A subdomain in the transmembrane domain is necessary for p185neu* activation.

The neu proto-oncogene encodes a protein highly homologous to the epidermal growth factor receptor. The neu protein (p185) has a molecular weight of 185,000 Daltons and, like the EGF receptor, possesses tyrosine kinase activity. neu is activated in chemically induced rat neuro/glioblastomas by substitution of valine 664 with glutamic acid within the transmembrane domain. The activated neu* protein (p185*) has an elevated tyrosine kinase activity and a higher propensity to dimerize, but the mechanism of this activation is still unknown. We have used site-directed mutagenesis to explore the role of specific amino acids within the transmembrane domain in this activation. We found that the lateral position and rotational orientation of the glutamic acid in the transmembrane domain does not correlate with transformation. However, the primary structure in the vicinity of Glu664 plays a significant role in this activation. Our results suggest that the Glu664 activation involves highly specific interactions in the transmembrane domain of p185.     

Multiple independent activations of the neu oncogene by a point mutation altering the transmembrane domain of p185

The neu oncogene, which is frequently activated in neuro- and glioblastomas of BDIX rats, was originally identified in the NIH 3T3 focus-forming assay. cDNA clones of the normal and transforming alleles of neu have been isolated. When these clones are inserted into the expression vector pSV2, they direct the synthesis of p185, the neu gene product. The transforming cDNA clone yields foci when transfected onto a NIH 3T3 monolayer, but the normal cDNA does not. The construction of in vitro recombinants between the normal and transforming cDNAs has allowed the determination of the mutation responsible for the activation of the neu proto-oncogene. A single point mutation changes a valine in the transmembrane domain of the predicted protein product insert to a glutamic acid. The DNAs from four independent cell lines containing activated neu oncogenes contain the identical mutation at this position.     

Conformational changes induced by the transforming amino acid substitution in the transmembrane domain of the neu oncogene-encoded p185 protein

Theneu oncogene is frequently found in certain types of human carcinomas and has been shown to be activated in animal models by nitrosourea-induced mutation. The activating mutation in theneu oncogene results in the substitution of a glutamic acid for a valine at position 664 in the transmembrane domain of the encoded protein product of 185 kda (designated p185), which, on the basis of homology studies, is presumed to be a receptor for an as yet unidentified growth factor. It has been proposed that activating amino acid substitutions in this region of p185 lead to a conformational change in the protein which causes signal transduction via an increase in tyrosine kinase activity in the absence of any external signal. Using conformational energy analysis, we have determined the preferred three-dimensional structures for the transmembrane decapeptide (residues 658–667) of the p185 protein with valine and glutamic acid at the critical position 664. The results indicate that the global minimum energy conformation of the decapeptide from the normal protein with Val at position 664 is an -helix with a sharp bend (CD* conformation at residues 664 and 665) in this region, whereas the global minimum conformation for the decapeptide from the mutant transforming protein with Glu at position 664 assumes an all -helical configuration. Furthermore, the second highest energy conformation for the decapeptide from the normal protein is identical to the global minimum energy conformation for the decapeptide from the transforming protein, providing a possible explanation why overexpression of the normal protein also has a transforming effect. These results suggest there may be a normal and a transforming conformation for theneu-encoded p185 proteins which may explain their differences in transforming activity.     

Vaccination with the extracellular domain of p185 neu prevents mammary tumor development in neu transgenic mice

The HER2/neu oncogene product, p185^{HER2/ neu} , is overexpressed on the surface of many human breast cancers. Strains of transgenic mice have been developed that express the rat neu oncogene in mammary epithelial cells and develop spontaneous mammary tumors that overexpress p185 ^neu . This model provides an ideal system for testing interventions to prevent tumor development. In this study, we immunized neu-transgenic mice with a vaccine consisting of the extracellular domain of p185 ^neu (NeuECD). Immunized mice developed Neu-specific humoral immune responses, as measured by circulating anti-Neu antibodies in their sera, and cellular immune responses, as measured by lymphocyte proliferation to NeuECD in vitro. In addition, the subsequent development of mammary tumors was significantly lower in immunized mice than in controls and vaccine treatment was associated with a significant increase in median survival. Received: 10 September 1998 / Accepted: 17 November 1998     

Deducing the transmembrane domain organization of presenilin-1 in gamma-secretase by cysteine disulfide cross-linking

Gamma-secretase is a founding member of membrane-embedded aspartyl proteases that cleave substrates within transmembrane domains, and this enzyme is an important target for the development of therapeutics for Alzheimer's disease. The structure of gamma-secretase and its precise catalytic mechanism still remain largely unknown. Gamma-secretase is a complex of four integral membrane proteins, with presenilin (PS) as the catalytic component. To gain structural and functional information about the nine-transmembrane domain (TMD) presenilin, we employed a cysteine mutagenesis/disulfide cross-linking approach. Here we report that native Cys92 is close to both Cys410 and Cys419, strongly implying that TMD1 and TMD8 are adjacent to each other. This structural arrangement also suggests that TMD8 is distorted from an ideal helix. Importantly, binding of an active site directed inhibitor, but not a docking site directed inhibitor, reduces the ability of the native cysteine pairs of PS1 to cross-link upon oxidation. These findings suggest that the conserved cysteines of TMD1 and TMD8 contribute to or allosterically interact with the active site of gamma-secretase.     

Oncogenic activation of p185neu stimulates tyrosine phosphorylation in vivo.

p185, the product of the neu/erbB2 proto-oncogene, is oncogenically activated by a point mutation that substitutes glutamic acid for valine in the transmembrane domain of the protein. We have found that the transforming form of p185 differs from its normal counterpart in inducing increased tyrosine phosphorylation of other proteins in vivo and in having a much shorter half-life. These results support the model that the transforming p185 resembles a ligand-activated receptor.     

The extracellular domain of p185/neu is released from the surface of human breast carcinoma cells, SK-BR-3.

The human breast carcinoma cell line SK-BR-3, expresses the neu oncogene product, p185, which is a receptor tyrosine kinase. Using a double monoclonal antibody capture enzyme-linked immunosorbent assay for p185, activity was detected in conditioned media from cultures of SK-BR-3 cells. Two monoclonal antibodies specific for the extracellular domain of p185/neu immunoprecipitated a protein with a molecular mass of approximately 105 kDa. p105 was further shown to compete with p185 for binding to monoclonal antibodies and pulse-chase experiments indicate that it was generated by post-translational processing. Peptide maps showed that p105 and p185 are related polypeptides. Since p105 is close to the predicted size for the extracellular domain of p185/neu, we propose that SK-BR-3 cells specifically process and release this portion of the receptor into the medium. The release of the extracellular domain may have implications in oncogenesis and its detection could prove useful as a cancer diagnostic.     

Conformational changes induced by the transforming amino acid substitution in the transmembrane domain of theneu oncogene-encoded p185 protein

Theneu oncogene is frequently found in certain types of human carcinomas and has been shown to be activated in animal models by nitrosourea-induced mutation. The activating mutation in theneu oncogene results in the substitution of a glutamic acid for a valine at position 664 in the transmembrane domain of the encoded protein product of 185 kda (designated p185), which, on the basis of homology studies, is presumed to be a receptor for an as yet unidentified growth factor. It has been proposed that activating amino acid substitutions in this region of p185 lead to a conformational change in the protein which causes signal transduction via an increase in tyrosine kinase activity in the absence of any external signal. Using conformational energy analysis, we have determined the preferred three-dimensional structures for the transmembrane decapeptide (residues 658–667) of the p185 protein with valine and glutamic acid at the critical position 664. The results indicate that the global minimum energy conformation of the decapeptide from the normal protein with Val at position 664 is an α-helix with a sharp bend (CD* conformation at residues 664 and 665) in this region, whereas the global minimum conformation for the decapeptide from the mutant transforming protein with Glu at position 664 assumes an all α-helical configuration. Furthermore, the second highest energy conformation for the decapeptide from the normal protein is identical to the global minimum energy conformation for the decapeptide from the transforming protein, providing a possible explanation why overexpression of the normal protein also has a transforming effect. These results suggest there may be a normal and a transforming conformation for theneu-encoded p185 proteins which may explain their differences in transforming activity.     

GM3 content modulates the EGF-activated p185c-neu levels, but not those of the constitutively activated oncoprotein p185neu

The functional relationship between ganglioside GM(3) and two tyrosine-kinase receptors, the normal protein p185(c-neu) and the mutant oncogenic protein p185(neu), was examined in HC11 cells and in MG1361 cells, respectively. In the former, p185(c-neu) expression and activation are controlled by EGF addition to the culture medium and by epidermal growth factor receptor (EGFR) activity, whereas the latter express unchangingly high levels of constitutively activated p185(neu). Studies were carried out using (+/-)-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride ([D]-PDMP), which inhibits ganglioside biosynthesis resulting in ganglioside depletion, and addition of exogenous GM(3) to the culture medium. In HC11 cells treated with only [D]-PDMP, p185(c-neu) levels remain similar to control cells, whereas levels of tyrosine-phosphorylated p185(c-neu) increase after treatment with [D]-PDMP in combination with EGF. When exogenous GM(3) is added in combination with [D]-PDMP and EGF, the enhanced phosphorylated-p185(c-neu) returns to control levels. Interestingly, EGFR levels also vary and, analogously to phosphorylated-p185(c-neu), the increase of EGFR content consequent to the [D]-PDMP and EGF addition is reversed by exogenous GM(3). In contrast, the addition of neither [D]-PDMP nor exogenous GM(3) modifies expression and tyrosine-phosphorylation levels of p185(neu) in MG1361 cells. These findings indicate that changes in GM(3) content modulate the tyrosine-phosphorylated p185(c-neu) levels in a reversible manner, but this is not specific for p185(c-neu) because EGFR levels are also modified. Furthermore, these data suggest that GM(3) may play a functional role by affecting the internalisation pathway of p185(c-neu)/EGFR heterodimers, but not of p185(neu) homodimers.     

EGF-stimulated tyrosine phosphorylation of p185neu: a potential model for receptor interactions.

p185neu is a receptor-like protein encoded by the neu/erbB-2 proto-oncogene. This protein is closely related to the epidermal growth factor (EGF) receptor, but does not bind EGF. We report here that incubation of Rat-1 cells with EGF stimulates tyrosine phosphorylation of p185. This effect is specific to EGF since neither platelet derived growth factor (PDGF) nor insulin, which also bind to receptors with ligand-stimulated tyrosine kinase activity, induced tyrosine phosphorylation of p185. The EGF-stimulated tyrosine phosphorylation of p185 and of the EGF receptor occurred with similar kinetics and EGF dose-responses, and both phosphorylations were prevented by down-regulation of the EGF receptor with EGF. Since p185 does not bind EGF, these results suggested that p185 is a substrate for the EGF receptor kinase. Incubation of cells with EGF before lysis stimulated the tyrosine phosphorylation of p185 in immune complexes. This suggested that EGF, acting through the EGF receptor, can regulate the intrinsic kinase activity of p185.     

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.     

基质金属蛋白酶-2和p185HER-2/neu蛋白在卵巢癌中的研究进展

基质金属蛋白酶-2(Matrix Metalloproteinase-2,MMP-2)是基质金属蛋白酶家族的重要成员,能降解明胶蛋白和Ⅳ型、V型胶原,在细胞外基质的降解过程中起着关键作用,能够促进肿瘤细胞发生侵袭和转移。p185HER-2/neu蛋白是一种相对分子质量185×103的跨膜糖蛋白,由HER-2/neu基因编码,属于酪氨酸激酶受体家族,p185HER-2/neu蛋白在人类多种癌症中存在扩增及过量表达,并与肿瘤的侵袭性表型及生存期短密切相关。就基质金属蛋白酶-2和p185HER-2/neu蛋白的生物学特性,与卵巢癌侵袭转移和预后的关系及MMP-2和p185HER-2/neu蛋白的研究情况等予以综述。     

Depolarization induces intersubunit cross-linking in a S4 cysteine mutant of the Shaker potassium channel

Voltage-gated potassium (K(v)) channels are integral membrane proteins, composed of four subunits, each comprising six (S1-S6) transmembrane segments. S1-S4 comprise the voltage-sensing domain, and S5-S6 with the linker P-loop forms the ion conducting pore domain. During activation, S4 undergoes structural rearrangements that lead to the opening of the channel pore and ion conduction. To obtain details of these structural changes we have used the engineered disulfide bridge approach. For this we have introduced the L361C mutation at the extracellular end of S4 of the Shaker K channel and expressed the mutant channel in Xenopus oocytes. When exposed to mild oxidizing conditions (ambient oxygen or copper phenanthroline), Cys-361 formed an intersubunit disulfide bridge as revealed by the appearance of a dimeric band on Western blotting. As a consequence, the mutant channel suffered a significant loss in conductance (measured by two-electrode voltage clamp). Removal of native cysteines failed to prevent the disulfide formation, indicating that Cys-361 forms a disulfide with its counterpart in the neighboring subunit. The effect was voltage-dependent and occurred during channel activation after Cys-361 has been exposed to the extracellular phase. Although the disulfide bridge reduced the maximal conductance, it caused a hyperpolarizing shift in the conductance-voltage relationship and reduced the deactivation kinetics of the channel. The latter two effects suggest stabilization of the open state of the channel. In conclusion, we report that during activation the intersubunit distance between the N-terminal ends of the S4 segments of the L361C mutant Shaker K channel is reduced.     

Destabilization of the transmembrane domain induces misfolding in a phenotypic mutant of cystic fibrosis transmembrane conductance regulator

Two phenotypic missense mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel pore (L346P and R347P in transmembrane (TM) segment 6) involve gain of a proline residue, but only L346P represents a significant loss of segment hydropathy. We show here that, for synthetic peptides corresponding to sequences of CFTR TM6 segments, circular dichroism spectra of wild type and R347P TM6 in membrane mimetic environments are virtually identical, but L346P loses approximately 50% helicity, implying a membrane insertion defect in the latter mutant. A similar defect was observed in the corresponding double-spanning ("hairpin") TM5/6-L346P synthetic peptide. Examination of the biogenesis of CFTR revealed that the full-length protein harboring the L346P mutation is rapidly degraded at the endoplasmic reticulum (ER), whereas the wild type and the R347P protein process normally. Furthermore, a second site mutation (R347I) that restores in vitro membrane insertion and folding of the TM5/6-L346P peptide also rescues the folding and cell surface chloride channel function of full-length L346P CFTR. The correlated in vitro/in vivo results demonstrate that destabilizing local hydrophobic character represents a sufficient signal for marking CFTR as a non-native protein by the ER quality control, with accompanying deleterious consequences to global protein folding events.     

p185, a product of the neu proto-oncogene, is a receptorlike protein associated with tyrosine kinase activity.

The neu oncogene was originally identified in cell lines derived from rat neuroectodermal tumors. neu is related to but distinct from the c-erbB gene, which encodes the epidermal growth factor (EGF) receptor. neu encodes a protein, designated p185, that is serologically related to the EGF receptor. Identification of the normal homolog of p185 encoded by the neu proto-oncogene enabled us to compare the product of the neu proto-oncogene with the mutated version specified by the neu oncogene and with the EGF receptor. The normal form of p185 was structurally similar to its transforming counterpart, indicating that activation of the neu oncogene did not cause major structural alterations in the gene product. Both normal and transforming forms of p185 were associated with tyrosine kinase activity, supporting the idea that normal p185 functions as a growth factor receptor. p185 differed both structurally and functionally from the EGF receptor. p185 and the EGF receptor had distinct electrophoretic mobilities when synthesized under normal culture conditions or in the presence of tunicamycin. EGF did not stimulate increased turnover of p185 and did not bind quantitatively to p185. A number of other growth factors failed to stimulate degradation of p185 or tyrosine phosphorylation of p185 and are therefore unlikely to be ligands for p185.     

Structure prediction of the dimeric neu/ErbB-2 transmembrane domain from multi-nanosecond molecular dynamics simulations

Dimerization of the neu/ErbB-2 receptor tyrosine kinase is a necessary but not a sufficient step for signaling. Despite the efforts expended to identify the molecular interactions responsible for receptor-receptor contacts and particularly those involving the transmembrane domain, structural details are still unknown. In this work, molecular dynamics simulations of the helical transmembrane domain (TM) of neu and ErbB-2 receptors are used to predict their dimer structure both in the wild and oncogenic forms. A global conformational search method, applied to define the best orientations of parallel helices, showed an energetically favorable configuration with the specific mutation site within the interface, common for both the nontransforming and the transforming neu/ErbB-2 TM dimers. Starting from this configuration, a total of 10 simulations, about 1.4 ns each, performed in vacuum, without any constraints, show that the two helices preferentially wrap in left-handed interactions with a packing angle at about 20°. The resulting structures are nonsymmetric and the hydrogen bond network analysis shows that helices experience π local distortions that facilitate inter-helix hydrogen bond interactions and may result in a change in the helix packing, leading to a symmetric interface. For the mutated sequences, we show that the Glu side chain interacts directly with its cognate or with carbonyl groups of the facing backbone. We show that the connectivity between interfacial residues conforms to the knobs-into-holes packing mode of transmembrane helices. The dimeric interface described in our models is discussed with respect to mutagenesis studies. Received: 12 March 1999 / Revised version: 23 August 1999 / Accepted: 23 August 1999     

Inhibition of human NTPDase 2 by modification of an intramembrane cysteine by p-chloromercuriphenylsulfonate and oxidative cross-linking of the transmembrane domains

Human NTPDase 2 is a cell surface integral membrane glycoprotein that is anchored to the membranes by two transmembrane domains while the bulk of the protein containing the active site faces the extracellular milieu. It contains 10 conserved cysteine residues in the extracellular domain that are involved in disulfide bond formation and one free cysteine residue, C26, which is located in the N-terminal transmembrane domain. The human NTPDase 2 activity is inactivated by membrane perturbation that disrupts interaction of the transmembrane domains and is inhibited by p-chloromercuriphenylsulfonate (pCMPS), a sulfhydryl reagent. In this report, we show that C26 is the target of pCMPS modification, since a mutant in which C26 was replaced with a serine was no longer inhibited by pCMPS. Mutants in which cysteine residues are placed in the C-terminal transmembrane domain near the extracellular surface were still modified by pCMPS, but the degree of inhibition of their ATPase activity was lower than that of the wild-type enzyme. Thus, loss of the ATPase activity of human NTPDase 2 in the presence of pCMPS probably results from the disturbance of both transmembrane domain interaction and its active site. Inhibition of human NTPDase 2 activity by pCMPS and membrane perturbation is attenuated when the enzyme is cross-linked by glutaraldehyde. On the other hand, NTPDase 2 dimers formed from oxidative cross-linking of the wild-type enzyme and mutants containing a single cysteine residue in the C-terminal transmembrane domain displayed reduced ATPase activity. A similar reduction in activity was also obtained upon intramolecular disulfide formation in mutants that contain a cysteine residue in each of the two transmembrane domains. These results indicate that the mobility of the transmembrane helices is necessary for maximal catalysis.     

Probing the two-gate mechanism of DNA gyrase using cysteine cross-linking.

Cross-linking a pair of novel cysteine residues on either side of the bottom dimer interface of DNA gyrase blocks catalytic supercoiling. Limited strand passage is allowed, but release of the transported DNA segment (T segment) via opening of the bottom dimer interface is prevented. In contrast, ATP-independent relaxation of negatively supercoiled DNA is completely abolished, suggesting that T-segment entry via the bottom gate is blocked. These findings support a two-gate model for supercoiling by DNA gyrase and suggest that relaxation by gyrase is the reverse of supercoiling. Cross-linking a truncated version of gyrase (A64(2)B2), which lacks the DNA wrapping domains, does not block ATP-dependent relaxation. This indicates that passage of DNA through the bottom dimer interface is not essential for this reaction. The mechanistic implications of these results are discussed.     

Interaction of the neu/p185 and EGF receptor tyrosine kinases: Implications for cellular transformation and tumor therapy

Growth factor receptors such as the epidermal growth factor receptor (EGFR) and the p185^c-neu protein serve vital roles in the transduction of differentiation, developmental, or mitogenic signaling within normal cells. Two methods of analysis suggest that the inappropriately high expression of either protein tyrosine kinase promotes malignant transformation. First, data from in vitro experiments indicate that overexpression of either EGFR or p185^c-neu (or the human homolog c-erbB-2) transforms cell-lines. Second, analysis of primary tumors and tumor cell-lines derived from many epithelial tissues (breast, stomach, ovary, and pancreas) show growth factor receptor gene amplification and elevated protein levels. The physical and functional interaction of p185^c-neu and EGFR leads to the formation of a highly active, heterodimeric tyrosine kinase complex which synergistically activates cellular transformation. Anti-receptor antibodies have shown potential utility for the down modulation of these cell-surface proteins and suppression of the malignant phenotype. Design of organic antibody “mimetics” based on the structure of antireceptor antibodies may provide useful therapies and biological reagents to affect growth factor receptor function.