Thyrotropin receptors in thyroid plasma membranes. Characteristics of thyrotropin binding and solubilization of thyrotropin receptor activity by tryptic digestion.

Biologically active bovine 125I-thyrotropin preparations have been prepared, characterized, and used to evaluate the optimal conditions for thyrotropin binding to bovine thyroid plasma membranes in vitro. Binding of 125I-TSH has a pH optimum around 6.0 and is sensitive to the choice and concentration of buffer. Binding is inhibited by salts, especially those containing magnesium and calcium ions; magnesium concentrations optimal for adenylate cyclase assays (2 to 5 mM) result in 85 to 98% inhibition of binding. Binding is temperature sensitive. At 37 degrees binding has its highest initial level; however, instability of the membrane at this temperature causes a rapid loss of binding activity. Binding at 0 degrees is optimal in 30 min and at the same level as initial binding at 37 degrees; since there is no decrease in binding activity, it has been chosen as the optimal temperature. Thyrotropin, luteinizing hormone, the beta subunit of thyrotropin, and the alpha subunit of thyrotropin have relative binding affinities for the thyrotropin receptors of 100, 10, 2, and less than 0.5, respectively. In all of these characteristics, 125I-thyrotropin at 1.5 x 10(-5) M concentrations has the same properties of binding to bovine plasma membranes as do [3H]thyrotropin preparations which have been previously characterized (Amir, S.M., Carraway, T.F., Jr., Kohn, L.D., and Winand, R.V. (1973) J. Biol. Chem. 248, 4092-4100) and used to study binding at 5 x 10(-6) M concentrations. 125I-TSH binding as a function of hormone concentration results in curved Scatchard plots; however, Hill plots of these same binding data are linear and have a slope of 0.65. Taken together, these data suggest that the heterogeneity in thyrotropin binding constants which is evident in the Scatchard plot reflects a negatively cooperative relationship among the thyrotropin receptor sites, i.e. decreased hormonal affinity as hormone concentrations increase. Adenylate cyclase studies yield kinetic plots which also exhibit negative cooperativity; corrections for thyrotropin bound under the adverse binding conditions of the adenylate cyclase assays suggest that Km values for thyrotropin in this enzymatic assay are compatible with binding constants measured by the 125I-thyrotropin preparations. Tryptic digestion destroys binding activity on the thyroid plasma membrane but releases specific thyrotropin receptor activity into the supernatant phase. Chromatography on Sephadex G-100 indicates that this solubilized receptor fragment has a molecular weight between 15,000 and 30,000.     

The hepatic angiotensin II receptor. II. Effect of guanine nucleotides and interaction with cyclic AMP production

Guanine nucleotides were observed to modify the binding of 125I-angiotensin II to rat hepatic plasma membrane receptors. GTP and its nonhydrolyzable analogues greatly increased the dissociation rate of bound 125I-angiotensin II and altered hormone binding to the receptor under equilibrium conditions. In the absence of GTP, 125I-angiotensin II labeled both high affinity sites (Kd1 = 0.46 nM, N1 = 650 fmol/mg) and low affinity sites (Kd2 = 4.1 nM, N2 = 1740 fmol/mg). In the presence of guanine nucleotides, the affinities of the two sites were unchanged, but the number of high affinity sites decreased markedly to 52 fmol/mg. In analogous experiments using the angiotensin II antagonist, 125I-sarcosine1,Ala8-angiotensin II (125I-saralasin), guanine nucleotides minimally affected the interaction of 125I-saralasin with its receptor, increasing the dissociation rate 1.9-fold and the Kd 1.4-fold. The guanine nucleotide inhibition of agonist binding required a cation such as Na+ or Mg2+, with a maximal effect occurring at about 1 mM Mg2+. In liver plasma membranes prepared in EDTA, angiotensin II inhibited basal and glucagon-stimulated adenylate cyclase activities by 30% and 10%, respectively. Angiotensin II also caused a 40% inhibition of glucagon-stimulated cyclic AMP accumulation in intact hepatocytes, with a half-maximal effect occurring at 1 nM. The inhibition by angiotensin II of adenylate cyclase in membranes and of cAMP levels in intact cells could be reversed by the antagonist sarcosine1,Ile8-angiotensin II. Vasopressin caused a smaller 26% inhibition of glucagon-stimulated cyclic AMP accumulation. The ability of angiotensin II to inhibit cyclic AMP synthesis may provide an explanation for the observed effects of guanine nucleotides on 125I-angiotensin II binding to plasma membranes.     

Relationship of prolactin receptors to concanavalin A binding.

Concanavalin A, which binds to specific carbohydrate determinants on the cell surface, was used to investigate the binding of prolactin to its receptors in liver membranes from female rats. The binding of 125I-labeled ovine prolactin to receptors was sharply inhibited by concanavalin A. This effect was reversed by the competitive sugar alpha-methyl-D-mannopyranoside and thus required the presence of specifically bound lectin. Concentrations of concanavalin A of up to 50 mu/ml caused a progressive decrease in the apparent affinity of the prolactin receptor for hormone. When higher concentrations were used, the number of available binding sites decreased. Concanavalin A-resistant receptors, about 30% of the total, had the same dissociation constant (Kd) as the controls. The binding of 125I-labeled concanavalin A in the same membrane preparations showed the presence of two distinct types of concanavalin A binding. At low concentrations, the lectin bound with high affinity (Kd approximately equal to 6.6 . 10(-8) M. At high lectin concentrations, low affinity (Kd approximately equal to 6.7 . 10(-5) M) binding predominated. Since high affinity concanavalin A binding was saturated at 50 microgram/ml, this class of binding most likely alters the affinity of the prolactin receptor for hormone; low affinity concanavalin A binding may mask prolactin receptors, making them inaccessible to the hormone. Binding sites for concanavalin A and prolactin appear to be independent but closely related since (i) concanavalin A did not displace bound prolactin from its receptor, and (ii) detergent-solubilized 125I-labeled prolactin-receptor complexes bound to concanavalin A-Sepharose and were eluted by alpha-methyl-D-mannopyranoside.     

High affinity angiotensin II receptors in myocardial sarcolemmal membranes. Characterization of receptors and covalent linkage of 125I-angiotensin II to a membrane component of 116,000 daltons

High affinity receptors for angiotensin II have been identified on purified cardiac sarcolemmal membranes. Equilibrium binding studies were performed with 125I-labeled angiotensin II and purified sarcolemmal vesicles from calf ventricle. The curvilinear Scatchard plots were evaluated by nonlinear regression analysis using a two-site model which identified a high affinity site Kd1 = 1.08 +/- 0.3 nM and N1 = 52 +/- 10 fmol/mg of protein and a low affinity site Kd2 = 52 +/- 16 nM and N2 = 988 +/- 170 fmol/mg of protein. Monovalent and divalent cations inhibited the binding of 125I-angiotensin II by 50%. The affinity of angiotensin II analogs for the receptor was determined using competitive binding assays; sarcosine, leucine-angiotensin II (Sar,Leu-angiotensin II), Kd = 0.53 nM; angiotensin II, Kd = 2.5 nM; des-aspartic acid-angiotensin II, Kd = 4.81 nM; angiotensin I, Kd = 77.6 nM. There is a positive correlation between potency in inducing positive inotropic response in myocardial preparations reported by others and potency for the hormone receptor observed in the binding assays. Pseudo-Hill plots of the binding data showed that agonists display biphasic binding with Hill numbers around 0.65 while antagonists recognized a single class of high affinity receptors with Hill numbers close to unity. These data were confirmed using 125I-Sar,Leu-angiotensin II in equilibrium binding studies which showed that this antagonist bound to a single class of receptor sites; Kd = 0.42 +/- 0.04 nM and N = 1050 +/- 110 fmol/mg of protein. Competition-binding experiments with this 125I-peptide yielded monophasic curves with Hill numbers close to unity for both agonists and antagonists. Membrane-bound 125I-angiotensin II was covalently linked to its receptor by the use of bifunctional cross-linking reagents such as dithiobis(succinimidyl propionate) and bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone. Analysis of the membranes showed the labeling of a component with an apparent Mr = 116,000. The affinity labeled species showed characteristics expected of a functional component of the high affinity receptor. The affinity labeling of this membrane component was inhibited by nanomolar angiotensin II or Sar,Leu-angiotensin II. Together these data indicate that high affinity receptors exist for angiotensin II that most likely mediate the positive inotropic effects of this hormone on myocardial cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparison of the ability of seven gonadotropin preparations from different mammalian sources to interact with the adenylyl cyclase system in corpora lutea from rabbits and rats

The effects of guanine nucleotides and magnesium (Mg2+) on the interaction of seven different gonadotropin preparations with their rabbit and rat luteal receptors were studied and compared to the ability of these gonadotropins to stimulate luteal adenylyl cyclase activity. In both the rabbit and rat, human chorionic gonadotropin (hCG) and human luteinizing hormone (hLH) were less efficacious than the other gonadotropin preparations in stimulating luteal adenylyl cyclase activity and thus behaved as partial agonists. Addition of 2 mM MgCl2 increased the affinity of the rat luteal receptors for all seven gonadotropins tested, while in the rabbit Mg2+ increased the affinities for porcine, bovine, ovine, rat and rabbit LH but did not significantly alter the affinities for hCG or hLH. In no instance did the addition of 100 microM GTP alter the affinity of the receptor from that observed in the absence or presence of Mg2+. A positive correlation existed for both species between the Kd values calculated from binding experiments and the Kact values obtained in adenylyl cyclase assays suggesting that the specific gonadotropin-binding sites present in rabbit and rat luteal membranes represent receptors which mediate the stimulatory effect of LH. The magnitude of the Mg2+-induced increase in affinity of a given gonadotropin preparation for its receptor was correlated with the efficacy with which that gonadotropin stimulated luteal adenylyl cyclase activity in both the rabbit and rat.     

Properties of the interaction between bovine thyrotropin and bovine thyroid plasma membranes.

Studies have been conducted to characterize further the interaction between 125I-labeled bovine thyrotropin (TSH) and bovine thyroid plasma membranes. Sequential subcellular fractionation of thyroid homogenates yielded preparations of progressively greater specific binding activity, highest activity being found in fractions previously shown to contain predominately plasma membranes (Amir, S. M., Carraway, T.F., Kohn, L.D., and Winand, R.J. (1973) J. Biol. Chem. 248, 4092-4100). Although binding of 125I-TSH by plasma membranes was greatest at pH 6.0, studies were conducted at pH 7.45 as well as pH 6.0, and results obtained differed quantitatively, but not qualitatively. Binding was maximal at 0 degrees, 15 degrees, and 22 degrees and steady state values remained unchanged for at least 22 hours. At 37 degrees, binding was decreased by 40% at 1 hour; the loss was even greater (65%) at 50 degrees. A similar loss of binding was evident when membranes were preincubated without TSH at 37 degrees or higher and were then incubated with 125I-TSH at 0 degrees. Lineweaver-Burk analysis indicated that preincubation resulted in loss of receptor sites without change in affinity of residual receptors. Addition of Ca2+ (1 to 10 mM) to the preincubation medium prevented the effect of preincubation at 37 degrees by preserving the number of receptor sites without altering their affinity. Under similar conditions, Na+ and K+ were without protective effect. Membranes bound 45Ca2+ in a specific and saturable manner. Scatchard plots indicated a dissociatiion constant (Kd) of 9 X 10(-5) M and a capacity (n) of 54 nmol/mg of membrane protein. 45Ca2+ was also displaced from membranes by Mg2+ and Mn2+. Ca2+ had a biphasic effect on binding; low concentrations (1 to 10 muM) added to the incubation mixture stimulated binding, while higher concentrations (0.1 mM) caused inhibition. Mg2+ and Mn2+, at comparable concentrations, were also inhibitory, Na+ and K+ less so. In the case of Ca2+, both the stimulatory and inhibitory concentrations were lower than those required to achieve saturation of Ca2+-binding sites. Proteolytic enzymes (trypsin, alpha-chymotrypsin, and pronase) sharply reduced binding of 125I-TSH, owing to a decrease in receptor sites. Phospholipases A and C enhanced binding of TSH, while neuraminidase and beta-galactosidase were without measurable effect.     

The interaction of radioiodinated thyrotropin with plasma membranes: Evidence for high affinity binding sites in the thyroid

The binding of biologically active [¹²⁵I]thyrotropin to purified plasma membranes prepared from bovine thyroid glands was studied. At 4°C, specific binding reached a maximum after 2 h of incubation and a plateau was maintained for up to 20 h. Degradation of [¹²⁵I]thyrotropin was undetectable after 2 h of incubation and was only 10% of the total after 20 h.At pH 6.0, at which binding was maximal, a single class of binding sites, having a dissociation constant of approx. 25 nM, was evident. Dissociation studies revealed first order kinetics with a half-time of 2–3 min. At pH 7.5, binding curves were complex, suggesting two orders of binding sites with dissociation constants of approx. 200 nM and 80 pM. Further, at this pH, dissociation of the thyrotropin from its receptor was also complex, suggesting the presence of two first order reactions, one with a half-time similar to that seen at pH 6.0 and another with a half-time of 4 h. At both pH 6.0 and 7.5, insulin, glucagon, growth hormone, and prolactin were without effect on [¹²⁵I]thyrotropin binding.Similar high affinity and low affinity binding sites were seen with porcine thyroid membranes, but only low affinity sites were seen with either rat liver membranes or human cultured lymphocytes.     

Properties and distribution of receptors for pituitary adenylate cyclase activating peptide (PACAP) in rat brain and spinal cord.

A high density (in the pmol/mg protein range) of specific functional receptors for PACAP (pituitary adenylate cyclase activating polypeptide) was observed in membranes from rat brain cortex, olfactory bulb, hypothalamus, hippocampus, striatum, cerebellum, pons and cervico-dorsal spinal cord, using [125I]PACAP-27 (PACAP 1-27). The tracer bound rapidly, specifically and reversibly. Competition binding curves were compatible with the coexistence, in the eight central nervous areas explored, of high and low affinity binding sites for PACAP-27 (Kd of 0.2 nM and 3.0 nM, respectively), and of only one class of binding sites for PACAP-38 (PACAP (1-38), Kd 0.2-0.9 nM). VIP inhibited only partially the binding of [125I]PACAP-27, and PHI, GRF(1-29)NH2 and secretin were ineffective at 1 microM. Chemical [125I]PACAP-27 cross-linking revealed a single specific 64 kDa protein species. In rat brain cortical membranes, saturation and competition experiments, using [125I]PACAP-38 as radioligand, indicated the presence of both high (Kd 0.13 nM) and low (Kd 8-10 nM) affinity binding sites for PACAP-38 and of low affinity (Kd 30 nM) binding sites for PACAP-27. These data taken collectively suggest the coexistence of PACAP-A receptors with a slight preference for PACAP-27 over PACAP-38 and of PACAP-B receptors that recognize PACAP-38 with a high affinity and PACAP-27 with low affinity. Both PACAP-27 and PACAP-38 stimulated adenylate cyclase with similar potency and efficacy. VIP was markedly less potent in this respect and also less efficient, except on cerebellar membranes.     

Ca2+-dependent regulation of calmodulin binding and adenylate cyclase activation in bovine cerebellar membranes

The binding parameters of 125I-labeled calmodulin to bovine cerebellar membranes have been determined and correlated with the activation of adenylate cyclase by calmodulin. In the presence of saturating levels of free Ca2+ calmodulin binds to a finite number of specific membrane sites with a dissociation constant (Kd) of 1.2 nM. Furthermore, Scatchard analysis reveals a second population of binding sites with a 100-fold lower affinity for calmodulin. The Ca2+-dependence of calmodulin binding and of adenylate cyclase activation varies with the amount of calmodulin present, as can be inferred from the model of sequential equilibrium reactions which describes the activation of calmodulin-dependent enzymes. On the basis of this model, a quantitative analysis of the effect of free Ca2+ and of free calmodulin concentration on both binding and activation of adenylate cyclase was carried out. This analysis shows that both processes take place only when calmodulin is complexed with at least three Ca2+ atoms. The concentration of the active calmodulin X Ca2+ species required for half-maximal activation of adenylate cyclase is very similar to the Kd of the high affinity binding sites on brain membranes. A Hill coefficient of approx. 1 was found for both processes indicating an absence of cooperativity. Phenothiazines and thioxanthenes antipsychotic agents inhibit calmodulin binding to membranes and calmodulin-dependent activation of adenylate cyclase with a similar order of potency. These results suggest that the Ca2+-dependent binding of calmodulin to specific high affinity sites on brain membranes regulates the activation of adenylate cyclase by calmodulin.     

Atrial natriuretic peptide binding, cross-linking, and stimulation of cyclic GMP accumulation and particulate guanylate cyclase activity in cultured cells

The stimulation of cyclic GMP accumulation and particulate guanylate cyclase activity by atrial natriuretic peptide (ANP) was compared to the affinity and number of ANP receptors in eight cultured cell types. At 100 nM, ANP increased cyclic GMP by 13-fold in bovine adrenal cortical, 35-fold in human lung fibroblast, 58-fold in canine kidney epithelial, 60-fold in bovine aortic smooth muscle, 120-fold in rat mammary epithelial, 260-fold in rat Leydig, 300-fold in bovine kidney epithelial, and 475-fold in bovine aortic endothelial cells. ANP (1 microM) increased particulate guanylate cyclase activity by 1.5-, 2.5-, 3.1-, 3.2-, 5.0-, 7.0-, 7.8-, and 8.0-fold in bovine adrenal cortical, bovine aortic smooth muscle, human lung fibroblast, canine kidney epithelial, rat mammary epithelial, rat Leydig, bovine kidney epithelial, and bovine aortic endothelial cells, respectively. Specific 125I-ANP binding to intact rat Leydig (3,000 sites/cell; Kd = 0.11 nM), bovine aortic endothelial (14,000 sites/cell; Kd = 0.09 nM), bovine adrenal cortical (50,000 sites/cell; Kd = 0.12 nM), human lung fibroblast (80,000 sites/cell; Kd = 0.32 nM), and bovine aortic smooth muscle (310,000 sites/cell; Kd = 0.82 nM) cells was saturable and high affinity. No specific and saturable ANP binding was detected in bovine and canine kidney epithelial and rat mammary epithelial cells. Two ANP-binding sites of 66,000 and 130,000 daltons were specifically labeled by 125I-ANP after cross-linking with disuccinimidyl suberate. The 130,000-dalton ANP-binding sites bound to a GTP-agarose affinity column, and the specific activity of guanylate cyclase was increased by 90-fold in this fraction. Our results demonstrate that the increase in cyclic GMP accumulation and particulate guanylate cyclase activity by ANP does not correlate with the affinity and number of ANP-binding sites. These results suggest that multiple populations of ANP receptors exist in these cells and that only one receptor subtype (130,000 daltons) is associated with particulate guanylate cyclase activity.     

Characterization of kinin binding sites: identity of B2 receptors in the epithelium and the smooth muscle of the guinea pig ileum.

Binding of [125I-Tyr8]bradykinin (BK) was measured in homogenates of epithelial and smooth muscle layers of the guinea pig ileum. Binding assays were performed at 4 degrees C for 40 min (smooth muscle) or 90 min (epithelium) in 25 mM PIPES buffer at pH 6.8 in the presence of 1 mM 1,10-phenanthroline, 140 micrograms/mL bacitracin, 1 mM captopril, 1 mM dithiothreitol, and 0.1% bovine serum albumin. Specific binding of [125I-Tyr8]BK (0.32 nM) to epithelial and smooth muscle cell membranes was linearly related to protein concentration between 0.05 and 0.5 mg/mL. Equilibrium experiments showed that specific binding of [125I-Tyr8]BK was saturable and Scatchard analysis indicated the presence of a high affinity site with a Kd value of 1.6 nM and a Bmax of 156 fmol/mg of protein in the epithelial cell membranes. In smooth muscle membranes, Kd was 1.8 nM and the maximum number of binding sites was 58 fmol/mg of protein. Unlabelled peptides, namely bradykinin, [Tyr8]BK, [Hyp3]BK, D-Arg[Hyp3]BK, [Hyp3,Tyr(Me8)]BK, and kallidin displaced [125I-Tyr8]BK binding while other peptides, angiotensin II and substance P, had no effect. A series of B2-receptor antagonists displaced [125I-Tyr8]BK from specific binding sites with IC50 values ranging from 16 to 152 nM on epithelial cell membranes; similar values were obtained from smooth muscle cell membranes. These findings suggest that the binding sites in both preparations are of the B2 type. B1-receptor agonists and antagonists were found to be inactive at concentrations up to 10(-4) M. Results obtained in the two preparations were compared and a positive highly significant correlation was demonstrated between the two sets of data.(ABSTRACT TRUNCATED AT 250 WORDS)     

Urokinase binding and receptor identification in cultured endothelial cells.

Recombinant human single-chain urokinase (rscu-PA), two-chain urokinase (tcu-PA), and diisopropyl-fluorophosphate-treated tcu-PA (DFP-tcu-PA) bound to cultured human and porcine endothelial cells in a rapid, saturable, dose-dependent and reversible manner. Analysis of specific binding results in cultured human umbilical vein endothelial cells (HUVECs) gave the following estimated values for Kd and Bmax: 0.57 +/- 0.08 nM (mean +/- S.E.) and 188,000 +/- 18,000 sites/cell for 125I-labeled rscu-PA; 0.54 +/- 0.10 nM and 132,000 +/- 23,900 sites/cells for 125I-labeled tcu-PA; 0.89 +/- 0.14 nM and 143,000 +/- 30,300 sites/cell for 125I-labeled DFP-tcu-PA, respectively. Values for Kd were similar for primary and subcultured (six passages) HUVECs, but Bmax values were lower in subcultured HUVECs. Similar Kd values were found in cultured porcine endothelial cells; however, Bmax values varied depending on the endothelial cell type. All 125I-labeled urokinase forms yielded similar cross-linked approximately 110-kDa ligand-receptor complexes with cultured HUVECs, and 125I-labeled DFP-tcu-PA bound to a single major approximately 55-kDa protein in whole-cell lysates (ligand blotting/autoradiography), suggesting the presence of a single major approximately 55-kDa urokinase receptor in cultured HUVECs. The approximately 55-kDa urokinase receptor, isolated from several separate batches of cultured HUVECs (3-5 micrograms of protein, approximately 1 x 10(9) cells), by ligand affinity chromatography, exhibited the following properties: retained biologic activity as evidenced by its ability to bind 125I-labeled rscu-PA by ligand blotting/autoradiography and formation of a cross-linked 125I-labeled approximately 110-kDa rscu-PA-receptor complex; single-chain approximately 55-kDa protein, following reduction; complete conversion to and formation of a single major deglycosylated approximately 35-kDa protein, following treatment with N-glycanase.     

Binding of [3H]forskolin to solubilized preparations of adenylate cyclase

The binding of [3H]forskolin to proteins solubilized from bovine brain membranes was studied by precipitating proteins with polyethylene glycol and separating [3H]forskolin bound to protein from free [3H]forskolin by rapid filtration. The Kd for [3H]forskolin binding to solubilized proteins was 14 nM which was similar to that for [3H]forskolin binding sites in membranes from rat brain and human platelets. Forskolin analogs competed for [3H]forskolin binding sites with the same rank potency in both brain membranes and in proteins solubilized from brain membranes. [3H]forskolin bound to proteins solubilized from membranes with a Bmax of 38 fmol/mg protein which increased to 94 fmol/mg protein when GppNHp was included in the binding assay. In contrast, GppNHp had no effect on [3H]forskolin binding to proteins solubilized from membranes preactivated with GppNHp. Solubilized adenylate cyclase from non-preactivated membranes had a basal activity of 130 pmol/mg/min which was increased 7-fold by GppNHp. In contrast, adenylate cyclase from preactivated membranes had a basal activity of 850 pmol/mg/min which was not stimulated by GppNHp or forskolin. Thus, the number of high affinity binding sites for [3H]forskolin in solubilized preparations correlated with the activation of adenylate cyclase by GppNHp via the guanine nucleotide binding protein (GS).     

Kinetics of receptor-mediated binding of tropoelastin to ligament fibroblasts

Soluble 125I-labeled tropoelastin bound to confluent cultures of bovine ligamentum nuchae fibroblasts and to fibroblast plasma membrane preparations in a time-dependent, saturable, and reversible manner. Scatchard analysis indicates that there are approximately 2 X 10(6) binding sites/cell with a binding efficiency (Kd) of 8 X 10(-9) M. Binding of tropoelastin to cells and membranes reached equilibrium by 90 min and was reversible with 50% of specifically bound material released by 40 min. Specific binding of tropoelastin to cells pre-treated with dilute trypsin solutions was reduced significantly when compared with controls. Four polypeptides of estimated molecular masses of 67, 61, 55, and 43 kDa were obtained from detergent extracts of plasma membranes by elution affinity chromatography on elastin-Affi-Gel. Our findings establish that elastin-specific binding proteins displaying characteristics of a true receptor are present on the surface of elastin-producing cells.     

Characterization of angiotensin II receptor subtypes in rat liver

Radioligand binding studies identified two classes of 125I-angiotensin II-binding sites in rat liver membranes. High affinity binding sites (Kd = 0.35 +/- 0.13 nM, N = 372 +/- 69 fmol/mg of protein) were inactivated by dithiothreitol (0.1-10 mM) without any apparent change in low affinity binding sites (Kd = 3.1 +/- 0.8 nM, N = 658 +/- 112 fmol/mg of protein). Dithiothreitol inactivation was readily reversible but could be made permanent by alkylation of membrane proteins with iodoacetamide. Angiotensin II stimulation of glycogen phosphorylase in isolated rat hepatocytes (maximal stimulation 780%, EC50 = 0.4 nM) was completely inhibited by 10 mM dithiothreitol, a concentration which also abolished high affinity site binding; phosphorylase stimulation by glucagon and norepinephrine under these conditions was unaltered. Angiotensin II inhibition of glucagon-stimulated adenylate cyclase activity in hepatocytes required higher angiotensin II concentrations (EC50 = 3 nM) than phosphorylase stimulation and was not affected by dithiothreitol. Fractional occupancy of high affinity binding sites by 125I-angiotensin II correlated closely with angiotensin II-mediated phosphorylase stimulation, whereas occupancy of low affinity sites paralleled inhibition of adenylate cyclase activity. These data indicate that the physiologic effects of angiotensin II in rat liver are mediated by two distinct receptors, apparently not interconvertible, and provide the first evidence for angiotensin II receptor subtypes with differing biochemical features and mechanisms of action.     

Expression and characterization of erythropoietin receptors on normal human bone marrow cells

We studied the specific binding of 125I-labeled bioactive recombinant human erythropoietin (Epo) to human bone marrow mononuclear cells (BMNC) obtained from normal subjects. The 125I-labeled Epo bound specifically to the BMNC. Scatchard analysis of the data showed two classes of binding sites; one high affinity (Kd 0.07 nM) and the other low affinity (Kd 0.38 nM). The number of Epo binding sites per BMNC was 46 +/- 16 high-affinity receptors and 91 +/- 51 low-affinity receptors. The specific binding was displaced by unlabeled Epo, but not by other growth factors. Receptor internalization was observed significantly at 37 degrees C, but was prevented by the presence of 0.2% sodium azide. These findings indicate that human BMNC possess two classes of specific Epo receptors with characteristics of a hormone-receptor association.     

Characterisation of heterologous and homologous low-density lipoprotein binding to apolipoprotein B,E receptors on porcine adrenal cortex membranes: enhanced binding of trypsin-modified human low-density lipoprotein

The characteristics of the binding of homologous and heterologous (human) LDL to membrane preparations from porcine adrenal cortex have been determined. The membranes displayed a single class of high-affinity, saturable binding site for both 125I-labelled porcine and human LDL, which was dependent on divalent cations, in addition to a low-affinity, non-saturable component(s). Porcine LDL displaced both 125I-labelled porcine and 125I-labelled human LDLs from the high-affinity binding site more effectively than human LDL, reflecting the lower Kd, (13.2 micrograms/ml) for porcine than human (Kd 19.2 micrograms/ml) LDL. These values are comparable to those obtained for half-maximal binding of human and bovine LDLs in a bovine adrenocortical membrane system (Kovanen, P.T., Basu, S.K., Goldstein, J.L. and Brown, M.S. (1979) Endocrinology 104, 610-616). Tryptic modification of porcine LDL (T-LDL) diminished its ability to compete with 125I-labelled native LDL for the high-affinity binding site; in contrast, 125I-labelled porcine T-LDL showed an elevated receptor affinity (Kd 9.7 micrograms/ml) and was more efficiently displaced by its unlabelled counterpart than by native porcine LDL. Tryptic treatment of human LDL similarly increased its binding affinity (Kd 8.3 micrograms/ml), although in this case, the unlabelled T-LDL displaced not only 125I-labelled human T-LDL but also 125I-labelled human LDL from the high-affinity site more effectively than native LDL. We conclude that (i) porcine adrenocortical membranes possess binding sites specific for LDL and resembling the apolipoprotein B,E receptors already demonstrated in murine, bovine and human adrenal cortex; (ii) tryptic modification of porcine LDL may remove or destroy segments of apolipoprotein B100 which contribute to receptor recognition sites on the surface of the particle; (iii) trypsinised porcine LDL may interact with the membrane binding site by a mechanism differing from that by which native LDL binds, and (iv) trypsinisation of human LDL may cleave or remove species-specific segments of the B100 protein at or close to the receptor recognition site(s) on the particle, thus decreasing structural differences between porcine and human LDL, and thereby enhancing its binding affinity for the porcine receptor.     

Interaction of clonidine and clonidine analogues with alpha-adrenergic receptors of neuroblastoma X glioma hybrid cells and rat brain: comparison of ligand binding with inhibition of adenylate cyclase

Clonidine and several analogues of clonidine are shown to be useful probes for alpha 2-adrenergic receptors in a comparative study of ligand binding and inhibition of adenylate cyclase. The alpha-adrenergic properties of a new potential probe, N-(4-hydroxyphenacetyl)-4-aminoclonidine hydrochloride, are described. [3H]Clonidine binds to alpha-receptors of NG108-15 neuroblastoma X glioma hybrid cell membranes with Kd values of 1.7 and 33 nM for putative high-affinity and low-affinity sites, respectively. p-Aminoclonidine and hydroxyphenacetyl aminoclonidine displace [3H]clonidine from the high-affinity sites with Kd values of 2.3 and 5.8 nM, respectively. Rat brain alpha 2-receptors also exhibit high affinity toward clonidine, p-aminoclonidine, and hydroxyphenacetyl aminoclonidine, as determined by displacement of specifically bound [3H]clonidine. Clonidine, p-amino-clonidine, and hydroxyphenacetyl aminoclonidine elicit modest inhibition (up to 24%) of NG108-125 adenylate cyclase by interaction with alpha 2-receptors (Kd,app 300, 30, and 130 nM, respectively); these compounds also partially reverse the inhibition elicited by (--)-norepinephrine. Components of the adenylate cyclase assay mixture, particularly ATP, GTP, sodium ions, and a nucleoside-triphosphate-regenerating system, decrease the high-affinity [3H]clonidine binding to NG108-15 membranes; in the presence of these components, alpha-receptors possess only low affinity (Kd 43 nM) for [3H]clonidine. The results are consistent with the concept that certain components required for the receptor-mediated inhibition of adenylate cyclase convert alpha 2-receptors from a high-affinity inactive state to a low-affinity active state.     

Characterization of the binding of human growth hormone to microsomal membranes from rat liver.

The binding of 125I-labelled human growth hormone to the 100000g microsomal membrane fraction prepared from the livers of normal female rats was dependent on time, temperature, pH, membrane concentration and concentration of 125I-labelled human growth hormone. At 22 degrees C binding reached a steady state after 16h, with the mean maximal specific binding being 20% of the tracer initially added. Dissociation of 125I-labelled human growth hormone from the membranes, after addition of excess of unlabelled hormone, was relatively slow with a half-time greater than 24h. Only minor degradation of the 125I-labelled human growth hormone was observed during incubation with membranes for 16 or 25h at 22 degrees C. Similarly, no significant change in the ability of membranes to bind human growth hormone was evident after preincubation of the membranes for 16 or 25h. Specificity studies showed that up to 90% of the 125I-labelled human growth hormone bound could be displaced by 1 mug of unlabelled hormone. Ovine prolactin also showed considerable competition for the binding site. Non-primate growth-hormone preparations (ovine, bovine, porcine and rat) and non-related hormones (insulin, thyrotropin, lutropin and follitropin) all showed negligible competition. Scatchard analysis of the binding data was consistent with two classes of binding site with binding affinities of 0.64 X 10(10) +/- 0.2 X 10(10)M-1 and 0.03 X 10(10) +/- 0.007 X 10(10)M-1 and corresponding binding capacities of 98.4 +/- 10 fmol/mg of protein and 314.6 +/- 46.3 fmol/mg of protein. These studies provide data which, in general, are consistent with the criteria required for hormone-receptor interaction. However, proof of the thesis that the human-growth-hormone-binding sites in female rat liver represent physiological receptors must await the demonstration of a correlation between hormone binding and a biological response.