Xiaolan Qian

Intermolecular association of the p185neu protein and EGF receptor modulates EGF receptor function

We have used cross-linking reagents on cell lines expressing both p185neu and EGFR. The lysates of the cells were precipitated with anti-p185neu or anti-EGFR antibodies. These precipitates included a high molecular weight complex that was identified as an EGFR-p185neu heterodimer. Heterodimerization was found to be induced by exposure to EGR. The EGFR of these cells displayed three affinity states for EGF: low (Kd, approximately 10(-9) M), high (Kd, 10(-9) to 10(-10) M), and very high (Kd, 10(-11) M), as determined by Scatchard analyses. Relatively small levels of EGF had a dramatic biological effect on cells expressing very high affinity EGFR. The very high affinity EGFR disappeared after the cells were treated with anti-p185neu monoclonal antibodies that selectively down-regulated p185neu. EGF and TPA had differential effects on down-modulation of the EGFR in cells that express either one or both species of receptor proteins.

Inhibition of a naturally occurring EGFR oncoprotein by the p185neu ectodomain : implications for subdomain contributions to receptor assembly

Mutant Epidermal Growth Factor Receptor (EGFR) oncoproteins lacking most of subdomains I and II of the extracellular region, a deletion which includes most of the first of two cysteine-rich sequences, have been observed in multiple human epithelial tumors, including malignant gliomas. These EGFR oncoproteins, designated ΔEGFR or EGFRvIII, confer increased tumorigenicity in vivo and are often coexpressed with full-length EGFR in human tumors. We have expressed an ectodomain-derived, carboxyl-terminal deletion mutant of the p185neu oncogene (T691stop) in human glioblastoma cells coexpressing endogenous EGFR and activated ΔEGFR oncoproteins. The p185neu ectodomain-derived mutant forms heterodimers with ΔEGFR proteins and reduces the phosphotyrosine content and kinase activity of ΔEGFR monomers. As a consequence of T691stop neu expression and surface localization, cell proliferation in conditions of full growth and reduced serum and anchorage-independent growth in soft agar was reduced in glioblastoma cells expressing either endogenous EGFR alone or coexpressing EGFR and elevated levels of ΔEGFRs. T691stop neu mutant receptors abrogate the dramatic growth advantage conferred by ΔEGFR in vivo, suggesting that physical associations primarily between subdomains III and IV of the p185neu and EGFR ectodomains are sufficient to modulate signaling from activated EGFR complexes. Receptor-based inhibitory strategies exploit the thermodynamic preference for erbB ectodomains to heterodimerize, thereby creating erbB receptor assemblies which are defective in signaling and do not internalize. Pharmaceuticals which mimic the p185neu ectodomain may therefore have important therapeutic applications in advanced human malignancies expressing erbB receptors.

Domain-specific Interactions between the p185 neu and Epidermal Growth Factor Receptor Kinases Determine Differential Signaling Outcomes

We expressed the epidermal growth factor receptor (EGFR) along with mutant p185 neu proteins containing the rat transmembrane point mutation. The work concerned the study of the contributions made by various p185 neu subdomains to signaling induced by a heterodimeric ErbB complex. Co-expression of full-length EGFR and oncogenic p185 neu receptors resulted in an increased EGF-induced phosphotyrosine content of p185 neu , increased cell proliferation to limiting concentrations of EGF, and increases in both EGF-induced MAPK and phosphatidylinositol 3-kinase (PI 3-kinase) activation. Intracellular domain-deleted p185 neu receptors (T691stop neu) were able to associate with full-length EGFR, but induced antagonistic effects on EGF-dependent EGF receptor down-regulation, cell proliferation, and activation of MAPK and PI 3-kinase pathways. Ectodomain-deleted p185 neu proteins (TΔ5) were unable to physically associate with EGFR, and extracellular domain-deleted p185 neu forms failed to augment activation of MAPK and PI 3-kinase in response to EGF. Association of EGFR with a carboxyl-terminally truncated p185 neu mutant (TAPstop) form did not increase transforming efficiency and phosphotyrosine content of the TAPstop species, and proliferation of EGFR·TAPstop-co-expressing cells in response to EGF was similar to cells containing EGFR only. Thus, neither cooperative nor inhibitory effects were observed in cell lines co-expressing either TΔ5 or TAPstop mutant proteins. Unlike the formation of potent homodimer assemblies composed of oncogenic p185 neu , the induction of signaling from p185 neu ·EGFR heteroreceptor assemblies requires the ectodomain for ligand-dependent physical association and intracellular domain contacts for efficient intermolecular kinase activation.

Sustained mitogen-activated protein kinase activation is induced by transforming erbB receptor complexes.

We used a genetic approach to characterize features of mitogen-activated protein kinase (MAPK) activation occurring as a consequence of expression of distinct erbB receptor combinations in transformed human cells. Kinase-deficient erbB proteins reduced epidermal growth factor (EGF)-induced tyrosine phosphorylation of endogenous Shc proteins and also reduced immediate and sustained EGF-induced ERK MAPK activities in human glioblastoma cells, although basal ERK MAPK activities were unaffected. Basal and EGF-induced JNK and p38 MAPK kinase activities were equivalent in parental cancer cells and EGFR-inhibited subclones. When ectopically overexpressed in murine fibroblasts and human glioblastoma cells, a constitutively activated human EGF receptor oncoprotein (deltaEGFR) induced EGF-independent elevation of basal ERK MAPK activity. Basal JNK MAPK kinase activity was also specifically induced by deltaEGFR, which correlated with increased phosphorylation of a 54-kDa JNK2 protein observed in deltaEGFR-containing cells. The JNK activities in response to DNA damage were comparably increased in cells containing wildtype EGFR or deltaEGFR. Consistent with the notion that transforming erbB complexes induce sustained and unregulated MAPK activities, coexpression of p185(neu) and EGFR proteins to levels sufficient to transform murine fibroblasts also resulted in prolonged EGF-induced ERK in vitro kinase activation. Transforming erbB complexes, including EGFR homodimers, deltaEGFR homodimers, and p185(neu)/EGFR heterodimers, appear to induce sustained, unattenuated activation of MAPK activities that may contribute to increased transformation and resistance to apoptosis in primary human glioblastoma cells.

Absence of autophosphorylation site Y882 in the p185neu oncogene product correlates with a reduction of transforming potential.

Autophosphorylation of type I receptor tyrosine kinases (RTKs) comprises one step in the signaling events mediated by erbB receptors such as p185neu and EGFR. Previous analysis of p185neu has indicated that there are at least five tyrosine autophosphorylation sites, Y882, Y1028, Y1143, Y1226/7 and Y1253, of which Y882 might be important because of its location in the kinase activity domain. We have specifically analysed the effect of a Y882F (phenylalanine substituted for tyrosine at position 882) mutation in the enzymatic active domain. We also deleted the carboxyl terminal 122 amino acids which contained three other autophosphorylation sites (TAPstop) and combined mutants of that deletion with Y882F (Y882F/APstop). Both in vitro and in vivo transformation assays showed that substitution of tyrosine882 by phenylalanine significantly decreased the transforming potential of activated, oncogenic p185neu, although no significant difference in the total phosphotyrosine levels of the mutant proteins were observed. To analyse mitogenic signaling in response to ligand, the intracellular domains of p185neu and Y882F were fused with the extracellular domain of the EGF receptor. The proliferation of cells expressing these chimeric receptors was EGF-dependent, and cells expressing EGFR/Y882F chimeric receptors were less responsive to EGF stimulation than those expressing EGFR/neu receptors. In vitro kinase assays demonstrated that abolishing the autophosphorylation site Y882 diminished the enzymatic tyrosine kinase activity of p185neu. These studies, taken together with the phenotypic inhibition observed with cells expressing Y882F, suggest that the tyrosine882 residue may be important for p185neu-mediated transformation by affecting the enzymatic kinase function of the p185neu receptor.

Identification of a 140 kDa protein of rat presynaptic terminal membranes encompassing the active zones

A polyclonal antiserum raised against the carboxy-terminal 17 amino acids of the rat p185c-neu (anct) reacted with a 140 kDa polypeptide in membranes of synaptosome fractions from neocortex and hippocampus of 11-day-old and adult rats. The same antiserum reacted with a 185 kDa polypeptide in microsome membranes from rat pheochromocytoma cells (PC12). By light microscopic immunocytochemistry, the anct antibodies against the 140 kDa protein were localized in the neuropile of brain, cerebellum and spinal cord of 11-day-old and adult rats. Especially prominent staining was obtained in the CA2-CA3 zones of the hippocampus, and in the substantia gelatinosa in the spinal cord. The finely granular and diffuse pattern of the immunostain was consistent with synaptic localizations. Interestingly, antibodies against the entire endodomain of p185c-neu (a-Bacneu) were localized in granular structures, probably representing axo-somatic and axo-dendritic synapses, on a subset of pyramidal neurons of the CA3 zone. By immunoelectron terminals in the giant mossy fiber type in the CA3 and CA4 regions. The immunolocalization of the anct antibodies was restricted in segments of the presynaptic membrane facing the synaptic cleft which include the active zone. The identify and function of the 140 kDa membrane protein of rat brain presynaptic terminals, detected by the anct antibodies, is unknown. The 140 kDa protein may be related to p185c-neu, a tyrosine kinase, or to other known or unknown kinases.