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.

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.

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.

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.

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.

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.

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.

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.

The Eph family in the patterning of neural development.

The Eph family represents the largest subfamily of receptor tyrosine kinases. Its members are predominantly expressed in the developing and adult nervous system. Besides playing an important role in the contact-mediated repulsion of axons, they have recently also been implicated in the control of cell migration. Characteristics of the Eph family are extended promiscuity in the interaction between receptors and ligands, the necessity of membrane attachment of the ligands to exert their function, the lack of induction of mitogenic responses, and the bi-directional signalling of receptors and ligands.

The Eph receptor family: axonal guidance by contact repulsion.

Eph receptors and their ligands have been implicated in axonal pathfinding during the formation of neuronal networks. Whereas other guiding molecules, such as netrins and most semaphorins, are diffusible factors that can guide axons over considerable distances, Eph receptors and their ligands are cell-surface bound and are, therefore, involved in short-range contact-mediated guidance. Those molecules that have been analyzed appear to guide axons by repelling the growth cone, rather than attracting it. Here, we summarize the known biological functions of Eph receptors and ligands focusing on the developing nervous system, and we review the data on possible mechanisms of action.

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.

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.

Trophic factors.

The various neurotrophic factors influence a wide range of cell functions in the developing, mature and injured nervous system. Recent studies have provided valuable insights on the receptors that mediate these effects and on the intracellular events that follow the binding of the ligand. Although growth factors were known to be expressed by non-neuronal cells in the targets and pathways of neuronal projections, it is now clear that the neurons themselves can also be a source of these molecules. A better understanding of the mechanisms of action of trophic factors on the survival and differentiation of neurons, coupled with advances in methods for the delivery of these molecules to the nervous system have provided an impetus for exploring their use as aids to the protection and regeneration of the injured nervous system.

RTKdb: database of Receptor Tyrosine Kinase.

Receptor Tyrosine Kinases (RTK) are transmembrane receptors specifically found in metazoans. They represent an excellent model for studying evolution of cellular processes in metazoans because they encompass large families of modular proteins and belong to a major family of contingency generating molecules in eukaryotic cells: the protein kinases. Because tyrosine kinases have been under close scrutiny for many years in various species, they are associated with a wealth of information, mainly in mammals. Presently, most categories of RTK were identified in mammals, but in a near future other model species will be sequenced, and will bring us RTKs from other metazoan clades. Thus, collecting RTK sequences would provide a good starting point as a new model for comparative and evolutionary studies applying to multigene families. In this context, we are developing the Receptor Tyrosine Kinase database (RTKdb), which is the only database on tyrosine kinase receptors presently available. In this database, protein sequences from eight model metazoan species are organized under the format previously used for the HOVERGEN, HOBACGEN and NUREBASE systems. RTKdb can be accessed through the PBIL (Pôle Bioinformatique Lyonnais) World Wide Web server at http://pbil.univ-lyon1.fr/RTKdb/, or through the FamFetch graphical user interface available at the same address.

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.

brt, a mouse gene encoding a novel receptor-type protein-tyrosine kinase, is preferentially expressed in the brain.

Using polymerase chain reaction (PCR)-mediated cloning procedure, we have isolated a novel gene encoding protein-tyrosine kinase from fetal mouse brain. This gene, named brt (from brain tyrosine kinase), had an open reading frame that encoded 856 amino acids. The deduced amino acid sequence revealed that the gene product had all the hallmarks of a receptor-type protein-tyrosine kinase. The brt protein showed significant homology to the axl/ufo protein that has an oncogenic potential. Northern blot analysis showed that brt was expressed preferentially in the brain of both embryo and adult. Thus, brt is a new member of the subfamily of receptor-type protein-tyrosine kinases and is suggested to play important roles in development and functions in the central nervous system.

Colon carcinoma kinase-4 defines a new subclass of the receptor tyrosine kinase family.

Complementary DNA (cDNA) encoding a novel member of the receptor tyrosine kinase (RTK) family has been isolated from colon carcinoma tissue. Colon carcinoma kinase 4 (CCK-4) mRNA is highly expressed in human lung tissue and at lower levels in the thyroid gland and ovary. While no mRNA was found in human adult colon tissues, expression varied remarkably in colon carcinoma-derived cell lines. CCK-4 cDNA encodes a chicken KLG-related, 1071 amino acid-long transmembrane glycoprotein containing several genetic alterations within the RTK consensus sequences. These define CCK-4 as a catalytically inactive member of the RTK family of proteins and, in analogy to HER3, suggest a potentially tumor-characteristic role as a signal amplifier or modulator for an as yet unidentified kinase-competent partner.

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.

The Drosophila VEGF receptor homolog is expressed in hemocytes.

Several signalling pathways have been defined by studies of genes originally characterised in Drosophila. However, some mammalian signalling systems have so far escaped discovery in the fly. Here, we describe the identification and characterisation of fly homologs for the mammalian vascular endothelial growth factor/platelet derived growth factor (VEGF/PDGF) and the VEGF receptor. The Drosophila factor (DmVEGF-1) gene has two splice variants and is expressed during all stages, the signal distribution during embryogenesis being ubiquitous. The receptor (DmVEGFR) gene has several splice variants; the variations affecting only the extracellular domain. The most prominent form is expressed in cells of the embryonic haematopoietic cell lineage, starting in the mesodermal area of the head around stage 10 of embryogenesis. Expression persists in hemocytes as embryonic development proceeds and the cells migrate posteriorly. In a fly strain carrying a deletion uncovering the DmVEGFR gene, hemocytes are still present, but their migration is hampered and the hemocytes remain mainly in the anterior end close to their origin. These data suggest that the VEGF/PDGF signalling system may regulate the migration of the Drosophila embryonic haemocyte precursor cells.