Identification of the interleukin-3 receptor using an iodinatable, cleavable, photoreactive cross-linking agent

Murine interleukin-3 (mIL-3) is a lymphokine that stimulates the proliferation and differentiation of both pluripotent hemopoietic stem cells and their committed progeny. However, very little is known about the mechanism by which this growth factor elicits its effects on responsive cell populations. To gain insight into early events following mIL-3 receptor interaction, we initiated studies to isolate the receptor and study its properties. In this report, we demonstrate the use of a new iodinatable, cleavable, photoreactive cross-linking agent, sulfosuccinimidyl 2-(p-azidosalicylamido)-1,3'-dithiopropionate to identify the mIL-3 receptor. These studies reveal the mIL-3 receptor to be a single polypeptide chain with a molecular weight of 67 kDa and an isoelectric point of approximately 6.2.     

Covalent crosslinking analysis of angiotensin receptors on differentiated NG108-15 cells

Neuroblastoma x glioma hybrid cells (NG108-15) were used as a model system to characterize neuronal-glial type angiotensin (ANG) receptors by covalent crosslinking analysis. After differentiation with 1.5% DMSO and 0.5% fetal bovine serum for four to five days, saturation analysis revealed a single high affinity site with a Kd = 1.35 +/- 0.42 nM and a Bmax = 468 +/- 106 fmol/mg protein. Using the homobifunctional crosslinking reagent bis(sulfosuccinimidyl) suberate (BS3), a site with an estimated Mr of 78 kDa was specifically labeled with 125I-ANG II as determined by SDS-polyacrylamide gel electrophoresis. Both ANG II and ANG III (10(-6) M) inhibited specific labeling. The Ki for ANG III binding was similar by both pharmacologic (Ki = 3.33 +/- 0.98 nM) and gel densitometric (Ki = 2.65 +/- 0.32 nM) analyses. We conclude that the 78 kDa protein represents a high affinity ANG binding site with similar affinities for both ANG II and ANG III.     

Amplified expression of tumor necrosis factor receptor in cells transfected with Epstein-Barr virus shuttle vector cDNA libraries

As an approach to isolate the cell-surface receptor for tumor necrosis factor (TNF), we have developed transfectants of human B-lymphoblastoid cells (UC cells) that overexpress the TNF receptor. These transfectants were isolated from UC cells transfected with cDNA libraries of HeLa or NG108 cells constructed in the mammalian expression vector EBO-pcD. This vector contains the Epstein-Barr virus origin of replication (ori-P) plus the EBNA-1 gene conferring replication function to ori-P and, therefore, the ability to replicate autonomously within the transfected cell (Margolskee, R.F., Kavathas, P., and Berg, P. (1988) Mol. Cell. Biol. 8, 2837-2947). Cells overexpressing the TNF receptor were identified and separated by the binding of fluoresceinated TNF and flow cytometric selection. Scatchard analysis of 125I-TNF binding data revealed a single class of high affinity receptors with a dissociation constant (Kd) of 0.2 to 2 nM and a receptor density of about 150,000 per cell, an increase of approximately 150-fold over UC cells. Cross-linking of receptor-ligand with bis-sulfosuccinimidyl suberate followed by polyacrylamide gel electrophoresis gave estimates of 87 and 104 kDa for the size of the complex. Based on its ability to bind TNF, a 68-kDa receptor protein was identified in cell extracts enriched for the receptor by using immobilized wheat germ agglutinin and TNF affinity chromatography. The difference in the estimated size of the receptor and the receptor-ligand complexes demonstrates that TNF binds to the receptor as a monomer or a dimer. Analysis of cDNA sequences conferring receptor amplification in transfectants revealed that plasmid DNA was present at 30 or more copies per cell, most likely integrated into the genomic DNA or organized into high molecular weight catenanes, and autonomously replicating units could not be recovered. Therefore, while this vector was useful in generating stable receptor-amplified cells, it was not maintained as a recoverable episome.     

Identification and distribution of two forms of the interleukin 1 receptor

Using affinity crosslinking techniques, we have biochemically characterized the interleukin-1 (IL1) receptor and investigated its distribution on a range of murine and human cell lines. We show that two forms of IL1 receptor can be identified on the basis of specific crosslinking with 125I-IL1 alpha and 125I-IL1 beta. The two receptor forms have an approximate molecular mass of approximately 80 and approximately 60 kDa, and were found on both murine and human cells. Their relative distribution shows no clear cell lineage restriction and does not correlate with preferential binding of IL1 alpha or IL1 beta. Some cells, such as the T helper cell line D10.G4.1, express both forms of the receptor. Iodine 125-IL1 was crosslinked to the two receptor forms and a partial peptide map analysis of the two receptor/ligand complexes was performed. Comigration of the major partial peptide fragments suggests that the approximately 80 and approximately 60 kDa forms of the receptor may be differentially processed forms of the same protein. Treatment of the approximately 60 kDa IL1 receptor on Raji cells with N-glycanase reduced its molecular mass by 12 kDa, showing that this lower molecular mass form is a glycoprotein; glycosylation differences alone probably do not account for the difference in mass between the two forms.     

Crosslinking of platelet glycoprotein Ib by N-succinimidyl(4-azidophenyldithio)propionate and 3,3′-dithiobis(sulfosuccinimidyl propionate)

To examine the relationship between glycoprotein Ib and other proteins in the platelet membrane and the interaction of this protein with thrombin, platelets were crosslinked by two cleavable reagents, SADP (N-succinimidyl(4-azidophenyldithio)propionate) and DTSSP (3,3′-dithiobis(sulfosuccinimidyl propionate)). Two-dimensional, unreduced-reduced sodium dodecyl sulphate (SDS)-polyacrylamide electrophoresis and staining by silver or wheat germ agglutinin-conjugated peroxidase, after protein transfer to nitrocellulose, demonstrated that SADP intramolecularly crosslinked glycoprotein Ib and formed intermolecular complexes of glycoprotein IIb and some high molecular weight proteins. DTSSP intermolecularly crosslinked glycoprotein Ib, glycoprotein IIb, and other high molecular weight proteins. With a low concentration of ¹²⁵I-labeled TLCK-thrombin (6 nM), crosslinking with SADP yielded a 200 000 Da complex containing radioactive-labeled thrombin, and high TLCK-thrombin concentration (0.1 μM) gave the complex and a 167 000 band. α- and TLCK-thrombin crosslinking with DTSSP also yielded the 200 000 complex, with the remaining radioactivity in a band corresponding to a highly crosslinked complex. The 200 000 complex formed by reaction with SADP or DTSSP was markedly reduced by preincubation of platelets with excess unlabeled TLCK-thrombin and had a pI similar to glycoprotein Il. These results suggest that glycoprotein Il is one of the proteins composing the high affinity receptor for thrombin.     

Recombinant 55-kDa tumor necrosis factor (TNF) receptor. Stoichiometry of binding to TNF alpha and TNF beta and inhibition of TNF activity.

The extracellular domain of the 55-kDa TNF receptor (rsTNFR beta) has been expressed as a secreted protein in baculovirus-infected insect cells and Chinese hamster ovary (CHO)/dhfr- cells. A chimeric fusion protein (rsTNFR beta-h gamma 3) constructed by inserting the extracellular part of the receptor in front of the hinge region of the human IgG C gamma 3 chain has been expressed in mouse myeloma cells. The recombinant receptor proteins were purified from transfected cell culture supernatants by TNF alpha- or protein G affinity chromatography and gel filtration. In a solid phase binding assay rsTNFR beta was found to bind TNF alpha with high affinity comparable with the membrane-bound full-length receptor. The affinity for TNF beta was slightly impaired. However, the bivalent rsTNFR beta-h gamma 3 fusion protein bound both ligands with a significantly higher affinity than monovalent rsTNFR beta reflecting most likely an increased avidity of the bivalent construct. A molecular mass of about 140 kDa for both rsTNFR beta.TNF alpha and rsTNFR beta.TNF beta complexes was determined in analytical ultracentrifugation studies strongly suggesting a stoichiometry of three rsTNFR beta molecules bound to one TNF alpha or TNF beta trimer. Sedimentation velocity and quasielastic light scattering measurements indicated an extended structure for rsTNFR beta and its TNF alpha and TNF beta complexes. Multiple receptor binding sites on TNF alpha trimers could also be demonstrated by a TNF alpha-induced agglutination of Latex beads coated with the rsTNFR beta-h gamma 3 fusion protein. Both rsTNFR beta and rsTNFR beta-h gamma 3 were found to inhibit binding of TNF alpha and TNF beta to native 55- and 75-kDa TNF receptors and to prevent TNF alpha and TNF beta bioactivity in a cellular cytotoxicity assay. Concentrations of rsTNFR beta-h gamma 3 equimolar to TNF alpha were sufficient to neutralize TNF activity almost completely, whereas a 10-100-fold excess of rsTNFR beta was needed for similar inhibitory effects. In view of their potent TNF antagonizing activity, recombinant soluble TNF receptor fragments might be useful as therapeutic agents in TNF-mediated disorders.     

HepG2 cells predominantly express the type II interleukin 1 receptor (biochemical and molecular characterization of the IL-1 receptor).

In this study we have characterized the cell surface interleukin 1 (IL-1) receptor in HepG2 hepatoma cells. We found that HepG2 cells bind both IL-1 alpha and beta with high affinity, KDs of 136 and 180 pM and receptor densities of 16,000 and 8500 binding sites/cell respectively. The binding sites appeared to be predominantly type II since phorbol ester treatment of the cells, which selectively downregulates type II IL-1 receptors, reduced binding by 68% while treatment of the cells with an inhibitory monoclonal antibody specific for the type I receptor had no significant effect on IL-1 binding. Competition studies with a modified IL-1 beta analog (Glu4) also revealed binding kinetics more consistent with binding to type II receptors than to type I. Crosslinking and ligand blotting with human 125I-IL-1 demonstrated the presence of two bands, a 78 kDa band typical of crosslinking to type II (p60) receptor, and a 98 kDa band, typical of crosslinking to the type I (p80) receptor. Low level expression of the type I receptor was consistent with molecular biological studies employing polymerase chain reaction (PCR) amplification which indicated that mRNA for the type I receptor was produced by the HepG2 cells. Functional receptors were demonstrated by the induction of IL-8 by IL-1 stimulated cells.     

Isolation and partial characterisation of immunoglobulin from southern bluefin tuna Thunnus maccoyii Castelnau

Specific and total serum immunoglobulins were extracted by immunoaffinity, mannan-binding protein and Protein A affinity chromatography from southern bluefin tuna (Thunnus maccoyii Castelnau) immunised with rabbit IgG, and from non-immunised southern bluefin tuna. SDS-PAGE in 10% reducing gels revealed two heavy chains with molecular weights of approximately 74.6 +/- 1.3 kDa and 71.2 +/- 0.9 kDa, and two light chains with molecular weights of approximately 29 +/- 1.2 kDa and 28 +/- 1.0 kDa. Under non-reducing, but denaturing, conditions in 4% and 5% SDS-PAGE gels, a high molecular weight and a low molecular weight fraction were demonstrated. By gel filtration using Sephacryl HR 300 a molecular weight of 845 kDa, consistent with a tetramer, was obtained for the high molecular weight fraction, and a molecular weight of 168 kDa, consistent with a monomer, was obtained for the low molecular weight fraction. The extinction coefficient at A280 for the purified immunoglobulin (Ig) was determined to be 1.24. Tuna a-rabbit IgG Ig was reactive with all non-reduced mammalian IgG antigens tested, suggesting that common conformational antigenic determinants were recognised.     

Characterisation of atrial natriuretic peptide receptors in bovine ventricular sarcolemma

Analysis of [125I]-ANP binding data in an isolated bovine ventricular sarcolemmal membrane fraction revealed a single high affinity binding site (Kd approximately 5 x 10(-11) M). The ring deleted ANP analogue des [QSGLG]-ANP (4-23)-NH2 bound with a 1000-fold lower affinity indicating the absence of C-type receptors in this preparation. ANP stimulated guanylate cyclase activity by up to 2-fold with half-maximal activation at approximately 10(-9) M. Crosslinking [125I]-ANP to its receptor with disuccinimidyl suberate (DSS) revealed two radiolabelled bands of 120 kDa and 65 kDa on non-denaturing SDS-PAGE. Radioactive signals from both bands were lost by reducing the sample with beta-mercaptoethanol prior to electrophoresis, in which case a radioactive fragment of less than 5 kDa migrated with the dye front. These results suggest that the binding of ANP to both high and low molecular weight "receptor" proteins may be associated with the hydrolysis of the peptide.     

Binding and crosslinking of 125I-labeled recombinant human tumor necrosis factor to cell surface receptors

Highly purified recombinant human tumor necrosis factor (TNF) (molecular mass determined as 17 kilodaltons (kDa) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as 36 kDa by Sephadex G-100 gel chromatography) was labeled with 125I to a specific activity of 5 microCi/micrograms without appreciable loss of activity. The binding of 125I-TNF to eighteen human and twelve animal cell lines was examined. The binding varied considerably among different cell lines. In most cell lines, the binding was inhibited up to greater than 90% by the addition of a 100-fold excess of unlabeled TNF. Some human and mouse cell lines showed no significant binding above background levels, suggesting that these cell lines had no receptors for TNF. Among the TNF receptor-positive cell lines, there was no direct correlation between the level of specific TNF binding and the level of sensitivity to the cytotoxic or cytostatic effect of TNF. Some cell lines were sensitive to TNF, whereas others were not affected at all by TNF. The TNF receptor-negative cell lines were also resistant to TNF. Therefore, although the existence of TNF receptor seems to be necessary, it does not alone determine cellular sensitivity to TNF. Scatchard analysis of the binding data revealed that human HeLa S3 and THP-1 had about 50,000 and 10,000 receptors/cell with a dissociation constant (KD) of 0.3-0.5 nM, respectively. Similarly, mouse L-929 and L-M cells had about 5,000 receptors/cell with KD of 3-5 nM. 125I-TNF bound to HeLa S3 cells was rapidly internalized at 37 degrees C, presumably by receptor-mediated endocytosis, and degraded to acid-soluble products. The turnover of TNF receptors on HeLA S3 cells seemed to be rapid, since the level of specific binding quickly decreased after treatment with 100 micrograms/ml of cycloheximide at 37 degrees C with a half-life of about 1.5 h. The crosslinking of the cell-bound 125I-TNF with the use of disuccinimidyl suberate yielded a complex of 105 kDa for HeLa S3 and THP-1 cells, and a complex of 100 kDa for U937 cells. The crosslinking was completely inhibited by the addition of a 100-fold excess of unlabeled TNF. Assuming that the complex was due to a one-to-one association of the dimeric form of TNF (34 kDa) with the receptor, we estimated the molecular size of the human TNF receptor to be 71 kDa for HeLa S3 and THP-1, and 66 kDa for U937.     

Purification of homogeneity and amino acid sequence analysis of a receptor protein for interleukin 1

The interleukin 1 (IL-1) receptor from mouse EL-4 thymoma cells was purified to homogeneity by a method which utilized ligand affinity chromatography and classical chromatographic techniques. After solubilization of the receptor from intact cells with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, the IL-1 binding activity was purified greater than 23,000-fold. Analysis of the purified protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot, and ligand blot demonstrated that a single protein of molecular mass of approximately 80 kDa is the IL-1 binding polypeptide. The purified protein bound IL-1 with a dissociation constant of approximately 1.1 X 10(-10) M, which is indistinguishable from the affinity of the cell-bound receptor. The amino acid composition of this protein is strikingly similar to the composition deduced from the sequence of a cDNA coding for an IL-1 receptor from EL-4 cells. Protein sequence analysis of Staphylococcus aureus V-8 protease-derived peptides yields data consistent with the sequence proposed from cloned cDNA. These studies have demonstrated that the high affinity IL-1 receptor on EL-4 cells is the 80-kDa protein.     

Evidence for Shared Receptor Proteins for Human Interleukin-3 and Granulocyte-Macrophage Colony-Stimulating Factor in the Human M-07 Cell Line

Abstract

The biologic response of the human leukemia cell line M-07 to granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL-3) and interleukin 4 (IL-4) is mediated by a low number of high affinity receptors. Cross-competition studies revealed that IL-3 and GM-CSF partially inhibited the specific binding of the heterologous radiolabeled ligand, whereas IL-4 binding was not affected by these cytokines. The molecular mechanism of cross-competition was investigated by chemical crosslinking and immuno-precipitation. Trimolecular receptor complexes consisting of a major 73kDa and two minor 120 and 128kDa membrane proteins for IL-3, and a major 84kDa and two minor 120 and 130 kDa proteins for GM-CSF were found on M-07 cells. The 73 and 84kDa proteins represent distinct and non-linked membrane proteins and are identical with the cloned, low affinity IL-3 and GM-CSF receptor proteins (Gearing et al, 1989, Hayashida et al, 1990). The higher molecular weight proteins share common binding sites as evidenced by immunoprecipitation of double-crosslinked membranes. The 120/128kDa proteins are most likely identical with the recently cloned and shared β-subunit of the IL-3 and GM-CSF receptor (Kitamura et al, 1991) containing a single or two IL-3 and/or GM-CSF molecules.     

Characterization of a recombinant extracellular domain of the type 1 tumor necrosis factor receptor: evidence for tumor necrosis factor-alpha induced receptor aggregation.

An expression plasmid encoding the extracellular portion of the human tumor necrosis factor (TNF) type 1 receptor (TNF-R1) was constructed and used to generate a stable cell line secreting soluble TNF-R1 (sTNF-R1). The sTNF-R1 was purified, and its biochemical properties and its interactions with human TNF-alpha were examined. SDS-PAGE resolved the purified sTNF-R1 into three bands of approximate Mr 24,200, 28,200, and 32,800. Sedimentation equilibrium analysis gave a molecular weight of 25,000 for sTNF-R1 whereas the molecular weight obtained by gel filtration chromatography was approximately 55,000-60,000. Scatchard analysis of [125I]TNF-alpha binding to sTNF-R1 revealed high-affinity binding (Kd = 93 pM), comparable to that observed for the intact receptor on whole cells. Competitive binding experiments showed that sTNF-R1 has a 50-60-fold higher affinity for TNF-alpha than for TNF-beta, in contrast to the equal affinities of TNF-alpha and TNF-beta for the full-length TNF-R1 transiently expressed in mammalian cells. The sTNF-R1 was found to block the cytotoxicity of TNF-alpha and TNF-beta on a murine L-M cell assay. The sizes of the sTNF-R1.TNF-alpha complex determined by gel filtration chromatography and sedimentation equilibrium were approximately 141 and 115 kDa, respectively. The stoichiometry of the complex was examined by Scatchard analysis, size-exclusion chromatography, HPLC separation, amino acid composition, sequence analysis, and sedimentation equilibrium. The data from these studies suggest that at least two molecules of sTNF-R1 can bind to a single TNF-alpha trimer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and functional significance of N-glycosylation of the 5-ht5A receptor

The presence and roles of N-glycosylation of the human (h) 5-ht(5A) receptor were investigated using a heterologous expression system. Following transient transfection of COS-7 cells with h5-ht(5A) receptor cDNA, SDS-PAGE/Western blot analysis of immunoreactivity demonstrated two protein species; a predominant species with a molecular weight of approximately 35-45 kDa and a minor species of approximately 45-55 kDa. Transfected cells grown in the presence of the N-glycosylation inhibitor tunicamycin, failed to express the minor immunoreactive species indicating this represented the N-glycosylated form of the h5-ht(5A) receptor. Comparison of the molecular weights of immunoreactive bands arising from the wild-type and each of the mutant 5-ht(5A) receptors with disruption of the predicted N-glycosylation sites (N6S and N21S) demonstrated that both identified asparagines were N-glycosylated. Immunocytochemical and ELISA studies demonstrated that the [N6S]h5-ht(5A) receptor mutation, but not the [N21S]h5-ht(5A) receptor mutation, reduced protein expression in the cell membrane, indicating that N-glycosylation of the N6 residue is important for the membrane expression of this neurotransmitter receptor; a requirement for receptor function.     

Isolation and partial characterization of the formyl peptide receptor components on human neutrophils

The receptor for formylated peptides such as FMLP has been reported to consist of glycoprotein components ranging from 24-95 kDa, and to exhibit both high and low affinity for ligand. Controversy exists on the molecular size and number of these components, and whether the different affinities represent distinct ligand binding sites. In this study, the receptor was found to be comprised of components, of 94, 68, and approximately 40 kDa molecular size. Competitive binding inhibition experiments showed that FMLP bound to the components in the following order from highest to lowest affinity: 68 kDa greater than approximately 40 kDa greater than 94 kDa. Our findings suggest that the FMLP receptor of human neutrophils contains at least three components, and that each component has a different affinity for FMLP.     

Covalent labeling of opioid receptors with radioiodinated human beta-endorphin. Identification of binding site subunit

125I-beta-Endorphin (human) binds with high affinity, specificity, and saturability to rat brain and neuroblastoma X glioma hybrid cell (NG 108-15) membranes. Dissociation constants and binding capacities were obtained from Scatchard plots and are 2 nM and 0.62 pmol/mg of protein for rat whole brain and 6 nM and 0.8 pmol/mg of protein for NG 108-15 cells. Results from competition experiments also indicate that this ligand interacts with high affinity with both mu and delta opioid binding sites, with a slight preference for mu sites, while exhibiting low affinity at kappa sites. We have demonstrated that human 125I-beta-endorphin is a useful probe for the investigation of the subunit structure of opioid receptors. The specific cross-linking of this ligand has revealed the presence of four reproducible bands or areas after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography at 65, 53, 38, and 25 kDa. All labeled bands seem to be opioid receptor related since they are eliminated when binding is carried out in an excess of various opiates. The evidence we have obtained using rat whole brain (delta congruent to mu), rat thalamus (largely mu), bovine frontal cortex (delta:mu congruent to 2:1), and NG 108-15 cells (delta) demonstrates that different labeling patterns are obtained when mu and delta binding sites are cross-linked. The pattern obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from cross-linked mu sites contains a major (heavily labeled) component of 65 kDa and a minor component of 38 kDa, while patterns from delta sites contain a major labeled component of 53 kDa. This 53-kDa band appears clearly in extracts from NG 108-15 cells and bovine frontal cortex, while in rat whole brain a diffusely labeled region is present between 55 and 41 kDa. In addition, NG 108-15 cells also display a minor labeled component at 25 kDa. The relationship of the minor bands to the major bands is not clear.     

Vasoactive intestinal peptide effects on GH3 pituitary tumor cells: high affinity binding, affinity labeling, and adenylate cyclase stimulation. Comparison with peptide histidine isoleucine and growth hormone-releasing factor

The vasoactive intestinal polypeptide (VIP) receptor was characterized on the GH3 rat pituitary tumor cell line using competitive binding studies with peptides having sequence homology with VIP. Further studies investigated receptor coupling to the adenylate cyclase complex by measurement of cAMP levels. Finally, the molecular weight of the receptor was estimated by affinity labeling techniques. Studies using 125I-VIP and unlabeled competing peptides revealed a single class of high affinity binding sites with a dissociation constant (KD) of 17 +/- 2 nM (mean +/- S.E.M.) for VIP, 275 +/- 46 nM for peptide histidine isoleucine (PHI), and 1380 +/- 800 nM for human pancreatic growth hormone releasing factor (GHRF). VIP and PHI each stimulated intracellular cAMP accumulation in a dose-dependent manner; both peptides demonstrated synergism with forskolin. In contrast, GHRF neither stimulated accumulation of cAMP nor demonstrated synergism with forskolin. VIP plus PHI (1 microM each) caused no significant increase in cAMP over either VIP or PHI alone, implying that the two peptides act through the same receptor. Covalent crosslinking of 125I-VIP to its binding site using either disuccinimidyl suberate (DSS) or ethylene glycol bis(succinimidyl succinate) (EGS) was followed by SDS-PAGE and autoradiography. The result is consistent with an Mr 47 000 VIP-binding subunit comprising or being associated with the VIP receptor of GH3 pituitary tumor cells.     

Characterization of beta-125I-endorphin cross-linked proteins in NG108-15 cell membranes. A 25-kilodalton protein with properties of delta-opioid-binding site.

Cross-linking of beta-125I-endorphin to NG108-15 cell membranes labeled bands with molecular masses of 55, 35, and 25 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We applied several criteria to evaluate the relevance of these cross-linked bands to delta-opioid receptors, including selectivity, stereospecificity, affinity, G-protein coupling, down-regulation, and correlation with opioid receptor level in different well-characterized cell lines. Only the 25 kDa protein adequately fulfilled all these criteria. Thus, cross-linking to the 25-kDa band was selectively inhibited by ligands with delta-opioid affinity, but not by mu-opioid, kappa-opioid, or optically inactive opioid ligands or by non-opioid ligands. Based on inhibition of cross-linking, we calculated an affinity of [D-Ala2,D-Leu5]enkephalin binding to the 25-kDa and (Kd = 6 nM) that is similar to that reported for [D-Ala2,D-Leu5]enkephalin binding to NG108-15 membranes; this affinity decreased approximately 10-fold in the presence of Na+/guanyl-5'-yl imidodiphosphate. Chronic agonist treatment of NG108-15 cells reduced cross-linking to the 25-kDa band, but not to others, in a manner parallel to down-regulation of opioid receptors. Finally, the amount of the 25-kDa band was roughly proportional to the level of opioid receptors present in N18TG2, NS20Y, ST7-3, and ST8-4 cells. The 25-kDa band was absent in PC12h, NIH3T3, and C6BU1 cells as well as in liver, all of which had no detectable opioid binding.     

Analysis of murine interferon-gamma binding to its receptor on intact cells and solubilized membranes. Identification of an 80 kDa receptor

The receptor for murine-interferon-gamma (Mu-IFN-gamma) has been characterized for its molecular size and equilibrium binding constant on a thymoma cell line, EL-4. Binding of 125I-IFN-gamma to intact cells and their solubilized membranes has shown a single class of receptor with Kd values of 1.9 x 10(-9) M and 1.3 x 10(-8) M, respectively. It was shown that solubilization of the Mu-IFN-gamma receptor with a Zwitterionic detergent (Chaps) preserves its binding activity. A direct comparison of the molecular mass of the Mu-IFN-gamma receptor on intact cells versus detergent-solubilized membranes was performed using a radiolabeled photoactivated crosslinking reagent and direct hybridization with 125I-labeled IFN-gamma on Western blots of solubilized receptor. The results indicate that both types of receptors have an identical molecular mass of approximately 80 kDa.