Insulin receptor interaction in human placental plasma membranes

Insulin-receptor interaction in partially purified preparations of human placental plasma membranes from normal mothers at term of pregnancy has been characterized. 125I-insulin became rapidly and reversibly bound to plasma membranes, being time and temperature dependent. The binding readily appeared at 1.0 ng/ml insulin concentration which falls within the physiological range of peripheral blood. Low levels of unlabeled insulin inhibited binding; 20 ng/ml insulin produced fifty per cent inhibition. Scatchard plots of data from competitive insulin binding proved to be curvilinear. The insulin greater ability for binding observed in this preparation can be explained by the purification degree achieved at the plasma membranes. 125I-insulin was less degraded by partially purified placental plasma membranes than by a microsomal-membrane preparation obtained without differential centrifugation in sucrose linear gradient. All these properties strongly suggest that the insulin-binding sites characterized in the plasma membrane fraction of the placenta represent biologically important receptors to hormone.     

Identification of the insulin receptor in undifferentiated and differentiated NB-15 mouse neuroblastoma cells by affinity labeling

Plasma membranes prepared from clonal NB-15 mouse neuroblastoma cells were sequentially incubated with 125I-labeled insulin (10 nM) and the bifunctional cross-linking agent disuccinimidyl suberate. This treatment resulted in the cross-linking of 125I-labeled insulin to a polypeptide that gave an apparent Mr of 135 000 on a sodium dodecyl sulfate-polyacrylamide gel electrophoresed in the presence of 10% beta-mercaptoethanol. Affinity labeling of this polypeptide was inhibited by the presence of 5 microM unlabeled insulin, but not by 1 microM unlabeled nerve growth factor. Using the same affinity labeling technique, 125I-labeled nerve growth factor (1 nM) did not label any polypeptide appreciably in the plasma membranes of NB-15 cells but labeled an Mr 145 000 and an Mr 115 000 species in PC-12 rat pheochromocytoma cells. The number of insulin binding sites per cell in the intact differentiated NB-15 mouse neuroblastoma cells was approx. 6-fold greater than that in the undifferentiated NB-15 mouse neuroblastoma cells as measured by specific binding assay, suggesting an increase of the number of insulin receptors in NB-15 mouse neuroblastoma cells during differentiation.     

Radioligand receptor assay for prolactin using chicken and turkey kidney membranes.

1. A sensitive and specific radioreceptor assay for measuring prolactin in tissue extracts, culture media and serum has been developed utilizing membrane fractions prepared from either chicken or turkey kidney, and an ovine prolactin standard and tracer. 2. Assay sensitivity was 1.0 +/- 0.1 ng per tube, 50% inhibition of binding occurred with 12.8 +/- 1.2 ng of unlabeled ovine prolactin standard, and intra- and interassay coefficients of variation were 3.9% and 8.8%, respectively. 3. The 47,800 g kidney membrane preparation, which yielded maximum specific binding of 20-40%, offered advantages over current methods including use of an easily available and inexpensive tissue, no need for pretreatment of the donor animal, a high yield of receptor protein, and a simplified method of membrane preparation.     

Stimulation of iodine organification in porcine thyroid cells by thyroid stimulators

Several Graves' sera were simultaneously assessed in a bioassay based on the ability of porcine thyroid cells to organify 125I and in a radioreceptor assay for TSH receptor binding activity. Both assay systems were sensitive to 1 mcU/ml (final concentration) of unlabelled bovine TSH. Six Graves' sera were studied in detail over a wide (0-1.0 mcl sera) dose response range in repeat determinations. Two sera exhibited parallel binding and stimulating. However, two sera revealed significant inhibition of 125I-TSH binding prior to the demonstration of stimulation and the other two sera showed stimulatory capabilities before significant binding was evident. IgG was prepared from one serum by ammonium sulphate precipitation and chromatography on Sepharose 6B and then subjected to preparative isoelectric focusing. The isoelectric distribution of the two activities were found to be identical with major peaks of activity at pl=9.5 and pl=8.5. In summary: 1) each Graves' sera exhibits different dose-response curves with respect to binding and stimulation, 2) at certain concentrations of sera, only binding or stimulation were evident, 3) neither assay was consistently more sensitive for the presence of Graves' immunoglobulins, 4) for one Graves' sera, binding and stimulation could not be separated by isoelectric focusing. These studies would suggest each Graves' immunoglobulin has inherently different characteristics in its interaction with the TSH receptor.     

The measurement of free nitrendipine in human serum by an equilibrium dialysis - radioreceptor assay

A radioreceptor assay using [³H]nitrendipine and rat cerebral cortical membranes, in conjunction with equilibrium dialysis, measures the unbound (free) level of nitrendipine in human sera. The sensitivity of the assay is 0.1–0.2 picomoles/ml and is linear from 4 × 10^?11 to 4 × 10^?9 M nitrendipine. Other dihydropyridine calcium channel antagonists may be measured using this assay if these compounds are used to generate the standard curve. Blank serum interferes with specific [³H]nitrendipine binding (24 percent inhibition per 20 μ1 serum) whereas serum dialysates do not. Total serum nitrendipine levels may be measured, but the sensitivity of the assay is decreased due to interference by serum. Nitrendipine is highly protein bound in serum (93 – 99 percent). This protein binding is essentially unchanged over a serum concentration from 1 to 100 ng/ml. This assay is suitable for pharmacokinetic and pharmacodynamic studies.     

Identification and characterization of a pituitary corticotropin-releasing factor binding protein by chemical cross-linking

A corticotropin-releasing factor (CRF) binding protein has been identified based on the chemical cross-linking of ovine [Nle21,m-125I-Tyr32]CRF (125I-oCRF) to bovine anterior pituitary membranes using disuccinimidyl suberate (DSS). The apparent molecular weight of the cross-linked complex determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography was approximately 75,000 and was slightly decreased in its nonreduced state, suggesting the presence of intramolecular disulfide bonds. Subtracting the molecular weight of 125I-oCRF, the binding protein appeared to have a molecular weight of approximately 70,000. The cross-linking was specific since an excess (1 microM) of an unrelated peptide (insulin) did not affect the appearance of the Mr 75,000 band. The concentration of CRF required to inhibit cross-linking by 50% was found to be similar to that determined for bovine pituitary CRF receptors by radioreceptor assay. The nonhydrolyzable GTP analogue 5'-guanylylimidodiphosphate dose dependently inhibited the cross-linking of 125I-oCRF to the Mr 70,000 protein. 50 nM of the inactive CRF analogue, [Ala14]oCRF, had no effect on the cross-linking, an observation which is consistent with this compound's low potencies in bioassays and radioreceptor assays. These results strongly suggest that this Mr 70,000 protein is the biological bovine anterior pituitary CRF receptor.     

A radioimmunoassay for human insulin receptor correlation between insulin binding and receptor mass.

We have developed a radioimmunoassay for human insulin receptor. Serum from a patient with Type B severe insulin resistance was used as anti-insulin receptor antiserum. Pure human placental insulin receptor was used as reference preparation and 125I labeled pure insulin receptor as trace. The radioimmunoassay was sensitive (limit of detection less than 17 fmol), reproducible (inter and intra-assay coefficients of variation 12.5% and 1.6% respectively) and specific (no crossreactivity with pure placental IGF-1 receptor, insulin and glucagon). The anti-insulin receptor antibody was, however, able to differentiate between insulin receptor from human placenta and from rat liver. To determine the number of insulin binding sites per receptor, we measured insulin binding (by insulin binding assay) and insulin receptor mass (by radioimmunoassay) in solubilized aliquots from 5 human placentas. The molar ratio of insulin binding to receptor mass was 0.86 +/- 0.12 when binding was determined with monoiodinated 125I-Tyr A 14-insulin. It was 1.94 +/- 0.27 when randomly iodinated 125I-insulin was used. In conclusion, using a sensitive, reproducible and specific radioimmunoassay, we have measured insulin receptor mass independent of insulin binding. Our data are most compatible with binding of one insulin molecule per human placental insulin receptor.     

Determination of insulin antibodies.

Insulin antibodies were determined as percentage binding of 125I-insulin in the sera of normal persons and of diabetic subjects treated and untreated with insulin. The effect of the dilution of the serum, circulating insulin and extraction of free and total insulin was evaluated. The determination of insulin antibodies in samples at a final dilution of 1:10 clearly discriminated between insulin-treated and untreated subjects. In insulin-treated subjects, the determination of insulin antibodies in samples at a final dilution of 1:100 gave false-negative results in 28 per cent. However, the determination of insulin antibodies at a final dilution of 1:100 discriminated between insulin-resistant and non-resistant diabetic subjects. Extraction of total insulin at pH 3.0 using 0.1 N HCl increased the percentage of 125I-insulin binding significantly. Extraction of free insulin by charcoal from the samples did not increase the binding of 125I-insulin. The injection of crystalline insulin 4 hours prior to withdrawing the samples did not decrease binding of 125I-insulin.     

Further evidence for the involvement of a membrane proteolytic step in insulin action.

The hypothesis that insulin action involves a membrane proteolytic step was further explored, by using isolated rat adipocytes and liver plasma membranes. (1) The maximal insulin stimulation of 2-deoxyglucose transport and lipogenesis in fat-cells was selectively inhibited (73-88%) by N alpha-p-tosyl-L-lysine chloromethyl ketone (Tos-Lys-CH2Cl; active-site inhibitor of trypsin; 30-125 microM), p-nitrophenyl p'-guanidinobenzoate (active-site inhibitor of serine proteinases; 30-125 microM) and p-tosyl-L-arginine methyl ester (arginine ester substrate analogue of proteinases; 1-2 mM), under conditions where neither the basal rate of each metabolic process nor insulin binding nor cellular ATP content were affected. In contrast, N-acetyl-L-alanyl-L-alanyl-L-alanine methyl ester (alanine ester substrate analogue of proteinases; 1-2 mM) was ineffective. (2) Endoproteinase Arg-C (0.25-40 micrograms/ml) exerted dose-dependent insulin-like effects on both 2-deoxyglucose transport and lipogenesis in fat-cells, whereas endoproteinase Lys-C (5-100 micrograms/ml) was ineffective. The maximal activation by endoproteinase Arg-C of both processes (200 and 177% of control values respectively) was shown to occur under conditions where membrane integrity (assessed by measurement of lactate dehydrogenase leakage and passive glucose diffusion) was preserved. This effect was inhibited by Tos-Lys-CH2Cl (125 microM) and was not additive with the maximal insulin effect. (3) Insulin (1-100 ng/ml) produced a dose-dependent increase in the trichloroacetic acid-soluble 125I radioactivity released after a 30 min incubation at 37 degrees C of 125I-labelled liver plasma membranes, but was ineffective on 125I-labelled bovine serum albumin. Insulin effects on both radio-labelled proteins were reproduced by wheat-germ agglutinin (20 micrograms/ml), an insulin mimicker shown to act through the insulin receptor. These data provide further evidence for the hypothesis that insulin bioeffects involve the activation of a membrane serine proteinase with arginine specificity.     

Ontogeny of ovine fetal liver and kidney plasma membrane insulin receptors and fetal growth

This investigation was performed to define certain characteristics of insulin-receptor interaction during the last 2 months of gestation in fetal sheep liver and kidney. Twenty-one sheep carrying a total of 46 fetuses were sacrificed at various gestational ages from 94 days to term; fetal and maternal livers and kidneys were analyzed by a radioreceptor assay for insulin binding characteristics. Specific binding of insulin to partially purified ovine fetal liver and kidney plasma membranes increased as gestation approached term, at which time specific binding was two- to fourfold greater to fetal than to maternal tissues. Associated with increased specific binding were late gestational increases in affinity of insulin for receptors in both fetal liver and kidney and an earlier increase in insulin receptor concentration in fetal kidney. These observations in fetal sheep liver and kidney are similar to reported observations in other species. However, the increase in specific binding of insulin to male fetal liver membranes was exponential; in contrast, there was no apparent increase in specific binding to female fetal liver membranes during the gestational interval surveyed. Both the weights and the vertebral column lengths of these fetuses were shown by multivariate analysis to be significantly affected by the interaction between specific binding of insulin and fetal sex. However, in 30 additional sheep fetuses we observed no difference between male and female fetuses in the increase with time in liver glycogen content. The lack of sex difference in this postreceptor event is consonant with the demonstrated dissociation between liver insulin receptors and glycogen synthesis in the late fetal rat. Our observations suggest that late gestational differences between male and female sheep fetuses in insulin specific binding to liver and, possibly, to other tissues such as cartilage, muscle, and/or fat, that are coupled to postreceptor events may account for differences in fetal growth between the sexes.     

Insulin-like growth factor binding to the atypical insulin receptors of a human lymphoid-derived cell line (IM-9).

The cells of the IM-9 human lymphocyte-derived line contain a sub-population of insulin-binding sites whose immunological and hormone-binding characteristics closely resemble those of the atypical insulin-binding sites of human placenta. These binding sites, which have moderately high affinity for multiplication-stimulating activity [MSA, the rat homologue of insulin-like growth factor (IGF) II] and IGF-I, are identified on IM-9 cells by 125I-MSA binding. They account for approximately 30% of the total insulin-receptor population, and do not react with a monoclonal antibody to the type I IGF receptor (alpha IR-3). The relative concentrations of unlabelled insulin, MSA and IGF-I required to displace 50% of 125I-MSA from these binding sites (1:4.7:29 respectively) are maintained for cells, particulate membranes, Triton-solubilized membranes precipitated either by poly(ethylene glycol) or a polyclonal antibody (B-10) to the insulin receptor, and receptors purified by insulin affinity chromatography. Because the atypical insulin/MSA-binding sites outnumber the type I IGF receptors in IM-9 cells by approximately 10-fold, they also compete with the latter receptors for 125I-IGF-I binding. Thus 125I-IGF-I binding to IM-9 cells is inhibited by moderately low concentrations of insulin (relative potency ratios for insulin compared with IGF-I are approx. 1/14 to 1/4) and is partially displaced (65-80%) by alpha IR-3. When type I IGF receptors are blocked by alpha IR-3 or removed by B-10 immunoprecipitation or insulin affinity chromatography, the hormone-displacement patterns for 125I-IGF-I binding resemble those of the atypical insulin/MSA-binding sites.     

An insulin-like growth factor I/insulin hybrid exhibiting high potency for interaction with the type I insulin-like growth factor and insulin receptors of placental plasma membranes

We have prepared by semisynthetic methods a two-chain insulin/insulin-like growth factor I hybrid that contains a synthetic peptide related to residues 22-41 of insulin-like growth factor I linked via peptide bond to ArgB22 of des-octapeptide-(B23-B30)-insulin and have applied the analog to the analysis of ligand interactions with the type I insulin-like growth factor and insulin receptors of placental plasma membranes. Relative potencies for the inhibition of 125I-labeled insulin-like growth factor I binding to type I insulin-like growth factor receptors were 1.0:0.20:0.003 for insulin-like growth factor I, the hybrid analog, and insulin, respectively. Corresponding relative potencies for the inhibition of 125I-labeled insulin binding to insulin receptors were 0.007:0.28:1 for the three respective peptides. Additional studies identified that the hybrid analog interacts with only one of two populations of insulin-like growth factor I binding sites on placental plasma membranes and permitted the analysis of insulin-like growth factor I interactions with the separate populations of binding sites. We conclude that (a) des-octapeptide-(B23-B30)-insulin can serve well as a scaffold to support structural elements of insulin-like growth factor I and insulin necessary for high affinity binding to their receptors, (b) major aspects of structure relevant to the conferral of receptor binding affinity lie in the COOH-terminal region of the insulin B chain and in the COOH-terminal region of the insulin-like growth factor I B domain and in its C domain, and (c) the evolution of ligand-receptor specificity in these systems has relied as much on restricting interactions (through the selective introduction of negative structural elements) as it has on enhancing interactions (through the introduction of affinity conferring elements of structure).     

Identification of the insulin receptor in undifferentiated and differentiated NB-15 mouse neuroblastoma cells by affinity labeling

Plasma membranes prepared from clonal NB-15 mouse neuroblastoma cells were sequentially incubated with ¹²⁵I-labeled insulin (10 nM) and the bifunctional cross-linking agent disuccinimidyl suberate. This treatment resulted in the cross-linking of ¹²⁵I-labeled insulin to a polypeptide that gave an apparent M_r of 135 000 on a sodium dodecyl sulfate-polyacrylamide gel electrophoresed in the presence of 10% β-mercaptoethanol. Affinity labeling of this polypeptide was inhibited by the presence of 5 μM unlabeled insulin, but not by 1 μM unlabeled nerve growth factor. Using the same affinity labeling technique, ¹²⁵I-labeled nerve growth factor (1 nM) did not label any polypeptide appreciably in the plasma membranes of NB-15 cells but labeled an M_r 145 000 and an M_r 115 000 species in PC-12 rat pheochromocytoma cells. The number of insulin binding sites per cell in the intact differentiated NB-15 mouse neuroblastoma cells was approx. 6-fold greater than that in the undifferentiated NB-15 mouse neuroblastoma cells as measured by specific binding assay, suggesting an increase of the number of insulin receptors in NB-15 mouse neuroblastoma cells during differentiation.     

Leprechaunism: an inherited defect in a high-affinity insulin receptor.

We examined in vivo oral glucose tolerance tests and in vitro insulin binding, cellular response, and insulin-receptor structure of fibroblasts cultured from the skin of a patient with leprechaun syndrome and her parents. In response to oral glucose, the proband exhibited marked hyperinsulinism (maximum plasma insulin = 4,120 microU/ml), the father had mild hyperinsulinism (maximum plasma insulin = 240 microU/ml), and the mother was normal. [125I]insulin binding to monolayers of intact fibroblasts demonstrated complex kinetics that were interpreted using a two-receptor model. Normal high-affinity binding had an apparent KA of 1.6 X 10(10)/molar with 1,100 sites/cell. The proposed low-affinity state receptor had an apparent KA of 6.8 X 10(7)/molar with approximately 30,000 sites/cell. Insulin binding to the proband's cells had no high-affinity binding but had normal low-affinity binding. Cells from the mother had 60%, and cells from the father, 2%, of control insulin binding to the high-affinity receptor, but normal, low-affinity site binding. Two different, insulin-stimulable responses were evaluated under experimental conditions identical with those used for insulin binding. Insulin stimulation of 2-methylaminoisobutyric acid uptake occurred with half-maximal responses between 25 and 50 ng/ml insulin. This response was similar in cells from controls and the patient. By contrast, the uptake and phosphorylation of 2-deoxy-D-glucose was stimulated at half-maximal insulin concentrations between 1 and 10 ng/ml in control cells but was not significantly increased in the proband's cells until 1,000 ng/ml concentrations of insulin were attained. In affinity crosslinking experiments, [125I]insulin was covalently bound to insulin receptors of fibroblast membranes using disuccinimidylsuberate. [125I]insulin specifically bound to 125,000 dalton monomeric subunits and 250,000 dalton dimers. In control cells, the ratio of monomer to dimer was approximately one, but significantly fewer dimers were crosslinked in insulin receptors from the patient's cells. We conclude that in this family two different recessive mutations impair high-affinity insulin-receptor binding and that the proband with leprechaunism is a compound heterozygote for these mutations. The two mutations produced structural changes in the receptor that altered subunit interactions and loss of high-affinity binding and cellular responsivity.     

Glucose regulation of insulin receptor affinity in primary cultured adipocytes

Treatment of primary cultured adipocytes with 20 mM glucose resulted in a progressive increase in specific 125I-insulin binding that began almost immediately (no lag period) and culminated in a 60% increase by 24 h. This effect was dose-dependent (glucose ED50 of 4.6 mM) and mediated by an increase in insulin receptor affinity. Moreover, it appears that glucose modulates insulin receptor affinity through de novo protein synthesis rather than through covalent modification of receptors, since cycloheximide selectively inhibited the glucose-induced increase in insulin binding capacity (ED50 of 360 ng/ml) and restored receptor affinity to control values. Importantly, insulin sensitivity of the glucose transport system was increased by glucose treatment (63%) to an extent comparable with the enhancement in receptor affinity, thus indicating a functional coupling between insulin binding and insulin action. When the long term effects of insulin were assessed (24 h), we found that insulin treatment reduced 125I-insulin binding by greater than 60% by down-regulating the number of cell surface receptors in a dose-dependent manner (insulin ED50 of 7.4 ng/ml). On the basis of these studies, we conclude that 1) insulin binding is subject to dual regulation (glucose controls insulin action by enhancing receptor affinity, whereas insulin controls the number of cell surface receptors); and 2) glucose appears to modulate insulin receptor affinity through the rapid biosynthesis of an affinity regulatory protein.     

Radioreceptor assay for lactogenic hormones based on membranes from rat mammary tumour

A highly sensitive radioreceptor assay (RRA) for human prolactin (hPRL) based on membrane preparations obtained from chemically induced rat mammary tumour is described. The binding of 125I-labelled, highly purified pituitary human prolactin was specific for lactogenic hormones and depending on time, temperature, and concentration of receptor protein. Optimal specific receptor binding (18-20%) was obtained by incubation at 21 degrees C for 18 h. The prolactin receptor was shown to have a single "class" of binding sites with an affinity constant (Ka) of 6.0 X 10(10) mol-1. The binding capacity was 8-33 fmol/mg membrane protein. The sensitivity of the radioreceptor assay was 0.5 ng/ml ovine prolactin (NIH-PS-10) or 0.84 ng/ml human prolactin (NIH-VLS-4). The receptor binding activity of various purified prolactin preparations from different species was comparable to the biological hormone activities, indicating that this in vitro assay system measures values which are biologically relevant.     

Increase of (Ca2+ +Mg2+)-ATPase activity of renal basolateral membranes by platelet-derived growth factor through a specific receptor

Studies were made on the direct effect of platelet-derived growth factor (PDGF) on the high-affinity (Ca2+ +Mg2+)-ATPase, a membrane bound Ca2+-extrusion pump enzyme of the basolateral membranes (BLM) of canine kidney (Km for free Ca2+ = 1.0 x 10(-7) M, Vmax = 180 nmol Pi/mg/min). At 1 x 10(-7) M free Ca2+, PDGF (10(-10)-10(-8) M) stimulated the enzyme activity significantly. Addition of 5 - 200 microM suramin, a compound that blocks binding of PDGF to its receptors on cell membranes, inhibited the stimulatory effect of PDGF dose-dependently (IC50 = 40 microM). A high affinity specific receptor for PDGF (Kd = 4.4 x 10(-10) M, Bmax = 460 fmol/mg protein) was detected on BLM preparations by radioreceptor assay with 125I-PDGF and unlabelled PDGF. Suramin (10-1000 microM) also inhibited the binding of PDGF to BLM preparations dose-dependently. From these results, it is proposed that PDGF stimulates (Ca2+ +Mg2+)-ATPase activity of kidney BLM preparations by enhancing its affinity for free Ca2+ through a specific receptor.     

A radioreceptor assay method for insulin

A sensitive and practical radioreceptor assay method for pharmaceutical insulin products has been developed with partially purified rat liver plasma membranes and the optimal conditions under which the best overall assay performance is obtainable have been defined. Intra- and inter-assay variations of the method averaged 7.3 and 12.2% of the man, respectively, when expressed as the coefficient of variation. Potency estimates of an insulin product obtained with the proposed method correlated well with those determined by the mouse convulsion bioassay method. Liver membranes prepared according to the method could be stored for up to ten weeks at 4 degrees C and for 6 months or more at - 18 degrees C without losing insulin-binding ability.     

Modification of the insulin receptor by diethyl pyrocarbonate: effect on insulin binding and action

Insulin binding to rat liver plasma membranes is inhibited in a time- and dose-dependent fashion by prior treatment of membranes with the histidine-specific reagent diethyl pyrocarbonate. If all receptors are occupied by unlabeled hormone during diethyl pyrocarbonate treatment, no inhibition of 125I-labeled insulin binding is observed folowing washout of unlabeled hormone and unreacted reagent. Scatchard analysis of the binding inhibtion due to diethyl pyrocarbonate reveals a loss in receptor number rather than a change in receptor affinity for hormone. Fat cells treated with diethyl pyrocarbonate exhibit a rightward shift in the dose-response relationship for insulin-stimulated glucose oxidation consistent with a loss in receptor number due to the reagent. The pH profile for inhibition of insulin binding by diethyl pyrocarbonate and the partial reversibility of this inhibition by hydroxylamine are consistent with modification of a histidine residue. These results suggest that a histidine residue at or near the receptor binding site is required for formation of the biologically relevant insulin - receptor complex.