[Show abstract][Hide abstract] ABSTRACT: Human colorectal cancer (CRC) cell lines are used widely to investigate tumor biology, experimental therapy and biomarkers. However, to what extent these established cell lines represent and maintain the genetic diversity of primary cancers is uncertain. In this study, we profiled 70 CRC cell lines for mutations and DNA copy-number by whole-exome sequencing and SNP microarray analyses, respectively. Gene expression was defined using RNA-Seq. Cell line data was compared to that published for primary CRCs in the Cancer Genome Atlas. Notably, we found that exome mutation and DNA copy-number spectra in CRC cell lines closely resembled those seen in primary colorectal tumors. Similarities included the presence of two hypermutation phenotypes, as defined by signatures for defective DNA mismatch repair and DNA polymerase ε (POLE) proofreading deficiency, along with concordant mutation profiles in the broadly altered WNT, MAPK, PI3K, TGFβ and p53 pathways. Further, we documented mutations enriched in genes involved in chromatin remodeling (ARID1A, CHD6, SRCAP) and histone methylation or acetylation (ASH1L, EP300, EP400, MLL2, MLL3, PRDM2, TRRAP). Chromosomal instability was prevalent in non-hypermutated cases, with similar patterns of chromosomal gains and losses. While paired cell lines derived from the same tumor exhibited considerable mutation and DNA copy-number differences, in silico simulations suggest that these differences mainly reflected a pre-existing heterogeneity in the tumor cells. In conclusion, our results establish that human CRC lines are representative of the main subtypes of primary tumors at the genomic level, further validating their utility as tools to investigate CRC biology and drug responses.
[Show abstract][Hide abstract] ABSTRACT: Most colon cancers arise from somatic mutations in the tumor suppressor gene APC (adenomatous polyposis coli), and these mutations cause constitutive activation of the Wnt-to-β-catenin pathway in the intestinal epithelium. Because Wnt-β-catenin signaling is required for homeostasis and regeneration of the adult intestinal epithelium, therapeutic targeting of this pathway is challenging. We found that genetic activation of the cytokine-stimulated pathway mediated by the receptor gp130, the associated Jak (Janus kinase) kinases, and the transcription factor Stat3 (signal transducer and activator of transcription 3) was required for intestinal regeneration in response to irradiation-induced damage in wild-type mice and for tumorigenesis in Apc-mutant mice. Systemic pharmacological or partial genetic inhibition of gp130-Jak-Stat3 signaling suppressed intestinal regeneration, the growth of tumors in Apc-mutant mice, and the growth of colon cancer xenografts. The growth of Apc-mutant tumors depended on gp130-Jak-Stat3 signaling for induction of the polycomb repressor Bmi-1, and the associated repression of genes encoding the cell cycle inhibitors p16 and p21. However, suppression of gp130-Jak-Stat3 signaling did not affect Wnt-β-catenin signaling or homeostasis in the intestine. Thus, these data not only suggest a molecular mechanism for how the gp130-Jak-Stat3 pathway can promote cancer but also provide a rationale for therapeutic inhibition of Jak in colon cancer.
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVES:Microsatellite instability (MSI) is an established marker of good prognosis in colorectal cancer (CRC). Chromosomal instability (CIN) is strongly negatively associated with MSI and has been shown to be a marker of poor prognosis in a small number of studies. However, a substantial group of "double-negative" (MSI-/CIN-) CRCs exists. The prognosis of these patients is unclear. Furthermore, MSI and CIN are each associated with specific molecular changes, such as mutations in KRAS and BRAF, that have been associated with prognosis. It is not known which of MSI, CIN, and the specific gene mutations are primary predictors of survival.METHODS:We evaluated the prognostic value (disease-free survival, DFS) of CIN, MSI, mutations in KRAS, NRAS, BRAF, PIK3CA, FBXW7, and TP53, and chromosome 18q loss-of-heterozygosity (LOH) in 822 patients from the VICTOR trial of stage II/III CRC. We followed up promising associations in an Australian community-based cohort (N=375).RESULTS:In the VICTOR patients, no specific mutation was associated with DFS, but individually MSI and CIN showed significant associations after adjusting for stage, age, gender, tumor location, and therapy. A combined analysis of the VICTOR and community-based cohorts showed that MSI and CIN were independent predictors of DFS (for MSI, hazard ratio (HR)=0.58, 95% confidence interval (CI) 0.36-0.93, and P=0.021; for CIN, HR=1.54, 95% CI 1.14-2.08, and P=0.005), and joint CIN/MSI testing significantly improved the prognostic prediction of MSI alone (P=0.028). Higher levels of CIN were monotonically associated with progressively poorer DFS, and a semi-quantitative measure of CIN was a better predictor of outcome than a simple CIN+/- variable. All measures of CIN predicted DFS better than the recently described Watanabe LOH ratio.CONCLUSIONS:MSI and CIN are independent predictors of DFS for stage II/III CRC. Prognostic molecular tests for CRC relapse should currently use MSI and a quantitative measure of CIN rather than specific gene mutations.Am J Gastroenterol advance online publication, 17 September 2013; doi:10.1038/ajg.2013.292.
The American Journal of Gastroenterology 09/2013; · 9.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: PURPOSE: PIK3CA and PTEN mutations are prevalent in colorectal cancer (CRC), and potential markers of response to MEK inhibitors and anti-EGFR antibody therapy. Relationships between PI3K pathway mutation, clinicopathological characteristics, molecular features and prognosis remain controversial. EXPERIMENTAL DESIGN: 1,093 stage I-IV CRCs were screened for PIK3CA (exons 9, 20), KRAS (codons 12-13), BRAF (codon 600) mutations and microsatellite instability (MSI). PTEN (exons 3-8) and CpG island methylator phenotype (CIMP) status were determined in 744 and 489 cases. PIK3CA data was integrated with 17 previous reports (n=5,590). RESULTS: PIK3CA and PTEN mutations were identified in 11.9% and 5.8% of CRCs. PTEN mutation was associated with proximal tumors, mucinous histology, MSI-high, CIMP-high and BRAF mutation (P<0.02). PIK3CA mutation was related to older age, proximal tumors, mucinous histology and KRAS mutation (P<0.04). In integrated cohort analysis, PIK3CA exon 9 and 20 mutations were overrepresented in proximal, CIMP-low and KRAS mutated cancers (P=0.011). Comparing PIK3CA exonic mutants, exon 20 mutation was associated with MSI-high, CIMP-high and BRAF mutation, and exon 9 mutation was associated with KRAS mutation (P=0.027). Disease-free survival for stage II/III CRCs did not differ by PI3K pathway status. CONCLUSIONS: PI3K pathway mutation is prominent in proximal colon cancers, with PIK3CA exon 20 and PTEN mutations associated with features of the sessile serrated pathway (MSI-high/CIMP-high/BRAF(mut)), and PIK3CA exon 9 (and to a lesser extent exon 20) mutation associated with features of the traditional serrated pathway (CIMP-low/KRAS(mut)) of tumorigenesis. Our data highlight the PI3K pathway as a therapeutic target in distinct CRC subtypes.
Clinical Cancer Research 04/2013; · 7.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Activation of the canonical transforming growth factor-β (TGF-β) signaling pathway provides growth inhibitory signals in the normal intestinal epithelium. Colorectal cancers (CRCs) frequently harbor somatic mutations in the pathway members, TGFBR2 and SMAD4, but to what extent mutations in SMAD2 or SMAD3 contribute to tumorigenesis is unclear. A cohort of 744 primary CRCs and 36 CRC cell lines were sequenced for SMAD4, SMAD2 and SMAD3, and analyzed for allelic loss by SNP microarray analysis. Mutation spectra were compared between the genes, the pathogenicity of mutations was assessed and relationships with clinicopathological features were examined. The prevalence of SMAD4, SMAD2 and SMAD3 mutations in sporadic CRC was 8.6% (64/744), 3.4% (25/744) and 4.3% (32/744), respectively. A significant overrepresentation of two genetic hits was detected for SMAD4 and SMAD3, consistent with these genes acting as tumor suppressors. SMAD4 mutations were associated with mucinous histology. The mutation spectra of SMAD2 and SMAD3 were highly similar to that of SMAD4, both in mutation type and location within the encoded proteins. In silico analyses suggested the majority of the mutations were pathogenic, with most missense changes predicted to reduce protein stability or hinder SMAD complex formation. The latter altered interface residues or disrupted the phosphorylation regulated Ser-Ser-X-Ser motifs within SMAD2 and SMAD3. Functional analyses of selected mutations demonstrated reductions in SMAD3 transcriptional activity and SMAD2-SMAD4 complex formation. Joint biallelic hits in SMAD2 and SMAD3 were overrepresented and mutually exclusive to SMAD4 mutation, underlining the critical roles of these three proteins within the TGF-β signaling pathway.
[Show abstract][Hide abstract] ABSTRACT: Biallelic protein-truncating mutations in the adenomatous polyposis coli (APC) gene are prevalent in sporadic colorectal cancer (CRC). Mutations may not be fully inactivating, instead producing WNT/β-catenin signalling levels 'just-right' for tumourigenesis. However, the spectrum of optimal APC genotypes accounting for both hits, and the influence of clinicopathological features on genotype selection remain undefined. We analysed 630 sporadic CRCs for APC mutations and loss of heterozygosity (LOH) using sequencing and single-nucleotide polymorphism microarrays, respectively. Truncating APC mutations and/or LOH were detected in 75% of CRCs. Most truncating mutations occurred within a mutation cluster region (MCR; codons 1282-1581) leaving 1-3 intact 20 amino-acid repeats (20AARs) and abolishing all Ser-Ala-Met-Pro (SAMP) repeats. Cancers commonly had one MCR mutation plus either LOH or another mutation 5' to the MCR. LOH was associated with mutations leaving 1 intact 20AAR. MCR mutations leaving 1 vs 2-3 intact 20AARs were associated with 5' mutations disrupting or leaving intact the armadillo-repeat domain, respectively. Cancers with three hits had an over-representation of mutations upstream of codon 184, in the alternatively spliced region of exon 9, and 3' to the MCR. Microsatellite unstable cancers showed hyper-mutation at MCR mono- and di-nucleotide repeats, leaving 2-3 intact 20AARs. Proximal and distal cancers exhibited different preferred APC genotypes, leaving a total of 2 or 3 and 0 to 2 intact 20AARs, respectively. In conclusion, APC genotypes in sporadic CRCs demonstrate 'fine-tuned' interdependence of hits by type and location, consistent with selection for particular residual levels of WNT/β-catenin signalling, with different 'optimal' thresholds for proximal and distal cancers.Oncogene advance online publication, 22 October 2012; doi:10.1038/onc.2012.486.
[Show abstract][Hide abstract] ABSTRACT: Studies employing mouse models have identified crypt base and position +4 cells as strong candidates for intestinal epithelial stem cells. Equivalent cell populations are thought to exist in the human intestine; however robust and specific protein markers are lacking. Here, we show that in the human small and large intestine, PHLDA1 is expressed in discrete crypt base and some position +4 cells. In small adenomas, PHLDA1 was expressed in a subset of undifferentiated and predominantly Ki-67-negative neoplastic cells, suggesting that a basic hierarchy of differentiation is retained in early tumorigenesis. In large adenomas, carcinomas, and metastases PHLDA1 expression became widespread, with increased expression and nuclear localization at invasive margins. siRNA-mediated suppression of PHLDA1 in colon cancer cells inhibited migration and anchorage-independent growth in vitro and tumor growth in vivo. The integrins ITGA2 and ITGA6 were downregulated in response to PHLDA1 suppression, and accordingly cell adhesion to laminin and collagen was significantly reduced. We conclude that PHLDA1 is a putative epithelial stem cell marker in the human small and large intestine and contributes to migration and proliferation in colon cancer cells.
Cancer Research 05/2011; 71(10):3709-19. · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BRAF(V600E) mutations are found in 10% of colorectal cancers (CRCs). The low frequency of this mutation therefore makes it a challenging target for drug development, unless subsets of patients with higher rates of BRAF(V600E) can be defined. Knowledge of the concordance between primary-metastasis pairs and the impact of BRAF(V600E) on outcome would also assist in optimal drug development. We selected primary CRCs from 525 patients (stages I-IV) evenly matched for age (<70 and ≥70), gender and tumor location (right, left and rectum), and 81 primary-metastasis pairs. BRAF(V600E), KRAS mutation and microsatellite instability (MSI) were determined and correlated with clinical features and patient outcomes. In multivariate analyses, increasing patient age (p = 0.04), female gender (p = 0.0005) and right-sided tumor location (p < 0.0001) were independently associated with BRAF(V600E). The prevalence of BRAF(V600E) was considerably higher in older (age > 70) females with KRAS wild-type right-sided colon cancers (50%) compared to the unselected cohort (10%). BRAF(V600E) was associated with inferior overall survival in metastatic CRC (HR = 2.02; 95% CI 1.26-3.26), particularly evident in patients treated with chemotherapy, and is independent of MSI status. BRAF status was concordant in all primary tumors and matched metastases (79 wild-type pairs and two mutant pairs). Clinicopathological and molecular features can identify CRC patients with a higher prevalence of BRAF(V600E). Patients with BRAF(V600E) wild-type primary tumor do not appear to acquire the mutation in their metastases, and BRAF(V600E) is associated with poorer outcomes in metastatic patients. Our findings are timely and will help inform the rational development of BRAF(V600E) inhibitors in CRC.
International Journal of Cancer 05/2011; 128(9):2075-84. · 6.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Oncogene mutations contribute to colorectal cancer development. We searched for differences in oncogene mutation profiles between colorectal cancer metastases from different sites and evaluated these as markers for site of relapse.
One hundred colorectal cancer metastases were screened for mutations in 19 oncogenes, and further 61 metastases and 87 matched primary cancers were analyzed for genes with identified mutations. Mutation prevalence was compared between (a) metastases from liver (n = 65), lung (n = 50), and brain (n = 46), (b) metastases and matched primary cancers, and (c) metastases and an independent cohort of primary cancers (n = 604). Mutations differing between metastasis sites were evaluated as markers for site of relapse in 859 patients from the VICTOR trial.
In colorectal cancer metastases, mutations were detected in 4 of 19 oncogenes: BRAF (3.1%), KRAS (48.4%), NRAS (6.2%), and PIK3CA (16.1%). KRAS mutation prevalence was significantly higher in lung (62.0%) and brain (56.5%) than in liver metastases (32.3%; P = 0.003). Mutation status was highly concordant between primary cancer and metastasis from the same individual. Compared with independent primary cancers, KRAS mutations were more common in lung and brain metastases (P < 0.005), but similar in liver metastases. Correspondingly, KRAS mutation was associated with lung relapse (HR = 2.1; 95% CI, 1.2 to 3.5, P = 0.007) but not liver relapse in patients from the VICTOR trial.
KRAS mutation seems to be associated with metastasis in specific sites, lung and brain, in colorectal cancer patients. Our data highlight the potential of somatic mutations for informing surveillance strategies.
Clinical Cancer Research 02/2011; 17(5):1122-30. · 7.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK) are endogenous inhibitors constraining the activity of the oncogenic Src-family kinases (SFKs) in cells. Both kinases suppress SFKs by selectively phosphorylating their consensus C-terminal regulatory tyrosine. In addition to phosphorylation, CHK can suppress SFKs by a unique non-catalytic inhibitory mechanism that involves tight binding of CHK to SFKs to form stable complexes. In this review, we discuss how allosteric regulators, phosphorylation, and inter-domain interactions interplay to govern the activity of CSK and CHK and their ability to inhibit SFKs. In particular, based upon the published results of structural and biochemical analysis of CSK and CHK, we attempt to chart the allosteric networks in CSK and CHK that govern their catalysis and ability to inhibit SFKs. We also discuss how the published three-dimensional structure of CSK complexed with an SFK member sheds light on the structural basis of substrate recognition by protein kinases.
[Show abstract][Hide abstract] ABSTRACT: We describe a statistical method for the characterization of genomic aberrations in single nucleotide polymorphism microarray data acquired from cancer genomes. Our approach allows us to model the joint effect of polyploidy, normal DNA contamination and intra-tumour heterogeneity within a single unified Bayesian framework. We demonstrate the efficacy of our method on numerous datasets including laboratory generated mixtures of normal-cancer cell lines and real primary tumours.
[Show abstract][Hide abstract] ABSTRACT: Frameshift mutations in microsatellite instability high (MSI-High) colorectal cancers are a potential source of targetable neo-antigens. Many nonsense transcripts are subject to rapid degradation due to nonsense-mediated decay (NMD), but nonsense transcripts with a cMS in the last exon or near the last exon-exon junction have intrinsic resistance to nonsense-mediated decay (NMD). NMD-resistant transcripts are therefore a likely source of expressed mutant proteins in MSI-High tumours.
Using antibodies to the conserved N-termini of predicted mutant proteins, we analysed MSI-High colorectal cancer cell lines for examples of naturally expressed mutant proteins arising from frameshift mutations in coding microsatellites (cMS) by immunoprecipitation and Western Blot experiments. Detected mutant protein bands from NMD-resistant transcripts were further validated by gene-specific short-interfering RNA (siRNA) knockdown. A genome-wide search was performed to identify cMS-containing genes likely to generate NMD-resistant transcripts that could encode for antigenic expressed mutant proteins in MSI-High colon cancers. These genes were screened for cMS mutations in the MSI-High colon cancer cell lines.
Mutant protein bands of expected molecular weight were detected in mutated MSI-High cell lines for NMD-resistant transcripts (CREBBP, EP300, TTK), but not NMD-sensitive transcripts (BAX, CASP5, MSH3). Expression of the mutant CREBBP and EP300 proteins was confirmed by siRNA knockdown. Five cMS-bearing genes identified from the genome-wide search and without existing mutation data (SFRS12IP1, MED8, ASXL1, FBXL3 and RGS12) were found to be mutated in at least 5 of 11 (45%) of the MSI-High cell lines tested.
NMD-resistant transcripts can give rise to expressed mutant proteins in MSI-High colon cancer cells. If commonly expressed in primary MSI-High colon cancers, MSI-derived mutant proteins could be useful as cancer specific immunological targets in a vaccine targeting MSI-High colonic tumours.
PLoS ONE 01/2010; 5(12):e16012. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: PURPOSE: Colorectal cancer prognosis is currently predicted from pathologic staging, providing limited discrimination for Dukes stage B and C disease. Additional markers for outcome are required to help guide therapy selection for individual patients. EXPERIMENTAL DESIGN: A multisite single-platform microarray study was done on 553 colorectal cancers. Gene expression changes were identified between stage A and D tumors (three training sets) and assessed as a prognosis signature in stage B and C tumors (independent test and external validation sets). RESULTS: One hundred twenty-eight genes showed reproducible expression changes between three sets of stage A and D cancers. Using consistent genes, stage B and C cancers clustered into two groups resembling early-stage and metastatic tumors. A Prediction Analysis of Microarray algorithm was developed to classify individual intermediate-stage cancers into stage A-like/good prognosis or stage D-like/poor prognosis types. For stage B patients, the treatment adjusted hazard ratio for 6-year recurrence in individuals with stage D-like cancers was 10.3 (95% confidence interval, 1.3-80.0; P = 0.011). For stage C patients, the adjusted hazard ratio was 2.9 (95% confidence interval, 1.1-7.6; P = 0.016). Similar results were obtained for an external set of stage B and C patients. The prognosis signature was enriched for downregulated immune response genes and upregulated cell signaling and extracellular matrix genes. Accordingly, sparse tumor infiltration with mononuclear chronic inflammatory cells was associated with poor outcome in independent patients. CONCLUSIONS: Metastasis-associated gene expression changes can be used to refine traditional outcome prediction, providing a rational approach for tailoring treatments to subsets of patients. (Clin Cancer Res 2009;15(24):7642-51).
Clinical Cancer Research 12/2009; 15(24):7642-7651. · 7.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract The kinetics, binding equilibria and stoichiometry of the interaction between epidermal growth factor and the soluble extracellular domain of the epidermal growth factor receptor (sEGFR), produced in CHO cells using a bioreactor, have been studied by three methods: analytical ultracentrifugation, biosensor analysis using surface plasmon resonance detection (BIAcore 2000) and fluorescence anisotropy. These studies were performed with an sEGFR preparation purified in the absence of detergent using a mild two step chromatographic procedure employing anion exchange and size exclusion HPLC. The fluorescence anisotropy and analytical ultracentrifugation data indicated a 1:1 molar binding ratio between EGF and the sEGFR. Analytical ultracentrifugation further indicated that the complex comprised 2EGF: 2sEGFR, consistent with the model proposed recently by Lemmon et al. (1997). Global analysis of the BIAcore binding data showed that a simple Langmuirian interaction does not adequately describe the EGF: sEGFR interaction and that more complex interaction mechanisms are operative. Furthermore, analysis of solution binding data using either fluorescence anisotropy or the biosensor, to determine directly the concentration of free sEGFR in solution competition experiments, yielded Scatchard plots which were biphasic and Hill coefficients of less than unity. Taken together our data indicate that in solution there are two sEGFR populations; one which binds EGF with a KD of 2–20 nM and the other with a KD of 400–550 nM.
[Show abstract][Hide abstract] ABSTRACT: About 15% of colorectal cancers harbor microsatellite instability (MSI). MSI-associated gene expression changes have been identified in colorectal cancers, but little overlap exists between signatures hindering an assessment of overall consistency. Little is known about the causes and downstream effects of differential gene expression.
DNA microarray data on 89 MSI and 140 microsatellite-stable (MSS) colorectal cancers from this study and 58 MSI and 77 MSS cases from three published reports were randomly divided into test and training sets. MSI-associated gene expression changes were assessed for cross-study consistency using training samples and validated as MSI classifier using test samples. Differences in biological pathways were identified by functional category analysis. Causation of differential gene expression was investigated by comparison to DNA copy-number data.
MSI-associated gene expression changes in colorectal cancers were found to be highly consistent across multiple studies of primary tumors and cancer cell lines from patients of different ethnicities (P < 0.001). Clustering based on consistent changes separated additional test cases by MSI status, and classification of individual samples predicted MSI status with a sensitivity of 96% and specificity of 85%. Genes associated with immune response were up-regulated in MSI cancers, whereas genes associated with cell-cell adhesion, ion binding, and regulation of metabolism were down-regulated. Differential gene expression was shown to reflect systematic differences in DNA copy-number aberrations between MSI and MSS tumors (P < 0.001).
Our results show cross-study consistency of MSI-associated gene expression changes in colorectal cancers. DNA copy-number alterations partly cause the differences in gene expression between MSI and MSS cancers.
Clinical Cancer Research 12/2008; 14(24):8061-9. · 7.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent crystallographic data on the isolated extracellular domain of the epidermal growth factor receptor (EGFR) have suggested a model for its activation by ligand. We have tested this model in the context of the full-length EGFR displayed at the cell surface, by introducing mutations in two regions (CR1 and CR2) of the extracellular domain thought to be critical for regulation of receptor activation. Mutations in the CR1 and CR2 domains have opposing effects on ligand binding affinity, receptor dimerization, tyrosine kinase activation, and signaling competence. Tyr(246) is a critical residue in the CR1 loop, which is implicated in the positioning and stabilization of the receptor dimer interface after ligand binding; mutations of Tyr(246) impair or abolish receptor function. Mutations in CR2, which weaken the interaction that restricts the receptor to the tethered (inactive) state, enhance responsiveness to EGF by increasing affinity for the ligand. However, weakening of the CR1/CR2 interaction does not result in spontaneous activation of the receptors' kinase. We have used an antibody (mAb 806), which recognizes a transition state of the EGF receptor between the negatively constrained, tethered state and the fully active back-to-back dimer conformation, to follow conformational changes in the wild-type and mutant EGF receptors after ligand binding. Our results suggest that EGFR on the cell surface can be untethered, but this form is inactive; thus, untethering of the receptor is not sufficient for activation, and ligand binding is essential for the correct positioning of the two receptor subunits to achieve kinase activation.
Journal of Biological Chemistry 06/2004; 279(21):22387-98. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The epidermal growth factor (EGF) receptor (EGFR) is one of four homologous transmembrane proteins that mediate the actions of a family of growth factors including EGF, transforming growth factor-alpha, and the neuregulins. We review the structure and function of the EGFR, from ligand binding to the initiation of intracellular signalling pathways that lead to changes in the biochemical state of the cell. The recent crystal structures of different domains from several members of the EGFR family have challenged our concepts of these processes.
Experimental Cell Research 04/2003; 284(1):31-53. · 3.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: ErbB2 does not bind ligand, yet appears to be the major signaling partner for other ErbB receptors by forming heteromeric complexes with ErbB1, ErbB3, or ErbB4. The crystal structure of residues 1-509 of ErbB2 at 2.5 A resolution reveals an activated conformation similar to that of the EGFR when complexed with ligand and very different from that seen in the unactivated forms of ErbB3 or EGFR. The structure explains the inability of ErbB2 to bind known ligands and suggests why ErbB2 fails to form homodimers. Together, the data suggest a model in which ErbB2 is already in the activated conformation and ready to interact with other ligand-activated ErbB receptors.
[Show abstract][Hide abstract] ABSTRACT: We report the crystal structure, at 2.5 A resolution, of a truncated human EGFR ectodomain bound to TGFalpha. TGFalpha interacts with both L1 and L2 domains of EGFR, making many main chain contacts with L1 and interacting with L2 via key conserved residues. The results indicate how EGFR family members can bind a family of highly variable ligands. In the 2:2 TGFalpha:sEGFR501 complex, each ligand interacts with only one receptor molecule. There are two types of dimers in the asymmetric unit: a head-to-head dimer involving contacts between the L1 and L2 domains and a back-to-back dimer dominated by interactions between the CR1 domains of each receptor. Based on sequence conservation, buried surface area, and mutagenesis experiments, the back-to-back dimer is favored to be biologically relevant.