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The epidermal growth factor (EGF) receptor is a transmembrane polypeptide of 170 000 daltons (Da) with a cytoplasmically facing protein kinase domain. The regulation of the tyrosine kinase activity of the EGF receptor by added EGF and by receptor association state was studied in an in vitro system. The rate of autophosphorylation of the solubilized and purified EGF receptor was found to be independent of receptor concentration. To determine whether the zero-order kinetics observed point to intrapeptide phosphorylation, we measured the sedimentation characteristics of the undenatured solubilized receptor. The receptor was found to exist in two association-dissociation states-a monomeric 7.7S form and a dimeric 12S form. The 7.7S form is an active tyrosine kinase; it has high basal activity, and the activity is not further stimulated by EGF; it appears to be an EGF-independent form of the receptor kinase. The 12S form is devoid of catalytic activity, but in the presence of EGF it dissociates into the active monomeric form. Freshly purified receptor preparations contain mainly the monomeric receptor, have high basal kinase activity, and show low EGF stimulatability (less than 1.3-fold). Aging of the receptor results in progressive dimerization and decay of EGF-independent kinase activity (and increase in EGF stimulatability). All of these processes are reversed in the presence of EGF or dithiothreitol.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract. This paper describes studies on the migratory behavior of epidermal growth factor (EGF) receptor kinase using antibodies that are specific for either the kinase domain or the extracellular domain of the receptor. Antiserum was raised to a 42,000-D subfragment of EGF receptor, which was shown earlier to carry the kinase catalytic site but not the EGF-binding site. Another antiserum was raised to the pure intact 170,000-D EGF receptor. The specificities of these antibodies were established by immunoprecipitation and immunoblotting experiments. The domain specificity was examined by indirect immunofluorescent staining of fixed cells. The anti-42-kD peptide antibody could bind specifically to EGF receptors of both human and murine origin and was found to be directed to the cytoplasmic part of the molecule. It did not bind to EGF receptor-negative cells, which contained other types of tyrosine kinases. The antibodies raised against the intact receptor recognized only EGF receptorspecific epitopes and were directed to the extracellular part of the molecule.The anti-receptor antibodies described above were used to visualize the cyclic locomotory behavior of EGF receptor kinase under various conditions of EGF stimulation and withdrawal. The receptor was examined in fixed and permeabilized cells by indirect immunofluorescent staining. The results demonstrate the following: (a) the receptor kinase domain migrates to the perinuclear region upon challenge with EGF; (b) both extracellular and cytoplasmic domains of the receptor are involved in migration as a unit; (c) withdrawal of EGF results in rapid recycling of the perinuclear receptors to the plasma membrane; (d) this return to the cell surface is inhibited by methylamine, chloroquine, and monensin; and (e) neither the internal migration nor the recycling process is blocked by inhibitors of protein biosynthesis.

Recently, we isolated a new peptide growth factor of Mr 34 000 from synctial membranes of human placenta. In its polypeptide molecular weight and receptor binding specificity it is unlike several known growth factors. In this paper we described immunocytochemical studies on its cellular location and biosynthesis. A rabbit antiserum was raised against a homogeneous preparation of the placental peptide. The specificity of the antibody was established by immunoprecipitation and immunoblot analyses. The antibody recognized both the native and denatured 34-kilodalton (kDa) peptide but showed no binding to a variety of other growth factors and hormones tested. The antibody was used to investigate the genesis and location of the 34-kDa membranous mitogen. Immunoperoxidase staining of placental tissue slices revealed a restricted localization of the antigen in the cytoplasmic organelles of cytotrophoblasts and in the brush border membranes of syncytiotrophoblasts. No other placental structures contained the antigen. A developmentally regulated appearance of the mitogen was suggested by the fact that first trimester placenta consistently stained far more strongly than term placenta. These studies show that the 34-kDa mitogenic protein originates in placenta from embryo-derived cellular structures and suggest that in its strategic location it may influence trophoblastic growth in an autocrine manner. In other studies we investigated the presence and biosynthesis of the 34-kDa peptide in the A431 vulval carcinoma cell line, which was shown earlier to contain a membrane-associated 34-kDa growth factor. The studies demonstrate that this cell line, as well as some other human carcinomas of breast and bladder origin, actively expresses this peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

SummaryWe have investigated the ability of a heterogeneous thymic stromal cell (HTSC) culture system to promote in vitro differentiation of CD3-4-8-thymocytes. Culture of purified murine CD3-4-8-thymocytes on HTSC for 1 d resulted in the appearance of CD4 + 8 + cells, which did not occur when the sorted cells were maintained in medium alone. It is remarkable that when the culture period was extended to 2 d, CD3-4-8-progenitors differentiated further to CD4 + 8-and CD4-8 + cells, which also expressed high levels of TCR-CD3. This rapid differentiation on stroma in vitro appears to outpace parallel development in vivo. The differentiation potential of a subset of CD3 -4-8-thymocytes that express high levels of a marker of normal and neoplastic thymic progenitors, the 1Cll antigen, was examined next. 1C11hiCD3-4-8 -cells also gave rise to CD4-8 + and CD4+8 + populations after 1 d of culture on HTSC. Extending the culture period to 2 d resulted in a significant percentage of CD3-expressing cells that were CD4+8 +, CD4+8 -and CD4-8 + cells. These results suggest that in the in vitro HTSC culture system, various subsets of immature thymocytes can differentiate into all the mature phenotypes of cells normally found in the adult mouse thymus. This may provide a novel and rapid assay for thymic progenitors.T he thymus is the major site of differentiation of T lymphocytes. Bone marrow-or fetal liver-derived thymic progenitors migrate to the thymus where they undergo rapid proliferation and differentiation, a process known as thymic maturation (1, 2). In the thymus, under the influence of the thymic stromal microenvironment, immature thymocytes acquire various cell surface molecules including the MHC coreceptors CD4 (MHC class II-restricted) and CD8 (MHC class I-restricted), and the TCR o~3 or 3'~ heterodimers associated with the invariant CD3 polypeptides (3-6). Based on their CD4 and CD8 expression, thymocytes are divided into four categories: CD4-8-, CD4+8 +, CD4+8 -, and CD4-8 + (1). The immature CD3-4-8-cells contain a subpopulation that proceeds through sequential CD3-4-8 + large cells (a rapidly cycling population) and CD31~ + large thymocyte intermediates, before giving rise to the phenotypically and functionally mature CD3Si4+8 -and CD3hi4-8 + T cells, as well as CD31~ + 8 + small thymocytes that die in situ (7-9). During the process of thymic education, maturing T cells acquire the property of responsiveness to foreign antigens presented in association with self-MHC molecules (10) by the APCs, and of tolerance or nonresponsiveness to self-antigens (10--13). Recently, it has been suggested (14) that interaction of CD31~ + 8 + cells with affinity for self-MHC complexed with unknown self-peptides may be required for positive selection. Negative selection to Mlsand V~17-detected antigens occurs at a later stage when CD3md4+8 + cells are committed to either the CD4 or the CD8 lineage (14).Although the lineage relationships between CD3-4-8-cells and the phenotypically and functionally mature T cell subsets have been est...

This paper describes the identification and characterization of a new peptide growth factor. The peptide was isolated from trophoblastic brush border membranes of human placenta. The purified preparation was homogeneous and consisted of a single polypeptide of Mr 34 000 with a pI of about 6.0. This peptide stimulated DNA replication in cultured fibroblasts. The following association was seen between activity and protein: During DEAE-cellulose chromatography, both the 34-kilodalton (kDa) protein and the mitogenic activity displayed identical binding and salt dependence of elution. Nondenaturing electrophoresis at pH 8.3 revealed a comigration of the 34-kDa protein and the DNA replication stimulatory activity. Identical electrophoretic mobilities were displayed for both activity and protein at pH 7.0. These results demonstrate that the preparation is homogeneous and show that growth factor activity is intrinsic to the 34-kDa polypeptide. Binding of the 125I-labeled 34-kDa mitogen to target fibroblastic cells was specific; i.e., nanomolar concentrations of the unlabeled 34-kDa protein competed effectively with the labeled protein, whereas a variety of well-characterized growth factors and hormones were unable to compete even at micromolar levels. Thus the 34-kDa protein interacts with target cells through highly specific surface receptors. Chemical cross-linking techniques were used to investigate the identity of the receptor for the 34-kDa mitogen. Cross-linking of fibroblastic cells containing bound 125I-labeled 34-kDa protein generated a radiolabeled complex of 86 kDa in all four cell types examined.(ABSTRACT TRUNCATED AT 250 WORDS)

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SummaryStrain C.B17 scidAcid (SCID) mice, which lack functional T and B lymphocytes, show heightened susceptibility to the induction of thymic lymphomas by x-irradiation. Susceptibility is highest in thymus-chimeric SCID-BL mice (thymectomized SCID mice bearing a C57BL thymus graft). All SCID-BL lymphomas originate in the cells of the thymic graft (C57BL type) and lack murine leukemia virus expression. Both SCID and SCID-BL lymphomas are phenotypically CD4-8 + and/or CD4+8 + , but only the SCID-BL tumors express CD3. Injection of C57BL or BALB/c bone marrow into irradiated SCID-BL mice prevents lymphoma development, but SCID marrow is completely ineffective. The results suggest that the scid condition enhances the activity of a putative lymphomagenic agent induced in the bone marrow by x-irradiation and that C57BL thymic cells are highly sensitive targets. Moreover, the failure of SCID bone marrow to protect against lymphomagenesis vs. the efficacy of marrow from immunocompetent donors points to involvement of T or B lineage cells in this process.