2009 Group 4 Project
- 1 'Tropomyosin-related Kinase(Trk): a novel family of cell surface receptor tyrosine-kinase'
- 2 Mechanism of Trk Activation via multiple Signalling pathways
- 3 Current Research
- 4 Timeline
- 5 Glossary
- 6 References
- 7 2009 Group Projects
'Tropmyosin-related kinase receptors (also known as Trk or colloqiually as "Terk" receptors), are a specific family of Tyrosine kinase receptors originating from the Trk oncogene. Trk's are present, expressed at low levels, throughout mammalian tissue types and bear particular signifcance in embryological development and maintainence and growth of neuronal cell types. During embryonic development expression is significantly higher than in mature mammals, as if neuronal expression. Thus, Trks appear to be a major component of a highly intricate system of receptor complexes that mediate neurotropin and growth factor control of selective cell death and differentiation. There are three particular sub-types of receptor within the Trk loci - Trk A, Trk B and Trk C - each of which is characterised by specific differences in structure and discrimation for different Neurotrophin ligands, and thus, distinct functions.
What are neurotrophins? Neurotrophins are signalling proteins (growth factors) which are expressed mostly during embryological development to regulate neuronal tissue development. There are four typical neurotropic factors – Nerve Growth Factor (NGF), Brain-derived Neurotrophic Factor (BDNF), Neurotrophin-3 (NT-3), and Neurotrophin-4 (NT-4). These factors are believed to regulate selective tissue death via the Trk receptors and the low-affinity neurotrophin receptor p75NTR. The Selectivty of NTs for various receptors is indicated in figure 1.
- Trk receptors are composed of an extracellular segment that binds polypeptide ligands, transmembrane helix, and cytoplasmic segment where the tyrosine kinase catalytic activity takes place.
- TrK receptors act as dimeric transmembrane proteins. Distally, three cysteine-rich motiffs and two Leucine rich regions form a conserved NGF binding region which is common to all TrK's near the amino terminus.
- Additionally, the common binding region is flanked by a pair of extracellular immunoglobulin-like domains located proximally to the cell membrane. The juxtamembrane complex contains a variable amino acid sequence suggested to determine ligand bindng affinity and specificity and may bind directly to some ligands; The localization of these peptides appears to be non-specific in tertiary NGF-TrKA complex structures however their conformation within this complex appears to indicate direct participation in complex binding.
- Further NGF binding site affinity is generated through collaboration with the p75 protein/neurotrophin receptor (p75NTR). Furthermore, p75NTR regulates NT-3-TrkA and NT-4/5-TrkB mediated receptor activation by in addition to massively increasing signal transduction sensitivity by circumnavigating the prequisite of these neurotropins for receptor activation. This is acheived through allosteric distortion of Trk binding regions.
- intracellularly, Trk shares many of the highy conserved motifs of other tyrosine kinases with some distinct variations. There are 10 conserved tyrosines in the intracellular domain, three of which, are known to contribute to the autoregulatory loop within the kinase region. Overall, 8 of the 10 tyrosines contribute to the kinase region- either by regulating kinase activity directly or by inducing adaptor protein binding including those of the Ras and phosphatidylinositol pathways. The two remaining tyrosine residues contribute to second-messenger events coupled by Shc and PLC-γ1 protein and are located outside the kinase domain.
TrK receptors achieve their function primarily via neuroptrophin signaling. There are three types of trk receptors, namely trkA, trkB and trkC. These receptors have different binding affinity to certain types of neurotrophins. Hence, the signalling by these distinct receptors will result in various cellular functions.Trk receptors regulate cell survival, proliferation, axon and dendrite growth and the expression and activity of ion channels and neurotransmitter receptors. TrK also regulate synaptic strength and plasticity in adult nervous system. As a family of tyrosine kinase receptors, TrK receptors are activated and regulated in a similar fashion. TrK receptor signaling is involved in G proteins activation and pathways regulated by MAP kinase, PI3K and PLC.
- Trk A
Trk A also known as Neurotrophic tyrosine kinase, receptor, type 1 is a high affinity receptor for nerve growth factor (NGF). Binding of NGF to TrK A leads to the recruitment of proteins that interact with specific phosphotyrosine residues in the cytoplasmic domains of Trk receptor A. Such interactions activate signaling pathways, such as the Ras, PI3K, and PLC pathways and result in activation of gene expression, neuronal growth and proliferation.
- Trk B
Trk B also known as Neurotrophic tyrosine kinase, receptor, type 2 is a high affinity receptor for brain derived growth factor (BDNF) and neurotrophin-4 (NT4). Trk B are consisted of 3 isoforms in human central nervous system. The isoform (TK+) is a typical tyrosine kinase receptor, and transduce the BDNF signal via the common signaling pathways such as Ras-ERK, PI3K, and PLCγ. The other two isoforms, TKT1 and TKT2 are distinct from the typical kinase receptors as the C-terminal sequences are T1 and T2 specific.
- Trk C
Trk C also known as Neurotrophic tyrosine kinase, receptor, type 3 is a high affinity receptor for neurotrophin-3 (NT3). Binding of NT3 on Trk C triggers the neuronal growth from neural stem cells in a process known as neurogenesis. NT3 is particularly important in proprioceptive neuron growth.
Demonstration of function in Knock-out mice:Multiple studies of utilizing transgenic mouse models have been employed to highlight the effects of Trk deficiency on embryological development of mice.
- Trk Defective: Overall, Trk defective mice demonstrate a significant reduction in survival over a 30 day period. Specifically, such knockouts result in impaired olfactory function, as well as poor pain and thermoception responses. This is attributed to a >70% reduction in neuronal cells in the dorsal root, trigeminal and sympathetic ganglia over this period. Multiple other pathological conditions exist but it is unclear as to whether these are attributable to Trk deficiency or secondary to extremely impaired nervous function.
- Trk A defective: TrkA defective mice lack the ability to perceive painful and thermoceptive stimuli. This is attributed to the malformation of applicable peripheral sensory neurons during embryonic and post-natal development.
- Trk B Defective: inactivation of the Trk B gene product results in significant neurological deficiencies; poor feeding responses, poor touch perception in addition other behavioural abnormalities. Overall, this is perceived to be due to a >80% depletion of catecholaminergic neurons known to relay sensory input from the gastrointestinal, cardiovascular and pulmonary systems.
- Trk C Defective: Defective mice demonstrate unusual postural responses as well as severely impaired proprioception. It is believed that these deficiencies are attributable to an absolute deficiency of Ia muscle afferents that innervate proprioceptive spindle fibres during embryological development.
NGF binding to ligation region facilitates dimerisation of Trk Receptor monomers into the signal transductive unit- activating the kinase unit. Phosphorylation of the autoregulatory loop tyrosines of the cytoplasmic domain further activates the kinase. Phosphorylation of an additional five tyrosines within this region promotes signalling by providing docking sites for adaptor proteins which regulate signalling-cascade couples. Overall, the purpose of this process appears to be to facilitate down-stream phosphorylation and gene expression regulation(Eg. upregulation of Neuronal Factor Kappa B) via multiple transductive pathways, many of which, are synonymous with tyrosine kinase receptor activity. Thus, the autophosphorylation of intracellular domain tyrosine residues precipitates the association of second-messenger adaptor proteins which may or may not themselves be phosphorylated in order to illicit the desired signalling event.(see dynamics overview image for some examples in addition to those below)
co-operation with p75NTR: Association with p75NTR(also known as the "low affinity neurotropin receptor") appears to be an integral component of regulation of Trk's. This combination of Trk and p75NTR proteins results in the formation of a high-affinity binding site within the Trk binding region, increasing the responsiveness of receptors for thier ligands. Additionally, coordination with p75NTR has been implicated in a reduction in ubiquitination, delay of internalisation of receptors during sensitisation, and overall decreases the apoptotic potential of cell lines  .
Mechanism of Trk Activation via multiple Signalling pathways
Tropomyosine receptor kinases (Trk) enhance neuronal cell growth and differentiation of neurites via the activation of various signalling pathways. PKC, PLCy1, Ras/MAPK and PI3 are the four well known pathways, which exhibit this activity. The latter process involves the activation of transcription factors that will bind onto a specific target gene, thus trigerring neuronal cell division and/ differentiation via production of specific proteins that inhibit apoptosis. Cite error: Closing
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DAG is necessary for the further activation of the PKC and it remains bound to the cell membrane. IP3, a water soluble protein , in turn is released into the cytoplasm and leads to the release of Ca2+ reserves from the endoplasmic reticulum. This increase of calcium ions actives the classic isoforms of [Protein Kinase C|Protein kinase C]. 
Protein Kinase C (PKC) is a family of nucleotide-independent, Ca2+-dependent serine kinases. . At least 11 isozymes have been identified,  most PKC isozymes are ubiquitous and multiple members of the family can be coexpressed within the same cell leading to complex pathways. However, the basic signal transduction pathway involves the allosteric activation of PKC by the intracellular messengers diacylglycerol and Calcium ions. The inactive PKC is mainly found in the cytosol. Upon stimulation the PKC proteins translocate to the cell membrane. This translocation is the result of a cascade initiated by the binding of a number of extracellular ligands, such as, growth factors, hormones, and neurotransmitters to a G protein-coupled receptor (GPRC) found on the plasma membrane. This in turn activates a stimulating G protein which in turn activates phospholipase C (PLC).  A G protein is then activated, which then activates phospholipase C (PLC). The PLC cleaves phosphoinositol-4,5-bisphosphate (PIP2) into 1,2-diacylglycerol (DAG) and inositol-1.,4,5-triphosphate (IP3).  The IP3, couples to Ca2+ channels found on the endoplasmic reticulum releasing Ca2+ reserves. The increased concentration of calcium ions bind to inactive PKC molecules and then translocates to the plasma membrane. Here is binds to DAG giving rise to the active PKC enzyme.  The PKC family plays a major role in cellular signal transduction. Their main roles consist in the regulation of cell proliferation, differentiation, survival and apoptosis. 
Ras-MAPK (mitogen-activated protein kinase) Pathway
Activation of Ras-MAPK signalling pathway is predominantly essential for promoting neuronal cell growth and differentiation. Several pathways derive from Trk receptors for activation of Ras molecules, which begin by formation of ligand-bound mitogen (BDNF or NGF) at an extracellular domain of the trk, causing receptor dimerization. This subsequently will lead to phosphorylation of the intracellular parts of the receptors, which activates Guanine Exchange factors (GEFs), such as sos, Grb2 and Shc. GEFs trigger the exchange of GDP bound to inactive Ras to GTP, resulting Ras to activate. The presence of activated Ras molecules, stimulate signaling via a major downstream pathway known as MAP kinase (mitogen-activated protein kinase)-although activated Ras, would be also capable of triggering several other downstream pathways such as PLC-y1 and PI3-kinases (Figure..). 
Mitogen-activated protein kinase (MAPK) pathway involves a series of signalling cascade. Raf is the first component of MAPK, activated by Ras-GTP on the plasma membrane.  Raf then phosphorylates MEK1/2, which in turn activates the ERK1/2 by phosphorylation. ERK1/2 kinase would further phosphorylate a variety of downstream targets. These pathways may include phosphorylation of MAP 2-kinase by MAP 3-kinase on its serine and threonine amino-acid residues. Further phosphorylation of MAP- kinase by MAP 2-kinase on its serine and tyrosine amino-acid residues subsequently yields activated MAP-kinase. This results in activation of transcription factors that will lead to changes in gene expression.  . (Click here for well illustration of MAP-kinase pathway on You-tube)Nonetheless, aberrant Ras-MAPK signaling has been identified to be a possible factor responsible for the uncontrolled proliferation and malignancy (See “Cancer Biotherapy”, current research).
PI3K (Phosphatidylinositol 3-kinase) Pathway
Phosphatidylinositides which promotes survival of many populations of neurons. PI3K directly activates Ras-dependent pathway through which Trk signaling promotes survival in many, but not all, neurons. PI3K has an array of main effectors which include the mitogen-transducing signal proteins such as protein kinase C, phosphoinositide-dependent kinases and mitogen activated protein kinases. They are activated either via their interaction with lipid products of PI3K or through PI3K-dependent phosphorylation of proteins. PI3K is activated by number of growth factor receptors with intrinsic and tyrosine kinase activity. Nerve growth factor (NGF) activates PI3K in TRK to regulate cell functions.
Cancer biotherapy via targeting ‘Ras / Ras-MAP-kinase’ signalling pathway
Recent studies have hypothesized that receptors responsible for Ras activation would be effective target for treating human cancers. Based on this hypothesis, since 1995, therapies utilizing the inhibition of Ras activation have aroused a major interest. Farnesylation is a process by which small G-proteins like Ras are activated and adhered to the cell membrane. In a recent paper , farnesyl transferase inhibitors were used in order to block the farnesylation of Ras proteins. The results revealed promising hopes in animal models but unfortunately have not been effective in human cancer treatment. The major downstream target of Ras-MAP-Kinase (MAPK) has also been taken as a subject for drug design. Sebolt and his colleagues showed that MAPK-inhibitors are effective in cancer therapy using mouse models  . It has been also suggested that Inhibition of the Ras-MAPK signaling pathway can serve as a potential therapy for NF1 in humans. 
Trk receptors can behave as signalling endosomes
In a recent publication, a hypothesized model was explained in which active Trk receptors are internalised into signaling endosomes during signalling transduction process.  The experiment that led to this understanding involved local application of NGF to axons and soma (cell bodies) of neuronal cells. It was shown that both ERK1/2 and ERK5 signaling cascades were activated. Surprisingly, application of NGF to neuronal cells at distance resulted only in activation of ERK5 pathway and NOT ERK1/2. It was then understood that activation of ERK5 in the cells somas required receptor internalisation due to inhibition by thermo-sensitive dynamin. Further investigation was conducted by local application of specific Trk-inhibitor (K252a) at either the distal axon or cell body, which blockaded the activation of ERK5 in the cell soma in the presence of NGF. Therefore, the hypothesized model in which active Trk receptors are internalised into signalling endosomes is inevitable. Nonetheless, the mechanism through which distance affects the specificity of signalling would be the future topic of investigation. 
MicroRNAs are tissue specific, small non-coding RNAs, which are involved in post-trancriptional-silencing mechanism (See also: miRNA Science Overview). In non-mammals, miRNAs have been discovered to be involved in developmental process of the nervous system. For instance, in Caenorhabditis elegans, miR-273 participate in a regulatory mechanism in which ensures for the morphgenesis and differentiation of receptor neurons.  Likewise, in mammalian brain tissue, recent studies show that certain miRNAs have been identified, in which suggest an important role of these small molecules in modulating Trk receptors and development of nervous system. In addition, functional regulation of these miRNAs is also linked to tumorgenesis of nervous system. For instance, three neuronal miRNAs (9,125a and 125b) are found to be involved in human neuroblastoma cell proliferation by down-modulating TrkC isoform. 
- Apoptosis- Programmed cell death.
- Autophosphorylation is the term used to define the phosphorylation of a kinase protein catalysed by its own enzymatic activity.
- BDNF- Brain-derived neurotrophic growth factor is a protein that promotes activation of tropomyosine receptor kinases (trk).
- Dynamin- GTPase enzyme responsible for endocytosis in eukaryotic cells.
- ERK- Extracellular regulated kinase.
- Exons-Expressed regions of eukaryotic genes.
- Farnesyl transferase inhibitors- a class of experimental cancer drug blocks farnesylation of Ras proteins.
- GTPases are a large family of enzymes that can bind and hydrolyse guanosine triphosphate(GTP).
- Neurofibromatosis (NF)- an inherited disorder that causes production of benign tumours on nerve tissues. There are two types of NF; NF1 and NF2. NF1 is the most common disease amongst infants, also known as Recklinghausen disease.
- NGF- Nerve growth factor is a protein that promotes activation of tropomyosine receptor kinases (trk).
- Phosphorylation- A process by which a phosphate group is added to a protein that is required for its cellular activation.
- Proto-oncogene- normal cellular proteins that have the potential to cause uncontrolled cell growth when mutated.
- Oncogene- a gene that enhances uncontrolled proliferation of cells when expressed at high levels.
- Ras- small G-proteins that belong to the superfamily of monomeric GTPases. They are involved in receptor-mediated signal transduction pathways.
- EJ, Reichardt LF. “Trk receptors: roles in neuronal signal transduction”. Annu Rev Biochem. 2003;72:609-42
- Manning.G,. Whyte DB, Martinez R, Hunter T, Sudarsanam S. “The protein kinase complement of the human genome”. Science. 2002;298(5600):1912-34.
- EJ, Reichardt LF. “Trk receptors: roles in neuronal signal transduction”. Annu Rev Biochem. 2003;72:609-42. Retrieved 2 May 2009 from Pubmed
- Barker PA. High affinity not in the vicinity? Neuron. 2007 Jan 4;53(1):1-4. Review retrieved from 
- Pyle AD, Lock LF, Donovan PJ. Neurotrophins mediate human embryonic stem cell survival. Nat Biotechnology 1996;24:344–50. Retrieved 13 May 2009 from Pubmed
- Melamed et al., A Novel Lymphocyte Singnaling Defect: trk A Mutation in the Syndrome of Congenital Insensitivity to Pain and Anhidrosis (CIPA). Journal of Clinical Immunology, 2004;24:(4)441-448. Retrieved on 13 May 2009 from Pubmed Cite error: Invalid
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- Patapoutian A, and Reichardt LF. Trk receptors: mediators of neurotrophin action. Current Opinion in Neurobiology 2001; 11:272–280; Retrieved May 20, 2009, from 
- Barker PA. High affinity not in the vicinity? Neuron. 2007 Jan 4;53(1):1-4. Review retrieved from 
- Barker PA. p75NTR is positively promiscuous: novel partners and new insights. Neuron. 2004 May 27;42(4):529-33. Review. Retrieved from 
- Mankidy, R., Hastings, J., & Thackeray, J. (2003). Distinct Phospholipase C-#-Dependent Signaling Pathways in the Drosophila Eye and Wing Are Revealed by a New small wing Allele. Genetics Society of America. 164(2): 553–563.
- Bioengineering; Research from University of Tokyo broadens understanding of bioengineering. (2009,May). Medical Devices & Surgical Technology Week,70.
- Takai, Y., Kishimoto, A., Inoue, M., & Nishizuka, Y. (1977). Studies on a cyclic nucleotide-independent protein kinase and its proenzyme in mammalian tissues. I. Purification and characterization of an active enzyme from bovine cerebellum. J. Biol. Chem., 252(21), 7603-7609. 
- Acs, P., Wang, Q., Bogi, K., Marquez, A., Lorenzo, P., Biro, T., Szallai, Z., Mushinski, F., & Blue, P. (1997). Both the Catalytic and Regulatory Domains of Protein Kinase C Chimeras Modulate the Proliferative Properties of NIH 3T3 Cells. J Biol Chem., 272(45), 28793-28799. Retrieved May 15, 2009, from Pubmed
- Dekker, L (2004). Protein Kinase C (Molecular Biology Intelligence Unit). New York: Springer.
- Signal Transduction Resource. (n.d.). Retrieved May 15, 2009, from Promega
- Steinberg, S. (2008). Structural Basis of Protein Kinase C Isoform Function. Physiol. Rev., 88, 1341-1378. Retrieved October 5, 2009, from Pubmed
- D Breitkreutz, L Braiman-Wiksman, N Daum, M F Denning, T Tennenbaum. (2007). Protein kinase C family: On the crossroads of cell signaling in skin and tumor epithelium. Journal of Cancer Research & Clinical Oncology, 133(11), 793-808. Retrieved May 16, 2009, from Pubmed
- Wiesmüller L, Wittinghofer F.,“Signal Transduction pathways involving Ras. Mini Review”.Cellular Signalling.1994;6(3) 247-267.
- Harvey, J., and Ashford, M.L.J. “Leptin in the CNS: much more than a satiety signal”. Neuropharmacology. 2003;44, 845-854.
- Krasilnikov MA. Phoshatidylinositol-3 kinase dependent pathways: the role in control of cell growth, survival, and malignant transformation. Biochemistry. Review 2000;65(1):59-67. Retrieved May 16, 2009, from Pubmed
- Kapeller R, and Cantley LC. Phosphatidylinositol 3-kinase. Bioessays. 1994;16(8):565-576. Retrieved May 16, 2009, from 
- Stephens LR, Jackson TR, and Hawkins PT. Agonist-stimulated synthesis of phosphatidylinosital(3,4,5)-triphosphate: a new intracellular signaling system? Biochim Biophys Acta 1993;1179(1):27-75. Retrieved May 16, 2009, from Pubmed
- Kohl,N.E., Omer C.A., Conner MW., Anthony NJ, Davide JP, deSolms SJ, Giuliani EA, Gomez RP., Graham SL., Hamilton K., “Inhibition of farnesyltransferase induces regression of mammary and salivary carcinomas in ras transgenic mice”. Nat Med (1995).1(8): 792-7.
- Sebolt,J.S,. Dudley D.T,. Herrera.R, Van Becelaere.K, Wiland A, Gowan RC, Tecle H, Barrett SD, Bridges A, Przybranowski.S,. Leopold WR. Saltiel A.R,. “Blockade of the MAP kinase pathway suppresses growth of colon tumors in vivo”. Nat Med. (1999).5(7): 810-6.
- Sebti S.M., Hamilton A.D,. “Farnesyltransferase and geranylgeranyltransferase I inhibitors and cancer therapy: lessons from mechanism and bench-to-bedside translational studies”. Oncogene.(2000).19(56): 6584-93.
- Watson, FL., Heerssen HM, Bhattacharyya A, Klesse L, Lin MZ, Segal RA.“Neurotrophins use the Erk5 pathway to mediate a retrograde survival response”.Nat Neurosci.2001;4(10):981-8
- Smirnova L, Gräfe A, Seiler A, Schumacher S, Nitsch R, Wulczyn FG. “Regulation of miRNA expression during neural cell specification”. Eur J Neurosci. 2005;21(6):1469-77
- Miska EA, Alvarez-Saavedra E, Townsend M, Yoshii A, Sestan N, Rakic P, Constantine-Paton M, Horvitz HR. “Microarray analysis of microRNA expression in the developing mammalian brain”. Genome Biol. 2004;5(9):R68
2009 Group Projects
--Mark Hill 14:02, 19 March 2009 (EST) Please leave these links to all group projects at the bottom of your project page.
Group 1 Meiosis | Group 2 Cell Death - Apoptosis | Group 3 Cell Division | Group 4 Trk Receptors | Group 5 The Cell Cycle | Group 6 Golgi Apparatus | Group 7 Mitochondria | Group 8 Cell Death - Necrosis | Group 9 Nucleus | Group 10 Cell Shape