The 56 kDa protein of human genital skin fibroblasts is identical to that radiolabelled by [3H]dihydrotestosterone 17 beta-bromoacetate

Analysis of soluble proteins from human genital skin fibroblasts by two-dimensional polyacrylamide gel electrophoresis reveals an abundant protein doublet of mol. wt 56,000 with isoelectric points (pI) of 6.7 and 6.5. This protein is absent in non-genital skin fibroblasts as well as in genital skin fibroblasts of most patients with complete forms of androgen insensitivity. The protein specifically binds androgen. A protein of similar estimated molecular weight (58,000) from human genital skin fibroblasts has recently been found to be covalently radiolabelled by the affinity ligand dihydrotestosterone 17 beta-bromoacetate (DHT-BA). In the present study these proteins have been found to be indistinguishable on one- and two-dimensional gel electrophoresis. Antibodies raised against the 56 kDa pI 6.7/6.5 protein also recognized the protein covalently radiolabelled by DHT-BA. A third protein of estimated mol. wt 59,000 has been found to be associated with several steroid hormone receptor complexes but has no known ligand binding activity. This protein was found to be clearly separable from the 56/58 kDa protein on two-dimensional gel electrophoresis as it has a more acidic pI of approximately 5.4. Furthermore, antibodies against the 59 kDa protein do not recognize the 56 kDa species, and vice versa.     

The vaccinia virus soluble alpha/beta interferon (IFN) receptor binds to the cell surface and protects cells from the antiviral effects of IFN

Poxviruses encode a broad range of proteins that interfere with host immune functions, such as soluble versions of receptors for the cytokines tumor necrosis factor, interleukin-1 beta, gamma interferon (IFN-gamma), IFN-alpha/beta, and chemokines. These virus-encoded cytokine receptors have a profound effect on virus pathogenesis and enable the study of the role of cytokines in virus infections. The vaccinia virus (VV) Western Reserve gene B18R encodes a secreted protein with 3 immunoglobulin domains that functions as a soluble receptor for IFN-alpha/beta. We have found that after secretion B18R binds to both uninfected and infected cells. The B18R protein present at the cell surface maintains the properties of the soluble receptor, binding IFN-alpha/beta with high affinity and with broad species specificity, and protects cells from the antiviral state induced by IFN-alpha/beta. VV strain Wyeth expressed a truncated B18R protein lacking the C-terminal immunoglobulin domain. This protein binds IFN with lower affinity and retains its ability to bind to cells, indicating that the C-terminal region of B18R contributes to IFN binding. The replication of a VV B18R deletion mutant in tissue culture was restricted in the presence of IFN-alpha, whereas the wild-type virus replicated normally. Binding of soluble recombinant B18R to cells protected the cultures from IFN and allowed VV replication. This represents a novel strategy of virus immune evasion in which secreted IFN-alpha/beta receptors not only bind the soluble cytokine but also bind to uninfected cells and protect them from the antiviral effects of IFN-alpha/beta, maintaining the cells' susceptibility to virus infections. The adaptation of this soluble receptor to block IFN-alpha/beta activity locally will help VV to replicate in the host and spread in tissues. This emphasizes the importance of local effects of IFN-alpha/beta against virus infections.     

Identification of the origin of the growth hormone-binding protein in rat serum

GH specifically interacts with a soluble binding protein in serum. The GH-binding protein (GHBP) has been shown to contain the extracellular portion of the cell surface GH receptor (GHR). In rats and mice there is a unique mRNA that encodes the GHBP. This mRNA contains an alternatively spliced exon that replaces the transmembrane and intracellular domains of the receptor with a short hydrophilic carboxy-terminus of 17 and 25 amino acids, respectively, in rats and mice. In humans and other species no mRNAs encoding the GHBP have been identified, suggesting that the GHBP is in these cases a proteolytically processed GHR. In this study a monoclonal antibody (GHBP 4.3) was raised to the rat GHBP using as immunogen a synthetic peptide containing the unique C-terminal 17 amino acids that are not found in the rat GHR. As predicted, this antibody is specific to rat GHBP and does not cross-react with rat GHR. In combination with polyclonal and monoclonal antibodies that recognize both GHBP and GHR, this antibody was used to show that all, or most, of the GHBP in rat serum is indeed derived from the alternatively spliced GHBP mRNA and not from proteolytic processing of the GHR. In addition, endogenous rat serum GHBP was found to exist in two forms, with apparent mol wt of 52 and 44 kDa, arising from a single protein core of 32 kDa by extensive glycosylation. The concentrations of GHBP in male and female rat plasma were also estimated to be 300 and 575 ng/ml, respectively (measured in nonglycosylated GHBP equivalents).     

C-terminal arginine cluster is essential for receptor binding of norovirus capsid protein

Noroviruses are the major viral pathogens of epidemic acute gastroenteritis affecting people worldwide. They have been found to recognize human histo-blood group antigens as receptors. The P domain of norovirus capsid protein was found to be responsible for binding to viral receptors, and the recombinant P protein forms P dimers and P particles in vitro. In this study, we demonstrate that a highly conserved arginine (R) cluster at the C terminus of the P domain is critical for receptor binding and P particle formation of the P proteins. Deletions of the R cluster abolished these functions. Replacement of the R cluster with histidines (another positively charged amino acid) resulted in low efficiency of receptor binding and P particle formation, while replacement with alanines led to loss of both functions completely. The R cluster also contains a highly conserved trypsin digestion site. A treatment of capsid protein or P domain mutants from both genogroup I (Norwalk virus) and genogroup II (VA387) noroviruses with trypsin resulted in a removal of the R cluster and the S domain, leaving a P polypeptide of 31.3 kDa (Norwalk virus) or 34.3 kDa (VA387), similar to the soluble P protein found in vivo. Our findings imply that the proteolytic process could be a necessary step for norovirus replication in the host.     

Expression of human soluble tissue factor in yeast and enzymatic properties of its complex with factor VIIa.

The extracellular domain of human tissue factor (TF, amino acids 1-217) was expressed in Saccharomyces cerevisiae, using the inducible yeast acid phosphatase promoter and the yeast invertase signal sequence to direct its secretion into the culture broth. Two active soluble forms sTF alpha (high molecular weight form) and sTF beta (low molecular weight form) were purified, the yield being approximately 10 and 1 mg/liter of culture supernatant, respectively. sTF alpha had an apparent molecular mass of 150 kDa on SDS-polyacrylamide gel electrophoresis and contained more than 200 residues of mannose/mol of protein. sTF beta had an apparent molecular mass of 37 kDa and contained 22 residues of mannose/mol of protein. N-Glycosidase F treatments of both rTFs reduced the apparent molecular mass to 35 kDa. The amino-terminal sequences and amino acid compositions of sTF alpha and sTF beta were consistent with those deduced from the cDNA sequence, thereby indicating that the difference in molecular mass is caused by heterogeneity of oligosaccharide structures. Of these recombinant TFs, sTF beta enhanced factor VIIa-amidolytic activity 40-fold toward the chromogenic substrate and 147-fold toward the fluorogenic substrate, affecting mainly the kcat value. The enhancement was comparable with that of TF purified from human placenta. The TF-mediated enhancement of factor VIIa-amidolytic activity was inhibited by heparin-activated antithrombin III, forming a high molecular weight complex. As treatment of sTF beta with denaturants such as guanidine hydrochloride or urea led to a biphasic loss of the activity, the extracellular domain of TF probably consists of two discrete domains. This expression system provides a significant amount of the extracellular domain of TF so that studies of interactions with factor VII are feasible.     

Single subunit structure of the human thyrotropin receptor.

We have produced rabbit antibody against a synthetic peptide corresponding to N-terminal region of the extracellular domain of human thyrotropin receptor (hTSH-R) (N peptide, aminoacid residues 29-57). Western blot analysis revealed that N-peptide antibody recognized recombinant hTSH-R stably expressing in CHO-K1 cells as a mol. wt. about 104 kDa regardless in the presence or absence of disulfide-reducing agent. The band was not detected in untransfected CHO-K1 cells and no band was also stained by the antibody absorbed with N-peptide. In a reducing condition, the antibody also bound the rat receptor from FRTL5 cells as the same molecular size (104 kDa). These results clearly indicate that TSH-R is composed of a single subunit and that two subunit model for the TSH-R may reflect artifactual proteolytic cleavage of the receptor during membrane preparation.     

Cleavage and release of a soluble form of the receptor tyrosine kinase ARK in vitro and in vivo

The receptor tyrosine kinase ARK (also called AXL or UFO) is the murine prototype of a small family of receptors with an extracellular domain resembling cell adhesion molecules and a conserved tyrosine kinase domain. ARK is capable of homophilic binding, as well as of binding to GAS6, a secreted member of the class of vitamin K dependent proteins whose expression is up-regulated in growth-arrested cells. To gain understanding of the physiological role of ARK signaling, we have investigated the ARK forms which are expressed by cells in culture as well as by mouse organs. We found that ARK is not only expressed as a transmembrane protein, but is also cleaved in the extracellular domain to generate a soluble ARK form of about 65 kDa, which is easily detected in conditioned media of ARK expressing cells, in serum and plasma and in mouse organs. Soluble ARK is also produced by tumor cells in vivo. The function of these molecules could be that of binding GAS6, thereby inhibiting the interaction of this ligand with its cell-associated receptor, or they could be involved in binding to ARK itself. © 1996 Wiley-Liss, Inc.     

A soluble form of the interleukin 4 receptor in biological fluids

Murine biological fluids and murine cell culture supernatants were analyzed for the presence of soluble murine interleukin 4 receptor (sIL4R) with the use of two monoclonal antibodies directed against the receptor. Mouse urine, serum, ascitic fluid, and cell culture supernatants contained varying levels of immunoreactive protein. All of the immunoreactive protein possessed interleukin 4 (IL 4) binding activity. Following partial purification of ascitic fluid a protein was isolated that binds IL 4 with high affinity. This data is consistent with the fact that murine biological fluids contain a soluble version of the murine IL 4 receptor that arises via secretion of the soluble receptor and/or via shedding of the extracellular portion of the full-length receptor from the cell surface.     

A new tagging system for production of recombinant proteins in Drosophila S2 cells using the third domain of the urokinase receptor

The use of protein fusion tag technology greatly facilitates detection, expression and purification of recombinant proteins, and the demands for new and more effective systems are therefore expanding. We have used a soluble truncated form of the third domain of the urokinase receptor as a convenient C-terminal fusion partner for various recombinant extracellular human proteins used in basic cancer research. The stability of this cystein-rich domain, which structure adopts a three-finger fold, provides an important asset for its applicability as a fusion tag for expression of recombinant proteins. Up to 20mg of intact fusion protein were expressed by stably transfected Drosophila S2 cells per liter of culture using this strategy. Purification of these secreted fusion proteins from the conditioned serum free medium of S2 cells was accompanied by an efficient one-step immunoaffinity chromatography procedure using the immobilized anti-uPAR monoclonal antibody R2. An optional enterokinase cleavage site is included between the various recombinant proteins and the linker region of the tag, which enables generation of highly pure preparations of tag-free recombinant proteins. Using this system we successfully produced soluble and intact recombinant forms of extracellular proteins such as CD59, C4.4A and vitronectin, as well as a number of truncated domain constructs of these proteins. In conclusion, the present tagging system offers a convenient general method for the robust expression and efficient purification of a variety of recombinant proteins.     

Structure and membrane topology of the high-affinity receptor for human IFN-gamma: requirements for binding IFN-gamma. One single 90-kilodalton IFN-gamma receptor can lead to multiple cross-linked products and isolated proteins

We analyzed the high affinity receptor for IFN-gamma of Raji cells and human placenta by combining Scatchard analysis, cross-linking experiments, and receptor purification. Only one high affinity binding site was found, Kd 2.1 X 10(-10). The receptor is a 90-kDa glycoprotein. However, multiple cross-linked products of 110 kDa to about 250 kDa could be generated and proteins of 90, 70, and 50 kDa could be obtained upon purification. These proteins all contained the same 90-kDa receptor, or part of it. We suggest that extensive cross-linking and/or proteolysis may explain many of the conflicting results published thus far. The extracellular domain of the 90-kDa receptor protein was highly resistant to digestion with trypsin or proteinase K. Trypsin digestion neither affected the number of binding sites per cell, nor the Kd for IFN-gamma. A cluster of sites for different proteases was found in the intracellular domain. The 50-kDa fragment created by trypsin digestion had the same characteristics as the isolated 50-kDa receptor fragment. It contained the IFN-gamma binding site and the receptor's extracellular and amino-terminal domain. N-linked glycosylation contributed about 15 kDa to its molecular mass, of which 4 kDa were attributable to sialic acid residues. O-Linked glycosylation was not detected. The number of binding sites per cell and the Kd for IFN-gamma were not affected by the presence or absence of N-linked glycosylation. The receptor contained at least one critical disulfide bridge and the reduced receptor could be reactivated in vitro.     

Expression of the full-length rabbit prolactin receptor and its specific domains in baculovirus infected insect cells

The prolactin receptor is a membrane protein mainly involved in the development of the mammary gland and in lactation in mammals. We used specific cDNA constructs and the insect/baculovirus expression system and produced independently and in large amounts several recombinant forms of the rabbit mammary gland prolactin receptor: the full-length receptor (L1, L2), a truncated membrane form (S), a secretable form of the extracellular domain (E) and two forms of the intracellular domain (I1, I2). Of these forms, the L1 and L2 are associated with the membrane fraction, the E is predominantly secreted into the medium and the I1 and I2 are expressed as soluble proteins and surprisingly, a great portion accumulates in the culture medium. The molecular mass (94 kDa) of the expressed full-length receptor corresponds to the translation product of the entire cDNA coding region. The receptor biochemically identified in the rabbit mammary gland is however much shorter. Thus, in the mammary gland, the receptor presumably undergoes post-translational modifications. The receptor forms L1, L2 and S bind prolactin with specificity and affinity similar to those reported for the native receptor. They also interact with two monoclonal antibodies, M110 and A917, specific for the native conformation of the hormone-binding site. The I1 and I2 forms do not bind prolactin, whereas the E form does. Thus, the hormone binding site is located in the extracellular domain which can function autonomously as a PRL-binding soluble protein. However, the E form binds prolactin with a higher affinity than the native receptor and it does not bind one of the two antireceptor monoclonal antibodies, known to be hormone binding-site specific. Thus, the conformation of the native receptor and that of the E form differ.     

Soluble fragment of P-cadherin adhesion protein found in human milk

Classical cadherins such as E- and P-cadherin are transmembrane proteins that mediate specific cell-to-cell adhesion and are important to tissue development and function. Cadherin function can be modulated by various means, including proteolytic cleavage of the extracellular adhesion domain from the cells' surface, yielding large soluble fragments termed (soluble) sE- or sP-cadherin. In people with certain carcinomas, sE-cadherin can be detected at elevated levels in the serum and sometimes can serve as a prognostic marker. Soluble E-cadherin also is found in urine of patients with bladder cancer. In addition to being present in bodily fluids of cancer patients, sE- and sP-cadherin are present in the serum of healthy people, suggesting that shedding of cadherins is a normal event. Here, we report high levels of 80 kDa sP-cadherin in human milk. In the lactating mammary gland tissue, P-cadherin appears to be a protein secreted by epithelial cells, rather than an adhesion protein. This is in contrast to the non-lactating mammary gland where P-cadherin is restricted to myoepithelial cells, and is present at sites of cell-cell contact.     

Human melanocortin 1 receptor-mediated ubiquitination of nonvisual arrestins. Role of Mahogunin Ring Finger 1 E3 ligase

Signaling from the melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor (GPCR) crucial for melanocyte proliferation and differentiation, is regulated by cytosolic β-arrestins (ARRBs). MC1R signaling is also negatively modulated by the E3-ubiquitin ligase Mahogunin Ring Finger-1 (MGRN1), whose mutation causes hyperpigmentation, congenital heart defects and neurodegeneration in mice. We showed previously that although MC1R interacts stably with human ARRB1 or ARRB2, only ARRB2 mediates receptor desensitization and internalization. We analyzed MC1R-dependent ARRB ubiquitination, and the possible role of MGRN1. ARRB1 expressed in heterologous cells or human melanoma cells migrated in SDS-PAGE as a 55 kDa protein whereas ARRB2 migrated as two major bands of apparent molecular weight near 45 and 55 kDa, with an intermediate mobility band occasionally detected. These forms were related by post-translational modification rather than by proteolysis. Presence of MC1R favored expression of the 45 kDa protein, the form that interacted preferentially with MC1R. MC1R also mediated poly- or multimonoubiquitination of ARRB2. Ubiquitination was agonist-independent, but required a native MC1R conformation and/or normal receptor trafficking to the plasma membrane, as it was not observed for loss-of-function MC1R variants. In a heterologous expression system, MC1R-dependent ARRB ubiquitination was enhanced by overexpression of MGRN1 and was impaired by siRNA-mediated MGRN1 knockdown thus pointing to MGRN1 as the responsible E3-ligase. Co-immunoprecipitation experiments demonstrated interaction of MGRN1 and ARRBs in the presence of MC1R, suggesting a scaffolding role for the GPCR that may determine the selectivity of E3-ubiquitin ligase engagement and the functional outcome of ARRB ubiquitination.     

One interferon gamma receptor binds one interferon gamma dimer

We investigated the stoichiometry of the interferon gamma and interferon gamma receptor interaction, using recombinant interferon gamma and recombinant soluble interferon gamma receptor, applying chemical cross-linking and chromatographic techniques, and analyzing the resulting products in denaturing polyacrylamide gels. Interferon gamma cross-linked to itself produced a major band of an apparent molecular mass of 34 kDa, which suggests that it exists as a dimer in physiological buffer and which agrees with published data. Soluble interferon gamma receptor cross-linked to itself produced mainly a 28-kDa band, suggesting that the interferon gamma receptor exists as a monomer. Interferon gamma cross-linked to the soluble interferon gamma receptor resulted in the formation of two main products of apparent molecular masses of 60 and 44 kDa. The predominant 60-kDa band resulted from the cross-linking of one interferon gamma dimer (34 kDa) to one interferon gamma receptor molecule (27 kDa). The 44-kDa band was formed by the cross-linking of one interferon gamma molecule to one interferon gamma receptor. Kinetic studies showed that the cross-linking of interferon gamma dimer to the soluble receptor proceeds through the intermediate formed by cross-linking one molecule of the interferon gamma dimer to the receptor. Reducing and dissociating agents inhibited complex formation. When chromatographed on Sephadex G-100, interferon gamma was eluted as a protein of 34-kDa molecular mass, the soluble interferon gamma receptor as a protein of 40 kDa, and their mixture was eluted in one peak corresponding to an apparent molecular mass of 73 kDa. Sodium dodecyl sulfate-polyacrylamide gel analysis of the eluted mixture showed the presence of both interferon gamma and interferon gamma receptor at a ratio of 2:1. The found results suggest that the interferon gamma receptor binds interferon gamma as a dimer.     

N-glycosylation of fibroblast growth factor receptor 1 regulates ligand and heparan sulfate co-receptor binding

The regulation of cell function by fibroblast growth factors (FGF) occurs through a dual receptor system consisting of a receptor-tyrosine kinase, FGFR and the glycosaminoglycan heparan sulfate (HS). Mutations of some potential N-glycosylation sites in human fgfr lead to phenotypes characteristic of receptor overactivation. To establish how N-glycosylation may affect FGFR function, soluble- and membrane-bound recombinant receptors corresponding to the extracellular ligand binding domain of FGFR1-IIIc were produced in Chinese Hamster Ovary cells. Both forms of FGFR1-IIIc were observed to be heavily N-glycosylated and migrated on SDS-PAGE as a series of multiple bands between 50 and 75 kDa, whereas the deglycosylated receptors migrated at 32 kDa, corresponding to the expected molecular weight of the polypeptides. Optical biosensor and quartz crystal microbalance-dissipation binding assays show that the removal of the N-glycans from FGFR1-IIIc caused an increase in the binding of the receptor to FGF-2 and to heparin-derived oligosaccharides, a proxy for cellular HS. This effect is mediated by N-glycosylation reducing the association rate constant of the receptor for FGF-2 and heparin oligosaccharides. N-Glycans were analyzed by mass spectrometry, which demonstrates a predominance of bi- and tri-antennary core-fucosylated complex type structures carrying one, two, and/or three sialic acids. Modeling of such glycan structures on the receptor protein suggests that at least some may be strategically positioned to interfere with interactions of the receptor with FGF ligand and/or the HS co-receptor. Thus, the N-glycans of the receptor represent an additional pathway for the regulation of the activity of FGFs.     

Tumor necrosis factor alpha-induced activation of c-jun N-terminal kinase is mediated by TRAF2.

Tumor necrosis factor alpha (TNF alpha) a pro-inflammatory cytokine is an endogenous mediator of septic shock, inflammation, anti-viral responses and apoptotic cell death. TNF alpha elicits its complex biological responses through the individual or cooperative action of two TNF receptors of mol. wt 55 kDa (TNF-RI) and mol. wt 75 kDa (TNF-RII). To determine signaling events specific for TNF-RII we fused the extracellular domain of the mouse CD4 antigen to the intracellular domain of TNF-RII. Crosslinking of the chimeric receptor using anti-CD4 antibodies initiates exclusively TNF-RII-mediated signals. Our findings show that: (i) TNF-RII is able to activate two members of the MAP kinase family: extracellular regulated kinase (ERK) and c-jun N-terminal kinase (JNK); (ii) TRAF2, a molecule that binds TNF-RII and associates indirectly with TNF-RI, is sufficient to activate JNK upon overexpression; (iii) dominant-negative TRAF2 blocks TNF alpha-mediated JNK activation and (iv) TRAF2 signals the activation of JNK and NF-kappaB through different pathways. Our findings suggest that TNF alpha-mediated JNK activation in fibroblasts is independent of the cell death pathway and that TRAF2 occupies a key role in TNF receptor signaling to JNK.     

Cell surface molecules that bind fibronectin's matrix assembly domain

The assembly of fibronectin into disulfide cross-linked extracellular matrices requires the interaction of mesenchymal cells with two distinct sites on fibronectin, the Arg-Gly-Asp cell adhesive site and an amino-terminal site contained within the first five type I homologous repeats (Quade, B. J., and McDonald, J. A. (1988) J. Biol. Chem. 263, 19602-19609). Proteolytically derived 29-kDa fragments of fibronectin (29kDa) containing these repeats bind to monolayers of cultured fibroblasts and inhibit fibronectin matrix assembly. The cell surface molecules interacting with fibronectin's 29-kDa matrix assembly domain have resisted purification using conventional methods such as affinity chromatography. Accordingly, in order to identify molecules which bind this fragment, 125I-labeled 29kDa was allowed to bind to fibroblast monolayers and chemically cross-linked to the cell surface with bis(sulfosuccinimidyl) suberate. Extraction of the cross-linked cell layer yielded radiolabeled complexes of 56, 150, and 280 kDa. Formation of these cross-linked complexes was specifically inhibited by the addition of excess unlabeled 29kDa but was unaffected by the presence of fibronectin fragments containing other type I repeats outside of the 29kDa matrix assembly domain. The cross-linked complexes were insoluble in nondenaturing detergents but soluble when denatured and reduced, suggesting that 29kDa may be cross-linked to components of the pericellular matrix. Immunoprecipitation of cross-linked cell extracts with a polyclonal antibody to fibronectin that does not recognize the amino terminus demonstrate that the 280-kDa band contains 29kDa cross-linked to fibronectin present on the cell surface. Formation of the 150-kDa complex was inhibited by EDTA, suggesting that divalent cations are required for its formation. Although the molecular mass and divalent cation requirement suggest that the 150-kDa complex may be related to an integrin, this complex was not immunoprecipitated by polyclonal antibodies generated to the alpha 5 beta 1 integrin fibronectin receptor.     

A soluble binding protein specific for interleukin 1 beta is produced by activated mononuclear cells

Soluble interleukin 1 (IL 1) binding proteins were identified by gel filtration and covalent cross-linking of 125I IL 1 in normal human serum and inflammatory exudate. High molecular weight 125I IL 1 protein complexes occurred with both IL 1 alpha and IL 1 beta, however, high molecular weight binding appeared to be non-specific. One specific IL 1 beta binding protein was observed to elute at approximately 100 kDa on gel filtration when bound to 125I IL 1 beta. This complex migrated as a broad band at 60 kDa when covalently cross-linked and analyzed by SDS-PAGE. The protein did not bind 125I IL 1 alpha and 125I IL 1 beta binding was only displaceable by excess cold IL-1 beta. The production of the specific IL 1 beta binding protein was assessed in a number of cell populations. Unstimulated peripheral blood mononuclear cells (PBMNC) did not produce the binding protein, but stimulation with phytohemagglutinin (PHA) caused production within 24 hr and binding protein levels remained elevated for up to 7 days. Stimulation with lipopolysaccharide (LPS) and IL 1 alpha did not consistently induce synthesis of the binding protein. Ligand-binding studies were performed to compare solubilized EL 4 NOB.1 cell membrane IL 1 receptor (sIL 1R) with semi-purified IL 1 beta binding protein from pooled synovial fluid. The sIL 1R preparation bound ligand with an affinity of 168 pM while the IL 1 beta binding protein bound 125I IL 1 beta with an affinity of 370 pM. This protein may function as an important carrier molecule for IL 1 beta and determine its distribution and kinetics in vivo.