Evidence from two transformed cell lines that the phosphorylations of peptide tyrosine and phosphatidylinositol are catalyzed by different proteins.

Two transformed rodent cell lines (RS-1 and LSTRA) were studied in vitro to determine if their major protein tyrosine kinases catalyzed the phosphorylation of phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P), or diacylglycerol. RS-1 cells, transformed by Rous sarcoma virus, contain high levels of pp60src; LSTRA cells, transformed by Moloney murine leukemia virus, contain a tyrosine kinase (pp56) that is the product of an unknown cellular gene. Rates of phosphorylation of peptide tyrosine were elevated more than 20-fold in RS-1 and LSTRA particulate fractions compared to fractions from suitable control cells (N2 and YAC-1), but there was not a proportional increase in rates of phosphorylation of PtdIns, PtdIns4P, or diacylglycerol. Heat (34 degrees C) completely inactivated the LSTRA tyrosine kinase, while it enhanced the phosphorylation of PtdIns and PtdIns4P and had no effect on the phosphorylation of diacylglycerol. PtdIns4P inhibited the phosphorylation of PtdIns but had no effect on tyrosine kinase activity. An antibody, raised against a peptide with a sequence homologous to the autophosphorylation site of pp60src, immunoprecipitated tyrosine kinase activity from RS-1 and LSTRA extracts but had no effect on PtdIns kinase or PtdIns4P kinase activity. These results provide evidence that the phosphorylations of tyrosine and PtdIns are catalyzed by different proteins. An additional observation was that a monoclonal antibody that binds to pp60src and pp56 removed PtdIns kinase as well as tyrosine kinase activity from RS-1 and LSTRA particulate extracts. This antibody also removed PtdIns kinase from N2 and YAC-1 extracts, in which tyrosine kinase activity was low or undetectable. Thus, the anti-pp60src monoclonal antibody may recognize the PtdIns kinase in addition to pp60src and pp56.     

Humanized antibodies for antiviral therapy.

Antibody therapy holds great promise for the treatment of cancer, autoimmune disorders, and viral infections. Murine monoclonal antibodies are relatively easy to produce but are severely restricted for therapeutic use by their immunogenicity in humans. Production of human monoclonal antibodies has been problematic. Humanized antibodies can be generated by introducing the six hypervariable regions from the heavy and light chains of a murine antibody into a human framework sequence and combining it with human constant regions. We humanized, with the aid of computer modeling, two murine monoclonal antibodies against herpes simplex virus gB and gD glycoproteins. The binding, virus neutralization, and cell protection results all indicate that both humanized antibodies have retained the binding activities and the biological properties of the murine monoclonal antibodies.     

Molekulare Mechanismen der Kardiotoxizität von Tyrosinkinaseinhibitoren

In several human malignancies activating mutations or amplifications of tyrosine kinase inhibitors are causal or contribute to tumorigenesis. Therefore, the addition of tyrosine kinase inhibitors to standard chemotherapy was an innovative and revolutionizing therapeutic approach. Unfortunately, the most serious adverse event is a higher incidence of dilated cardiomyopathy seen in patients treated with tyrosine kinase inhibitors. This review deals with the molecular mechanisms of cardiotoxicity of tyrosine kinase inhibition. For this purpose we employ trastuzumab, a humanized monoclonal antibody directed against the tyrosine kinase receptor HER2/erbB2.     

Monoclonal Antibodies as Therapeutic Agents: Advances and Challenges

Despite the major advances in conventional forms of treatment (i.e. surgical techniques, radiotherapy and chemotherapy) and improved survival rates, cancer is still the second leading cause of death in developing countries. One major limitation of cytotoxic drugs and radiation in the treatment of cancer patients is their inability to discriminate between malignant and normal tissues. This in turn prevents the delivery of the optimal (therapeutic) dose of such agents to malignant tissues for their eradication. With the advent of hybridoma technology in 1975, it has been possible for the first time to produce large amounts of an antibody (i.e. monoclonal antibody) against any antigens of interest. Since each antibody is highly specific for a particular antigen, this typical feature of the antibodies has resulted in their widespread use in diagnostic kits, medical research (e.g. to unravel the function of the antigen in physiological and pathological conditions), and more recently, for the management of a wide range of human diseases such as autoimmune disease and human cancers. Thanks to recent advances in genetic engineering, the immunogenicity of rodent antibodies was reduced by producing the chimeric or humanized version of such antibodies or by developing the fully human antibodies. In other instances, as intact antibodies are too large for rapid penetration into solid tumours, it has been possible to develop a smaller fragment of such antibodies (e.g. Fab, scFv, VHH) with greater potential for use in cancer imaging and therapy. Depending on the target antigens and the antibody format, monoclonal antibodies can induce their anti-tumour activities by several mechanisms including activation of the host effector cells. To date, several mAbs have been approved for management of human cancers including: anti-EGFR antibody cetuximab and anti-VEGF antibody bevacizumab for treatment of metastatic colorectal cancer, anti-HER-2 antibody trastuzumab for metastatic breast cancer, anti-CD20 antibodies rituximab and ibritumomab tituxetan for non-Hodgkin lymphoma, anti- CD52 antibody alemeutumab for chronic lymphocytic leukaemia, and anti-CD33 antibody gemutuzumab ozogamicin for the treatment of acute myeloid leukaemia patients. Monoclonal antibodies currently account for about 30% of all new drugs in development, with more than 500 antibodies at different stages of clinical trials worldwide. In this review, the characteristic features of some of the therapeutic antibodies and the antigens recognised by such antibodies will be discussed as well as several challenges that need to be addressed in order to facilitate their widespread use as “magic bullets” in the management of human diseases and in particular human cancers.     

Monoclonal antibodies for treating cancer

PURPOSE: To assess the current status of in-vivo use of monoclonal antibodies for treating cancer. DATA IDENTIFICATION: Publications appearing between 1980 and 1988 were identified by computer searches using MEDLINE and CANCERLIT, by reviewing the table of contents of recently published journals, and by searching bibliographies of identified books and articles. STUDY SELECTION: More than 700 articles, including peer-reviewed articles and book chapters, were identified and selected for analysis. DATA EXTRACTION: The literature was reviewed and 235 articles were selected as relevant and representative of the current issues and future applications for in-vivo monoclonal antibodies for cancer therapy and of the toxicity and efficacy which has been associated with clinical trials. RESULTS OF DATA SYNTHESES: Approaches include using antibody alone (interacting with complement or effector cells or binding directly with certain cell receptors) and immunoconjugates (antibody coupled to radioisotopes, drugs, toxins, or other biologicals). Most experience has been with murine antibodies. Trials of antibody alone and radiolabeled antibodies have confirmed the feasibility of this approach and the in-vivo trafficking of antibodies to tumor cells. However, tumor cell heterogeneity, lack of cytotoxicity, and the development of human antimouse antibodies have limited clinical efficacy. Although the immunoconjugates are very promising, heterogeneity and the antimouse immune response have hampered this approach as has the additional challenge of chemically or genetically coupling antibody to cytotoxic agents. CONCLUSIONS: As a therapeutic modality, monoclonal antibodies are still promising but their general use will be delayed for several years. New approaches using human antibodies and reducing the human antiglobulin response should facilitate treatment.     

False serum calcitonin high levels using a non-competitive two-site IRMA.

Dual site antibody-base immunoassays are commonly used in clinical laboratories to quantify the CT serum concentrations as a specific and sensitive marker of medullary thyroid carcinoma (MTC). Heterophile antibodies can interfere with these assays, however, and cause erroneous results. In order to avoid this interference, immobilized and conjugated antibodies from two different animal species or immunoreactive antibody fragments, as well as the addition of non-immune globulins, are generally included among the assay reagents. We describe the case of a 73-year-old man affected by a multinodular goiter, who showed high basal CT plasma levels as measured by a monoclonal antibody based IRMA. The finding of negative results for the presence of MTC at fine needle aspiration (FNA) and the mild increase observed in plasma CT during a pentagastrin (Pg) stimulation test, suggested that the high CT levels might depend on a cross-reaction with heterophilic antibodies. In fact, after the addition of the heterophilic blocking tube (HBT) to each specimen, the CT levels markedly decreased by more than 80% (average decrease+/-SE= 87.6+/-2.668%). Such a decrease strongly suggests that in our case the routinely used F(ab')2 fragments were unable to eliminate all of the interference and that the elevated serum CT levels might have been caused by human heterophilic antibodies. In conclusion, these results indicate a novel cause of CT false positivity, suggesting that high serum CT levels, when combined with a slight increase during Pg stimulation, should be critically interpreted in view of the possible presence of heterophilic antibodies in the specimens.     

Utilization of monoclonal antibodies in the treatment of leukemia and lymphoma

The generation of murine monoclonal antibodies reactive with human leukemia and lymphoma cells has recently led to clinical trials that have begun to evaluate the use of these reagents in the treatment of various leukemias and lymphomas. Several of these studies have demonstrated that infusion of monoclonal antibody can cause the rapid and specific clearance of leukemic cells from the peripheral blood. Intravenously administered antibody also rapidly binds to bone marrow lymphoblasts, and in one instance, has resulted in the partial regression of tumor cell infiltrates in lymph nodes and skin. Unfortunately, clinically significant responses have not in general been achieved, but these clinical studies have identified specific factors that result in the development of resistance to antibody- mediated lysis in vivo. These factors include the presence of circulating antigen, antigenic modulation, reactivity of monoclonal antibody with normal cells, immune response to murine antibody, and the inefficiency of natural immune effector mechanisms. Current research is now being directed towards developing methods to circumvent each of these obstacles. Future clinical studies utilizing antibodies in vitro or with different specificity may demonstrate greater therapeutic efficacy. In addition, monoclonal antibodies can be used as carriers of other cytotoxic agents and in conjunction with other agents that will reduce the total load. Monoclonal antibodies represent new and powerful reagents that may in the near future become an additional therapeutic modality for patients with malignant disease.     

A candidate targeting molecule of insulin-like growth factor-Ⅰ receptor for gastrointestinal cancers

Advances in molecular research in cancer have brought new therapeutic strategies into clinical usage.One new group of targets is tyrosine kinase receptors,which can be treated by several strategies,including small molecule tyrosine kinase inhibitors(TKIs) and monoclonal antibodies(mAbs).Aberrant activation of growth factors/receptors and their signal pathways are required for malignant transformation and progression in gastrointestinal(GI) carcinomas.The concept of targeting specif ic carcinogenic receptors...     

Tyrosine phosphorylation in T cells is regulated by phosphatase activity: studies with phenylarsine oxide.

Activation of T cells induces rapid tyrosine phosphorylation on the T-cell receptor zeta chain and other substrates. These phosphorylations can be regulated by a number of protein-tyrosine kinases (ATP: protein-tyrosine O-phosphotransferase, EC 2.7.1.112) and protein-tyrosine-phosphatases (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48). In this study, we demonstrate that phenylarsine oxide can inhibit tyrosine phosphatases while leaving tyrosine kinase function intact. We use this reagent to investigate the effect of tyrosine phosphatase inhibition in a murine T-cell hybridoma. Increasing concentrations of phenylarsine oxide result in an increase in tyrosine phosphate on a number of intracellular substrates in unstimulated T cells, suggesting that a protein-tyrosine kinase is constitutively active in these cells. The effect of phenylarsine oxide on T cells stimulated with an anti-Thy 1 monoclonal antibody is more complex. At low concentrations of drug, there is a synergistic increase in the level of tyrosine phosphate on certain cellular substrates. At higher concentrations, anti-Thy 1-stimulated tyrosine phosphorylation is inhibited. These results indicate that tyrosine phosphorylation in T cells is tightly regulated by tyrosine phosphatases. Partial inhibition of these enzymes results in enhanced substrate phosphorylation. Inhibition of all stimulated tyrosine phosphorylation by high doses of phenylarsine oxide suggests that tyrosine kinase activity is regulated by tyrosine phosphatases.     

Drug insight: antiangiogenic therapies for gastrointestinal cancers--focus on monoclonal antibodies

Tumor angiogenesis is strongly induced by vascular endothelial growth factor (VEGF), which is overexpressed in most human gastrointestinal cancers. VEGF overexpression is known to be associated with poor prognosis and survival in patients with various solid tumors. The humanized monoclonal anti-VEGF antibody bevacizumab (Avastin, Genentech Inc., South San Francisco, CA) is a prototypic antiangiogenic compound, and has proven therapeutic benefit combined with conventional chemotherapy-namely, significantly improved progression-free survival in patients with metastatic colorectal cancer. Bevacizumab is the only anti-VEGF antibody that has been approved by the FDA and the European Medicines Agency for the treatment of metastatic colorectal cancer. Several ongoing clinical studies are evaluating the potential of bevacizumab therapy for other gastrointestinal cancers, in combination with chemotherapy, other targeted therapies and/or radiation. Soluble chimeric receptors, tyrosine kinase inhibitors, and monoclonal antibodies against VEGF and molecular targets in the integrin and Delta-like protein 4-Notch pathways are being developed. As tumors acquire resistance to anti-VEGF therapy, further development of antiangiogenic and vascular targets and therapy is warranted.     

An anti-idiotopic antibody-based enzyme-linked immunosorbent assay for the quantification of the monoclonal anti-D BRAD-5

OBJECTIVES: We aimed to develop an anti-idiotopic antibody-based enzyme-linked immunosorbent assay (ELISA) to quantify human monoclonal anti-D using BRAD-5 as a model system. MATERIALS AND METHODS: One of the anti-idiotopic antibodies 2E6 was used to capture BRAD-5 with the other anti-idiotopic antibody 3B1 biotinylated for detection. RESULTS: The assay developed can detect BRAD-5 at < 2 ng/ml. Assay interference caused by heterophilic antibodies in some human sera was removed by preincubation with bovine serum. The assay is reproducible with intra- and inter-assay coefficient of variation (CV) < 10%. CONCLUSION: This ELISA should prove of benefit in developing a monoclonal anti-D for prophylactic use.     

Introduction to antibody engineering and phage display

The vertebrate immune system, capable of rapidly producing highly specific antibodies upon immunisation, has been used to produce murine monoclonal antibodies (mAbs) via immortalisation and isolation of antibody-secreting cells. These mAbs have had a profound impact in the fields of diagnostics and therapeutics. However, the therapeutic use of murine mAbs is complicated by their immunogenicity. To circumvent this immunogenicity, antibody engineering techniques which render murine mAbs more compatible with the human immune system have been devised. Over the last decade, the technique of antibody phage display, which facilitates the production of human mAbs, has been developed. This review serves as an introduction to antibody engineering, phage display and the development of antibody fragments into viable diagnostic and therapeutic reagents.     

Human bradykinin B2 receptors isolated by receptor-specific monoclonal antibodies are tyrosine phosphorylated.

We report the immunoaffinity isolation of bradykinin B2 receptors in a tyrosine-phosphorylated state from WI-38 human lung fibroblasts. We generated six monoclonal antibodies directed against B2 bradykinin receptor biologic activity mediating prostaglandin E2 production in WI-38. These cells express a repertoire of bradykinin receptor affinity forms with closely correlated biologic activity and [3H]bradykinin binding. Some of the monoclonal antibodies selectively recognize intermediate-affinity (Kd = 5.6 nM) or low-affinity (Kd = 42 nM) receptor forms, whereas others recognize epitopes common to both. The monoclonal antibodies block bradykinin binding and biologic activity. Immunoaffinity chromatography on an immobilized monoclonal antibody of intermediate- plus low-affinity specificity yields WI-38 B2 receptors with intact [3H]bradykinin binding activity and a molecular mass of 78 kDa. The same band is immunoblotted by all the monoclonal antibodies, indicating a similar molecular mass for receptor forms of different affinity. Anti-phosphotyrosine antibodies demonstrate that the receptors are tyrosine phosphorylated, with implications for receptor function and regulation. Genistein completely inhibits bradykinin-mediated prostaglandin E2 production with an IC50 of 8 microM, indicating that tyrosine kinase activity is critical for the signal transduction leading to arachidonic acid release.     

pp60c-src kinase expression in brain of adult rats in relation to age.

Although there is evidence of alterations in brain protein phosphorylation patterns with age, it is not known if the protein kinases that phosphorylate only at tyrosine residues are involved in these changes. For this reason, we examined the age-related expression of pp60c-src, a tyrosine protein kinase enriched in neural tissues, in whole brain of adult Fischer-344 rats. The pp60c-src kinase activity was immunoprecipitated using a monoclonal antibody and the incorporation of [32P] from radiolabeled ATP into an exogenous substrate (casein) measured. The results showed that there was a substantial amount of pp60c-src kinase activity in brain of the adult animals ranging in age from 4 to 23 months and that it was not significantly different among these groups. Also, immunoprecipitates obtained under conditions of monoclonal antibody excess and utilized for immunoblot analysis indicated that the relative levels of the pp60c-src protein were unchanged in the same animals. These results suggest that, at the whole brain level, the pp60c-src kinase has a stable turnover and that a high amount of activity is biologically important in brain of adult rats through early senescence.     

Studies of human polyclonal and monoclonal antibodies binding to lupus autoantigens and cross-reactive antigens

BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune rheumatic disease serologically characterized by production of a variety of autoantibodies. Antibodies to double-stranded (ds) DNA are considered to be a diagnostic marker in SLE and their presence often correlates with active disease. The murine R4A anti-dsDNA antibody was found to cross-react with a peptide, D/EWD/EYS/G (R4A peptide), identified by analysing decapeptides selected from a peptide library. The R4A peptide inhibited binding of antibody to dsDNA and antibody deposition in kidneys in vivo. In other previous work, mice immunized with the peptide in a decapeptide form bound to a polylysine backbone, multiple antigenic peptide, were found to develop both anti-DNA and anticardiolipin antibodies. METHODS: To determine if human anti-DNA antibodies bind R4A peptide, we investigated the binding of monoclonal and polyclonal anti-dsDNA and anticardiolipin antibodies to the R4A peptide from patients with SLE. RESULTS: DNA binding by four immunoglobulin (Ig) G and two IgM human monoclonal anti-DNA antibodies was inhibited by the R4A peptide. While monomeric peptide was unable to inhibit affinity-purified polyclonal anti-DNA antibodies, serum anti-DNA reactivity was inhibited by an octameric form of the peptide in 10 SLE patients. CONCLUSIONS: Human anti-DNA reactivity includes the same fine specificity as that present in murine anti-DNA reactivity. Peptide binding might be a useful surrogate marker for SLE.     

Amelioration of murine immune thrombocytopenia by CD44 antibodies: a potential therapy for ITP?

To explore the potential for monoclonal antibodies as a treatment for immune thrombocytopenia (ITP) and to further explore their mechanisms of action, we tested 8 monoclonal CD44 antibodies in murine ITP and found 4 antibodies that could successfully ameliorate ITP; 2 of these antibodies function at a full 3-log fold lower dosage compared with IVIg. Further characterization of the 2 most successful antibodies (5035-41.1D and KM114) demonstrated that, similar to IVIg: (1) the presence of the inhibitory IgG receptor FcγRIIB was required for their ameliorative function, (2) complement-deficient mice responded to anti-CD44 treatment, and (3) human transgenic FcγRIIA-expressing mice also responded to the CD44 therapeutic modality. Dissimilar to IVIg, the Fc portion of the CD44 antibody was not required. These data demonstrate that CD44 antibodies can function therapeutically in murine ITP and that they could potentially provide a very-low-dose recombinant therapy for the amelioration of human ITP.     

The murine stk gene product, a transmembrane protein tyrosine kinase, is a receptor for macrophage-stimulating protein.

Macrophage-stimulating protein (MSP) was originally identified as an inducer of murine resident peritoneal macrophage responsiveness to chemoattractants. We recently showed that the product of RON, a protein tyrosine kinase cloned from a human keratinocyte library, is the receptor for MSP. Similarity of murine stk to RON led us to determine if the stk gene product is the murine receptor for MSP. Radiolabeled MSP could bind to NIH 3T3 cells transfected with murine stk cDNA (3T3/stk). Binding was saturable and was inhibited by unlabeled MSP but not by structurally related proteins, including hepatocyte growth factor and plasminogen. Specific binding to STK was demonstrated by cross-linking of 125I-labeled MSP to membrane proteins of 3T3/stk cells, which resulted in a protein complex with a molecular mass of 220 kDa. This radiolabeled complex comprised 125I-MSP and STK, since it could be immunoprecipitated by antibodies to the STK beta chain. Binding of MSP to stk cDNA-transfected cells induced tyrosine phosphorylation of the 150-kDa STK beta chain within 1 min and caused increased motile activity. These results establish the murine stk gene product as a specific transmembrane protein tyrosine kinase receptor for MSP. Inasmuch as the stk cDNA was cloned from a hematopoietic stem cell, our data suggest that in addition to macrophages and keratinocytes, a cell in the hematopoietic lineage may also be a target for MSP.     

Characterization of murine monoclonal antibodies directed against the Kell blood group glycoprotein

Murine monoclonal antibodies (MoAbs) were produced against the blood group KEL1 glycoprotein (93 kD component) immunopurified from human erythrocytes. One monoclonal antibody, 5A11 (IgGa, kappa), detects by immunoblotting a 93 and 184 kD component from KEL: 1,-2 or KEL: -1,2 red cell membrane preparations, separated by SDS polyacrylamide gel electrophoresis (PAGE) under non-reducing conditions. The 184 kD component was not detected under reducing conditions, suggesting that it represented a dimer of the 93 kD KEL glycoprotein. Neither the 93 nor the 184 kD could be detected from K0 or McLeod erythrocyte membrane preparations, indicating that the monoclonal antibody reacts with the KEL glycoprotein previously identified as a 93 kD species. Since this antibody does not agglutinate native or protease-treated erythrocytes, it is likely that it reacts with the cytoplasmic domain of the KEL glycoprotein. This was also substantiated by showing that 5A11 could immunoprecipitate the 93 kD component from intact membranes and inside-out vesicles but not from right-side-out vesicles. Immunostaining of membrane proteins prepared from human blood cells (platelets, lymphocytes, monocytes and granulocytes) and non-human erythrocytes revealed that the 93 kD molecule was only present on human red cells. Several other murine monoclonal antibodies obtained from the same fusion experiment gave identical results, but competition analyses on immunoblots indicated that these antibodies reacted with distinct epitopes on the KEL glycoprotein.     

Concurrent HER2 vaccination and inhibition of kinase activity: safety and immunogenicity

Evaluation of: Hamilton E, Blackwell K, Hobeika AC et al. Phase I clinical trial of HER2-specific immunotherapy with concomitant HER2 kinase inhibition. J. Transl. Med. 10, 28 (2012). Passive immunotherapy with the monoclonal antibody trastuzumab and tyrosine kinase activity inhibition with lapatinib are HER2-targeted therapies used in the clinic for the treatment of HER2-overexpressing breast cancers. Unfortunately, the therapeutic efficacy of both these therapies is abolished by primary and acquired tumor resistance. Active immunotherapy against HER2, which, thanks to trastuzumab, is a clinically validated tumor-associated antigen, might provide an alternative therapeutic strategy for HER2-overexpressing breast cancers. This Phase I study of HER2 immunotherapy with concomitant lapatinib treatment in 12 patients with metastatic breast cancer resistant to trastuzumab demonstrates the feasibility and safety of concurrent vaccination against HER2 and inhibition of HER1 and HER2 kinases. However, it is inconclusive regarding the effect of lapatinib on the immune responses induced by dHER2/AS15; vaccination triggered variable levels of anti-HER2 antibodies in all the patients, but a HER2-specific T-cell response was detected in one patient only. Since the presence of Tregs in these patients was not assessed, it remains unclear whether lapatinib and/or Tregs account for the near absence of a T-cell response.     

The murine c-fgr gene product associated with Ly6C and p70 integral membrane protein is expressed in cells of a monocyte/macrophage lineage.

The c-fgr gene is a member of the Src family of protooncogene tyrosine kinases. A monoclonal antibody (2H2) that recognizes the specific region of the N-terminal domain of the murine c-fgr gene product (Fgr) has been established. With an immune complex kinase assay in a monocytic leukemia cell line, 2H2 monoclonal antibody was shown to precipitate a 59-kDa protein that corresponds in molecular mass to murine Fgr. Fgr was expressed highly in lymph nodes, slightly in spleen and peripheral blood leukocytes, and barely in the thymus and was not detected in bone marrow. In the presence of a mild detergent, Fgr was coimmunoprecipitated with a 70-kDa protein (p70) or with p70 plus several other molecules that were expressed on the cell-surface membrane of macrophage tumor cell lines PU5-1.8 and J774.1, respectively. By contrast, Fgr was not coimmunoprecipitated with a low-affinity receptor for the Fc portion of IgG that is associated with human Fgr. The molecule was also coimmunoprecipitated with the Ly6C molecule from a macrophage cell line (J774.1) that showed protein-tyrosine kinase activity. Peptide mapping revealed that this kinase activity was derived from Fgr. The similarity of relationship between this intramembrane p70 and/or Ly6C and cytoplasmic Fgr to relationships previously reported between T-cell antigen receptor complex, including CD4 and CD8 coreceptors, and Lck or Fyn in T cells and between surface IgM and Lyn or Blk in B cells, suggests that the Fgr and p70 or Ly6C are, indeed, associated with each other and in the murine system may be responsible for recognition of extracellular substances (either cellular or noncellular) and for signal transduction in cells of monocyte/macrophage lineage.