Specificity of photolabeling of beta-cell membrane proteins with an 125I-labeled glyburide analog.

The interaction between sulfonylureas and membrane proteins from a hamster insulin-secreting tumor (HIT) cell line has been examined. Four HIT cell membrane proteins were covalently linked to an 125I-labeled glyburide analog by photolabeling. Three photolabeled polypeptides of M(r) 65,000, 55,000, and 30,000 were identified as low affinity "glyburide receptors." These proteins appear to be of similar abundance, when quantitated by photolabeling, with half-maximal displacements (Ki values) by glyburide, glipizide, and tolbutamide in the low micromolar range. The glyburide analog is more tightly bound to a M(r) 140,000 protein with dissociation constants, determined by filtration binding assays and by photolabeling, of 7 and 9.0 nM, respectively. The labeled analog was displaced from the M(r) 140,000 protein by glyburide, glipizide and tolbutamide with Ki values of 3.3 nM, 103 nM, and 25 microM, respectively, as estimated by photolabeling. Optimal conditions established for visualizing the M(r) 140,000 band on autoradiograms prepared after UV cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis include irradiating the radioligand-receptor complex at 1.5 J/cm2 at 312 nm, followed by heating samples in pH 9.0 sodium dodecyl sulfate-gel sample buffer. With receptor sites partially occupied (5 nM radioligand), approximately 0.75% of the protein is photocoupled to the radioligand and visualized by autoradiography. Our results confirm that the M(r) 140,000 polypeptide contains the beta-cell high affinity glyburide binding site and show that the second generation sulfonylurea antidiabetic drugs have a selective increase in affinity for this receptor.     

Characterization of the sulfonylurea receptor on beta cell membranes

Specific, high affinity sulfonylurea receptors were characterized on membranes of an insulin-secreting hamster beta cell line (HIT cells). Saturable binding of the sulfonylurea, [3H]glyburide, was linear up to 0.8 mg/ml membrane protein. Scatchard analysis of equilibrium binding data at room temperature indicated the presence of a single class of saturable, high affinity binding sites with a Kd of 0.76 +/- 0.04 nM and a Bmax of 1.09 +/- 0.13 pmol/mg protein, n = 9. The insulin secretory potency of glyburide, glipizide, tolbutamide, tolazamide, and carboxytolbutamide was compared to the ability of these ligands to displace [3H]glyburide from the sulfonylurea receptor. Tolbutamide, tolazamide, and glipizide demonstrated reasonable agreement with ED50 values of 15 microM, 3 microM, and 30 nM and Ki values of 25.3 microM, 7.2 microM, and 45 nM, respectively. The inactive tolbutamide metabolite, carboxytolbutamide, at the highest concentration tested, only partially displaced [3H]glyburide from the receptor and was a very poor secretagogue. At 37 degrees C the affinity of [3H]glyburide binding, Kd = 2.0 nM, was similar to the ED50 of 5.5 nM when the free glyburide concentrations were corrected for binding of the drug to albumin. These studies suggest that sulfonylureas initiate their biologic effect through a high affinity, specific interaction with sulfonylurea receptors on the beta cell membrane.     

Increased cytosolic calcium. A signal for sulfonylurea-stimulated insulin release from beta cells

The mechanisms by which glyburide and tolbutamide signal insulin secretion were examined using a beta cell line (Hamster insulin-secreting tumor (HIT) cells). Insulin secretion was measured in static incubations, free cytosolic Ca2+ concentration ([Ca2+]i) was monitored in quin 2-loaded cells, and cAMP quantitated by radioimmunoassay. Insulin secretory dose-response curves utilizing static incubations fit a single binding site model and established that glyburide (ED50 = 112 +/- 18 nM) is a more potent secretagogue than tolbutamide (ED50 = 15 +/- 3 microM). Basal HIT cell [Ca2+]i was 76 +/- 7 nM (mean +/- S.E., n = 141) and increased in a dose-dependent manner with both glyburide and tolbutamide with ED50 values of 525 +/- 75 nM and 67 +/- 9 microM, respectively. The less active tolbutamide metabolite, carboxytolbutamide, had no effect on [Ca2+]i or insulin secretion. Chelation of extracellular Ca2+ with 4 mM EGTA completely inhibited the sulfonylurea-induced changes in [Ca2+]i and insulin release and established that the rise in [Ca2+]i came from an extracellular Ca2+ pool. The Ca2+ channel blocker, verapamil, inhibited glyburide- or tolbutamide-stimulated insulin release and the rise in [Ca2+]i at similar concentrations with IC50 values of 3 and 2.5 microM, respectively. At all concentrations tested, the sulfonylureas did not alter HIT cell cAMP content. These findings provide direct experimental evidence that glyburide and tolbutamide allow extracellular Ca2+ to enter the beta cell through verapamil-sensitive, voltage-dependent Ca2+ channels, causing a rise in [Ca2+]i which is the second messenger that stimulates insulin release.     

Sulfonylurea binding to a low-affinity site inhibits the Na/K-ATPase and the KATP channel in insulin-secreting cells

We have used hamster insulinoma tumor (HIT) cells, an insulin-secreting tumor cell line, to investigate modulation of the Na/K-ATPase and of the ATP-sensitive K channel (K(ATP)) by the sulfonylurea glyburide. Membrane proteins from cells cultured in RPMI with 11 mM glucose have at least two glyburide receptor populations, as evidenced by high and low binding affinity constants, (K(d) = 0.96 and 91 nM, respectively). In these cells K(ATP) channel activity was blocked by low glyburide concentrations, IC(50) = 5.4 nM. At 12.5 nM glyburide the inhibition developed slowly, tau = 380 s, and caused reduction of channel activity by 75 percent. At higher concentrations, however, inhibition occurred at a fast rate, tau = 42 s at 100 nM, and was almost complete. Na/K- ATPase activity measured enzymatically and electrophysiologically was also suppressed by glyburide, but higher concentrations were needed, IC(50) = 20-40 nM. Inhibition occurred rapidly, tau = 30 s at 50 nM, when maximum, activity was reduced by 40 percent. By contrast, cells cultured in RPMI supplemented with 25 mM glucose exhibit a single receptor population binding glyburide with low affinity, K(d)= 68 nM. In these cells inhibition of the Na/K-ATPase by the sulfonylurea was similar to that observed in cells cultured in 11 mM glucose, but K(ATP) channel inhibition was markedly altered. Inhibition occurred only at high concentrations of glyburide and at a fast rate; maximum inhibition was observed at 100 nM. Based on these data, we propose that glyburide binding to the high affinity site affects primarily K(ATP) channel activity, while interaction with the low affinity site inhibits both Na/K-ATPase and K(ATP) channel activities. The latter observation suggests possible functional interactions between the Na/K-ATPase and the K(ATP) channel.     

Co-expression of sulfonylurea receptors and KATP channels in hamster insulinoma tumor (HIT) cells. Evidence for direct association of the receptor with the channel.

Cell membranes isolated from hamster insulinoma (HIT T15) cells at passages 65-74 contain high and low affinity receptors for a sulfonylurea derivative, 5-[125I]iodo,2-hydroxyglyburide (KD values of approximately 7 nM and 16 microM). Between passages 75 and 85, the estimated B(max) for the high affinity receptor decreases approximately 10-fold from approximately 1.6 to 0.16 pmol/mg membrane protein. By contrast, the density of low affinity binding sites, 800-1000 pmol/mg, is unaltered. The drop in high affinity receptors is paralleled by a decrease in the density of KATP channels assessed using patch-clamp and 86Rb(+)-efflux techniques. These results strongly support the idea that the high affinity sulfonylurea receptor is an integral part of the KATP channel.     

Bovine Chromaffin Cells Have Insulin-Like Growth Factor-I (IGF-I) Receptors: IGF-I Enhances Catecholamine Secretion

The binding of 125I-insulin-like growth factor-I (125I-IGF-I) to bovine chromaffin cells was measured. Chromaffin cell cultures contained 111,000 +/- 40,000 IGF-I binding sites/cell. These sites bound IGF-I with a KD of 1.1 +/- 0.3 nM and had a much lower affinity for insulin. Cross-linking studies showed that 125I-IGF-I bound to a protein that had an Mr of approximately 125,000, similar to the Mr of the alpha subunit of the IGF-I receptor in other tissues. Cells cultured with IGF-I (10 nM) for 4 days exhibited an almost twofold increase in high K+-evoked catecholamine secretion. Insulin was much less potent than IGF-I in enhancing catecholamine secretion. These data indicate that binding of IGF-I to its receptors on chromaffin cells can modulate the function of these cells.     

Lack of Usefulness of DN-1417 for Characterization of a CNS Receptor for Thyrotropin-Releasing Hormone

CNS receptors for thyrotropin-releasing hormone (TRH) and its analogs are likely to mediate the experimentally and clinically observed net excitatory effect of these peptides on lower motor neurons. Previous findings suggest that several types of TRH receptors with distinct TRH analog specificities may be present in rat CNS. In particular, based on competition isotherm assays with unlabeled analog gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-prolineamide (DN-1417). Funatsu et al. claim the existence of a limbic forebrain site that binds this peptide and TRH with high affinity but that does not bind [3-methyl-histidyl2]-TRH (MeTRH). Using saturation and competition isotherm experiments, we have examined the binding of [3H]TRH and [3H]DN-1417 in three regions of rat CNS: pyriform cortex/amygdala, limbic forebrain, and lumbosacral spinal cord. In all three regions, saturation assays with [3H]TRH (0.4-100 nM) resolved only a single, saturable receptor with high affinity (KD = 12-14 nM) for TRH; in no case could more than one saturable site be identified. When [3H]DN-1417 was substituted as the assay ligand, no high-affinity binding component for this analog could be detected in the three regions. Competition curves for the binding of unlabeled DN-1417 to limbic forebrain and lumbosacral spinal cord ([3H]TRH as assay ligand) were monophasic (not biphasic like those of Funatsu et al.) and indicative of low-affinity binding of DN-1417 in these regions (Ki values = 2-3 microM; in agreement with values obtained in similar assays with [3H]MeTRH).(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of the receptor for insulin-like growth factor I

The receptor for insulin-like growth factor I (IGF-I) was purified from the rat liver cell line BRL-3A by a combination monoclonal anti-receptor antibody column and a wheat germ agglutinin column. Analyses of these receptor preparations on reduced sodium dodecyl sulfate-polyacrylamide gels yielded protein bands of Mr 136K (alpha subunit) and Mr 85K and 94K (beta subunit). These receptor preparations bound 5 times more IGF-I than insulin, and the binding of both labeled ligands was more potently inhibited by unlabeled IGF-I than by insulin. These results indicate that these receptor preparations contained predominantly the IGF-I receptor. This highly purified receptor preparation was found to possess an intrinsic kinase activity; autophosphorylation of the receptor beta subunit was stimulated by low concentrations of IGF-I (half-maximal stimulation at 0.4 nM IGF-I). Twentyfold higher concentrations of insulin were required to give comparable levels of stimulation. A monoclonal antibody that inhibits the insulin receptor kinase was found to inhibit the IGF-I receptor kinase with the same potency with which it inhibits the insulin receptor. In contrast, monoclonal antibodies to other parts of the insulin receptor only poorly recognized the IGF-I receptor. A comparison of V8 protease digests of the insulin and IGF-I receptors again revealed some similarities and also some differences in the structures of these two receptors. Thus, the IGF-I receptor is structurally, antigenically, and functionally similar to but not identical with the insulin receptor.     

Use of the heterobifunctional cross-linker m-maleimidobenzoyl N-hydroxysuccinimide ester to affinity label cholecystokinin binding proteins on rat pancreatic plasma membranes

The binding of 125I-cholecystokinin-33 (125I-CCK-33) to its receptors on rat pancreatic membranes was decreased by modification of membrane protein sulfhydryl groups. Sulfhydryl modifying reagents also caused an accelerated release of bound 125I-CCK-33 from its receptor. Because of the presence of an essential sulfhydryl group(s) in CCK receptor binding we studied the application of the heterobifunctional (SH,NH2) cross-linker, m-maleimidobenzoyl N-hydroxysuccinimide ester (MBS), to affinity label 125I-CCK-33 binding proteins on rat pancreatic plasma membranes. Analysis of the cross-linked products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that this heterobifunctional cross-linker affinity labeled a major Mr = 80,000-95,000 protein previously identified as part of the CCK receptor on the basis of affinity labeling using homobifunctional and heterobifunctional photoreactive cross-linkers. Additional proteins of Mr greater than 200,000, and Mr = 130,000-140,000 were affinity labeled using MBS. The efficiency of the cross-linking reaction between 125I-CCK-33 and its membrane binding proteins with MBS was significantly greater than that obtained with NH2-directed homobifunctional reagents such as disuccinimidyl suberate. The efficiency of cross-linking could be dramatically improved by reduction of membrane proteins with low-molecular weight thiols prior to binding and cross-linking. The differential labeling patterns of the CCK binding proteins obtained with chemical cross-linkers of similar length but different chemical reactivity underscores the need for caution in predicting native receptor structure from affinity labeling data alone. Using the same pancreatic plasma membrane preparation and 125I-insulin, the Mr = 125,000 alpha-subunit of the insulin receptor was affinity labeled using MBS as cross-linker, demonstrating its utility in identifying other peptide hormone receptors.     

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.     

Characterization of bombesin receptors in a rat pituitary cell line

Bombesin is a tetradecapeptide which stimulates prolactin secretion in rats and man and in cultures of GH4C1 cells, a clonal strain of rat pituitary tumor cells. We have utilized [125I-Tyr4]bombesin to identify and characterize specific high affinity receptors in GH4C1 cells. Scatchard analysis of equilibrium binding data at 4 degrees C indicated the presence of a single class of non-interacting binding sites for bombesin (RT = 3600 +/- 500 sites/cell). The value for the equilibrium dissociation constant (Kd = 1.2 +/- 0.4 nM) agreed closely with the ED50 (0.5 nM) for bombesin stimulation of prolactin release. [125I-Tyr4]Bombesin binding at steady state at 37 degrees C was inhibited by increasing concentrations of unlabeled bombesin in a dose-dependent manner, with an ID50 = 1.4 +/- 0.2 nM. However, binding of [125I-Tyr4] bombesin was not inhibited by 100 nM thyrotropin-releasing hormone, vasoactive intestinal peptide, epidermal growth factor, or somatostatin. Therefore, [125I-Tyr4]bombesin binds to a receptor distinct from the receptors for other peptides which regulate hormone secretion by GH4C1 cells. The analog specificity for high affinity binding showed that the receptors for bombesin recognize the COOH-terminal octapeptide sequence in the molecule. Among five pituitary cell strains tested, two which contained saturable binding sites for [125I-Tyr4]bombesin (GH4C1 and GH3) had previously been shown to respond to bombesin with increased hormone secretion, whereas three which lacked receptors (GC, F4C1, and AtT20/D16v) were unresponsive. Therefore, the [125I-Tyr4]bombesin binding sites appear to be necessary for the biological actions of bombesin. Examination of the processing and metabolism of receptor-bound peptide demonstrated that at 4 degrees C [125I-Tyr4]bombesin binds to receptors on the surface of GH4C1 cells. At 37 degrees C, receptor-bound peptide is rapidly internalized and subsequently degraded in lysosomes. In summary, we have characterized for the first time specific, high affinity pituitary bombesin receptors which are necessary for the biological action of bombesin.     

Covalent labeling of a high-affinity, guanyl nucleotide sensitive parathyroid hormone receptor in canine renal cortex

Putative parathyroid hormone (PTH) receptors in canine renal membranes were affinity labeled with 125I-bPTH(1-34) using the heterobifunctional cross-linking reagent N-hydroxysuccinimidyl 4-azidobenzoate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a major 85,000 molecular weight (Mr) PTH binding component, the labeling of which was inhibited by nanomolar concentrations of unlabeled PTH and by micromolar concentrations of 5'-guanylyl imidodiphosphate [Gpp-(NH)p]. Labeling was not influenced by the unrelated peptides insulin and arginine vasopressin. Minor PTH binding components of Mr 55,000 and 130,000 were also seen, and labeling of these was likewise sensitive to unlabeled PTH and to Gpp(NH)p. Omission of protease inhibitors during the isolation of plasma membranes resulted in the loss of the Mr 85,000 PTH binding species and the appearance of an Mr 70,000 form. Several minor PTH binding components also were observed. Equilibrium binding studies showed that such membranes had an affinity for PTH indistinguishable from that in membranes isolated with protease inhibitors and displaying a major Mr 85,000 PTH binding species. We conclude that the major form of the adenylate cyclase coupled PTH receptor in canine renal membranes is an Mr 85,000 protein. An endogenous enzyme, probably a lysosomal cathepsin, can cleave this form to produce an Mr 70,000 receptor that retains full functional activity with respect to high-affinity, guanyl nucleotide sensitive PTH binding. The ability to covalently label the PTH receptor in high yield represents a major step toward the structural characterization of this important detector molecule.     

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.     

The beta subunit of the insulin receptor is an insulin-activated protein kinase

In the presence of adenosine 5'-[gamma-32P]triphosphate ([gamma-32P]ATP) and a partially purified human placental insulin receptor preparation, insulin stimulates the phosphorylation of an Mr 94000 protein in a time- and dose-dependent manner. Half-maximal stimulation of 32P incorporation occurs at (2-3) X 10(-9) M insulin, a concentration identical with the Kd for insulin binding in this preparation. Immunoprecipitations with monoclonal anti-insulin receptor antibody demonstrate that the Mr 94000 protein kinase substrate is a component of the insulin receptor, the beta subunit. If the partially purified, soluble placental receptor preparation is immunoprecipitated and then exposed to [gamma-32P]ATP and insulin, phosphorylation of the Mr 94000 protein is maintained. The photoincorporation of 8-azido[alpha-32P]ATP into placental insulin receptor preparations was carried out to identify the ATP binding site responsible for the protein kinase activity. Photoincorporation into numerous proteins was observed, including both subunits of the insulin receptor. However, when photolabeling was performed in the presence of excess adenosine 5'-(beta, gamma-imidotriphosphate), a nonhydrolyzable ATP derivative, the beta subunit of the insulin receptor was the only species protected from label incorporation. These data indicate that the beta subunit of the insulin receptor has insulin-dependent protein kinase activity. Phosphotyrosine formation is the primary result of this activity in placental insulin receptor preparations.     

Solubilization and molecular weight estimation of atrial natriuretic factor receptor from bovine adrenal cortex

Receptors for atrial natriuretic factor (ANF) have been solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate from bovine adrenal cortex and characterized. The detergent extract retained specific high-affinity binding sites for 125I-ANF. Scatchard analysis of the equilibrium binding data revealed a single class of binding site with a K-d of 1.8 nM and a maximum binding capacity of 2.5 pmol/mg of protein. The size of the 125I-ANF X receptor complexes was estimated to be 140,000 daltons by gel filtration on TSK gel G3000SW. Affinity labeling followed by electrophoresis under nonreducing conditions and autoradiography also revealed a single band of a similar size (Mr = 130,000); this band, however, migrated as a Mr = 70,000 species under reducing electrophoretic conditions. These results indicate that the ANF receptor, having a Mr of 130,000 - 140,000, is composed of disulfide-linked subunits and the ANF-binding site is located on the 70-kDa component.     

Identification and affinity labeling of very high affinity binding sites for the phenylalkylamine series of Ca+ channel blockers in the Drosophila nervous system

The interaction of putative Ca2+ channels of Drosophila head membranes with molecules of the phenylalkylamine series was studied from binding experiments using (-)-[3H]D888 and (+/-)-[3H]verapamil. These ligands recognize a single class (Kd = 0.1-0.4 nM; Bmax = 1600-1800 fmol/mg of protein) of very high affinity binding sites. The most potent molecule in the phenylalkylamine series was (-)-verapamil with a Kd value as exceptionally low as 4.7 pM. Molecules in the benzothiazepine and diphenylbutylpiperidine series of Ca2+ channel blockers as well as bepridil inhibited (-)-[3H]D888 binding in a competitive way with Kd values between 12 and 190 nM, suggesting a close correlation, as in the mammalian system, between these receptor sites and those recognizing phenylalkylamines. A tritiated (arylazido)phenylalkylamine with high affinity for the Drosophila head membranes, phenylalkylamine receptor Kd = 0.24 nM), was used in photoaffinity experiments. A protein of Mr 135,000 +/- 5,000 was specifically labeled after ultraviolet irradiation.     

Secretion of soluble functional insulin receptors by transfected NIH3T3 cells

In order to determine whether the human insulin receptor ectodomain can be expressed as a functional protein, the coding regions for the transmembrane and cytoplasmic domain of a full-length human insulin receptor cDNA were deleted by site-directed mutagenesis, and the resultant construct was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH3T3 cells, a cell line secreting an insulin binding protein was isolated. The insulin binding alpha subunit had an Mr of 138,000 and a beta subunit of Mr 48,000 (compared to 147,000 and 105,000 for the full-length human insulin receptor expressed in NIH3T3 cells). This difference in size of the alpha subunit was due to a difference in glycosylation as N-glycanase digestion reduced the apparent size of the alpha subunits of secreted and normal membrane-bound receptors to identical values. The secreted receptor formed disulfide-linked heterotetrameric structures with an Mr of 280,000. It was synthesized as an Mr 160,000 precursor which was cleaved into mature subunits with a t1/2 of 3 h. Increasing expression of the cDNA by induction with sodium butyrate lead to the appearance of an Mr 180,000 protein in the medium as well as the mature alpha and beta subunits. A Scatchard plot of insulin binding to the secreted receptor was curvilinear with a Kd of 7 X 10(-10) M for the high affinity sites and 10(-7) M for the low affinity site (compared to Kd values of 1.1 X 10(-9) M and 10(-7) M, respectively, for human insulin receptors expressed in these cells.     

The insulin-like growth factors (IGFs) I and II bind to articular cartilage via the IGF-binding proteins

Bovine articular cartilage discs (3 mm diameter x 400 micrometer thick) were equilibrated in buffer containing (125)I-insulin-like growth factor (IGF)-I (4 degrees C) +/- unlabeled IGF-I or IGF-II. Competition for binding to cartilage discs by each unlabeled IGF was concentration-dependent, with ED(50) values for inhibition of (125)I-IGF-I binding of 11 and 10 nM for IGF-I and -II, respectively, and saturation by 50 nM. By contrast, an analog of IGF-I with very low affinity for the insulin-like growth factor-binding proteins (IGF-BPs), des-(1-3)-IGF-I, was not competitive with (125)I-IGF-I for cartilage binding even at 100-400 nM. Binding of the (125)I-labeled IGF-II isoform to cartilage was competed for by unlabeled IGF-I or -II, with ED(50)s of 160 and 8 nM, respectively. This probably reflected the differential affinities of the endogenous IGF-BPs (IGF-BP-6 and -2) for IGF-II/IGF-I. Transport of (125)I-IGF-I was also measured in an apparatus that allows diffusion only across the discs (400 micrometer), by addition to one side and continuous monitoring of efflux on the other side. The time lag for transport of (125)I-IGF was 266 min, an order of magnitude longer than the theoretical prediction for free diffusion in the matrix. (125)I-IGF-I transport then reached a steady state rate (% efflux of total added (125)I-IGF/unit time), which was subsequently accelerated approximately 2-fold by addition of an excess of unlabeled IGF-I. Taken together, these results indicate that IGF binding to cartilage, mostly through the IGF-BPs, regulates the transport of IGFs in articular cartilage, probably contributing to the control of their paracrine activities.     

Binding of human recombinant 125I-interferon gamma to receptors on human cells

Recombinant human interferon gamma (rIFN-gamma) produced in Escherichia coli was labeled with 125I to study its binding to receptors of HeLa and lymphoblastoid cells. All the cell lines examined had receptors for rIFN-gamma, although the binding varied considerably among different cell lines. The binding of 125I-rIFN-gamma was competed up to 90% by the addition of unlabeled rIFN-gamma, although not by the addition of IFN-alpha or -beta. By adding increasing concentrations of unlabeled rIFN-gamma to binding assays containing a constant amount of 125I-rIFN-gamma, we determined a KD of 3.7 and 6.3 X 10(-10) M for its binding to Daudi and HeLa cells, respectively. About 13,000 receptors per cell were present in Daudi and 5,000 in HeLa cells. The Mr of the IFN-gamma/receptor complex was determined by cross-linking experiments to be about 125,000. This complex is smaller than the IFN-alpha/receptor complex that has a Mr of about 140,000. The rIFN-gamma receptor was down-regulated when HeLa cells were treated with this interferon, but not when these cells were treated with IFN-beta. These findings suggest that the receptors for IFN-alpha and -gamma differ in several characteristics. The turnover of the rIFN-gamma receptor was measured by inhibiting protein synthesis with cycloheximide and the half-life of this receptor was found to be 2 h. The unglycosylated rIFN-gamma was bound to cellular receptors with an affinity similar to that previously reported for natural IFN-gamma. The lymphoblastoid cell lines examined had high affinity receptors for rIFN-gamma, but did not respond to treatment with this IFN with an induction of the synthesis of the enzyme (2'-5')oligo(A) synthetase, whereas HeLa cells responded to rIFN-gamma. The reason for the lack of response of lymphoblastoid cells is presently unknown.     

Purification of vasoactive intestinal peptide receptor from porcine liver by a newly designed one-step affinity chromatography

Vasoactive intestinal peptide (VIP) receptors were solubilized from porcine liver membrane using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. The solubilized VIP receptor has been purified approximately 50,000-fold to apparent homogeneity by a one-step affinity chromatography using a newly designed VIP-polyacrylamide resin. The purified receptor bound 125I-VIP with a Kd of 22.3 +/- 0.7 nM and retained its peptide specificity toward VIP-related peptides. The specific activity of the purified receptor (16,400 pmol/mg of protein) was very close to the theoretical value (18,900 pmol/mg of protein) calculated assuming one binding site/protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified receptor revealed a single band with an Mr of 53,000 after either silver staining or radioiodination. Affinity labeling of the purified receptor with 125I-VIP using dithiobis(succinimidyl propionate) gave a single radioactive band, the labeling of which was completely inhibited by an excess of unlabeled VIP. In conclusion, an Mr 53,000 protein containing the VIP-binding site was purified to homogeneity by a one-step affinity chromatography using immobilized VIP.