAb nos 1 and 19, which are directed towards acid residues 95-134 and the C-terminus, inhibited SOM binding more potently than LAC binding. Thus, it seems that these mid-molecule and C-terminus regions are also important in hGH binding, and that they play a role in the partial overlap of SOM and LAC binding.     

Insulin binding to cultured adult hepatocytes. Effects of bacitracin and chloroquine on the nature of cell-associated radioactivity.

Sephadex (G-50 fine grade)-gel chromatography and trichloroacetic acid (TCA) precipitation were used to investigate the effects of chloroquine and bacitracin on the nature of cell-associated radioactivity in studies on the binding and degradation of 125I-insulin in cultured rat hepatocytes. Sephadex peak I, eluted with the void volume, increased with hepatocyte incubation time and comprised 6% of total cell-bound radioactivity at 120 min. However, all radioactivity in this peak was due to unspecific binding. Peak II, corresponding to intact insulin, represented 95% of specifically cell-associated label at 5 min and decreased to 77% at 120 min. Peak III, containing the final low-Mr degradation products, increased with incubation time (22% of specifically bound label at 120 min). The TCA-precipitable and TCA-soluble fractions of hepatocytes extracted with 0.1% SDS were within 4-7% of the proportions of radioactivity in peaks II and III respectively. Scatchard plots based on insulin-binding data from Sephadex chromatography or TCA precipitation were identical. Dissociation studies revealed that at least 75% of the intact insulin associated with the hepatocytes was bound to receptors at the cell surface. Bacitracin increased the proportion of cell-associated intact hormone and decreased that of ligand degraded when analysed by either Sephadex chromatography or TCA precipitation. The proportion of surface-bound to internalized intact hormone remained unaltered, indicating that bacitracin acted predominantly at the cell surface. In the presence of chloroquine, which dramatically increased the contribution of peak I to specific binding, 'intact' insulin was substantially overestimated when determined as the TCA-precipitable fraction. In addition, all peak I material and 50% of cell-associated label in peak II was trapped intracellularly, thereby pointing to the lysosomal or prelysosomal site of action of this drug.     

Binding inhibition by monoclonal antibodies of ovine placental lactogen to growth hormone receptors in human liver

In the radioreceptor assay for growth hormone (RRA-GH) using [125I]iodo-hGH, hGH and human liver membrane particulate fractions as tracer, hormone standard and receptors, respectively, ovine placental lactogen (oPL) is capable of inhibiting the binding of [125I]iodo-hGH in a parallel manner with hGH and in equipotency. Similarly, in the RRA-GH by employing [125I]iodo-oPL, oPL and human liver membrane particulate fractions as tracer, hormone standard and receptors, respectively, hGH is also equipotent as oPL in inhibiting the binding of [125I]iodo-oPL in a parallel fashion. The addition of monoclonal antibodies against oPL in the assay was effective in inhibiting the binding of [125I] iodo-oPL to human liver, but could not, however, inhibit the binding of [125I]iodo-hGH to human liver. Furthermore, the addition of the monoclonal antibodies in the RRA-GH did not affect the parallelism of the oPL standard but lowered the total binding of oPL. Our studies indicate that the structure of the binding sequence in oPL which binds to the GH receptor of human liver is not identical to the equivalent sequence of hGH and that the monoclonal antibodies compete with GH receptors in human liver for the binding of oPL.     

α-Bungarotoxin Binds to Low-Affinity Nicotine Binding Sites in Rat Brain

Reported differences in the pharmacology and distribution of [3H]nicotine and [125I]alpha-bungarotoxin binding sites in mammalian brain suggest that these ligands label separate receptor sites. Affinity purification of an alpha-bungarotoxin binding protein from rat brain failed to copurify the high-affinity nicotine binding site, which remained in the nonbound soluble fraction after the affinity chromatography step. This confirms the independence of these putative receptor sites. Nevertheless, the binding of [125I]alpha-bungarotoxin to P2 membranes was inhibited by (-)-nicotine (Ki = 9 X 10(-6) M), and this sensitivity was preserved after affinity purification. It is proposed that alpha-bungarotoxin binds to a population of low-affinity nicotine binding sites. Comparison of the enantiomers of nicotine in competition studies at both radioligand binding sites revealed an 80-fold preference for the (-) form at the high-affinity [3H]nicotine binding site, whereas the site labelled by [125I]alpha-bungarotoxin displayed little stereoselectivity. In this respect, the brain alpha-bungarotoxin binding site resembles the nicotinic acetylcholine receptor from Torpedo electric organ.     

Alteration by concanavalin A of the slow dissociable component in the human growth hormone-receptor interaction

In mice liver plasma membranes (PM), the binding affinity of receptors for [125I] human growth hormone (hGH) was dependent on the association time: after 18 hours, a high affinity receptor form with KA = 6.8 X 10(9) M-1 accumulated and, as compared to after 1 hour, an increase up to 88%, in a slow dissociating component was observed. Preincubation of PM with concanavalin A (Con A) or other lectins from Lens culinaris (LCA), Ricinus communis (RCA I), Wheat germ agglutinin (WGA) specifically inhibited the binding of hGH to receptors by 54, 28, 50 and 25%, respectively. Furthermore, PM pretreatment with Con A concomitantly increased the rate of dissociation of the hormone-receptor (H-R) complex to 92 or 65% after association for 1 or 18 hours. These Con A-induced alterations resulted from a reduced fraction of the slow dissociable component together with an increased rate constant. The treatment of PM with Con A subsequent to incubation with the hormone did not decrease hormone binding but caused the conversion of the class of hGH receptors exhibiting fast dissociation kinetics towards a form exhibiting slow ones. These data strongly suggest a role for glycoproteins of the N-acetyllactosaminic type in the affinity state of liver membrane hGH receptors.     

The quantitation of human growth hormone by a radioreceptor assay using an established human cell line

Membrane receptors on cultured human lymphocytes (IM-9) have been shown to bind human growth hormone (hGH) in a specific manner. The aim of the present study was to develop an in vitro assay of hGH based on this binding. The assay should fulfil established pharmacopoeial requirements for quantitation of hormones. The binding of [125I]hGH was studied as a function of time, temperature, cell density, tracer concentration and the concentration of unlabelled hGH and other related hormones. Also, the dissociation of bound hGH and the chemical stability of hGH in the incubation medium were studied. From these studies, the conditions for an appropriate radioreceptor assay were determined. Briefly, 1.5-3.0 X 10(7) cells ml-1 were incubated with 5-20 X 10(-12) M [125I]hGH and three different concentrations of unlabelled hGH chosen from the linear part of the [125I]hGH displacement curve. The results were analyzed according to general pharmacopoeial principles. The mean values for growth hormone activity tested by radioreceptor assay were within the fiducial limits (P = 0.05) of the corresponding activity determined by the hypophysectomized rat body-weight gain assay. The in vitro assay was found to be more precise and less resource demanding than the in vivo bioassay of hGH. It is concluded that the in vitro bioassay described here is well suited as a screening method for potency determination of hGH preparations.     

Growth hormone binding and stimulation of amino acid uptake in epithelial cells of rat ventral prostate

The binding of [125I]-human growth hormone (hGH) was studied in epithelial cells isolated from rat ventral prostate. Binding and degradation were dependent on time and temperature. The effect of a lysosomotropic agent suggested internalization and lysosomal degradation of the hormone. Dissociation and stoichiometric studies indicated the existence of a single class of GH receptors with a Kd of 0.7 nM and a binding capacity of 46 fmol hGH bound mg-1 cell protein. The receptor appeared to possess a somatotrophic nature since lactogenic hormones such as human placental lactogen and rat prolactin exhibited a very low degree of competition (whereas a variety of unrelated hormones and neuropeptides showed no effect). GH-stimulated leucine uptake by the cells in a time- and dose-dependent manner, half maximal effect being observed at 0.32 nM GH thus suggesting a direct relationship with the binding step.     

Effect of cell density on induction of growth hormone receptors by dexamethasone in primary cultured rat hepatocytes

The effect of cell density on the regulation of growth hormone (GH) receptors was studied by measuring specific binding of [125I]hGH to primary cultured hepatocytes with or without dexamethasone, which induces GH receptors. In cell cultures without dexamethasone, the cell density did not affect the level of binding of labeled GH appreciably. On the other hand, in the presence of dexamethasone, which induced an increase in the level of GH receptors on the cells, GH-binding by cultured cells at low cell density (3.3 x 10(4) cells/cm2) was about one-third of that of cells at high cell density (10(5) cells/cm2). Scatchard plot analysis showed that the cell-density dependent change in induction of GH binding, by dexamethasone was due to change in the number of binding sites without significant change in their affinity. The binding capacity of glucocorticoid receptors, measured as specific binding of [3H]dexamethasone to the hepatocytes, was not significantly affected by cell density. These results suggest that cell density modulates GH receptor induction by dexamethasone via events after glucocorticoid receptor binding.     

The effects of diphenyleneiodonium on mitochondrial reactions. Relation of binding of diphenylene[125I]iodonium to mitochondria to the extent of inhibition of oxygen uptake.

1. Several ring-substituted derivatives of diphenyleneiodonium catalyse the exchange of Cl- and OH- ions across the inner membrane of rat liver mitochondria. They also inhibit state 3 and state 3u oxidations of glutamate plus malate in the presence of Cl- more than in its absence. Most have activities similar to diphenyleneiodonium, although 2,4-dichlorodiphenyleneiodonium is up to 50 times more active. 2. Diphenyleneiodonium inhibits soluble rat liver NADH dehydrogenase and NADH oxidation by rat liver sub-mitochondrial particles directly; 2,4-dichlorodiphenyleneiodonium is only about twice as inhibitory. 3. Liver mitochondria contain two classes of binding sites for diphenylene[125I]iodonium, namely high-affinity sites with an affinity constant of 3 X 10(5) M-1 (1--2 nmol/mg of protein), and low-affinity sites with an affinity constant of 1.3 X 10(3) M-1 (80 nmol/mg of protein). Both sites occur in hepatocytes with a relative enrichment of the low-affinity site. Nadh dehydrogenase preparations only apparently contain high-affinity binding sites. Only low-affinity sites occur in erythrocytes. 4. 2,4-Dichlorodiphenyleneiodonium competes with diphenylene[125I]iodonium for both low- and high-affinity sites, whereas tri-n-propyltin only competes for the low-affinity sites. 5. The high-affinity sites are apparently associated with NADH dehydrogenase and the low-affinity sites probably represent electrostatic binding of diphenylene[125I]iodonium to phospholipids. The high-affinity site does not appear to be associated with a rate-limiting stage of NADH oxidation.     

Analysis of photoaffinity label derivatives to probe thyroid hormone receptor in human fibroblasts, GH1 cells, and soluble receptor preparations

The regulation of growth hormone gene expression by thyroid hormone in cultured GH1 cells is mediated by a chromatin-associated receptor. We have previously described a photoaffinity label derivative of 3,5,3'-triiodo-L-thyronine (L-T3) in which the alanine side chain was modified to form N-2-diazo-3,3,3-trifluoropropionyl-L-T3 (L-[125I]T3-PAL). On exposure to 254 nm UV light, L-[125I]T3-PAL generates a carbene which covalently modifies two thyroid hormone receptor forms in intact GH1 cells; an abundant 47,000 Mr species and a less abundant 57,000 Mr form. We have now synthesized similar photoaffinity label derivatives of 3,5,3',5'-tetraiodo-L-thyronine (L-T4) and 3,3',5'-triiodo-L-thyronine (L-rT3). Both compounds identify the same receptor forms in intact cells and in nuclear extracts in vitro as L-[125I]T3-PAL. Labeling by L-[125I]rT3-PAL was low and consistent with the very low occupancy of receptor by L-rT3. Underivatized L-[125I]T3 and L-[125I]T4 labeled the same receptor forms at 254 nm but at a markedly lower efficiency than their PAL derivatives. In contrast, N-bromoacetyl-L-[125I]T3, a chemical affinity labeling agent, did not derivatize either receptor form in vitro. The relative efficiency of coupling to receptor at 254 nm was L-[125I]T4-PAL greater than L-[125I]T3-PAL greater than L-[125I]T4 greater than L-[125I]T3. Although L-[125I]T4-PAL has a lower affinity for receptor than L-[125I]T3-PAL, its coupling efficiency was 5-10-fold higher. This suggests that the alanine side chain of L-[125I]T4-PAL is positioned in the ligand binding region near a residue which is efficiently modified by photoactivation. With L-[125I]T4-PAL we were able to identify three different molecular weight receptor species in human fibroblast nuclei.     

Binding sites for angiotensin II in human mononuclear leucocytes.

Angiotensin II binding sites were demonstrated in human mononuclear leucocytes by use of [125I]angiotensin II. The binding of [125I]angiotensin II to mononuclear leucocytes was rapid and reversible. The abilities of unlabeled compounds to displace [125I]angiotensin II were proportional to their abilities to displace labeled hormone in adrenal and smooth muscle membrane preparations. The Scatchard plot revealed two apparent orders of binding sites. The affinity constants were comparable with those for binding sites in other main target tissues of angiotensin II.     

Presence of NK2 binding sites in the rat brain

Attempts were made to label tachykinin NK2 binding sites in the adult rat brain using [125I]neurokinin A (NKA) as ligand in the presence of NK1 and NK3 agonist or antagonist to avoid labelling of NK1 and NK3 binding sites, respectively. A high-affinity, specifically NK2-sensitive, [125I]NKA-binding, temperature-dependent, reversible, sensitive to GTPgammaS and correspondence to a single population of binding sites (K(D) and B(max) values: 2.2 nM and 7.3 fmol/mg protein) was demonstrated on hippocampal membranes. Competition studies performed with tachykinins and tachykinin-related compounds indicated that the pharmacological properties of these NK2-sensitive [125I]NKA binding sites were identical to those identified in the rat urinary bladder and duodenum. NKA, neuropeptide K, and neuropeptide gamma, as well as the potent and selective NK2 antagonists SR 144190, SR 48968 and MEN 10627, presented a nanomolar affinity for these sites. The regional distribution of these NK2-sensitive [125I]NKA binding sites differs markedly from those of NK1 and NK3 binding sites, with the largest labeling being found in the hippocampus, the thalamus and the septum. Binding in other brain structures was low or negligible. A preliminary autoradiographic analysis confirmed [125I]NKA selective binding in hippocampal CA1 and CA3 areas, particularly, and in several thalamic nuclei.     

Characterization of histamine H1-receptor on rat hepatocytes

The binding sites for [3H]pyrilamine in isolated rat hepatocytes were characterized. Scatchard analysis revealed two kinds of binding sites in hepatocytes, a high-affinity site and a low-affinity one. The rates of binding of the radioligand with the high-affinity binding site and its dissociation were rapid. The specificity of the sites for various histamine antagonists indicated that the high-affinity [3H]pyrilamine binding site is representative of the histamine H1 receptor. Treatment of hepatocytes with protease or phospholipase A2 significantly decreased the maximum binding capacity of the high-affinity site without affecting its dissociation constant, suggesting that the binding site is proteinaceous and is sensitive to a change in the lipid moiety of the membrane. Hepatocytic cyclic AMP and cyclic GMP were not significantly modulated by incubating hepatocytes with histamine. Thus, the action of histamine on hepatocytes might not be mediated by the cyclic nucleotides.     

Localization of insulin degradation products to an intracellular site in isolated rat hepatocytes

In the present study, we have examined whether insulin degradation products are present on the surface of isolated rat hepatocytes and can be removed by an acid dissociation technique. Hepatocytes were incubated with [125I]insulin for 30 minutes, rapidly washed to remove unbound insulin, and then briefly exposed to acidic conditions (pH 5.0) to remove bound hormone from the cell surface. The radioactive material removed from the cell by acid dissociation and that remaining with the cells were separately analyzed by high performance liquid chromatography. The two primary degradation products of insulin present in control cell extracts were found only with the cell-associated material after acid dissociation. The insulin-sized radioactive material in the extract of acid-dissociable material consisted of only intact [125I]insulin. These results show that the two primary degradation products of insulin in rat hepatocytes are found only intracellularly and suggest that the degradation of the hormone begins after it is internalized.     

Insulin processing and signal transduction in rat adipocytes

A glycine-HCl buffer (glycine, 50 mM/NaCl, 0.15 M/HCl, pH 3.5) was used to strip insulin bound to adipocyte cell surfaces. Adipocytes retained their integrity in the glycine buffer and their binding capacity for [125I]iodoinsulin could be completely recovered on transfer of the cells to physiological media. At 37 degrees C, [125I]iodoinsulin binds rapidly to plasma membrane receptors; maximal binding occurs within 10 min. At this temperature, the initial binding is followed by rapid internalization, degradation of the hormone and subsequent loss of label. Insulin treatment, at 37 degrees C, induced internalization of 37% of the plasma membrane insulin receptors. Phenylarsine oxide (PAO), a confirmed inhibitor of protein internalization, allowed insulin binding but completely inhibited degradation of the hormone. Monensin, a carboxylic ionophore which impairs uncoupling hormone-receptor complexes, effectively restricted insulin degradation over short time periods (less than 30 min). Addition of monensin to insulin-stimulated cells did not impair D-glucose uptake. It has previously been reported that PAO inhibits hexose transport through the direct interaction with the glucose transporters and low concentrations of PAO (1 microM) transiently inhibit insulin-stimulated glucose uptake. This recovery phenomenon was again observed when PAO was added to insulin-stimulated, monensin-treated adipocytes. The data suggests that lysosomal degradation of insulin is not requisite for signal transduction.     

Topography of the high-affinity lysine binding site of plasminogen as defined with a specific antibody probe

An antibody population that reacted with the high-affinity lysine binding site of human plasminogen was elicited by immunizing rabbits with an elastase degradation product containing kringles 1-3 (EDP I). This antibody was immunopurified by affinity chromatography on plasminogen-Sepharose and elution with 0.2 M 6-aminohexanoic acid. The eluted antibodies bound [125I]EDP I, [125I]Glu-plasminogen, and [125I]Lys-plasminogen in radioimmunoassays, and binding of each ligand was at least 99% inhibited by 0.2 M 6-aminohexanoic acid. The concentrations for 50% inhibition of [125I]EDP I binding by tranexamic acid, 6-aminohexanoic acid, and lysine were 2.6, 46, and 1730 microM, respectively. Similar values were obtained with plasminogen and suggested that an unoccupied high-affinity lysine binding site was required for antibody recognition. The antiserum reacted exclusively with plasminogen derivatives containing the EDP I region (EDP I, Glu-plasminogen, Lys-plasminogen, and the plasmin heavy chain) and did not react with those lacking an EDP I region [miniplasminogen, the plasmin light chain or EDP II (kringle 4)] or with tissue plasminogen activator or prothrombin, which also contain kringles. By immunoblotting analyses, a chymotryptic degradation product of Mr 20,000 was derived from EDP I that retained reactivity with the antibody. The high-affinity lysine binding site was equally available to the antibody probe in Glu- and Lys-plasminogen and also appeared to be unoccupied in the plasmin-alpha 2-antiplasmin complex. alpha 2-Antiplasmin inhibited the binding of radiolabeled EDP I, Glu-plasminogen, or Lys-plasminogen by the antiserum, suggesting that the recognized site is involved in the noncovalent interaction of the inhibitor with plasminogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photoaffinity labeling of dopamine D1 receptors

A high-affinity radioiodinated D1 receptor photoaffinity probe, (+/-)-7-[125I]iodo-8-hydroxy-3-methyl-1-(4-azidophenyl)-2,3,4,5-tetra hyd ro- 1H-3-benzazepine ([125I]IMAB), has been synthesized and characterized. In the absence of light, [125I]IMAB bound in a saturable and reversible manner to sites in canine brain striatal membranes with high affinity (KD approximately equal to 220 pM). The binding of [125I]IMAB was stereoselectively and competitively inhibited by dopaminergic agonists and antagonists with an appropriate pharmacological specificity for D1 receptors. The ligand binding subunit of the dopamine D1 receptor was visualized by autoradiography following photoaffinity labeling with [125I]IMAB and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Upon photolysis, [125I]IMAB incorporated into a protein of apparent agents in a stereoselective manner with a potency order typical of dopamine D1 receptors. In addition, smaller subunits of apparent Mr 62,000 and 51,000 were also specifically labeled by [125I]IMAB in these species. Photoaffinity labeling in the absence or presence of multiple protease inhibitors did not alter the migration pattern of [125I]IMAB-labeled subunits upon denaturing electrophoresis in both the absence or presence of urea or thiol reducing/oxidizing reagents. [125I]IMAB should prove to be a useful tool for the subsequent molecular characterization of the D1 receptor from various sources and under differing pathophysiological states.     

A novel and sensitive radioreceptor assay for serum melatonin levels.

A simple and sensitive radioreceptor assay (RRA) has been developed to measure melatonin levels in serum. The assay is based on competition between 2-[125I]iodomelatonin ([125I]MEL) and melatonin for binding to high-affinity binding sites in chick forebrain. To measure the amount of melatonin present in a serum sample, it was extracted with dichloromethane and added to the assay medium. The percentage inhibition of radioligand binding in the presence of the extracted serum was determined and compared to the percent displacement by known amounts of melatonin in a standard curve. There was little or no cross-reactivity with other structurally related compounds. The sensitivity of the assay is approximately 1.5pg/0.15 mL and the intra- and inter-assay variations are approximately 8%. Since the RRA results are comparable to that of an established radioimmunoassay (RIA), it provides a sensitive and rapid alternative to the more time consuming RIA.     

Differences in degradation processes for insulin and its receptor in cultured foetal hepatocytes.

Binding and degradation of 125I-labelled insulin were studied in cultured foetal hepatocytes after exposure to the protein-synthesis inhibitors tunicamycin and cycloheximide. Tunicamycin (1 microgram/ml) induced a steady decrease of insulin binding, which was decreased by 50% after 13 h. As the total number of binding sites per hepatocyte was 20000, the rate of the receptor degradation could not exceed 13 sites/min per hepatocyte. Cycloheximide (2.8 micrograms/ml) increased insulin binding by 30% within 6 h, an effect that persisted for up to 25 h. This drug had a specific inhibitory effect on the degradation of proteins prelabelled for 10 h with [14C]glucosamine, without affecting the degradation of total proteins. Chronic exposure to 10 nM-insulin neither decreased insulin binding nor modified the effect of the drugs. The absence of down-regulation of insulin receptors cannot be attributed to rapid receptor biosynthesis in foetal hepatocytes. Cellular insulin degradation, which is exclusively receptor-mediated, was determined by two different parameters. First, the rate of release of degraded insulin into the medium was 600 molecules/min per hepatocyte with 1 nM labelled hormone, and increased (preincubation with cycloheximide) or decreased (tunicamycin) as a function of the amount of cell-bound insulin. Secondly, the percentage of cell-bound insulin degraded was not changed by the presence of protein-synthesis inhibitors (25-30%). The stability of insulin degradation suggested that this process was dependent on long-life proteinase systems. Such differences in degradation rates and cycloheximide sensitivity imply that hormone- and receptor-degradation processes utilize distinct pathways.     

Characterization and regional distribution of calcitonin binding sites in the rat brain

Binding sites for calcitonin (CT), as assayed by the displacable binding of [125-I] iodo salmon CT ([125-I]sCT), were found on a membrane fraction prepared from rat brain. The half times of association varied between 23 and 7 min as a function of the temperatures used in the incubation medium, ranging from 6° to 37°C. Salmon CT in amounts as low as 10^?10 M inhibited the binding of [125-I]sCT to the membranes, whereas the virtually biologically inactive free acid of human CT and human CT sulfone did not affect the binding. The specific binding of [125-I]sCT to the membranes was directed to structural and/or conformational features in the COOH-terminal half of salmon CT. 133 to 8,900 times higher amounts of porcine CT and human CT and analogues thereof were required to achieve an inhibition of binding equal to that produced by salmon CT. Sixty-seven percent of specific binding of labeled hormone was not dissociable, even after 6 h of incubation with an excess of unlabeled hormone. [125-I]sCT extracted from the membranes was not degraded, as judged by gel permeation chromatography, and retained binding activity. Specific binding was highest in the hypothalamus, followed by the brainstem. It was intermediate in the midbrain-thalamus and the striatum, lower in the cortex and negligible in the hippocampus, and cerebellum and the spinal cord.