Differential associations between the cytoplasmic regions of the interleukin-12 receptor subunits beta1 and beta2 and JAK kinases.

The role of the cytoplasmic regions of interleukin-12 receptors (IL-12R) beta1 and beta2 in stimulating proliferation was examined. The transmembrane and cytoplasmic regions of IL-12Rbeta1 or IL-12Rbeta2 were fused to the extracellular domain of the epidermal growth factor (EGF) receptor, yielding chimeric receptors E12R1 and E12R2, respectively. These chimeras were stably transfected into BaF3 cells, a factor-dependent murine pro-B cell line. Only E12R2 or E12R1+E12R2 transfectants were capable of EGF-dependent proliferation. EGF-dependent phosphorylation of E12R2, JAK2, Tyk2, and STAT3 was observed. JAK2 was phosphorylated in E12R1-, E12R2-, and E12R1+E12R2-expressing cells. However, direct associations were detectable only between E12R2 and JAK2. Tyk2 phosphorylation was observed only in cells expressing E12R1 or E12R1+E12R2. In parallel with this activation pattern, direct interactions only between Tyk2 and E12R1 were demonstrable. Phosphorylation of STAT3 was observed in cells expressing E12R1, E12R2, and E12R1+E12R2. The expression levels of STAT4 protein in BaF3 cells are undetectable by the methods employed here; therefore, STAT4 phosphorylation was not observed. Taken together, the data indicate that differential interactions take place between the cytoplasmic regions of the two IL-12R subunits and JAK2/Tyk2 and that the cytoplasmic region of IL-12Rbeta2 alone is capable of delivering a proliferative signal.

A functional interleukin 12 receptor complex is composed of two beta-type cytokine receptor subunits.

We have identified a cDNA from a human phytohemagglutinin-activated lymphoblast library encoding a protein that binds 125I-labeled human interleukin 12 (125I-huIL-12) with a Kd of about 5 nM when expressed in COS-7 cells. When coexpressed in COS-7 cells with the previously identified IL-12 beta receptor (IL-12R beta) protein, two classes of 125I-huIL-12 binding sites were measured with Kds of about 55 pM and 8 nM, corresponding to the high- and low-affinity binding sites seen on phytohemagglutinin-activated lymphoblasts. This newly identified huIL-12R subunit is a member of the cytokine receptor superfamily, with closest resemblance to the beta-type cytokine receptor gp130 and the receptors for leukemia inhibitory factor and granulocyte colony-stimulating factor. Consequently, we have reclassified the previously identified IL-12R beta subunit as huIL-12R beta 1 and designated the newly identified subunit as huIL-12R beta 2. huIL-12R beta 2 is an 862-amino acid type I transmembrane protein with a 595-amino-acid-long extracellular domain and a cytoplasmic tail of 216 amino acids that contains three tyrosine residues. A cDNA encoding the mouse homolog of the huIL12R beta 2 protein has also been isolated. Human and mouse IL-12R beta 2 proteins show a 68% amino acid sequence identity. When expressed in COS-7 cells, huIL-12R beta 2 exists as a disulfide-linked oligomer with an apparent monomeric molecular weight of 130 kDa. Coexpression of the two identified IL-12R subunits in Ba/F3 cells conferred IL-12 responsiveness, and clones of these cotransfected Ba/F3 cells that grew continuously in the presence of IL-12 were isolated and designated LJM-1 cells. LJM-1 cells exhibited dose-dependent proliferation in response to huIL-12, with an ED50 of about 1 pM huIL-12. Interestingly, Ba/F3 cells transfected with IL-12R beta 2 alone proliferated in response to huIL-12 with an ED50 of about 50 pM, although a role for endogenous mouse IL-12R beta 1 in IL-12 signal transduction in these transfectants cannot be ruled out. These results demonstrate that the functional high-affinity IL-12R is composed of at least two beta-type cytokine receptor subunits, each independently exhibiting a low affinity for IL-12.

Developmental commitment to the Th2 lineage by extinction of IL-12 signaling.

Developmental-commitment to Th1 or Th2 responses critically influences host susceptibility to particular pathogens. We describe a novel mechanism governing stable commitment to Th2 differentiation. Naive T cells develop strongly polarized Th1 and Th2 profiles by 7 days after activation. However, commitment of these developing cells differs substantially. Although IL-4 reverses early Th1 differentiation, IL-12 cannot reverse early Th2 differentiation. Th1 reversibility results from maintenance of IL-4 signal transduction, whereas Th2 commitment results from rapid loss of IL-12 signaling. The IL-12 signaling defect in Th2 cells results in failure to phosphorylate Jak2, Stat3, and Stat4. Since Th2 cells express the mRNA for the cloned murine IL-12 receptor beta subunit, the signaling defect may involve expression or function of unidentified receptor components. The rapid extinction of IL-12 signaling in Th2 cells provides a demonstration of a mechanism for the stable commitment to a T helper phenotype.

Effective eradication of established murine tumors with IL-12 gene therapy using a polycistronic retroviral vector.

Our recent studies using IL-12 protein or fibroblasts genetically engineered to secrete IL-12 have demonstrated profound antitumor effects of IL-12 in murine models. The antitumor effects of local, high level IL-12 expression were examined using a retroviral vector, which can express both IL-12 subunits (p35 and p40) and the neomycin phosphotransferase (Neo)-marker gene from a polycistronic message utilizing internal ribosome entry site sequences. All animals intradermally (i.d.) receiving MCA207 murine sarcoma cell line nontransfected or Neo-transfected had progressively growing tumor, whereas all animals injected with MCA207 transfected with IL-12 were tumor free and were subsequently determined to be immune to a rechallenge of nontransfected MCA207 i.d. Similar results were obtained in experiments using the poorly immunogenic MCA102 murine sarcoma cell line. The inoculation of live MCA207-IL-12 tumor cells also caused the regression of contralateral nontransfected MCA207 inoculated either at the same time (80% protection) or up to 3 days before (33% protection) to the therapeutic tumor inoculation. In vivo depletion studies suggest that NK cells and IFN-gamma play important roles in the development of the early phase of the antitumor response, but that T cells (both CD4+ and CD8+) play the major role in the subsequent events, leading to long-term immunity. The potent antitumor effects observed for paracrine gene-delivered administration of IL-12 have thus been confirmed for multiple tumor cell types and in multiple murine strains. We believe that these results support the feasibility of IL-12 gene therapy for the treatment of human cancer.

Mouse interleukin-12 (IL-12) p40 homodimer: a potent IL-12 antagonist.

Interleukin-12 (IL-12) is a cytokine that has regulatory effects on T and natural killer (NK) cells and is composed of two disulfide-bonded subunits, p40 and p35. It was recently reported that supernatants from cultures of mouse IL-12 (moIL-12) p40-transfected COS cells could inhibit IL-12-dependent responses in vitro (Mattner, F., et al., Eur. J. Immunol. 1993. 23: 2202). We have further characterized the nature of the inhibitory substance. Purified mouse p40 produced in a baculovirus expression system was found to consist of two species: the p40 monomer and a disulfide-linked p40 dimer [(p40)2]. The (p40)2 was 25- to 50-fold more active than the p40 monomer in causing specific, dose-dependent inhibition of IL-12-induced mouse concanavalin A (Con A) blast proliferation and could also inhibit IL-12-induced interferon-gamma (IFN-gamma) secretion by mouse splenocytes and IL-12-dependent activation of mouse NK cells. Competitive binding studies on mouse Con A blasts showed that (p40)2 was equally effective as moIL-12 in competing with 125I-labeled moIL-12 ([125I]moIL-12) for binding to mouse Con A blasts. However, in contrast to moIL-12, mouse (p40)2 displayed little ability to compete with 125I-labeled human IL-12 (huIL-12) for binding to high-affinity IL-12 receptors (IL-12R) on human phytohemagglutinin (PHA) blasts and caused little or no inhibition of huIL-12-induced human PHA blast proliferation. Nonetheless, mouse (p40)2 was equally effective as moIL-12 in competing with [125I] huIL-12 for binding to COS cells transfected with the human IL-12R beta subunit and expressing low-affinity IL-12 binding sites. These results suggest that (i) the majority of the structural determinants required for binding of IL-12 to its receptor are contained within the p40 subunit, but p35 is required for signaling, (ii) the p40 subunit of IL-12 interacts with the beta subunit of IL-12R, and (iii) (p40)2 may be a suitable IL-12 antagonist for studying the role of IL-12 in various immune responses in vivo as well as in vitro. Further studies are required to determine whether or not (p40)2 is produced by normal lymphoid cells and is a physiologic regulator of IL-12 activity.

Interleukin-12: a cytokine with therapeutic potential in oncology and infectious diseases.

IL-12 is a cytokine that promotes cell-mediated immunity by promoting Th1-type cytokine responses, enhancing the lytic activity of NK/LAK cells, augmenting specific CTL responses, and inducing the production of IFN-gamma. On the other hand, IL-12 suppresses the development of Th2-type cytokine responses and humoral immunity, particularly IgGl and IgE responses. It is likely that IL-12 normally plays an important role in the host defense against intracellular microbial pathogens. In addition, the administration of rIL-12 to mice has been shown to have potent therapeutic effects in several tumour and infectious disease models. IL-12 has been shown to be more efficacious than IL-2 in several murine tumour models, and toxicology studies suggest that it may have a substantially better therapeutic index. In addition, the long serum half-life of IL-12 relative to other cytokines will allow more flexibility in dosing schedules. However, future clinical trials are required to determine whether the efficacy of IL-12 seen in these experimental models is predictive for its use as an immunomodulatory drug in humans.

Fibroblasts genetically engineered to secrete interleukin 12 can suppress tumor growth and induce antitumor immunity to a murine melanoma in vivo.

Interleukin 12 (IL-12), a disulfide-linked heterodimeric cytokine produced primarily by macrophages, is composed of light (p35) and heavy (p40) chains. It binds to a receptor on T-cells and natural killer cells, promoting the induction of primarily a TH1 response in vitro and in vivo. To determine whether paracrine IL-12 secretion can alter tumor cell growth or promote antitumor immunity, we have developed a delivery system using genetically engineered fibroblasts in murine tumor models. NIH3T3 cells were stably transfected to express 100-240 units/10(6) cells/48 h of IL-12 using expression plasmids carrying both the murine p35 and p40 genes of murine IL-12. The effects of paracrine secretion of IL-12 on tumor establishment and vaccination models were examined using the poorly immunogenic murine melanoma cell line (BL-6) in C57BL/6 mice. To determine the effects of IL-12 on tumor formation, nonirradiated BL-6 cells were inoculated s.c. into C57BL/6 mice admixed with NIH3T3 cells transfected with both subunits of mIL-12 (3T3-IL-12) or with cells transfected with only the neomycin phosphotransferase gene (3T3-Neo). Compared to mice given injections of BL-6 alone, the day of emergence of detectable tumors was significantly delayed in mice given injections of BL-6 admixed with 3T3-IL-12, but not in mice with BL-6 admixed with 3T3-Neo. Effectiveness in this system was related to the amount of IL-12 expressed by the 3T3-IL-12. To determine the ability of locally secreted IL-12 at the tumor site to induce antitumor immunity, 10(6) irradiated tumor cells mixed with 3T3-IL-12 or 3T3-Neo were injected as a vaccine, and the response to a tumor challenge was subsequently examined. With a tumor challenge of less than 1 x 10(5) nonirradiated BL-6 cells, significant delay of establishment of tumor was noted with a relatively small amount of IL-12 secretion (1.2 units/5 x 10(5) cells/48 h). Larger amounts of secreted IL-12 provided no additional therapeutic benefit. Histological examination of tumor inoculum with 3T3-IL-12 secreting a high level of IL-12 showed peritumoral accumulation of macrophages, a characteristic capsule around the tumor composed of palisades of fibroblasts, and decreased numbers of CD4+ cells in the tumor. These results suggest that local delivery of IL-12 inhibits tumor growth in a dose dependent manner but leads to the development of an antitumor immune response when IL-12 is expressed at the tumor site at the relatively small amount indicated above.(ABSTRACT TRUNCATED AT 400 WORDS)

Two human TNF receptors have similar extracellular, but distinct intracellular, domain sequences.

Tumor necrosis factor (TNF) is a cytokine with a wide range of biological activities in inflammatory and immunologic responses. These activities are mediated by specific cell surface receptors of 55 kDa and 75 kDa apparent molecular masses. A 75-kDa TNF receptor cDNA was isolated using partial amino acid sequence information and the polymerase chain reaction (PCR). When expressed in COS-1 cells, the cDNA transfers specific TNF-binding properties comparable to those of the native receptor. The predicted extracellular region contains four domains with characteristic cysteine residues highly similar to those of the 55-kDa TNF receptor, the nerve growth factor (NGF) receptor, and the CDw40 and OX40 antigens. The consensus sequence of the TNF receptor extracellular domains also has similarity to the cysteine-rich sequence motif LIM. In marked contrast to the extracellular regions, the intracellular domains of the two TNF receptors are entirely unrelated, suggesting different modes of signaling and function.

Two high-affinity interleukin 1 receptors represent separate gene products.

Interleukin 1 (IL-1) is a polypeptide hormone that mediates a broad range of biological activities and interacts with surface receptors on numerous cell types. Equilibrium binding studies have identified a class of IL-1 receptors on T cells, fibroblasts, and epithelial cells that have 2- to 5-fold higher affinity than the receptors on bone marrow cells, pre-B cells, and macrophage cell lines. Affinity cross-linking with human 125I-labeled IL-1 alpha (125I-IL-1 alpha) labels an approximately 100-kDa protein on T cells and fibroblasts and an approximately 80-kDa protein on pre-B cells and macrophage cell lines. Monoclonal and polyclonal antibodies specific for the IL-1 receptor on T cells and fibroblasts block human 125I-IL-1 alpha binding to T cells, fibroblasts, and epithelial cells but cannot block IL-1 binding to bone marrow cells, pre-B cells, and macrophages. These antibodies immunoprecipitate the IL-1 receptor-human 125I-IL-1 alpha complex from T cells and fibroblasts but not from pre-B cells and macrophage cell lines. An S1 nuclease protection assay demonstrated that T cells and fibroblasts contain identical IL-1 receptor mRNA but that pre-B cells and macrophages do not contain this receptor mRNA. Taken together, the data demonstrate that mouse T cells, fibroblasts, and epithelial cells express an identical IL-1 receptor, whereas the IL-1 receptor on pre-B cells, macrophages, and bone marrow cells represents a different gene product.

A sensitive technique to monitor gene transfer and expression in bone marrow stem cells.

The polymerase chain reaction technique (PCR), a primer-mediated enzymatic amplification of specific target sequences, was used to monitor gene transfer into hematopoietic progenitor stem cells. A gene coding for human interleukin 1 alpha (IL-1 alpha) was cotransfected with the rous sarcoma virus (RSV) CAT plasmid into mouse bone marrow cells by electroporation. Individual chloramphenicol (CAM)-resistant bone marrow progenitor colonies (granulocyte-macrophage colony-forming units; CFU-GM) containing 50-100 cells were analyzed by PCR for the presence and expression of IL-1 alpha and CAT sequences. Amplified IL-1 alpha DNA sequences were detected from a 50-cell CFU-GM colony. CAT and IL-1 alpha RNA expression was demonstrated from the CAM-resistant CFU-GM colonies.

Interleukin-1-independent activation of human T lymphocytes stimulated by anti-CD3 and a Hodgkin's disease cell line with accessory cell activity.

Antibodies directed against the human T cell receptor or the closely associated CD3 molecule stimulate polyclonal T cell proliferation via mechanisms that mimic a primary immune response. We have investigated the requirement for IL-1 production in anti-CD3 (OKT3)-mediated mitogenesis using a Hodgkin's disease cell line (L428) as the accessory cell. L428 cells did not produce detectable IL-1 following stimulation with lipopolysaccharide or phorbol ester (PMA), nor did they transcribe detectable levels of mRNA for IL-1 alpha or beta after such treatment. Despite their inability to produce IL-1, as few as 1 X 10(4) L428 cells reconstituted the proliferative response of accessory cell-depleted T cells to anti-CD3. Although larger numbers of non-rosette-forming (E-) cells were required for maximal responsiveness to anti-CD3, the maximal degree of proliferation was higher with E- cells than with L428 cells. L428-mediated T cell proliferation did not result from residual accessory cells in the responding population or an allogeneic effect since L428 cells were also capable of providing accessory cell activity for the anti-CD3-dependent generation of IL-2 by the Jurkat T cell line. Although the mechanism by which L428 cells provide accessory functions remains incompletely characterized, the ability of anti-HLA-DR F(ab')2 fragments to completely abrogate L428 and monocyte-mediated anti-CD3 mitogenesis, despite the addition of exogenous IL-1, provides evidence for the participation HLA-DR molecules in this response. These data indicate that anti-CD3-induced proliferation of unprimed human T lymphocytes can occur independently of IL-1 production by accessory cells and may involve the participation of HLA-DR molecules.

Expression of porcine cholecystokinin cDNA in a murine neuroendocrine cell line. Proteolytic processing, sulfation, and regulated secretion of cholecystokinin peptides.

The cDNA for porcine preprocholecystokinin (pre-pro-CCK) was engineered for expression in mammalian cells under the control of the Rous sarcoma virus-long terminal repeat promoter. This expression construct was transfected into the murine anterior pituitary cell line, AtT-20. A stable cell line (AtT-20/CCK) was derived that expresses CCK mRNA indistinguishable from the CCK mRNA found in pig brain or gut. The AtT-20/CCK cells carry out proteolytic processing and sulfation reactions to generate authentic sulfated CCK8 from pro-CCK. The cells also store and secrete CCK-immunoreactive peptides. This secretion can be stimulated with corticotropin releasing factor, the natural secretagogue for anterior pituitary cells. In contrast, monkey kidney epithelial cells (COS cells), which are transiently transfected to express CCK, predominantly secrete nonsulfated pro-CCK into the medium. These studies show that a murine neuroendocrine cell line contains the complete processing machinery required to generate authentic porcine CCK8. The processing events include simultaneous proteolytic processing at one and two basic amino acid sites and sulfation of tyrosine residues. The cell line thus duplicates exactly the processing patterns found to occur in pig brain cortex.

Interleukin 1 gene expression in adult T cell leukemia.

The adult T cell leukemia (ATL) is a T cell neoplasm etiologically associated with human T lymphotropic virus type I (HTLV-I) infection. ATL cells often abnormally express interleukin 2 (IL-2) receptors, and ATL patients may show clinical evidence of hypercalcemia, osteolytic bone lesions, or increased bone turnover. Whereas interleukin 1 (IL-1) is not generally recognized as a product of T cells, this cytokine is capable of both altering IL-2 receptor expression and activating osteoclasts. Thus, we investigated the possibility that primary ATL leukemic T cells and HTLV-I-infected long-term ATL cell lines produce IL-1. S1 nuclease protection assays demonstrated that primary leukemic ATL cells from five out of six patients, as well as one patient with T4+ chronic lymphocytic leukemia, contained considerable quantities of IL-1 beta messenger RNA (mRNA) and small amounts of IL-1 alpha mRNA. These primary leukemic T cells also released biologically active IL-1 protein as evaluated in the murine thymocyte comitogenesis bioassay. In contrast to primary tumor cells, four out of six long-term ATL cell lines produced variable amounts of IL-1 alpha mRNA in the absence of detectable IL-1 beta mRNA as measured by S1 nuclease protection. These data demonstrate that IL-1 gene (especially IL-1 beta) expression occurs in many primary HTLV-I-infected leukemic T cells raising the possibility that this mediator may play a role in the pathological changes associated with this leukemia. Also, these studies show that the pattern of IL-1 alpha and IL-1 beta gene expression differs between primary ATL tumor cells and long-term cultured ATL cell lines, indicating an interesting biological difference in these two HTLV-I-infected cell populations.

Interleukin 1 gene expression in cultured human keratinocytes is augmented by ultraviolet irradiation.

Interleukin 1 (IL-1) is a family of polypeptides initially found to be produced by activated monocytes and macrophages that mediate a wide variety of cellular responses to injury and infection. Epidermal epithelial cells (keratinocytes) produce "epidermal cell-derived thymocyte activating factor" or ETAF, which has been recently shown to be identical to IL-1. Human epidermis is normally exposed to significant amounts of solar ultraviolet radiation. Certain ultraviolet wavelengths (UVB, 290-320 nm) are thought to be responsible for most of the immediate and long-term pathological consequences of excessive exposure to sunlight. In this study, we asked whether exposure to UVB irradiation induced IL-1 gene expression in cultured human keratinocytes. Cultured human keratinocytes contain detectable amounts of IL-1 alpha and beta mRNA and protein in the absence of apparent stimulation; these levels could be significantly enhanced 6 h after exposure to 10 ng/ml of 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Exposure to UVB irradiation with an emission spectrum comparable to that of sunlight (as opposed to that of an unfiltered artificial UV light source) significantly increased the steady state levels IL-1 alpha and beta mRNA in identical populations of human keratinocytes. This was reflected in the production of increased IL-1 activity by these cultures in vitro. In the same cell population, exposures to UVB irradiation did not alter the level of actin mRNA; therefore, the effect of UV irradiation on IL-1 represents a specific enhancement of IL-1 gene expression. Local increases of IL-1 may mediate the inflammation and vasodilation characteristic of acute UVB-injured skin, and systemic release of this epidermal IL-1 may account for fever, leukocytosis, and the acute phase response seen after excessive sun exposure.

Induction of membrane-associated interleukin 1 by tumor necrosis factor in human fibroblasts.

Highly purified recombinant human tumor necrosis factor (TNF) was found to induce interleukin 1 (IL 1) production in diploid human FS-4 fibroblasts. Demonstration of IL 1 activity was based on the ability of TNF-treated FS-4 cells, subsequently fixed with formaldehyde, to stimulate thymocyte proliferation in the presence of phytohemagglutinin. Incubation of FS-4 cells with the optimal dose of TNF (10 ng/ml) resulted in a marked increase in [3H] thymidine uptake by thymocytes co-cultured with formaldehyde-fixed FS-4 cells. Induction of this apparently membrane-associated IL 1 (MA-IL 1) activity was demonstrable at 6 hr and reached a plateau after 48 hr of incubation with TNF. FS-4 cells did not secrete soluble IL 1 in response to TNF. Dexamethasone suppressed the synthesis of TNF-induced MA-IL 1. A monoclonal antibody specific for TNF neutralized MA-IL 1 induction, indicating that the induction is due to TNF, and not to a contaminant in the TNF preparation. The ability of TNF to induce IL 1 synthesis in FS-4 fibroblasts at the transcriptional level was confirmed by S1 nuclease protection assay. Cytoplasmic RNA from uninduced FS-4 cells contained no demonstrable RNA hybridizing with a human IL 1-alpha cDNA probe and low levels of RNA hybridizing with an IL 1-beta cDNA. Induction with TNF resulted in the appearance of IL 1-alpha mRNA and a very significant increase in IL 1-beta mRNA, indicating that TNF induces the synthesis of both IL 1-alpha and IL 1-beta in FS-4 cells.

Human keratinocytes contain mRNA indistinguishable from monocyte interleukin 1 alpha and beta mRNA. Keratinocyte epidermal cell-derived thymocyte-activating factor is identical to interleukin 1.

Keratinocytes produce an IL-1 like factor termed epidermal cell-derived thymocyte-activating factor (ETAF). In this study, we show that ETAF and IL-1 are identical by the following criteria: Both normal and malignant human keratinocytes contain mRNAs identical to monocytic IL-1 alpha and IL-1 beta mRNA, as determined by an S1 nuclease protection assay; and IL-1 activity in medium conditioned by these cells can be neutralized by antibodies specific for human IL-1. The IL-1 alpha and IL-1 beta mRNAs can be identified in cultured human keratinocytes in the absence of identifiable stimulation; this basal level of mRNA can be further induced to accumulate with certain defined stimuli. Cultured normal human keratinocytes (HFKs) contain 2-4 times more IL-1 alpha than IL-1 beta mRNA; in contrast, human peripheral blood monocytes contain 10-20 times more IL-1 beta than IL-1 alpha mRNA. The IL-1 activity released by these HFK can be neutralized by an antibody that neutralizes both alpha and beta IL-1, but not by an antibody that neutralizes only IL-1 beta. While human monocytes produce a large excess of IL-1 beta after appropriate stimulation, these data suggest that IL-1 alpha is a major (and may be the predominant) form of IL-1 produced by human keratinocytes.

Nucleotide sequence of rat preputial gland beta-glucuronidase cDNA and in vitro insertion of its encoded polypeptide into microsomal membranes.

We have selected the rat preputial gland beta-glucuronidase as a model protein to study the sorting of newly synthesized lysosomal hydrolases to the lysosome. The complete coding sequence of beta-glucuronidase messenger RNA was determined from the sequences of a group of overlapping cDNA clones isolated from preputial gland cDNA libraries. The beta-glucuronidase mRNA primary translation product contains 648 amino acids, including an amino-terminal signal sequence of 22 residues. The polypeptide has four potential sites for the addition of asparagine-linked core oligosaccharides. A 376-residue segment of beta-glucuronidase shows extensive homology (23% sequence identity) to a portion of Escherichia coli beta-galactosidase. This homology most likely reflects an evolutionary relationship between the bacterial and eukaryotic enzymes and the conservation of structural features necessary for the glycosidase activity of both proteins. Translation of mRNA synthesized in vitro by transcription of a cDNA containing the entire beta-glucuronidase coding region yielded a polypeptide that was immunoprecipitated with anti-beta-glucuronidase antiserum and had the same electrophoretic mobility as the primary translation product of natural beta-glucuronidase mRNA. In the presence of microsomal membranes, the in vitro-synthesized beta-glucuronidase underwent cotranslational incorporation into the microsomes, as indicated by removal of the signal sequence and the addition of several oligosaccharide chains. The beta-glucuronidase cDNA will provide a useful tool to study the mechanism of mannose phosphorylation and other aspects of the sorting of lysosomal enzymes to lysosomes.

Interleukin 1 alpha and interleukin 1 beta bind to the same receptor on T cells.

Pure, E. coli-derived recombinant murine interleukin 1 alpha (IL 1 alpha) was labeled with 125I and used for receptor binding studies. The 125I-IL 1 binds to murine EL-4 thymoma cells in a specific and saturable manner. Scatchard plot analysis for binding studies carried out at 4 degrees C reveals a single type of high affinity binding site with an apparent dissociation constant of approximately 2.6 X 10(-10) M and the presence of approximately 1200 binding sites per cell. The rate of association of the 125I-IL 1 with EL-4 cells is slow, requiring more than 3 h to reach apparent steady state at 4 degrees C. Cell-bound 125I-IL 1 cannot be dissociated from EL-4 cells upon removal of unbound 125I-IL 1 and incubation of the cells at 4 degrees C in the presence or absence of unlabeled IL 1. Unlabeled recombinant murine IL 1 competes for 125I-IL 1 binding in a dose-dependent manner, whereas interferon-alpha A, interleukin 2 (IL 2), epidermal growth factor, and nerve growth factor have no effect. The 125I-IL 1 binding site is sensitive to trypsin, suggesting that it is localized on the cell surface. We have also examined the ability of purified recombinant human IL 1 alpha and IL 1 beta to compete for binding of the radiolabeled murine IL 1 to its receptor and to stimulate IL 2 production by EL-4 cells. Previous reports have shown that human IL 1 alpha is approximately 60% homologous in amino acid sequence with murine IL 1, but that human IL 1 beta is only about 25% homologous with either murine IL 1 or human IL 1 alpha. Despite these marked differences, however, we report here that both human IL 1 proteins are able to recognize the same binding site as mouse IL 1. In addition, murine as well as both human IL 1 proteins stimulate IL 2 production by EL-4 cells.