Full-length Receptor Tyrosine Kinase Research Articles

Structural studies of full-length receptor tyrosine kinases and their implications for drug design.

Receptor tyrosine kinases (RTKs) are important drug targets for cancer and immunological disorders. Crystal structures of individual RTK domains have contributed greatly to the structure-based drug design of clinically used drugs. Low-resolution structures from electron microscopy are now available for the RTKs, EGFR, PDGFR, and Kit. However, there are still no high-resolution structures of full-length RTKs due to the technical challenges of working with these complex, membrane proteins. Here, we review what has been learned from structural studies of these three RTKs regarding their mechanisms of ligand binding, activation, oligomerization, and inhibition. We discuss the implications for drug design. More structural data on full-length RTKs may facilitate the discovery of druggable sites and drugs with improved specificity and effectiveness against resistant mutants.

The First 3D Model of the Full-Length KIT Cytoplasmic Domain Reveals a New Look for an Old Receptor

Receptor tyrosine kinases (RTKs) are key regulators of normal cellular processes and have a critical role in the development and progression of many diseases. RTK ligand-induced stimulation leads to activation of the cytoplasmic kinase domain that controls the intracellular signalling. Although the kinase domain of RTKs has been extensively studied using X-ray analysis, the kinase insert domain (KID) and the C-terminal are partially or fully missing in all reported structures. We communicate the first structural model of the full-length RTK KIT cytoplasmic domain, a crucial target for cancer therapy. This model was achieved by integration of ab initio KID and C-terminal probe models into an X-ray structure, and by their further exploration through molecular dynamics (MD) simulation. An extended (2-µs) MD simulation of the proper model provided insight into the structure and conformational dynamics of the full-length cytoplasmic domain of KIT, which can be exploited in the description of the KIT transduction processes.

Downstream signaling and genome-wide regulatory effects of PTK7 pseudokinase and its proteolytic fragments in cancer cells

BackgroundThe full-length membrane protein tyrosine kinase 7 (PTK7) pseudokinase, an important component of the planar cell polarity and the Wnt canonical and non-canonical pathways, is a subject of step-wise proteolysis in cells and tissues. The proteolysis of PTK7 involves membrane type-matrix metalloproteinase (MT1-MMP), members of the Disintegrin Domain and Metalloproteinase (ADAM) family, and γ-secretase. This multi-step proteolysis results in the generation of the digest fragments of PTK7. These fragments may be either liberated into the extracellular milieu or retained on the plasma membrane or released into the cytoplasm and then transported into the nucleus.ResultsWe employed the genome-wide transcriptional and kinome array analyses to determine the role of the full-length membrane PTK7 and its proteolytic fragments in the downstream regulatory mechanisms, with an emphasis on the cell migration-related genes and proteins. Using fibrosarcoma HT1080 cells stably expressing PTK7 and its mutant and truncated species, the structure of which corresponded to the major PTK7 digest fragments, we demonstrated that the full-length membrane 1–1070 PTK7, the N-terminal 1–694 soluble ectodomain fragment, and the C-terminal 622–1070 and 726–1070 fragments differentially regulate multiple genes and signaling pathways in our highly invasive cancer cell model. Immunoblotting of the selected proteins were used to validate the results of our high throughput assays.ConclusionsOur results suggest that PTK7 levels need to be tightly controlled to enable migration and that the anti-migratory effect of the full-length membrane PTK7 is linked to the down-regulation of multiple migration-related genes and to the activation of the Akt and c-Jun pathway. In turn, the C-terminal fragments of PTK7 act predominantly via the RAS-ERK and CREB/ATF1 pathway and through the up-regulation of cadherin-11. In general, our data correlate well with the distinct functionality of the full-length receptor tyrosine kinases and their respective intracellular domain (ICD) proteolytic fragments.

Benzyl Isothiocyanate: Double Trouble for Breast Cancer Cells

An inverse association between dietary intake of cruciferous vegetables and cancer risk has been established for different types of malignancies, including breast cancer. The anticarcinogenic effect of cruciferous vegetables has been attributed to chemicals with an isothiocyanate (ITC) functional moiety. Research over the past three decades has provided extensive preclinical evidence for the efficacy of various ITCs against cancer in preclinical models. Benzyl isothiocyanate (BITC) is one such compound with the ability to inhibit chemically induced cancer, oncogenic-driven tumor formation, and human tumor xenografts in rodent cancer models. Prior work also has established that BITC has the ability to influence carcinogen metabolism and signaling pathways relevant to tumor progression and invasion. In this issue, Kim and colleagues show that BITC inhibits breast cancer stem cell growth, both in vitro and in vivo, in association with suppression of the full-length receptor tyrosine kinase RON as well as its activated truncated form (sfRon), both of which seem to drive stemness in breast cancer cells. Overexpression of RON or sfRon prevented the BITC effect. These data complement prior work from this group showing elimination of mammary tumor cells via tumor cell apoptosis by BITC administration. The inhibition of breast cancer stem cells is observed at pharmacologic concentrations of BITC. This perspective briefly reviews epidemiologic evidence, preclinical efficacy data, and molecular and cellular mechanistic attributes of BITC. Critical issues relevant to clinical development of BITC are discussed briefly.

Abstract 3881: BacMam-enabled biochemical and cellular assays to assess inhibitors of full-length receptor tyrosine kinases

Abstract Receptor tyrosine kinases (RTKs) are important pharmacological targets in oncology. Current biochemical assays designed to assess compound efficacy utilize truncated forms of the kinase lacking the transmembrane or extracellular domains and fail to address how these domains might affect the observed pharmacology. Purification of the full-length recombinant receptor can be both costly and difficult to scale for large screening campaigns. Additionally, because of the generic nature of most substrates used in activity assays for RTKs, small amounts of contaminating kinases can interfere with assay results. Numerous cell-based assays for pathway analysis can also serve as readouts for these targets, but they are not a direct measurement of kinase activity. To address these limitations, we have employed a BacMam-mediated gene delivery system to express full-length receptor tyrosine kinases with C-terminal green fluorescent protein (GFP) fusions in a variety of cellular backgrounds. Expression of these full-length RTKs has enabled the development of two complimentary assay platforms. First, we present a competitive displacement assay that uses a europium (Eu)-labeled anti-GFP antibody and an AlexaFluor® 647-labeled active site probe to characterize compound binding to the RTK in a lysate-based format. Here, the GFP moiety serves as an epitope tag to allow for very specific and sensitive detection of compound binding, discriminating compounds with sub-nanomolar affinity. Using the same BacMam RTK-GFP constructs, we have also developed a simple, addition-only, cell-based method to detect auto-phosphorylation of the RTK via TR-FRET between a terbium (Tb)-labeled anti-phospho-tyrosine antibody and the GFP moiety. Together, these tools can be used in a high-throughput screening format with a fluorescence-based readout to more fully characterize compound efficacy against full-length RTKs expressed in native cellular contexts. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3881. doi:1538-7445.AM2012-3881

Excitotoxicity downregulates TrkB.FL signaling and upregulates the neuroprotective truncated TrkB receptors in cultured hippocampal and striatal neurons.

Brain-derived neurotrophic factor (BDNF) plays an important role in neuronal survival through activation of TrkB receptors. The trkB gene encodes a full-length receptor tyrosine kinase (TrkB.FL) and its truncated (T1/T2) isoforms. We investigated the changes in TrkB protein levels and signaling activity under excitotoxic conditions, which are characteristic of brain ischemia, traumatic brain injury, and neurodegenerative disorders. Excitotoxic stimulation of cultured rat hippocampal or striatal neurons downregulated TrkB.FL and upregulated a truncated form of the receptor (TrkB.T). Downregulation of TrkB.FL was mediated by calpains, whereas the increase in TrkB.T protein levels required transcription and translation activities. Downregulation of TrkB.FL receptors in hippocampal neurons correlated with a decrease in BDNF-induced activation of the Ras/ERK and PLCγ pathways. However, calpain inhibition, which prevents TrkB.FL degradation, did not preclude the decrease in signaling activity of these receptors. On the other hand, incubation with anisomycin, to prevent the upregulation of TrkB.T, protected to a large extent the TrkB.FL signaling activity, suggesting that truncated receptors may act as dominant-negatives. The upregulation of TrkB.T under excitotoxic conditions was correlated with an increase in BDNF-induced inhibition of RhoA, a mediator of excitotoxic neuronal death. BDNF fully protected hippocampal neurons transduced with TrkB.T when present during excitotoxic stimulation with glutamate, in contrast with the partial protection observed in cells overexpressing TrkB.FL or expressing GFP. These results indicate that BDNF protects hippocampal neurons by two distinct mechanisms: through the neurotrophic effects of TrkB.FL receptors and by activation of TrkB.T receptors coupled to inhibition of the excitotoxic signaling.

Signaling Through Truncated Receptors

Trk family neurotrophin receptors include not only full-length receptor tyrosine kinases but also truncated isoforms that lack kinase activity. Although the truncated isoforms were initially believed to act mainly by inhibiting the function of the full-length isoforms, distinct functions have begun to emerge. Esteban et al. used a yeast two-hybrid system to screen mouse brain cDNA for proteins that interact with a unique C-terminal fragment of TrkCT1, the truncated TrkC isoform, and identified the scaffolding protein tamalin. This interaction was confirmed in an in vitro binding assay. In situ hybridization indicated that expression of TrkCT1 and tamalin in mouse brain overlapped; moreover, when the two proteins were transfected into human embryonic kidney (HEK) 293 cells, immunofluorescence analysis indicated that they colocalized. Neurotrophin-3 (NT3) enhanced coimmunoprecipitation of TrkCT1 and tamalin from HEK293 cells and promoted colocalization of the endogenous proteins in cultured mouse hippocampal neurons. Tamalin forms a complex with the guanine nucleotide exchange factor ARNO (for Arf nucleotide-binding site opener) and, in a HEK293 line with endogenous tamalin that was stably transfected with TrkCT1, NT3 promoted translocation of the guanosine triphosphatase (GTPase) Arf6 to actin-rich membrane ruffles. Arf6 translocation was blocked by tamalin dominant negatives and enhanced by tamalin overexpression. Moreover, NT3-dependent membrane ruffling (monitored by actin polymerization) was blocked by a mutant form of ARNO that cannot promote Arf6 GTP exchange. NT3 stimulated Rac1 activation in HEK293 cells transfected with TrkCT1 and tamalin, and this was blocked by a dominant-negative form of Arf6. Thus, these data define a signaling pathway mediated through the truncated TrkCT1 receptor in which NT3 activates Arf6 and Rac1. P. F. Esteban, H.-Y. Yoon, J. Becker, S. G. Dorsey, P. Caprari, M. E. Palko, V. Coppola, H. U. Saragovi, P. A. Randazzo, L. Tessarollo, A kinase-deficient TrkC receptor isoform activates Arf6-Rac1 signaling through the scaffold protein tamalin. J. Cell Biol. 173 , 291-299 (2006). [Abstract] [Full Text]

In vivo role of truncated trkb receptors during sensory ganglion neurogenesis

The mammalian trkB locus undergoes alternative splicing to produce two different types of brain-derived neurotrophic factor receptors. The first type is the full-length receptor tyrosine kinase (TrkB Tk+; the second type is a truncated receptor lacking the intracellular tyrosine kinase domain (TrkB Tk−). To investigate the function of both types of TrkB receptor in vivo, we have generated knockout mice lacking all isoforms of the TrkB receptor ( trkB −/−) and compared sensory neuron survival in these mice to that in the previously described TrkB kinase domain knockout mice ( trkB k −/−). We observed that the presence of truncated TrkB receptors in trkB k −/− mice results in more severe sensory neuron losses. Increased neuron losses associated with the presence of truncated TrkB were most severe in regions where neuron survival is most dependent on brain-derived neurotrophic factor and neurotrophin-3. Our data suggest that truncated TrkB receptors negatively influence neuron survival by interfering with the function of catalytic TrkB receptors.

Tyrosine kinase activity of purified recombinant cytoplasmic domain of platelet-derived growth factor β-receptor (β-PDGFR) and discovery of a novel inhibitor of receptor tyrosine kinases

Aberrant expression of platelet-derived growth factor and its receptor (PDGFR) has been implicated in various human disorders, including cardiovascular disease and certain types of cancer. Inhibitors of the tyrosine kinase activity of PDGFR are leads in the development of novel agents to combat these diseases. We describe here a novel, potent inhibitor of PDGFR tyrosine kinase, 3-(4-dimethylamino-benzylidenyl)-2-indolinone (DMBI). The compound also inhibits signal transduction through fibroblast growth factor receptor 1 (FGFR1), but is not active towards epidermal growth factor receptor (EGFR) or c-Src tyrosine kinase. The activity of DMBI and other tyrosine kinase inhibitors was compared in a cell-based assay as well as in an assay based on purified recombinant platelet-derived growth factor β-receptor (β-PDGFR) lacking the transmembrane and ligand-binding domain. We showed that this truncated β-PDGFR could dimerize, and that dimerization was required for tyrosine kinase activity. Tyrosine kinase activity was modulated by inhibitors of β-PDGFR autophosphorylation in cells, but not by specific inhibitors of EGFR or c-Src tyrosine kinase. We conclude that β-PDGFR lacking the transmembrane and ligand-binding domain retains the essential properties of the full-length receptor tyrosine kinase.

The Kinase Domain and Membrane Localization Determine Intracellular Interactions between Epidermal Growth Factor Receptors

Receptor tyrosine kinases play a central role in cellular growth, differentiation, and oncogenesis. All of these responses are triggered by growth factors interacting with the extracellular domain of transmembrane-spanning receptors, leading to dimerization and activation of an intrinsic tyrosine-specific kinase activity by an allosteric mechanism. Precise mechanisms of receptor dimerization remain poorly understood, and current models suggest that the ligand binding domain plays a major determining role. To examine the role of the intracellular domain in the association of juxtaposing receptor molecules, the full-length epidermal growth factor receptor was transiently co-expressed in human 293 fibroblasts with a truncated receptor that lacks the extracellular domain. After metabolic labeling with [35S]methionine, the association of these receptor constructs was monitored by co-immunoprecipitation with an extracellular domain-specific antibody. Specific interactions found between these receptors were independent of ligand binding or an intact ATP-binding site. Truncated receptors that had sequences necessary for membrane localization, and that were capable of interacting with full-length receptor tyrosine kinase, also displayed constitutive kinase activity as well as the capacity to transphosphorylate kinase-negative receptors. Receptor co-immunoprecipitation occurred between constructs that comprise the intracellular domains of the epidermal growth factor and beta-platelet-derived growth factor receptors, and HER-2. Subsequent deletion analysis has identified the major region of epidermal growth factor receptor intracellular interaction to be within the kinase domain.

The rat trkC locus encodes multiple neurogenic receptors that exhibit differential response to neurotrophin-3 in PC12 cells

Members of the Trk tyrosine kinase family have recently been identified as functional receptors of the NGF family of neurotrophins. Here we show the rat trkC locus to be complex, encoding at least four distinct polypeptides. Three of the encoded polypeptides are full-length receptor tyrosine kinases that differ by novel amino acid insertions in the kinase domain. A fourth protein is a truncated receptor that lacks the catalytic domain. Tyrosine phosphorylation, cross-linking, and ligand binding assays indicate that TrkC receptors interact with NT-3 and not with the related neurotrophins NGF, BDNF, xNT-4, or hNT-5. Furthermore, high and low affinity NT-3-binding sites are associated with the TrkC receptors. Stable and transient expression of TrkC receptors in PC12 cells indicates that the neurite outgrowth response elicited by NT-3 is dramatic in receptors lacking the novel kinase insert (gp150trkC) but absent in receptors containing the 14 amino acid insert in the kinase domain (gp150trkC14). These data suggest that the trkC locus encodes receptors that may be capable of mediating different biological responses within the cell. This could have important implications in understanding the role of neurotrophins in the development of the vertebrate nervous system.