Identification of a gp130 cytokine receptor critical site involved in oncostatin M response

Gp130 cytokine receptor is involved in the formation of multimeric functional receptors for interleukin-6 (IL-6), IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor, and cardiotrophin-1. Cloning of the epitope recognized by an OSM-neutralizing anti-gp130 monoclonal antibody identified a portion of gp130 receptor localized in the EF loop of the cytokine binding domain. Site-directed mutagenesis of the corresponding region was carried out by alanine substitution of residues 186-198. To generate type 1 or type 2 OSM receptors, gp130 mutants were expressed together with either LIF receptor beta or OSM receptor beta. When positions Val-189/Tyr-190 and Phe-191/Val-192 were alanine-substituted, Scatchard analyses indicated a complete abrogation of OSM binding to both type receptors. Interestingly, binding of LIF to type 1 receptor was not affected, corroborating the notion that in this case gp130 mostly behaves as a converter protein rather than a binding receptor. The present study demonstrates that positions 189-192 of gp130 cytokine binding domain are essential for OSM binding to both gp130/LIF receptor beta and gp130/OSM receptor beta heterocomplexes.     

Competitive inhibition of T3 binding to alpha 1 and beta 1 thyroid hormone receptors by fatty acids.

This study was undertaken to investigate whether fatty acids inhibit the binding of T3 to the alpha 1 and beta 1 form of the thyroid hormone receptor. Fatty acids inhibited the binding of T3 to both receptor proteins isolated from a bacterial expression system. The effectiveness of inhibition depends on the chain length and degree of saturation of the fatty acids. The inhibition of T3 binding to the alpha 1 and beta 1 receptor by oleic acid is competitive in nature; the Ki value was 5.4 10(-6) M for the c-erbA alpha 1 protein and 3.3 10(-6) M for the c-erb beta 1 protein. The findings indicate a direct interaction of fatty acids with T3 receptor proteins.     

Effects of beta subunit acylation on lutropin receptor site binding.

The effects of various modifications on the beta subunit of lutropin have been studied using the binding characteristics of the reconstituted hormone in the rat testicular radioligand assay. Conditions for iodinating lutropin and lutropin derivatives were determined which resulted in 15 per cent specific binding when tested immediately and retention of 6 to 7 per cent specific binding even after storage for 6 months. Acetimidinyl, acetyl, and carbamyl derivatives of the beta subunit were prepared and combined with unmodified alpha subunit to form reconstituted lutropin. Modification of the beta subunit was shown to have no effect on the time course of binding to testicular receptors or, with one exception, on the extent of receptor saturation. Very high concentrations of lutropin reconstituted with acetylated beta subunit showed an anomalous binding behavior. Scatchard plots of the binding data support the view that the native hormone has a unique receptor affinity which is irreversibly disrupted by separation of subunits and that derivatization of the beta subunit does not alter this parameter further. These data also suggest that there are no significant differences in the amino groups modified on the beta subunit. Competition and preincubation tests for receptor sites that reacted only with modified lutropin and not with the native hormone were negative.     

Intrinsic signaling functions of the beta4 integrin intracellular domain

A key issue regarding the role of alpha6beta4 in cancer biology is the mechanism by which this integrin exerts its profound effects on intracellular signaling, including growth factor-mediated signaling. One approach is to evaluate the intrinsic signaling capacity of the unique beta4 intracellular domain in the absence of contributions from the alpha6 subunit and tetraspanins and to assess the ability of growth factor receptor signaling to cooperate with this domain. Here, we generated a chimeric receptor composed of the TrkB extracellular domain and the beta4 transmembrane and intracellular domains. Expression of this chimeric receptor in beta4-null cancer cells enabled us to assess the signaling potential of the beta4 intracellular domain alone or in response to dimerization using brain-derived neurotrophic factor, the ligand for TrkB. Dimerization of the beta4 intracellular domain results in the binding and activation of the tyrosine phosphatase SHP-2 and the activation of Src, events that also occur upon ligation of intact alpha6beta4. In contrast to alpha6beta4 signaling, however, dimerization of the chimeric receptor does not activate either Akt or Erk1/2. Growth factor stimulation induces tyrosine phosphorylation of the chimeric receptor but does not enhance its binding to SHP-2. The chimeric receptor is unable to amplify growth factor-mediated activation of Akt and Erk1/2, and growth factor-stimulated migration. Collectively, these data indicate that the beta4 intracellular domain has some intrinsic signaling potential, but it cannot mimic the full signaling capacity of alpha6beta4. These data also question the putative role of the beta4 intracellular domain as an "adaptor" for growth factor receptor signaling.     

Up-regulation of nicotinic receptors by nicotine varies with receptor subtype

Recent evidence suggests that in addition to alpha4beta2 and alpha3-containing nicotinic receptors, alpha6-containing receptors are present in midbrain dopaminergic neurons and involved in the nicotine reward pathway. Using heterologous expression, we found that alpha6beta2, like alpha3beta2 and alpha4beta2 receptors, formed high affinity epibatidine binding complexes that are pentameric, trafficked to the cell surface, and produced acetylcholine-evoked currents. Chronic nicotine exposure up-regulated alpha6beta2 receptors with differences in up-regulation time course and concentration dependence compared with alpha4beta2 receptors, the predominant high affinity nicotine binding site in brain. The alpha6beta2 receptor up-regulation required higher nicotine concentrations than for alpha4beta2 but lower than for alpha3beta2 receptors. The alpha6beta2 up-regulation occurred 10-fold faster than for alpha4beta2 and slightly faster than for alpha3beta2. Our data suggest that nicotinic receptor up-regulation is subtype-specific such that alpha6-containing receptors up-regulate in response to transient, high nicotine exposures, whereas sustained, low nicotine exposures up-regulate alpha4beta2 receptors.     

Role of the cytoplasmic domain of the beta-subunit of integrin alpha(v)beta6 in infection by foot-and-mouth disease virus

Field isolates of foot-and-mouth disease virus (FMDV) are believed to use RGD-dependent integrins as cellular receptors in vivo. Using SW480 cell transfectants, we have recently established that one such integrin, alpha(v)beta6, functions as a receptor for FMDV. This integrin was shown to function as a receptor for virus attachment. However, it was not known if the alpha(v)beta6 receptor itself participated in the events that follow virus binding to the host cell. In the present study, we investigated the effects of various deletion mutations in the beta6 cytoplasmic domain on infection. Our results show that although loss of the beta6 cytoplasmic domain has little effect on virus binding, this domain is essential for infection, indicating a critical role in postattachment events. The importance of endosomal acidification in alpha(v)beta6-mediated infection was confirmed by experiments showing that infection could be blocked by concanamycin A, a specific inhibitor of the vacuolar ATPase.     

alpha(6) Integrin is the main receptor of human papillomavirus type 16 VLP

The present study was performed to determine the specific receptor of type HPV-16 using recombinant human papillomavirus-like particle (HPV-16 L1-VLP). The expression levels of alpha(6), beta(1), and beta(4) integrins were determined and compared with the amount of HPV-VLP binding in ten cell lines by flow cytometry. Our results show that the amount of VLP binding and the expression level of alpha(6) integrin are correlated, which was confirmed by an inhibition experiment using antibodies and by immunocytochemistry. Both the expression level of alpha(6) integrin and the amount of HPV-VLP binding were high in cervical cancer cell lines, as the type HPV-16 is the main cause of cervical cancer. The degree of binding of HPV-VLP matched the alpha(6) integrin expression level in cell lines but was not correlated with beta(1) and beta(4) levels, which suggests that alpha(6) integrin is the main receptor of HPV type 16.     

Beta 2-adrenoceptor agonist-induced down-regulation after short-term exposure.

We examined the effect of duration of beta 2-adrenergic receptor (beta 2AR) occupancy by isoproterenol on specific binding of 125I-lodocyanopindolol (125I-ICYP) in membranes from rat L6 myoblasts. Ten minute exposure caused a time-and concentration-dependent maximal decrease in 125I-ICYP binding 24 hours after exposure equal to that following continuous exposure (p < 0.05). Low temperature, concanavalin A, H89 and ICl 118,551 blocked the decline in 125I-ICYP binding during the first hour following exposure probably representing receptor sequestration to a compartment or change to a form incapable of ligand binding. Compared to controls, receptor binding 4 and 24 hours following exposure was reduced 56 +/- 8.7% and 72 +/- 8.8%, respectively (p < 0.05), and was blocked by ICl 118,551 but not CGP12177. Isoproterenol-induced, but not forskolin-stimulated, cAMP accumulation was reduced 35% 24 hours following exposure (p < 0.05). 125I-ICYP binding in intact L6 cells 4 and 24 hours after exposure were respectively 56 +/- 8.9 and 61 +/- 13% of controls (p < 0.05). Following agonist exposure, CHO cell membranes expressing human beta 2ARs exhibited 125I-ICYP binding 85 +/- 2.0% and 6 +/- 2.8% of control values 4 and 24 hours, respectively (p < 0.05). A model predicting that full occupation of the beta 2AR activates receptor degradation explains our results that agonist-induced down-regulation of beta 2AR does not require continuous presence of the agonist.     

Protein kinase C-linked inactivation of the interleukin-1 receptor in a human transformed B-cell line

The effect of tumor-promoting phorbol ester treatment on the binding of interleukin-1 beta (IL-1 beta) to specific cell surface receptors was investigated. A 1 h exposure of Raji human B lymphoma cells with the protein kinase C-activating phorbol ester, phorbol dibutyrate (PDBu), reduced IL-1 beta binding by up to 90% of control cells. This effect was dose-dependent and was not observed with 4-alpha-phorbol, an inactive tumor promoter. Analysis of 125I-labeled IL-1 beta binding to intact cells revealed that PDBu caused a 91% decrease in high-affinity cell-surface receptor number without an effect on receptor affinity. The phorbol ester response was rapid (30 min), observed both at 4 and 37 degrees C, and was preceded by the rapid translocation (t much less than 6 min) of protein kinase C (PKC) from the cytosol to the cell membrane. The PDBu-induced decrease in IL-1 beta receptor number was inhibited by prior incubation of cells for 30 min with the PKC inhibitor 1-(5-Isoquinoline sulfonyl)-2-methylpiperazine (H7). The decrease in receptor binding was not due to enhanced IL-1 beta receptor internalization or shedding into the extracellular medium, since a similar effect was observed with solubilized IL-1 beta receptor. The most likely explanation for the phorbol ester effect appears to be cell surface inactivation of IL-1 receptors. These data suggest that modulation of PKC activity could play a role in the regulation of the IL-1 beta receptor.     

Binding of recombinant-produced interferon beta ser to human lymphoblastoid cells. Evidence for two binding domains

Human interferon beta (IFN beta ser), produced by recombinant DNA technology, was radiolabeled to approximately one atom of iodine-125/molecule of interferon without detectable loss of antiviral activity. At 37 degrees C, binding of 125I IFN beta ser occurred rapidly (t1/2max less than or equal to 15 min) followed by internalization and degradation of bound ligand. Kinetic analysis at 4 degrees C indicated diffusion-limited association kinetics independent of 125I IFN beta ser concentration. Dissociation of bound 125I IFN beta ser from Daudi cells was slow (t1/2 = 1.2 h) of bound radiolabeled ligand was observed in the presence of unlabeled IFN beta ser, naturally produced IFN beta, and IFN alpha 6, but was not observed with unlabeled IFN gamma or nonspecific proteins. Concomitantly, equilibrium analysis indicated heterogeneous binding of 125I IFN beta ser to six cell lines of lymphoid origin consistent with either negative cooperativity or two populations of receptors. Analysis of binding of 125I IFN beta ser to Daudi cell receptors in the presence of unlabeled IFN alpha 6 suggested that one receptor served both ligands. The latter conclusion was supported by results of chemical cross-linking experiments in which an 125I IFN beta ser/receptor complex (Mr 120,000-130,000) was observed following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This complex was absent when binding occurred in the presence of either excess unlabeled IFN beta ser or IFN alpha 6.     

Purification and characterization of human recombinant precursor interleukin 1 beta

We have purified the 31-kDa precursor of human interleukin 1 beta (proIL1 beta) from recombinant Escherichia coli expressing the protein. The recombinant precursor was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, spectroscopy, Western blot, and for biological and receptor binding activity. The protein migrates at the expected molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical gel filtration columns. The specific activity of the recombinant precursor is less than 10(2) units/mg in the EL4 thymoma assay compared with 5 x 10(8) units/mg for the recombinant 17-kDa mature protein. The inactivity of the precursor is attributable to the inability of the protein to bind the IL1 receptor on EL4 cells as shown by receptor competition studies using 125I-labeled 17-kDa IL1 beta. Inactivity of the IL1 beta precursor is not due to degradation of the protein in either the bioactivity or receptor binding assays. The inactive IL1 beta precursor is converted to an active form following proteolysis with chymotrypsin which generates a carboxyl-terminal fragment of 17 kDa that is 6 orders of magnitude more active than the starting IL1 beta precursor. Removal of the first 114 amino acids from proIL1 beta generates a fully active molecule. In contrast, removal of the first 77 amino acids by treatment with trypsin only partially restores activity. The resultant 22-kDa protein exhibits a 600-fold increase in both biological and receptor binding activity, demonstrating a direct correlation between the ability of sequences within the pro-region to inhibit biological activity and inhibit binding to the IL1 receptor. Far-UV circular dichroism spectroscopy indicates that proIL1 beta is similar in secondary structure to mature IL1 beta; both proteins are nonhelical beta sheet proteins.     

Analysis of the AU-rich elements in the 3'-untranslated region of beta 2-adrenergic receptor mRNA by mutagenesis and identification of the homologous AU-rich region from different species

The 35000-Mr beta-adrenergic receptor mRNA binding protein (beta ARB) is induced by beta-adrenergic agonists and binds to G-protein-linked receptor mRNAs that exhibit agonist-induced destabilization. Recently, we identified a 20-nucleotide, AU-rich region in the 3'-untranslated region of the hamster beta 2-adrenergic receptor mRNA consisting of an AUUUUA hexamer flanked by U-rich regions, which constitutes the binding domain for beta ARB. U to G substitution in the hexamer region attenuates the binding of beta ARB, whereas U to G substitution of hexamer and flanking U-rich domains abolishes binding of beta ARB and stabilizes beta 2-adrenergic receptor mRNA levels in transfectant clones challenged with either isoproterenol or cyclic AMP. In the study presented here, we mutated the 20-nucleotide ARE region to establish the minimal AU-rich sequence required for beta ARB binding. U to G substitutions of flanking poly(U) regions and of the hexamer established the nature of the binding properties. Using various mutants, we demonstrated also that binding of beta ARB correlates with the extent of destabilization of beta 2-adrenergic receptor mRNA in response to agonist stimulation. High-affinity binding of hamster, rat, mouse, porcine, and human ARE sequences to beta ARB was revealed by SDS-polyacrylamide gel electrophoresis following UV-catalyzed cross-linking and by gel mobility shift assays. Further, beta ARB was shown to bind more avidly to the 20-nucleotide ARE region than to well-established mRNA destablization sequences of tandem repeats of five pentamers. Thus, for beta 2-adrenergic receptor, mRNA destabilization likely occurs via conserved AU-rich elements present in the 3'-untranslated regions of receptor mRNAs.     

Internalization of transforming growth factor-beta and its receptor in BALB/c 3T3 fibroblasts

The fate of 125I-labeled transforming growth factor-beta (125I-TGF beta) after binding to its cells surface receptor has been investigated in BALB/c 3T3 mouse fibroblasts. Binding of 125I-TGF beta to cellular receptors at 4 degrees C is pH-sensitive, being markedly decreased at pH less than 6. Most (approximately 90%) of the 125I-TGF beta bound to cells at 4 degrees C can be removed by a brief treatment with acidic medium but is converted into an acid-resistant state rapidly after shifting the cells to 37 degrees C. Cell-bound 125I-TGF beta is degraded at 37 degrees C and the degradation products are released into the medium. The lysosomotropic bases chloroquine, methylamine, and ammonium and the carboxylic ionophore monensin inhibit the degradation and release of 125I-TGF beta from the cells. Cells allowed to accumulate 125I-TGF beta intracellularly by the action of chloroquine or monensin were treated with the bifunctional agent disuccinimidyl suberate in the presence of detergent Triton X-100; this treatment caused the cross-linking of internalized 125I-TGF beta with the 280-kilodalton TGF beta receptor component. Under conditions in which sustained binding and degradation of saturating 125I-TGF beta concentrations occurs, there is no marked decrease in the binding capacity of the cells even when protein synthesis is blocked with cycloheximide. These results indicate that after TGF beta binding the TGF beta:receptor complex becomes rapidly internalized and that TGF beta is directed towards lysosomes where it is degraded and released. However, the cell surface is replenished with TGF beta receptors recycled after internalization or supplied by a large intracellular pool.     

None of the integrins known to be present on the mouse egg or to be ADAM receptors are essential for sperm-egg binding and fusion

Antibody inhibition and alpha6beta1 ligand binding experiments indicate that the egg integrin alpha6beta1 functions as a receptor for sperm during gamete fusion; yet, eggs null for the alpha6 integrin exhibit normal fertilization. Alternative integrins may be involved in sperm-egg binding and fusion and could compensate for the absence of alpha6beta1. Various beta1 integrins and alphav integrins are present on mouse eggs. Some of these integrins are also reported to be receptors for ADAMs, which are expressed on sperm. Using alpha3 integrin null eggs, we found that the alpha3beta1 integrin was not essential for sperm-egg binding and fusion. Oocyte-specific, beta1 integrin conditional knockout mice allowed us to obtain mature eggs lacking all beta1 integrins. We found that the beta1 integrin null eggs were fully functional in fertilization both in vivo and in vitro. Furthermore, neither anti-mouse beta3 integrin function-blocking monoclonal antibody (mAb) nor alphav integrin function-blocking mAb inhibited sperm binding to or fusion with beta1 integrin null eggs. Thus, function of beta3 or alphav integrins does not seem to be involved in compensating for the absence of beta1 integrins. These results indicate that none of the integrins known to be present on mouse eggs or to be ADAM receptors are essential for sperm-egg binding/fusion, and thus, egg integrins may not play the role in gamete fusion previously attributed to them.     

3-(4-Aminobutyn-1-yl)pyridines: binding at alpha 4 beta 2 nicotinic cholinergic receptors

The binding of a series pyridylbutynylamines 6 was examined at alpha4beta2 nACh receptors. Structural modifications, comparing 6 with pyridyl ethers 2, did not consistently result in parallel effects on receptor affinity, suggesting possible differences in their modes of binding. Furthermore, the binding of amine 6a seemed to be accounted for by the newer vector pharmacophore models.     

Fully flexible docking models of the complex between alpha7 nicotinic receptor and a potent heptapeptide inhibitor of the beta-amyloid peptide binding

The heptapeptide IQTTWSR (IQ), recently reported as inhibitor of the beta-amyloid (Abeta) binding to nicotinic acetylcholine receptors (nAChrs), was docked to the homology model of the alpha7 nicotinic acetylcholine receptor. The most representative models were further subjected to molecular dynamics simulations. The data obtained here suggest that Abeta needs highly specific structural motifs to bind to the alpha7nAChR. These structural motifs are located principally in the upper and lower surroundings of loop C, including loop F and sheets beta1, beta2, beta6, beta9, and beta10 of the receptor. Overall, these results suggest that IQ can be mimicked by more bioavailable, stable compounds that would be helpful for the understanding of the Abeta binding site and its dynamics, and for the design of novel agents to be used as an effective alternative against Alzheimer's disease.     

Down-regulation of interleukin 1 (IL 1) receptor expression by IL 1 and fate of internalized 125I-labeled IL 1 beta in a human large granular lymphocyte cell line

The regulation of interleukin 1 (IL 1) receptor expression on a human large granular lymphocyte cell line, YT, and fate of internalized 125I-labeled IL 1 beta (125I-IL 1 beta) were studied. YT cells were selected for this study, because this cell line expresses a large number of specific high-affinity receptor for IL 1, responds biologically to exogenously added IL 1 by expressing high-affinity IL 2 receptors, and does not produce IL 1. YT cells constitutively express approximately 7 X 10(3) IL 1 receptors/cell with a Kd approximately 10(-10) M. Neither IL 2, phorbol myristic acid, nor lipopolysaccharide affected the total binding of 125I-IL 1 beta by YT cells. In contrast, the capacity of YT cells to bind 125I-IL 1 beta when incubated at 37 degrees C for 3 to 16 hr with a low dose of purified IL 1 beta (approximately 6 U/ml) was reduced by greater than 80%. The loss of binding capability gradually recovered by 16 hr after removal of IL 1 beta from cultured YT cells. The apparent loss of IL 1 receptor expression was accompanied by the internalization of 125I-IL 1 beta into cells. Acid treatment of YT cells to remove bound 125I-IL 1 beta at 4 degrees C showed that 50% of the 125I-IL 1 beta bound to cells could no longer be recovered after 30 min at 37 degrees C, and this increased to 80% after 3 hr at 37 degrees C. Fractionation of cell extracts on Percoll gradient additionally showed 125I-IL 1 beta to appear intracellularly after receptor binding on plasma membranes, and to be successively transferred to some membranous organelles (d approximately equal to 1.037) through an intermediate density organelle (d approximately equal to 1.050), and to finally end up in lysosomal cell fractions (d approximately equal to 1.05 to 1.08) after approximately 3 hr at 37 degrees C. Only approximately 5% of internalized 125I-IL 1 beta was released into culture media by 6 hr of incubation at 37 degrees C. However, the radioactivity in the TCA soluble fraction of the culture media increased gradually by 6 hr and a lysosomotropic enzyme, ethylamine, significantly inhibited both the transfer of internalized 125I-IL 1 beta to the lysosomal fraction and the degradation of 125I-IL 1 beta. This study represents the first evidence of autoregulation of IL 1 receptors by IL 1 and internalization of IL 1 molecules after binding to receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pre-and Postnatal Developmental Changes of Adrenoceptor Subtypes in Rat Brain

beta-Adrenergic receptor subtypes, beta 1 and beta 2, were studied during pre- and postnatal development in the rat brain. [125I]Iodocyanopindolol (6-300 pmol/L) binding assays in the presence of 5-hydroxytryptamine (0.6-6 mumol/L) were used to measure exclusively beta-adrenergic receptors. In forebrain tissue, saturable and stereoselective binding was detected on gestational day 13. The amount of beta-adrenergic binding increased until postnatal day 23, when adult values were reached. The dissociation constants of [125I]iodocyanopindolol binding remained the same throughout development, as did the affinity of several beta-adrenergic and non-beta-adrenergic compounds. The proportion of the beta 2-adrenergic receptors was determined using the beta 1-selective antagonist ICI-89406 (7-150 nmol/L) and was found to change from 65% in prenatal forebrain tissue to 28% in adulthood. In cerebellum/medulla pons tissue, however, the proportion of beta 2-receptor binding (80%) remained unchanged during the whole developmental period.     

High affinity ligand binding is not essential for granulocyte-macrophage colony-stimulating factor receptor activation.

The high affinity receptor of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is a heterodimer composed of two members of the cytokine receptor superfamily. GM-CSF binds to the alpha-subunit (GM-R alpha) with low affinity and to the receptor alpha beta complex (GM-R alpha beta) with high affinity. The GM-CSF.GM-R alpha beta complex is responsible for biological activity. Interactions of the N-terminal helix of mouse GM-CSF with mGM-R alpha beta were examined by introducing single alanine substitutions of hydrophilic residues in this region of mGM-CSF. The consequences of these substitutions were evaluated by receptor binding and biological assays. Although all mutant proteins exhibited near wild-type biological activity, most were defective in high affinity receptor binding. In particular, substitution of Glu-21 with alanine abrogated high affinity binding leaving low affinity binding unaffected. Despite near wild-type biological activity, no detectable binding interaction of this mutant with mGM-R beta in the context of mGM-R alpha beta was observed. Cross-linking studies showed an apparent interaction of this mutant protein with mGM-R alpha beta. The deficient receptor binding characteristics and near wild-type biological activity of this mutant protein demonstrate that mGM-CSF receptor activation can occur independently of high affinity binding, suggesting that conformational changes in the receptor induced by mGM-CSF binding generate an active ligand-receptor complex.