Quantifying the strength of heterointeractions among receptor tyrosine kinases from different subfamilies: Implications for cell signaling.

Receptor tyrosine kinases (RTKs) are single-pass membrane proteins that control vital cell processes such as cell growth, survival, and differentiation. There is a growing body of evidence that RTKs from different subfamilies can interact and that these diverse interactions can have important biological consequences. However, these heterointeractions are often ignored, and their strengths are unknown. In this work, we studied the heterointeractions of nine RTK pairs, epidermal growth factor receptor (EGFR)-EPH receptor A2 (EPHA2), EGFR-vascular endothelial growth factor receptor 2 (VEGFR2), EPHA2-VEGFR2, EPHA2-fibroblast growth factor receptor 1 (FGFR1), EPHA2-FGFR2, EPHA2-FGFR3, VEGFR2-FGFR1, VEGFR2-FGFR2, and VEGFR2-FGFR3, using a FRET-based method. Surprisingly, we found that RTK heterodimerization and homodimerization strengths can be similar, underscoring the significance of RTK heterointeractions in signaling. We discuss how these heterointeractions can contribute to the complexity of RTK signal transduction, and we highlight the utility of quantitative FRET for probing multiple interactions in the plasma membrane.

Pan-HER-targeted approach for cancer therapy: Mechanisms, recent advances and clinical prospect.

The Human Epidermal Growth Factor Receptor family is composed of 4 structurally related receptor tyrosine kinases that are involved in many human cancers. The efficacy and safety of HER inhibitors have been compared in a wide range of clinical trials, suggesting the superior inhibitory ability of multiple- HER-targeting blockade compared with single receptor antagonists. However, many patients are currently resistant to current therapeutic treatment and novel strategies are warranted to conquer the resistance. Thus, we performed a critical review to summarize the molecular involvement of HER family receptors in tumour progression, recent anti-HER drug development based on clinical trials, and the potential resistance mechanisms of anti-HER therapy.

Neurotrophin, p75, and Trk Signaling Module in the Developing Nervous System of the Marine Annelid Platynereis dumerilii.

In vertebrates, neurotrophic signaling plays an important role in neuronal development, neural circuit formation, and neuronal plasticity, but its evolutionary origin remains obscure. We found and validated nucleotide sequences encoding putative neurotrophic ligands (neurotrophin, NT) and receptors (Trk and p75) in two annelids, Platynereis dumerilii (Errantia) and Capitella teleta (Sedentaria, for which some sequences were found recently by Wilson, 2009). Predicted protein sequences and structures of Platynereis neurotrophic molecules reveal a high degree of conservation with the vertebrate counterparts; some amino acids signatures present in the annelid Trk sequences are absent in the basal chordate amphioxus, reflecting secondary loss in the cephalochordate lineage. In addition, expression analysis of NT, Trk, and p75 during Platynereis development by whole-mount mRNA in situ hybridization supports a role of these molecules in nervous system and circuit development. These annelid data corroborate the hypothesis that the neurotrophic signaling and its involvement in shaping neural networks predate the protostome-deuterostome split and were present in bilaterian ancestors.

Evolutionary Dynamics of the Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) Subfamily in Angiosperms.

Gene duplications are an important factor in plant evolution, and lineage-specific expanded (LSE) genes are of particular interest. Receptor-like kinases expanded massively in land plants, and leucine-rich repeat receptor-like kinases (LRR-RLK) constitute the largest receptor-like kinases family. Based on the phylogeny of 7,554 LRR-RLK genes from 31 fully sequenced flowering plant genomes, the complex evolutionary dynamics of this family was characterized in depth. We studied the involvement of selection during the expansion of this family among angiosperms. LRR-RLK subgroups harbor extremely contrasting rates of duplication, retention, or loss, and LSE copies are predominantly found in subgroups involved in environmental interactions. Expansion rates also differ significantly depending on the time when rounds of expansion or loss occurred on the angiosperm phylogenetic tree. Finally, using a dN/dS-based test in a phylogenetic framework, we searched for selection footprints on LSE and single-copy LRR-RLK genes. Selective constraint appeared to be globally relaxed at LSE genes, and codons under positive selection were detected in 50% of them. Moreover, the leucine-rich repeat domains, and specifically four amino acids in them, were found to be the main targets of positive selection. Here, we provide an extensive overview of the expansion and evolution of this very large gene family.

Genome-wide screening reveals the emergence and divergence of RTK homologues in basal Metazoan Hydra magnipapillata.

Receptor tyrosine kinases (RTKs) are key components of cell-cell signalling required for growth and development of multicellular organisms. It is therefore likely that the divergence of RTKs and associated components played a significant role in the evolution of multicellular organisms. We have carried out the present study in hydra, a diploblast, to investigate the divergence of RTKs after parazoa and before emergence of triploblast phyla. The domain-based screening using Hidden Markov Models (HMMs) for RTKs in Genomescan predicted gene models of the Hydra magnipapillata genome resulted in identification of 15 RTKs. These RTKs have been classified into eight families based on domain architecture and homology. Only 5 of these RTKs have been previously reported and a few of these have been partially characterized. A phylogeny-based analysis of these predicted RTKs revealed that seven subtype duplications occurred between 'parazoan-eumetazoan split' and 'diploblast-triploblast split' in animal phyla. These results suggest that most of the RTKs evolved before the radiata-bilateria divergence during animal evolution.

Transmembrane collagen receptors.

Collagen, the most abundant protein in animals, is a key component of extracellular matrices. Not only do collagens provide essential structural support for connective tissues, but they are also intimately involved in controlling a spectrum of cellular functions such as growth, differentiation, and morphogenesis. All collagens possess triple-helical regions through which they interact with a host of other proteins including cell surface receptors. A structurally diverse group of transmembrane receptors mediates the recognition of the collagen triple helix: integrins, discoidin domain receptors, glycoprotein VI, and leukocyte-associated immunoglobulin-like receptor-1. These collagen receptors regulate a wide range of behaviors including cell adhesion and migration, hemostasis, and immune function. Here these collagen receptors are discussed in terms of their molecular basis of collagen recognition, their signaling and developmental functions, and their roles in disease.

Unusual duplication of the insulin-like receptor in the crustacean Daphnia pulex.

BACKGROUND: The insulin signaling pathway (ISP) has a key role in major physiological events like carbohydrate metabolism and growth regulation. The ISP has been well described in vertebrates and in a few invertebrate model organisms but remains largely unexplored in non-model invertebrates. This study is the first detailed genomic study of this pathway in a crustacean species, Daphnia pulex. RESULTS: The Daphnia pulex draft genome sequence assembly was scanned for major components of the ISP with a special attention to the insulin-like receptor. Twenty three putative genes are reported. The pathway appears to be generally well conserved as genes found in other invertebrates are present. Major findings include a lower number of insulin-like peptides in Daphnia as compared to other invertebrates and the presence of multiple insulin-like receptors (InR), with four genes as opposed to a single one in other invertebrates. Genes encoding for the Dappu_InR are likely the result of three duplication events and bear some unusual features. Dappu_InR-4 has undergone extensive evolutionary divergence and lacks the conserved site of the catalytic domain of the receptor tyrosine kinase. Dappu_InR-1 has a large insert and lacks the transmembranal domain in the ß-subunit. This domain is also absent in Dappu_InR-3. Dappu_InR-2 is characterized by the absence of the cystein-rich region. Real-time q-PCR confirmed the expression of all four receptors. EST analyses of cDNA libraries revealed that the four receptors were differently expressed under various conditions. CONCLUSIONS: Duplications of the insulin receptor genes might represent an important evolutionary innovation in Daphnia as they are known to exhibit extensive phenotypic plasticity in body size and in the size of defensive structures in response to predation.

Cytoplasmic ACK1 interaction with multiple receptor tyrosine kinases is mediated by Grb2: an analysis of ACK1 effects on Axl signaling.

ACK1 (activated Cdc42-associated kinase 1), a cytoplsmic tyrosine kinase, is implicated in metastatic behavior, cell spreading and migration, and epidermal growth factor receptor (EGFR) signaling. The function of ACK1 in the regulation of receptor tyrosine kinases requires a C-terminal region that demonstrates a significant homology to the EGFR binding domain of MIG6. In this study, we have identified additional receptor tyrosine kinases, including Axl, leukocyte tyrosine kinase, and anaplastic lymphoma kinase, that can bind to the ACK1/MIG6 homology region. Unlike the interaction between MIG6 and EGFR, our data suggest that these receptor tyrosine kinases require the adaptor protein Grb2 for efficient binding, which interacts with highly conserved proline-rich regions that are conserved between ACK1 and MIG6. We have focused on Axl and compared how ACK1/Axl differs from the ACK1/EGFR axis by investigating effects of knockdown of endogenous ACK1. Although EGFR activation promotes ACK1 turnover, Axl activation by GAS6 does not; interestingly, the reciprocal down-regulation of GAS6-stimulated Axl is blocked by removing ACK1. Thus, ACK1 functions in part to control Axl receptor levels. Silencing of ACK1 also leads to diminished ruffling and migration in DU145 and COS7 cells upon GAS6-Axl signaling. The ability of ACK1 to modulate Axl and perhaps anaplastic lymphoma kinase (altered in anaplastic large cell lymphomas) might explain why ACK1 can promote metastatic and transformed behavior in a number of cancers.

Evolution of Xmrk: an oncogene, but also a speciation gene?

Genes that exert their function when they are introduced into a foreign genetic background pose many questions to our current understanding of the forces and mechanisms that promote either the maintenance or divergence of gene functions over evolutionary time. The melanoma inducing Xmrk oncogene of the Southern platyfish (Xiphophorus maculatus) is a stable constituent of the genome of this species. It displays its tumorigenic function, however, almost exclusively only after inter-populational or, even more severely, interspecific hybridization events. The Xiphophorus hybrid melanoma system has gained attention in biomedical research as a genetic model for studying tumor formation. From an evolutionary perspective, a prominent question is: how could this gene persist over millions of years? An attractive hypothesis is that Xmrk, acting as a detrimental gene in a hybrid genome, could be a speciation gene that shields the gene pool of its species from mixing with other closely related sympatric species. In this article, I briefly review our current knowledge of the molecular genetics and biochemical functions of the Xmrk gene and discuss aspects of its evolutionary history and presence with respect to this idea. While Xmrk as a potentially injurious oncogene has clearly survived for millions of years, its role as a speciation gene has to be questioned.

3D QSAR studies on a series of potent and high selective inhibitors for three kinases of RTK family.

For targets belonging to the same family of receptors, inhibitors often act at more than one biological target and produce a synergistic effect. Separate CoMFA and CoMSIA models were developed from our data set for the KDR, cKit and Tie-2 inhibitors. These models showed excellent internal predictability and consistency, and validation using test-set compounds yielded a good predictive power for the pIC(50) value. The field contour maps (CoMFA and CoMSIA) corresponding to the KDR, cKit and Tie-2 kinase subtypes reflected the characteristic similarities and differences between these types. These maps provided valuable information to facilitate structural modifications of the inhibitor to increase selectivity for the KDR over cKit and Tie-2.

Tracing the evolution and function of the Trk superfamily of receptor tyrosine kinases.

Most growth factors and their receptors have been strongly conserved during evolution. In contrast, Trks (Tropomyosin-related kinases) and related receptors in the Trk superfamily, Rors (receptor tyrosine kinase-like orphan receptors), Musks (muscle specific kinases) and Ddrs (discoidin domain receptor family), appear to be ancient, but their function has been lost in multiple lineages and the roles for the receptors have been modified over time. We will trace the evolution of the Trk superfamily and discuss possible conserved functional roles, including a unifying theme of target recognition by growing axons. We present an analogy between the evolution of G-protein-coupled receptors and receptor tyrosine kinases (RTKs), proposing that an important driving force for the divergence of receptors is the ease of divergence of their ligands.

Signalling and functional diversity within the Axl subfamily of receptor tyrosine kinases.

The related Axl, Sky and Mer receptor tyrosine kinases (RTKs) are increasingly being implicated in a host of discrete cellular responses including cell survival, proliferation, migration and phagocytosis. Furthermore, their ligands Gas6 and protein S are characteristically dependent on vitamin K for expression of their functions. The Gas6/Axl system is implicated in several types of human cancer as well as inflammatory, autoimmune, vascular and kidney diseases. Each member of the Axl RTK subfamily possesses distinct expression profiles as well as discrete functions. In this article, we review the knowledge so far on the intracellular signalling interactions and pathways concerning each of the Axl RTKs. In this way, we hope to gain a greater understanding of the mechanisms that set each of them apart, and that relay their associated functions.

Molecular regulation of receptor tyrosine kinases in hematopoietic malignancies.

Dysregulation of receptor tyrosine kinase (RTK) activity has been implicated in the progression of a variety of human leukemias. Most notably, mutations and chromosomal translocations affecting regulation of tyrosine kinase activity in the Kit receptor, the Flt3 receptor, and the PDGFbeta/FGF1 receptors have been demonstrated in mast cell leukemia, acute myeloid leukemia (AML), and chronic myelogenous leukemias (CML), respectively. In addition, critical but non-overlapping roles for the Ron and Kit receptor tyrosine kinases in the progression of animal models of erythroleukemia have been demonstrated [Persons, D., Paulson, R., Loyd, M., Herley, M., Bodner, S., Bernstein, A., Correll, P. and Ney, P., 1999. Fv2 encodes a truncated form of the Stk receptor tyrosine kinase. Nat. Gen. 23, 159-165.; Subramanian, A., Teal, H.E., Correll, P.H. and Paulson, R.F., 2005. Resistance to friend virus-induced erythroleukemia in W/Wv mice is caused by a spleen-specific defect which results in a severe reduction in target cells and a lack of Sf-Stk expression. J. Virol. 79 (23), 14586-14594.]. The various classes of RTKs implicated in the progression of leukemia have been recently reviewed [Reilly, J., 2003. Receptor tyrosine kinases in normal and malignant haematopoiesis. Blood Rev. 17 (4), 241-248.]. Here, we will discuss the mechanism by which alterations in these receptors result in transformation of hematopoietic cells, in the context of what is known about the molecular regulation of RTK activity, with a focus on our recent studies of the Ron receptor tyrosine kinase.

Leucine motif-dependent tyrosine autophosphorylation of type III receptor tyrosine kinases.

Activation loop tyrosine autophosphorylation is an essential requirement for full kinase activation of receptor tyrosine kinases (RTKs). However, mechanisms involved are not fully understood. In general, kinase domains of RTKs are folded into two main lobes, NH2- and COOH-terminal lobes. The COOH-terminal lobe of vascular endothelial growth factor receptor-2 (VEGFR-2) is folded into seven alpha-helices (alphaD-alphaI). In the studies presented here we demonstrate that leucine residues of helix I (alphaI) regulate tyrosine autophosphorylation and phosphotransferase activity of VEGFR-2. The presence of leucines 1158, 1161, and 1162 are essential for tyrosine autophosphorylation and kinase activation of VEGFR-2 and are involved in helix-helix packing via hydrophobic interactions. The presence of leucine 1158 is critical for kinase activation of VEGFR-2 and appears to interact with alphaE, alphaF, alphaH, and beta7. The analogous residue, leucine 957 on platelet-derived growth factor receptor-beta and leucine 910 on colony stimulating factor-1R are also found to be critical for tyrosine autophosphorylation of these receptors. Leucines 1161 and 1162 are also involved in helix-helix packing but they play a less critical role in VEGFR-2 activation. Thus, we conclude that leucine motif-mediated helix-helix interactions are critical for kinase regulation of type III RTKs. This mechanism is likely to be shared with other kinases and might provide a basis for the design of a novel class of tyrosine kinase inhibitors.

The role of tropomyosin-related kinase receptors in neurotrophin-induced rapid eye movement sleep in the cat.

The microinjection of nerve growth factor and neurotrophin-3 into the rostro-dorsal pontine tegmentum of the cat evokes a state that is comparable to naturally-occurring rapid eye movement sleep. Using two experimental paradigms, we tested the hypothesis that neurotrophin high-affinity receptors (trkA and trkC, tropomyosin-related kinase A and C, respectively) mediate this effect. First, trk and fos immunohistochemistry were combined to determine whether tyrosine kinase receptor-containing neurons in the dorsal pontine tegmentum are active in cats that exhibit long-lasting periods of rapid eye movement sleep following the local microinjection of nerve growth factor. During approximately two hours of recording, nerve growth factor-treated cats spent 59.8% of the time in a rapid eye movement sleep-like state; vehicle-injected (control) animals remained in quiet wakefulness and non-rapid eye movement sleep. Whereas control and nerve growth factor-treated cats exhibited a similar mean number of trkA- and trkC-immunoreactive neurons in the dorsal pontine tegmentum, the number of trkA- and trkC-immunoreactive neurons that expressed Fos, i.e. double-labeled cells that are presumably activated, was significantly larger in cats that were injected with nerve growth factor. Axon terminals contained tyrosine kinase receptor immunoreactivity in this region; many were apposed to Fos-immunoreactive neurons. In addition, patterns of tyrosine kinase receptor and Fos immunoreactivity similar to those observed in nerve growth factor-injected cats were present, in conjunction with long-lasting rapid eye movement sleep, following the microinjection of carbachol into the dorsal pons. In a second series of studies, nerve growth factor or neurotrophin-3 was injected alone or after K-252a, a blocker of tyrosine kinase receptors, into the rostro-dorsal pontine tegmentum. Nerve growth factor or neurotrophin-3 alone produced, with a mean latency of 4 min, a rapid eye movement sleep-like state. However, neurotrophin injections preceded by K-252a were not effective in inducing rapid eye movement sleep. These results indicate that the activation of trkA and trkC receptors in neurons in the pontine tegmentum is responsible, at least in part, for the rapid eye movement sleep-inducing effect of nerve growth factor and neurotrophin-3. Furthermore, the data suggest that these neurotrophins are capable of acting both pre- and postsynaptically to activate pontine neurons that are involved in the generation of rapid eye movement sleep.

Differential Trk expression in explant and dissociated trigeminal ganglion cell cultures.

During embryonic development, expression of neurotrophin receptor tyrosine kinases (Trks) by sensory ganglia is continuously and dynamically regulated. Neurotrophin signaling promotes selective survival and axonal differentiation of sensory neurons. In embryonic day (E) 15 rat trigeminal ganglion (TG), NGF receptor TrkA is expressed by small diameter neurons, NT-3 receptor TrkC and BDNF receptor TrkB are expressed by large diameter neurons. Organotypic explant and dissociated cell cultures of the TG (and dorsal root ganglia) are commonly used to assay neurotrophin effects on developing sensory neurons. In this study, we compared Trk expression in E15 rat TG explant and dissociated cell cultures with or without neurotrophin treatment. Only a subset of TG cells express each of the three Trk receptors in wholemount explant cultures as in vivo conditions. In contrast, all TG neurons co-express all three Trk receptors upon dissociation, regardless of neurotrophin treatment. Neurons cultured in low concentrations of one neurotrophin first, and switched to higher concentrations of another after 1 day, survive and display morphological characteristics of neurons cultured in a mixture of both neurotrophins for 3 days. Our results indicate that wholemount explant cultures of sensory ganglia represent in vivo conditions in terms of Trk expression patterns; whereas dissociation dramatically alters Trk expression by primary sensory neurons.

Activation of STAT5-dependent transcription by the neurotrophin receptor Trk.

Neurotrophins exert many of their biological effects via the Trk receptor tyrosine kinases and require the regulated activation of distinct transcriptional and post-translational cellular events. Here we provide evidence for a novel signaling cascade from activated Trks to the transcription factor STAT5. Utilizing the STAT5 responsive element derived from the p21(WAF1/Cip1) promoter to modulate luciferase expression, neurotrophin-dependent activation of Trk A, B, and C was found to induce STAT5-mediated transcriptional response. Structure-function analysis using Trk A mutants in heterologous cells further revealed that the kinase activity and an intact phospholipase C-gamma binding site are required for STAT5 activation. In most cytokine responsive cell systems, STAT5 function is modulated by JAK2-dependent tyrosine phosphorylation. However, reconstitution studies using a JAK2 deficient cell line indicate that neurotrophin-induced STAT5 activation does not require the cognate upstream kinase JAK2. In contrast, the Src kinase inhibitor PP1 significantly abolishes STAT5-dependent transcription in Trk A expressing 293T cells and in BDNF-treated primary cortical neurons. Together these results suggest that neurotrophins may regulate neuronal gene expression via STAT5 in a JAK2 independent manner.

Receptor protein tyrosine phosphatases regulate neural development and axon guidance.

The regulation of tyrosine phosphorylation is recognized as an important developmental mechanism. Both addition and removal of phosphate moieties on tyrosine residues are tightly regulated during development. Originally, most attention focused on the role of tyrosine kinases during development, but more recently, the developmental importance of tyrosine phosphatases has been gaining interest. Receptor protein tyrosine phosphatases (RPTPs) are of particular interest to developmental biologists because the extracellular domains of RPTPs are similar to those of cell adhesion molecules (CAMs). This suggests that RPTPs may have functions in development similar to CAMs. This review focuses on the role of RPTPs in development of the nervous system in processes such as axon guidance, synapse formation, and neural tissue morphogenesis.

TRK neurotrophin receptor-like proteins in the kidney of frog (Rana esculenta) and lizard (Podarcis sicula): an immunohistochemical study.

The present study investigates the occurrence of Trk-like neurotrophin receptor proteins in the lizard and frog kidney. In lizard rare TrkB-like immunoreactive cells in intermediate and distal tubules were found. TrkC-like immunoreactive cells were numerous in collecting tubules and became less numerous in collecting ducts. No TrkC-like immunoreactivity was detected in the ureteric duct. In the frog, we observed numerous TrkC-like immunoreactive cells in collecting tubules and ducts while they were scattered among negative epithelial cells in the wolffian duct. TrkB- and TrkA-like immunoreactivity was never found. Western blot analysis demonstrated that the frog and lizard kidney contains TrkC-like protein; TrkB-like protein was present only in the lizard kidney. These results demonstrate for the first time the occurrence of Trk-like proteins in the kidney of amphibians and reptiles, and aid in the assessment of the role of Trk receptor-like proteins in the kidney physiology of vertebrates.