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Biomedical subjects

M I Greene

Publications and source records attributed to M I Greene.

At least 145 records · Page 8Linked to original sources
PubMed1989

A point mutation in the neu oncogene mimics ligand induction of receptor aggregation.

The rat neu gene, which encodes a protein closely related to the epidermal growth factor receptor, is a proto-oncogene that can be converted into an oncogene by a point mutation. Both genes encode proteins with a relative molecular mass of 185,000 but the question of why the neu gene product, p185neu, is oncogenic, whereas the product of c-neu, p185c-neu, is not, remains unanswered. The proteins have several features common to the family of tyrosine kinase growth-factor receptors, including cysteine-rich external domains, a hydrophobic transmembrane region and a cytoplasmic tyrosine kinase domain. The oncogenic p185neu differs from p185c-neu by an amino-acid substitution in the transmembrane region of the glycoprotein: this replacement of valine by glutamic acid at position 664 induces increased intrinsic tyrosine kinase activity which is associated with transformation. Many glycoproteins with charged amino acids in the transmembrane region exist as multimeric complexes at the plasma membrane. We have therefore investigated the association state of both products of the neu gene and show that the oncoprotein p185neu is organized at the plasma membrane primarily in an aggregated form, but that p185c-neu is not. Induction of an aggregated state may mimic aspects of ligand-induced receptor aggregation resulting in enzymatic activation that leads to cellular transformation.

Animals↗
PubMed1989

Inhibition of cellular DNA synthesis by reovirus occurs through a receptor-linked signaling pathway that is mimicked by antiidiotypic, antireceptor antibody.

Mammalian reovirus type 3 binds to a 67-kD surface glycoprotein on the membrane of neuronal cells. This interaction initiates the infective reovirus cycle. The physiological function of this virus receptor is not known, however, initial studies illustrate a striking structural and antigenic homology to the beta adrenergic receptor family. The earliest known pathologic effect of reovirus type 3 is an inhibition of host cell DNA synthesis within 8-10 h after virus attachment. The studies reported here demonstrate that binding and aggregation of reovirus receptor molecules provides the signal for this inhibitory process. Inhibition of DNA synthesis in the neuroblastoma cell line B104.G4 was unaffected by using replication-defective virus or when lysosomal processing of normal virus was blocked. Inhibition was mimicked by an antiidiotypic, antireceptor mAb. Inhibition was not observed when monovalent mAb fragments were bound to receptors, but was reconstituted when these fragments were aggregated by the addition of anti-Ig. The signal for the inhibitory effect was generated within the first 60 min after mAb binding. These observations demonstrate that reovirus and antiidiotypic pathogenicity can result from the perturbation of membrane proteins that may perform normal physiological functions.

Adrenergic beta-Agonists↗
PubMed1989

Development of biologically active peptides based on antibody structure.

Antibody molecules are composed of several functional domains, including a variable domain that contacts antigen and a constant domain. The hypervariable regions of antibody molecules play an integral role in determining their specificity. However, the delineation of specific residues most critical in binding is difficult. We have been studying a monoclonal antibody (87.92.6) that binds to the reovirus type 3 receptor on a number of cell types, down-modulates the receptor, and inhibits DNA synthesis in the cells. We have shown that a peptide derived from the second complementarity-determining region of the monoclonal antibody 87.92.6 light-chain variable region can reproduce both of these effects. We were also able to demonstrate specific amino acid residues and structural features involved in producing these effects. The study of antibody structure, coupled with molecular synthetic techniques, can lead to the development of biologically active substances with potential clinical use.

Amino Acid Sequence↗
PubMed1989

Major histocompatibility complex class I-specific cytolytic T cells, derived from gld mice, lacking Thy-1, CD4, and CD8.

Thy-1 is a major cell surface molecule expressed on murine thymocytes and peripheral T cells. Its physiological function is unknown, but in vitro studies suggest that Thy-1 may transmit activation signals to T cells and may play a role in the growth and/or differentiation of thymocytes [Kroczek, R. A., Gunter, K. C., Seligmann, B. & Shevach, E. M. (1986) J. Immunol. 136, 4379-4384; Kroczek, R. A., Gunter, K. C., Germain, R. N. & Shevach, E. M. (1986) Nature (London) 322, 181-184]. However, not all mouse thymocytes are Thy-1+ [Scollay, R., Wilson, A., D'Amico, A., Kelly, K., Egerton, M., Pearse, M., Wu, L. & Shortman, K. (1988) Immunol. Rev. 104, 81-120]. In addition, C3H-gld/gld mice accumulate large numbers of Thy-1- (and Thy-1+) T-cell antigen receptor-positive CD8- CD4- (double negative) T cells in peripheral lymphoid organs. Our previous studies of these Thy-1- and Thy-1+ double negatives suggested that lack of Thy-1 expression correlated with diminished capacity to respond to T-cell stimuli. In this report, we describe a Thy-1- alpha/beta T-cell receptor-positive major histocompatibility complex-specific cytotoxic T-cell clone derived from C3H-gld/gld lymph node-residing cells. The data show that, at least in this system, Thy-1 (and CD8/CD4) expression is not required for growth, cytolytic activity, or expression of functional T-cell receptor complexes in vitro and raise the possibility that Thy-1 expression may not be obligatory in vivo for development of cytotoxic T-lymphocyte precursors in gld mice.

Animals↗
PubMed1989

Differential regulation of oncogenic and cellular p185 by serine/threonine kinases.

185c-neu is a member of a family of growth factor receptors with tyrosine kinase activity. A point mutation in the transmembrane region leads to activation of the enzymatic domain. We demonstrate that TPA (phorbol-12-myristate-13-acetate) stimulates the phosphorylation of p185c-neu on serine and threonine residues coincident with the inhibition of its intrinsic tyrosine kinase and the proliferation of cells that express it. The tyrosine kinase activity as well as the phosphorylation pattern of serine and threonine residues of oncogenic p185 (p185neu) and the growth of p185neu-expressing cells are not influenced by TPA. These observations indicate that the functional activity of p185c-neu can be regulated through protein kinase C (PKC) but the transmembrane point mutation present in p185neu renders it refractory to serine/threonine kinase regulation.

Cell Division↗
PubMed1989

Three dimensional structure of a functional internal image.

An anti-idiotypic antibody has been characterized that forms a functional internal image of the cell-attachment site of reovirus type 3. This antibody shares sequence similarity with the reovirus type 3 cell attachment protein. Studies utilizing synthetic peptides have shown that the region of sequence similarity corresponds to the cell-attachment site of the virus and the antibody. Studies are described demonstrating that these peptides possess biological activity similar to that of the virus and the antibody. Three-dimensional models of the cell-attachment site of the virus are presented and compared to a similar model for the antibody. These studies indicate that anti-idiotypic antibodies can be utilized to understand the three-dimensional basis of ligand - receptor interactions, and can lead to the development of novel biologically active substances.

Amino Acid Sequence↗
PubMed1989

Distribution of neu (c-erbB-2) protein in human skin.

The neu (c-erbB-2) gene encodes a transmembrane protein with tyrosine kinase activity that appears to be a growth factor receptor. Antibody was generated by immunization of rabbits with a synthetic polypeptide that was based on an internal sequence at the carboxy terminus of the molecule. This antibody was used to survey the expression of neu in human skin by immunohistochemistry. Significant protein was found in the squamous cell layer of the surface epidermis, in squamous cell carcinomas, in the external root sheath of hair follicles, and in eccrine gland secretory cells; it was poorly expressed in the basal cell layer and in a basal cell carcinomas. Increased neu expression appears to be associated with the differentiation of keratinocytes.

Antibody Specificity↗
PubMed1989

Subtype-selective immunoprecipitation of the beta 2-adrenergic receptor.

Most antibodies known to interact with beta-adrenergic receptors do not exhibit subtype selectivity, nor do they provide quantitative immunoprecipitation. A monoclonal antibody, G27.1 raised against a synthetic peptide corresponding to the C-terminus of the beta 2-adrenergic receptor of hamster, is selective for the beta 2 subtype. G27.1 provides nearly quantitative immunoprecipitation of the beta 2-adrenergic receptor from hamster lung that has been photoaffinity-labeled and solubilized with sodium dodecyl sulfate. Immunoprecipitation is completely blocked by nanomolar concentrations of the immunizing peptide. This antibody interacts with beta 2-adrenergic receptors from three rodent species, but not with those from humans. When C6 glioma cells, which contain both beta 1- and beta 2-adrenergic receptors, are photoaffinity-labeled in the absence or presence of subtype-selective antagonists, subtype-selective photoaffinity-labeling results. G27.1 can immunoprecipitate beta 2-, but not beta 1-, adrenergic receptors from these cells. Similar results were obtained following subtype-selective photoaffinity-labeling of membranes from rat cerebellum and cerebral cortex. The beta-adrenergic receptors from C6 glioma cells and rat cerebral cortex exist as a mixture of two molecular weight species. These species differ in glycosylation, as shown by endoglycosidase F digestion of crude and immunoprecipitated receptors.

Affinity Labels↗
PubMed1989

Expression pattern of the neu (NGL) gene-encoded growth factor receptor protein (p185neu) in normal and transformed epithelial tissues of the digestive tract.

The neu gene (also called NGL, erbB-2, and HER-2) encodes a 185-190 kDa transmembrane glycoprotein, p185neu, which has tyrosine-specific kinase activity and is homologous to but distinct from the epidermal growth factor receptor. The normal expression of neu mRNA and protein has been demonstrated in epithelial tissues of adult animals. Also, activation of the neu oncogene has been implicated in a variety of human adenocarcinomas. In the present study, we examined the expression of the p185neu protein in normal and transformed digestive tract tissues and in a panel of digestive tract-derived cell lines. By immunohistochemistry, strong reactivity was observed in the mucosal epithelium of the stomach, small intestine, and colon of both rodents and humans. In the small intestine, there was prominent p185neu expression by mucosal epithelium of the villus, with little or no staining in the crypts. Prominent expression was observed in the liver parenchyma, the endocrine and exocrine portions of the pancreas, and in the salivary gland. Immunoreactive p185neu was also demonstrated in fetal human intestinal epithelium. Tissue sections of selected benign and malignant colonic neoplasms were also examined. Immunoreactivity was consistently greater in adenomatous polyps than in adjacent normal colonic epithelium or areas showing malignant degeneration. By radioimmunoprecipitation, there was decreased expression in cell lines derived from more anaplastic colonic tumors. The p185neu protein is expressed widely in normal and transformed epithelial tissues of the digestive tract of the adult rat and human. This finding suggests that p185neu, a putative growth factor receptor, may play a role in the regulation of normal growth and function or in the malignant transformation of these cells.

Animals↗
PubMed1989

The neu (c-erbB-2) oncogene.

The neu gene was first identified in rat tumors that had been induced by the carcinogen ethyl nitrosourea. The human homolog of neu, usually designated c-erbB-2, is located on chromosome 17, q21. It specifies a transmembrane receptor-like phosphoglycoprotein that is closely related to the EGFr (c-erbB-1). The ligand for c-erbB-2 is not known. A significant proportion of adenocarcinomas (especially of the breast, colon, and pancreas) have amplification and/or overexpression of c-erbB-2. The unique qualities associated with the subset of tumors that overexpress c-erbB-2 have not yet been firmly identified.

Adenocarcinoma↗
PubMed1989

Linkage of tyrosine kinase activity with transforming ability of the p185neu oncoprotein.

The neu oncogene encodes a 185 kd glycoprotein (p185neu) with intrinsic tyrosine kinase activity. Sequencing data has demonstrated that oncogenic p185neu differs from c-neu by a single point mutation within the transmembrane region of the glycoprotein. This mutation results in the substitution of a glutamic acid residue for a nonpolar valine residue at amino acid position 664 of the rat neu gene product. Recent studies have demonstrated that this mutation results in specific aggregation of the p185neu oncoprotein mimicking ligand induced dimerization events. The cellular consequences of the aggregated phenotype may include the enzymatic activation of p185neu. We demonstrate that the oncoprotein p185neu possesses higher intrinsic tyrosine kinase activity and that this increase in enzymatic activity is apparent within the plasma membrane, manifesting itself through the increased tyrosine phosphorylation of substrates. Furthermore, the neu oncoprotein itself is also phosphorylated on tyrosine to a higher extent than the proto-oncoprotein. These results strongly link enzymatic activation of p185neu to cellular transformation events. To test directly the effect of p185neu tyrosine kinase activity on cellular transformation we constructed mutant p185neu devoid of ATP binding ability. This mutant protein is expressed at high levels, but is unable to induce the transforming phenotype. The point mutation within the transmembrane region of p185neu mimics aspects of ligand induced activation events including increases in the specific tyrosine kinase activity of the molecule leading to cellular transformation.

Adenosine Triphosphate↗
PubMed1988

Reovirus type 3 and [125I]-iodocyanopindolol bind to distinct domains on the beta-adrenergic like receptor.

Antireceptor antibodies have been developed as a probe to study the cellular receptor for reovirus type 3. Using this probe, a glycoprotein with a molecular weight of 65-67 kilodaltons and a pI of 5.8-6.0 was isolated and identified as the reovirus receptor. This protein was also structurally similar to the affinity-purified beta-adrenergic receptor from calf lung. In this report, we employ [125I]-iodocyanopindolol, a high affinity beta-adrenergic antagonist, to further characterize this protein. We show that R1.1, a murine thymoma cell line, possesses about 2,000 receptors per cell with high affinity for ICYP (kD = 3.3 X 10(-11) M). Competitive inhibition studies suggest that the receptor is of the beta-2 subtype. Solubilized receptor proteins from R1.1 cells bound to the antireceptor antibody were further purified by SDS-PAGE and electroelution from the gel. Five percent of the proteins thus obtained could bind ICYP with high affinity (kD = 1.6 X 10(-10) M). This suggests that the purification procedure produced a collection of forms of this 65- to 67-kilodalton protein, some of which retained the conformation for binding the beta ligands. We also demonstrate that the isolated receptor protein was able to bind ICYP even when the virus binding site was occupied by the anti-idiotype, suggesting that reovirus type 3 and the beta ligands bind to distinct domains on the receptor protein.

Adrenergic beta-Agonists↗
PubMed1988

The antigen-major histocompatibility complex-T cell receptor interaction. A structural analysis.

In summary, we wish to propose that regions on MHC molecules interact with complementary regions on processed peptide antigens, and that the resultant Ag-MHC complex forms a conformation with separate functional regions that are able to interact with similarly complementary areas on T cell receptors. It is the product of these interactions that determines whether a given peptide Ag is capable of binding the MHC molecule, and whether a given Ag-MHC complex is capable of stimulating a particular T cell. As more becomes known about the molecular aspects of MHC-restricted, Ag-specific T cell activation, it will become clear which amino acid residues on the Ag, MHC, and TCR are involved in these interactions.

Animals↗