This report describes the isolation and recombmiant expression of a cDNA clone encoding HER4, the fourth member of the human epidermal growth factor receptor (EGFR) family. The HER4/erbB4 gene encodes a 180-kDa transmembrane tyrosine kinase (HER4/p18WbB) whose extraceilular domain is most similar to the orphan receptor HER3/pl6OerbD3, whereas its cytoplasmic kinase domain exhibits 79% and 77% identity with EGFR and HER2/pj85erbB2, respectively. HER4 is most predominaudy expressed in several breast carcinoma cell lines, and in normal skeletal muscle, heart, pituitary, brain, and cerebellum. In addition, we describe the partial purification of a heparin-binding HER4-stimulatory factor from HepG2 cells. This protein was found to specifically stimulate the intrinsic tyrosine kinase activity of HER4/plW'rbB4 while having no direct effect on the phosphorylation of EGFR, HER2, or HER3. Furthermore, this heparin-binding protein induces phenotypic differentiation, and tyrosine phosphorylation, of a human mammary tumor cell line that overexpresses both HER4 and HER2. These findings suggest that this ligand-receptor interaction may play a role in the growth and differentiation of some normal and transformed cells.
Epidermal growth factor (EGF), transforming growth factor a (TGF-a), and amphiregulin are structurally and functionally related growth regulatory proteins. These secreted polypeptides all bind to the 170-kDa cell-surface EGF receptor, activating its intrinsic kinase activity. However, amphiregulin exhibits different activities than EGF and TGF-a in a number of biological assays. Amphiregulin only partially competes with EGF for binding EGF receptor, and amphiregulin does not induce anchorage-independent growth of normal rat kidney cells (NRK) in the presence of TGF-.8. Amphiregulin also appears to abrogate the stimulatory effect of TGF-a on the growth of several aggressive epithelial carcinomas that overexpress EGF receptor. These findings suggest that amphiregulin may interact with a separate receptor in certain cell types. Here we report the cloning of another member of the human EGF receptor (HER) family of receptor tyrosine kinases, which we have named "HER3/ERRB3." The cDNA was isolated from a human carcinoma cell line, and its 6-kilobase transcript was identified in various human tissues. We have generated peptide-specific antisera that recognizes the 160-kDa HER3 protein when transiently expressed in COS cells. These reagents will allow us to determine whether HER3 binds amphiregulin or other growth regulatory proteins and what role HER3 protein plays in the regulation of cell growth.
The negative regulation of T-or B-cell antigen receptor signaling by CD5 was proposed based on studies of thymocytes and peritoneal B-1a cells from CD5-deficient mice. Here, we show that CD5 is constitutively associated with phosphotyrosine phosphatase activity in Jurkat T cells. CD5 was found associated with the Src homology 2 (SH2) domain containing hematopoietic phosphotyrosine phosphatase SHP-1 in both Jurkat cells and normal phytohemagglutinin-expanded T lymphoblasts. This interaction was increased upon T-cell receptor (TCR)-CD3 cell stimulation. CD5 co-cross-linking with the TCR-CD3 complex down-regulated the TCR-CD3-increased Ca 2؉ mobilization in Jurkat cells. In addition, stimulation of Jurkat cells or normal phytohemagglutinin-expanded T lymphoblasts through TCR-CD3 induced rapid tyrosine phosphorylation of several protein substrates, which was substantially diminished after CD5 cross-linking. The CD5-regulated substrates included CD3, ZAP-70, Syk, and phospholipase C␥l but not the Src family tyrosine kinase p56 lck . By mutation of all four CD5 intracellular tyrosine residues to phenylalanine, we found the membrane-proximal tyrosine at position 378, which is located in an immunoreceptor tyrosine-based inhibitory (ITIM)-like motif, crucial for SHP-1 association. The F378 point mutation ablated both SHP-1 binding and the down-regulating activity of CD5 during TCR-CD3 stimulation. These results suggest a critical role of the CD5 ITIM-like motif, which by binding to SHP-1 mediates the down-regulatory activity of this receptor.CD5 is a 67-kDa cell surface glycoprotein expressed on thymocytes, mature peripheral T cells, and a subpopulation of peritoneal B cells (B-1a cells) which are increased in some autoimmune diseases and are associated with the production of autoantibodies (7). Molecular cloning of mouse and human CD5 (mCD5 and hCD5) (16, 17) revealed that it belongs to the scavenger receptor cysteine-rich (SRCR) family group B, which comprises a group of leukocyte membrane or soluble proteins with one or more domains homologous to the aminoterminal domain of type I macrophage SRCR domain (21). Thus far, 10 members of this group of proteins have been identified: CD5, CD6, WC1, M130, Sp␣, Pema-SREG, Ebnerin, CPR-ductin, hensin, and gallbladder mucin (2).Biochemical studies suggest that CD5 is associated with CD3 in the T-cell receptor (TCR)-CD3 complex and with the B-cell receptor (BCR) complex (6,24,32). Two different ligands for CD5 have been reported: CD72, a 42-kDa type II constitutively expressed glycoprotein on B cells (28, 51); and CD5L, an activation antigen expressed on splenocytes (3). The physiologic roles of CD5-CD72 and CD5-CD5L interactions are not known but would be consistent with a potential T-cell-B-cell cooperation during antibody-mediated immune responses (8).Early in vitro studies of T lymphocytes and thymocytes demonstrated that monoclonal antibodies (MAbs) to CD5 were costimulatory for T-cell proliferation (9, 18, 42). However, in vivo studies showed that CD5 down-modulation by spe...
Oxidoreductases of the thioredoxin superfamily possess the C-X-X-C motif. The redox potentials vary over a wide range for these proteins. A crucial determinant of the redox potential has been attributed to the variation of the X-X dipeptide. Here, we substitute Lys for Gly at the first X of Escherichia coli thioredoxin to investigate how a positive charge would affect the redox potential. The substitution does not affect the protein's redox potential. The equilibrium constant obtained from pairwise reaction between the mutant and wild-type proteins equals 1.1, indicating that the replacement does not significantly affect the thiol-disulfide redox equilibrium. However, the catalytic efficiency of thioredoxin reductase on the G33K mutant decreases approximately 2.8 times compared to that of the wild type. The mutation mainly affects K(m), with little effect on k(cat). The mutation also inhibits thioredoxin's ability to reduce insulin disulfide by approximately one-half. Whether the mutant protein supports the growth of phages T3/7 and f1 was tested. The efficiency of plating (EOP) of T3/7 on the mutant strain decreases 5 times at 37 degrees C and 3 x 10(4) times at 42 degrees C relative to that of the wild-type strain, suggesting that interaction between phage gene 5 protein and thioredoxin is hindered. The mutation also reduces the EOP of phage f1 by 8-fold at 37 degrees C and 1.5-fold at 42 degrees C. The global structure of the mutant protein does not change when studied by CD and fluorescence spectra. Therefore, G33K does not significantly affect the overall structure or redox potential of thioredoxin, but primarily interferes with its interaction with other proteins. Together with the G33D mutation, the overall results show that a charged residue at the first X has a greater influence on the molecular interaction of the protein than the redox potential.
Binding studies with a CD6 immunoglobulin fusion protein (CD6 Rg) resulted in the identification and cloning of a CD6 ligand. This ligand was found to be a member of the immunoglobulin supergene family and was named ALCAM (activated leukocyte cell adhesion molecule). Cell adhesion assays showed that CD6-ALCAM interactions mediate thymocyte-thymic epithelium cell binding. ALCAM is also expressed by activated leukocytes and neurons and may be involved in interactions between T cells and activated leukocytes and between cells of the immune and nervous systems, respectively. Herein we describe the preparation of domain-specific murine CD6 Rg fusion proteins and show that the membrane-proximal SRCR (scavenger receptor cysteine-rich) domain of CD6 contains the ALCAM binding site. We also show that mAbs which bind to this domain preferentially block CD6-ALCAM binding. These results demonstrate that the membrane-proximal SRCR domain of CD6 is necessary for CD6 binding to ALCAM and provide the first direct evidence for the interaction of an SRCR domain with a ligand.
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