Novel non-isotopic method for the localization of receptors in tissue sections.

We describe a novel fluorescent method for the detection of receptors for chimeric proteins in tissue sections. The technique was developed using a recombinant human insulin-like growth factor (IGF-1) chimera, bearing six additional histidine residues at the carboxy-terminal end (IGF-1-His). We demonstrated that dehydration of the tissue sections was detrimental for binding and that its prevention dramatically increased sensitivity. The specificity of IGF-1-His interaction was shown by gradual abolition of the fluorescent signal in the presence of increasing concentrations of IGF-1. Combining immunofluorescence with in situ ligand binding, we showed that IGF-1-His binding corresponded to the IGF-1 receptor (IGFR-1) distribution in human fetal kidney. Moreover, incubation of the tissue sections with an anti-IGFR-1 blocking antibody abolished IGF-1-His binding, demonstrating that the interaction was mediated by the IGFR-1. The method was also used to localize the IGFR-1 in E18 rat embryo sagittal sections. The IGF-1-His binding pattern was observed in brain, cartilage, lung, skin, heart, diaphragm, and tongue, and paralleled the previously reported IGFR-1 distribution. We believe that this new non-isotopic in situ ligand binding method will facilitate rapid and accurate localization of receptors in tissue sections.

Nonclinical studies addressing the mechanism of action of trastuzumab (Herceptin).

HER2 is a ligand-less member of the human epidermal growth factor receptor or ErbB family of tyrosine kinases. In normal biological systems, HER2 functions as a co-receptor for a multitude of epidermal growth factor-like ligands that bind and activate other HER family members. HER2 overexpression is observed in a number of human adenocarcinomas and results in constitutive HER2 activation. Specific targeting of these tumors can be accomplished with antibodies directed against the extracellular domain of the HER2 protein. One of these antibodies, 4D5, has been fully humanized and is termed trastuzumab (Herceptin; Genentech, San Francisco, CA). Treatment of HER2-overexpressing breast cancer cell lines with trastuzumab results in induction of p27KIP1 and the Rb-related protein, p130, which in turn significantly reduces the number of cells undergoing S-phase. A number of other phenotypic changes are observed in vitro as a consequence of trastuzumab binding to HER2-overexpressing cells. These phenotypic changes include downmodulation of the HER2 receptor, inhibition of tumor cell growth, reversed cytokine resistance, restored E-cadherin expression levels, and reduced vascular endothelial growth factor production. Interaction of trastuzumab with the human immune system via its human immunoglobulin G1 Fc domain may potentiate its antitumor activities. In vitro studies demonstrate that trastuzumab is very effective in mediating antibody-dependent cell-mediated cytotoxicity against HER2-overexpressing tumor targets. Trastuzumab treatment of mouse xenograft models results in marked suppression of tumor growth. When given in combination with standard cytotoxic chemotherapeutic agents, trastuzumab treatment generally results in statistically superior antitumor efficacy compared with either agent given alone. Taken together, these studies suggest that the mechanism of action of trastuzumab includes antagonizing the constitutive growth-signaling properties of the HER2 system, enlisting immune cells to attack and kill the tumor target, and augmenting chemotherapy-induced cytotoxicity.

Vaccination with the extracellular domain of p185neu prevents mammary tumor development in neu transgenic mice.

The HER2/neu oncogene product, p185(HER2/neu), is overexpressed on the surface of many human breast cancers. Strains of transgenic mice have been developed that express the rat neu oncogene in mammary epithelial cells and develop spontaneous mammary tumors that overexpress p185neu. This model provides an ideal system for testing interventions to prevent tumor development. In this study, we immunized neu-transgenic mice with a vaccine consisting of the extracellular domain of p185neu (NeuECD). Immunized mice developed Neu-specific humoral immune responses, as measured by circulating anti-Neu antibodies in their sera, and cellular immune responses, as measured by lymphocyte proliferation to NeuECD in vitro. In addition, the subsequent development of mammary tumors was significantly lower in immunized mice than in controls and vaccine treatment was associated with a significant increase in median survival.

Mutational analysis of thrombopoietin for identification of receptor and neutralizing antibody sites.

Thrombopoietin (TPO) is a hematopoietin important for megakaryocyte proliferation and production of blood platelets. We sought to characterize how TPO binds and activates its receptor, myeloproliferative leukemia virus receptor. The erythropoietin-like domain of TPO (TPO1-153) has been fused to the gIII coat protein of M13 bacteriophage. Forty residues were chosen for mutation to alanine using the criteria that they were charged residues or predicted to be solvent-exposed, based on a homology model. Phage enzyme-linked immunosorbent assay was used to determine affinities for binding to both the TPO receptor and five anti-TPO1-153 monoclonal antibodies. Mutations at mostly positively charged residues (Asp8, Lys14, Lys52, Lys59, Lys136, Lys138, Arg140) caused the greatest reduction in receptor-binding affinity. Most of these residues mapped to helices-1 and -4 and a loop region between helix-1 and helix-2. Two of the monoclonal antibodies that blocked TPO binding and bioactivity had determinants in helix-4. In contrast, the other three monoclonal antibodies, which were effective at blocking TPO activity but did not block initial binding of TPO to its receptor, had epitopes predominantly on helix or 3. These results suggest that TPO has two distinct receptor-binding sites that function to dimerize TPO receptors in a sequential fashion.

Growth regulation of human breast and ovarian tumor cells by heregulin: Evidence for the requirement of ErbB2 as a critical component in mediating heregulin responsiveness.

Alterations in the expression of the epidermal growth factor (EGF) receptor ErbB family are frequently encountered in a number of human cancers. Two of these receptors, ErbB3 and ErbB4, are known to bind a family of related proteins termed heregulins (HRGs) or neu differentiation factors. In biologically relevant systems, interaction of HRG with ErbB3 or ErbB4 results in the transactivation of ErbB2. In this report, we show that ErbB2 is a critical component in mediating HRG responsiveness in a panel of human breast and ovarian tumor cell lines. Because HRGs have been reported to elicit diverse biological effects on cultured cells, including growth stimulation, growth inhibition, and induction of differentiation, we systematically examined the effect of rHRG beta 1 on tumor cell proliferation. HRG binding studies were performed with a panel of breast and ovarian tumor cell lines expressing a range of levels of ErbB2. The biological responses to HRG were also compared to EGF and to the growth-inhibitory anti-ErbB2 antibody, 4D5. In most cases, HRG stimulation of DNA synthesis correlated with positive effects on cell cycle progression and cell number and with enhancement of colony formation in soft agar. On each cell line tested, the HRG effects were distinguishable from EGF and 4D5. Our findings indicate that HRG induces cell proliferation in a number of tumor cell lines. In addition, we show that methods for measuring cell proliferation, as well as experimental conditions, are critical for determining HRGs effect on tumor cell growth in vitro.

Development and characterization of murine monoclonal antibodies to the latency-associated peptide of transforming growth factor beta 1.

Transforming growth factor beta (TGF-beta) is a multifunctional peptide that controls proliferation, differentiation, and other functions in a variety of cell types. Transforming growth factor beta activities have been implicated in a variety of diseased states including arthritis, prostate cancer, and AIDS, and in the repair of tissue injury caused by trauma, burns, and surgery. We describe the development and characterization of novel murine monoclonal antibodies (MAbs) to the latency-associated peptide (LAP) of TGF-beta 1, and the subsequent development of an ELISA for the detection and quantitation of TGF-beta 1-LAP in buffer and serum matrices. Fusion of immune splenocytes with myeloma cells yielded 576 hybridomas, 110 of which were antibody secreting. Five were selected for extensive characterization. Clinically, the MAbs described here should be valuable for studying potentially abnormal production and/or function of the LAP, and its relationship to TGF-beta.

Natriuretic peptide receptor-B (guanylyl cyclase-B) mediates C-type natriuretic peptide relaxation of precontracted rat aorta.

The most potent known agonist for the natriuretic peptide receptor-B (NPR-B)/guanylyl cyclase-B is C-type natriuretic peptide (CNP). A homologous ligand-receptor system consists of atrial natriuretic peptide (ANP) and NPR-A/guanylyl cyclase-A. A third member of this family is NPR-C, a non-guanylyl cyclase receptor. Monoclonal antibodies were raised against NPR-B by immunizing mice with a purified receptor-IgG fusion protein consisting of the extracellular domain of NPR-B and the Fc portion of human IgG-gamma 1. One monoclonal antibody, 3G12, did not recognize NPR-A or NPR-C and bound to human and rat NPR-B. CNP binding to NPR-B and stimulation of cGMP synthesis were inhibited by 3G12. With cells isolated from either the media or adventitia layers of rat thoracic aorta, 3G12 did not interfere with ANP-stimulated cGMP synthesis, but it inhibited CNP-stimulated cGMP levels in cells from both layers. CNP (IC50 = 10 nM) and ANP (IC50 = 1 nM) caused relaxation of phenylephrine-contracted rat aortic rings. 3G12 caused a marked increase in the IC50 for CNP, from 10 nM to 140 nM, but failed to affect ANP-mediated relaxation. Therefore, our results for the first time demonstrate that CNP relaxes vascular smooth muscle by virtue of its binding to NPR-B.

Activation of TNF-R1 receptor in the presence of copper kills TNF resistant CEM leukemic T cells.

The cytotoxic effects of TNF on malignant cells are known to be mediated through high affinity surface receptors. The precise mechanism by which transformed cells are selectively killed by the activation of these receptors is yet unknown, but several intracellular signaling pathways are known to be involved. Phospholipase A2 activation by TNF-alpha has been shown to be important in the transduction of signals leading to cell death. We have used monitoring of extracellular acidification rate as a measure of cellular metabolism to follow the early time course of TNF effects on a human leukemic T cell line (CEM-SS cells). CEM-SS cells were relatively resistant to TNF cell killing but TNF caused an early stimulation of metabolism within 2-4 hr, followed by a suppression of metabolic activity occurring over 20 hr. In contrast, a TNF sensitive subclone of CEM cells (C1Ca) showed a rapid and dramatic decrease in metabolic activity corresponding to cytotoxicity within 18 hr. It was discovered that cupric o-phenanthroline markedly potentiated the effects of TNF on the resistant CEM-SS cells leading to cell death. This observation was specific for copper because ferric o-phenanthroline was without effect at the same concentration. The copper cytotoxic effect was shown to be mediated through the TNF-R1 receptor and independent of phospholipase A2 signaling.

Antibody to HER-2/neu receptor blocks DNA repair after cisplatin in human breast and ovarian cancer cells.

Approximately 30% of human breast and ovarian cancers have amplification and/or overexpression of HER-2/neu gene which encodes a cell surface growth-factor receptor. Overexpression of this receptor, p185HER-2/neu, is associated with poor outcome and may predict clinical response to chemotherapy. Antibodies to HER-2/neu receptor have a cytostatic effect in suppressing growth of cells with overexpression of p185HER-2/neu. To elicit a cytocidal effect, therapy with antireceptor antibody was used in combination with the DNA-damaging drug, cisplatin, and this combined treatment produced a synergistic decrease in cell growth. In addition, antibody mediated an increased sensitivity to cisplatin in drug-resistant ovarian carcinoma cells containing multiple copies of HER-2/neu gene. To evaluate the mechanism for this synergy, unscheduled DNA synthesis was measured in cancer cells using incorporation of [3H]thymidine and autoradiography, and formation and repair of cisplatin-induced DNA adducts was also measured. Treatment with cisplatin led to a marked, dose-dependent increase in unscheduled DNA synthesis which was significantly reduced by combined treatment with antireceptor antibody in HER-2/neu-overexpressing cells. Therapy with antibody to HER-2/neu receptor also led to a 35-40% reduction in repair of cisplatin-DNA adducts after cisplatin exposure and, as a result, promoted drug-induced killing in target cells. This phenomenon which we term receptor-enhanced chemosensitivity may provide a rationale for more selective targeting and exploitation of overexpressed growth factor receptors in cancer cells, thus leading to new strategies for clinical intervention.

Coexpression of erbB2 and erbB3 proteins reconstitutes a high affinity receptor for heregulin.

The heregulin/neu differentiation factor gene products were purified and cloned based on their ability to stimulate the phosphorylation of a 185-kDa protein in human breast carcinoma cell lines known to express erbB2. However, not all cells that express erbB2 respond to heregulin, indicating that other components besides erbB2 may be required for heregulin binding. Cells that are transfected with the closely related receptor, erbB3, display a single class of lower affinity heregulin binding sites than has been previously observed on breast carcinoma cell lines. Little or no stimulation of tyrosine phosphorylation in response to heregulin occurs in cells that are transfected with erbB3 alone. Transfection of cells with erbB3 and erbB2 reconstitutes a higher affinity binding receptor, which is also capable of generating a tyrosine phosphorylation signal in response to heregulin. A monoclonal antibody to erbB2 will inhibit heregulin activation of tyrosine phosphorylation and binding in cells transfected with both receptors but not with erbB3 alone. In cells expressing erbB2 and erbB3, both proteins become tyrosine-phosphorylated upon interaction with heregulin. Direct interaction between heregulin and the two proteins was demonstrated by chemical cross-linking experiments using 125I-heregulin followed by immunoprecipitation with antibodies specific for erbB2 or erbB3.

Anti-transforming growth factor (TGF)-beta antibodies inhibit breast cancer cell tumorigenicity and increase mouse spleen natural killer cell activity. Implications for a possible role of tumor cell/host TGF-beta interactions in human breast cancer progression.

TGF-beta effects on angiogenesis, stroma formation, and immune function suggest its possible involvement in tumor progression. This hypothesis was tested using the 2G7 IgG2b, which neutralizes TGF-beta 1, -beta 2, and -beta 3, and the MDA-231 human breast cancer cell line. Inoculation of these cells in athymic mice decreases mouse spleen natural killer (NK) cell activity. Intraperitoneal injections of 2G7 starting 1 d after intraperitoneal inoculation of tumor cells suppressed intraabdominal tumor and lung metastases, whereas the nonneutralizing anti-TGF-beta 12H5 IgG2a had no effect. 2G7 transiently inhibited growth of established MDA-231 subcutaneous tumors. Histologically, both 2G7-treated and control tumors were identical. Intraperitoneal administration of 2G7 resulted in a marked increase in mouse spleen NK cell activity. 2G7 did not inhibit MDA-231 primary tumor or metastases formation, nor did it stimulate NK cell-mediated cytotoxicity in beige NK-deficient nude mice. Finally, serum-free conditioned medium from MDA-231 cells inhibited the NK cell activity of human blood lymphocytes. This inhibition was blocked by the neutralizing anti-TGF-beta 2G7 antibody but not by a nonspecific IgG2. These data support a possible role for tumor cell TGF-beta in the progression of mammary carcinomas by suppressing host immune surveillance.

Stimulation of human T-cell proliferation by specific activation of the 75-kDa tumor necrosis factor receptor.

TNF-alpha can enhance the proliferation of human thymocytes stimulated by the comitogen Con A. To determine which of the two different TNF receptors is responsible for signaling this cellular response, we investigated the proliferation of human thymocytes in response to agonistic antibodies specific for the two TNF receptor types. In contrast to previously examined TNF activities in human cells, thymocyte proliferation was stimulated in response to rabbit polyclonal antibodies directed against the 75-kDa TNF receptor (TNF-R2), but not those directed against the 55-kDa TNF receptor (TNF-R1). Lymphotoxin (TNF-beta) was also shown to stimulate human thymocyte proliferation, demonstrating that TNF-beta can initiate a biologic response that is mediated by TNF-R2. TNF-R2-mediated T-cell proliferation was not restricted to the immature T cells within the thymus, as the anti-TNF-R2 antibodies also stimulated the proliferation of peripheral T cells. As a first step toward identifying a specific agonist of TNF-R2 with therapeutic potential, 10 anti-TNF-R2 mAb were examined for potential agonist activity. Nine of these significantly stimulated human thymocyte proliferation with maximal responses ranging from twofold to significantly greater than that obtained with TNF-alpha by itself.

Strain specificity and binding affinity requirements of neutralizing monoclonal antibodies to the C4 domain of gp120 from human immunodeficiency virus type 1.

The binding properties of seven CD4-blocking monoclonal antibodies raised against recombinant gp120 of human immunodeficiency virus type 1 strain MN (HIV-1MN) and two CD4-blocking monoclonal antibodies to recombinant envelope glycoproteins gp120 and gp160 of substrain IIIB of HIVLAI were analyzed. With a panel of recombinant gp120s from seven diverse HIV-1 isolates, eight of the nine antibodies were found to be strain specific and one was broadly cross-reactive. Epitope mapping revealed that all nine antibodies bound to epitopes located in the fourth conserved domain (C4) of gp120. Within this region, three distinct epitopes could be identified: two were polymorphic between HIV-1 strains, and one was highly conserved. Studies with synthetic peptides demonstrated that the conserved epitope, recognized by antibody 13H8, was located between residues 431 and 439. Site-directed mutagenesis of gp120 demonstrated that residue 429 and/or 432 was critical for the binding of the seven antibodies to gp120 from HIV-1MN. Similarly, residues 423 and 429 were essential for the binding of monoclonal antibody 5C2 raised against gp120 from HIV-1IIIB. The amino acids located at positions 423 and 429 were found to vary between strains of HIV-1 as well as between molecular clones derived from the MN and LAI isolates of HIV-1. Polymorphism at these positions prevented the binding of virus-neutralizing monoclonal antibodies and raised the possibility that HIV-1 neutralization serotypes may be defined on the basis of C4 domain sequences. Analysis of the binding characteristics of the CD4-blocking antibodies demonstrated that their virus-neutralizing activity was directly proportional to their gp120-binding affinity. These studies account for the strain specificity of antibodies to the C4 domain of gp120 and demonstrate for the first time that antibodies to this region can be as effective as those directed to the principal neutralizing determinant (V3 domain) in neutralizing HIV-1 infectivity.

Humanization of an antibody directed against IgE.

IgE antibodies bind to specific high-affinity receptors on mast cells, leading to mast cell degranulation and release of mediators, such as histamine, which produce symptoms associated with allergy. Hence, anti-IgE antibodies that block binding of IgE to its high-affinity receptor are of potential therapeutic value in the treatment of allergy. These antibodies must also not bind to IgE once it is bound to the receptor because this would trigger histamine release. This study describes the humanization of a murine antibody, MaE11, with these characteristics. Variants of the humanized antibody were evaluated to probe the importance of framework residues on antibody binding and to determine which charged residues in the CDR interacted with IgE. We found that only five changes in human framework residues were required to provide for binding comparable to that of the original murine antibody.

Development of a specific and sensitive two-site enzyme-linked immunosorbent assay for measurement of inhibin-A in serum.

A polyclonal chicken antiserum against purified 32-kilodalton (kDa) recombinant inhibin-A (rh-InhA) and two monoclonal antibodies (mAb) against either rh-InhA (11B5) or 28-kDa recombinant activin-A (rh-ActA; 9A9) were used to develop three sensitive InhA enzyme-linked immunosorbent assays (ELISAs). The sensitivity of an ELISA using affinity-purified chicken anti-rh-InhA (Ck) for both coat and capture (Ck/Ck) averaged 78 +/- 3 pg/ml, while the mAb/Ck ELISAs (11B5/Ck or 9A9/Ck) averaged 100 +/- 6 pg/ml in a 10% serum matrix, with intra-and interassay coefficients of variation of 2-5% and 8-10%, respectively, for all assays. The ELISA formats did not cross-react with purified rh-ActA or recombinant human transforming growth factor-beta 1 or detect any immunoreactive proteins in medium conditioned by cell lines expressing rh-ActA or recombinant human transforming growth factor-beta 1. The Ck/Ck ELISA detected significant amounts of immunoreactivity in medium from cells expressing the free alpha-subunit of inhibin and recombinant inhibin-B (rh-InhB). In contrast, the mAb/Ck ELISAs showed no cross-reactivity to medium conditioned by these two cell lines. All three ELISA formats detected rh-InhA added to either human or rat serum in vitro or serum from rats injected with rhInhA. The Ck/Ck and 9A9/Ck ELISAs successfully quantitated inhibin in sera from patients undergoing ovulation induction and in rats (with or without sc administration of pregnant female serum gonadotropin). The 11B5/Ck ELISA appeared to be specific for the 32-kDa form of inhibin, while the 9A9/Ck ELISA was useful in quantitating inhibin-A in biological fluids, with little cross-reactivity to free alpha-chain or inhibin-B.

A truncated intracellular HER2/neu receptor produced by alternative RNA processing affects growth of human carcinoma cells.

Cloned sequences encoding a truncated form of the HER2 receptor were obtained from cDNA libraries derived from two HER2-overexpressing human breast cancer cell lines, BT-474 and SK-BR-3. The 5' 2.1 kb of the encoded transcript is identical to that of full-length 4.6-kb HER2 transcript and would be expected to produce a secreted form of HER2 receptor containing only the extracellular ligand binding domain (ECD). The 3' end of the truncated transcript diverges 61 nucleotides before the receptor's transmembrane region, reads through a consensus splice donor site containing an in-frame stop codon, and contains a poly(A) addition site, suggesting that the truncated transcript arises by alternative RNA processing. S1 nuclease protection assays show a 40-fold variation in the abundance of the truncated 2.3-kb transcript relative to full-length 4.6-kb transcript in a panel of eight HER2-expressing tumor cell lines of gastric, ovarian, and breast cancer origin. Expression of this truncated transcript in COS-1 cells produces both secreted and intracellular forms of HER2 ECD; however, immunofluorescent labeling of HER2 ECD protein in MKN7 tumor cells that natively overexpress the 2.3-kb transcript suggests that transcriptionally generated HER2 ECD is concentrated within the perinuclear cytoplasm. Metabolic labeling and endoglycosidase studies suggest that this HER2 ECD (100 kDa) undergoes differential trafficking between the endoplasmic reticulum and Golgi compartments compared with full-length (185-kDa) HER2 receptor. Transfection studies indicate that excess production of HER2 ECD in human tumor cells overexpressing full-length HER2 receptor can result in resistance to the growth-inhibiting effects of anti-HER2 monoclonal antibodies such as muMAb4D5. These findings demonstrate alternative processing of the HER2 transcript and implicate a potentially important growth regulatory role for intracellularly sequestered HER2 ECD in HER2-amplified human tumors.

Monoclonal antibodies to the extracellular domain of HIV-1IIIB gp160 that neutralize infectivity, block binding to CD4, and react with diverse isolates.

Ten monoclonal antibodies prepared against a soluble, recombinant form of gp160, derived from the IIIB isolate of HIV-1, were characterized. Four of the antibodies neutralized HIV-1IIIB infectivity in vitro, three blocked the binding of recombinant gp120 to CD4, three were reactive with gp41, and one preferentially reacted with an epitope on gp120 within the gp160 precursor. All three CD4 blocking antibodies bound to distinct epitopes, with one mapping to the C1 domain, one mapping to the C4 domain, and one reactive with a conformation-dependent, discontinuous epitope. Of these, the antibody reactive with the discontinuous epitope exhibited neutralizing activity against homologous and heterologous strains of HIV-1. The binding of these monoclonal antibodies to a panel of seven recombinant gp120s prepared from diverse isolates of HIV-1 was measured, and monoclonal antibodies with broad cross reactivity were identified. The epitopes recognized by 7 of the 10 monoclonal antibodies studied were localized by their reactivity with synthetic peptides and with fragments of gp120 expressed as fusion proteins in a lambda gt-11 gp160 epitope library.

Human natriuretic peptide receptor-A guanylyl cyclase. Hormone cross-linking and antibody reactivity distinguish receptor glycoforms.

Most of the physiological actions of atrial natriuretic peptide (ANP) may be attributed to activation of the natriuretic peptide receptor-A (NPR-A) guanylyl cyclase. We report here that truncation of the NPR-A cytoplasmic domain results in increased expression of cell surface ANP binding sites. The truncated receptor exhibited a hyperbolic time course for ANP binding and had a high affinity for [125I]hANP, Kd = 8 pM. Cells expressing truncated NPR-A were used as an immunogen to obtain monoclonal antibodies against the native conformation of the extracellular domain. These antibodies were used to select for high levels of stable NPR-A expression in 293 cells, by fluorescence-activated cell sorting. Disuccinimidyl suberate cross-linked [125I]ANP to 135-kDa NPR-A on intact cells. Monoclonal antibody immunoprecipitation of 35S-labeled proteins revealed NPR-A size heterogeneity, with 135- and 125-kDa species. A synthetic peptide antibody directed against the extracellular domain immunoprecipitated 125-kDa NPR-A, but recognized both sizes of receptor by Western blotting. The 125-kDa NPR-A did not bind to or cross-link ANP. NPR-A size variants were expressed on the cell surface, and heterogeneity was removed by deglycosylation with protein:N-glycosidase F. Our results suggest that the degree of N-linked glycosylation of the NPR-A extracellular domain influences the ability to bind ANP.

Biochemical properties of the 75-kDa tumor necrosis factor receptor. Characterization of ligand binding, internalization, and receptor phosphorylation.

An expression plasmid encoding the human 75-kDa tumor necrosis factor (TNF) type 2 receptor (TNF-R2) was constructed and used to generate a stable human cell line (293/TNF-R2) overexpressing TNF-R2. Ligand binding analysis revealed high affinity binding (Kd = 0.2 nM) with approximately 94,000 +/- 7,500 sites/cell for 125I-TNF-alpha and approximately 5-fold lower affinity for TNF-beta (Kd = 1.1 nM) with 264,000 +/- 2,000 sites/cell. Cross-linking of 125I-TNF-alpha and 125I-TNF-beta to 293/TNF-R2 cells yielded predominant complexes with apparent molecular weights of 211,000 for TNF-alpha and 205,000 and 244,000 for TNF-beta, suggesting these complexes contain two or three TNF-R2 molecules. Immunoprecipitation of TNF-R2 from 32P-labeled 293/TNF-R2 cells demonstrated that the receptor is phosphorylated. The majority (97%) of 32Pi incorporation was found in serine residues with a very low level of incorporation (3%) in threonine residues. TNF-alpha treatment of 293/TNF-R2 cells did not significantly affect the degree or pattern of phosphorylation. Cell surface-bound 125I-TNF-alpha was slowly internalized by the 293/TNF-R2 cell line with a t1/2 = 25 min. Shedding of the extracellular domain of TNF-R2 was induced by 4 beta-phorbol 12-myristate 13-acetate but not by TNF-alpha or TNF-beta.