Neurotrophin & Receptor Proteins

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Neurotrophin & Receptor Proteins

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Neurotrophin & Receptor Proteins Background

Neurotrophins are a group of structurally related dimeric proteins from the neurotrophic factor family. Nerve growth factor (NGF) isolated from male mouse submandibular gland, was the first member of the neurotrophin family to be discovered. It was found to provide trophic support for sensory and sympathetic neurons by Levi-Montalcini in the early 1950s. Following that, brain-derived neurotrophic factor (BDNF) was identified in 1982 and purified later from pig brain. Other neurotrophins including neurotrophin-3 and neurotrophin-4/5 were described later. 
The mature form of neurotrophins consists of approximately 120 amino acids, only 28 residues of which vary between each neurotrophin. Because of this similarity, neurotrophins have similar conformations and are able to form heterodimers with each other. All neurotrophins share a common three dimensional structure composed of two pairs of antiparallel β-strands and a cysteine knot motif composed of 6 cysteine residues.
Neurotrophins regulate neuronal survival, neurite outgrowth and differentiation in the peripheral and central nervous systems. Each neurotrophin supports specific populations of neurons depending on which receptors are expressed by these neurons. During development, innervating neurons are dependent on neurotrophic factors expressed by target tissues. Neurons which do not receive sufficient amounts of neurotrophic factors undergo apoptosis. Neurotrophins are essential for survival not only during development but also in adulthood. They have roles in neuronal maintenance, synaptic plasticity, learning and memory in adults.
Neurotrophins act by binding to two types of cell surface receptors: 1) tropomyosin-related kinase (Trk) of the receptor tyrosine kinase family (TrkA to NGF and NT3, TrkB to BDNF, NT3, NT4/5, and TrkC to NT3); 2) A tumor necrosis factor family receptor called p75 Neurotrophin Receptor (NTR), which binds all neurotrophins with almost equal affinity. Neurotrophin binding causes Trk to dimerize, which leads to the phosphorylation of the tyrosine kinases located on the intracellular domain of the receptor. The intracellular kinase domain can trigger various signaling cascades such as Ras/ERK (extracellular signal-regulated kinase) protein kinase pathway, the phosphatidylinositol-3-kinase (PI-3 kinase)/Akt kinase pathway, and phospholipase C (PLC)-γ1. Some of the signaling pathways triggered by neurotrophin binding to p75NTR are involved in NF-kB activation leading to cell survival and Jun kinase activation leading to programmed cell death.

Nerve growth factor
Purified NGF is a multimeric protein composed of α, β, and γ subunits (α2β2γ). The β subunit is associated with the biological activity of NGF and the γ subunit is involved in β subunit processing. The gene encoding the β subunit is located on chromosome 1p22 in human and chromosome 3 in mouse and is about 45 kilobases in length. 
There are four messenger ribonucleic acid (mRNA) transcripts produced through two separate promoters and alternative splicing of four exons in the NGF gene. Two major transcripts are translated from initiation sites -187 and -121 to form 34 and 27 kDa preproNGF, respectively. In the endoplasmic reticulum, the signal peptide is removed to produce 32 and 25 kDa pro nerve growth factor (proNGF). ProNGF may undergo post-translational modification and protease cleavage to yield 13kDa mature NGF. Mature NGF is also known as β-NGF or 2.5S NGF depending on the isolation procedure. 
Mature NGF forms a non-covalent homodimer and binds with high affinity (kd ≈ 10-11 M) to TrkA and with low affinity ((kd ≈ 10-9 M) to the common neurotrophin receptor p75NTR. NGF is retrogradely transported from nerve terminals to soma. Both TrkA and p75NTR are involved in the retrograde transport of NGF. NGF promotes cell survival in cells expressing TrkA through activation of the Ras/PI-3 kinase/AKT pathway and the Ras/Mitogen-activated protein kinase (MAPK) pathway.

Brain-derived neurotrophic factor (BDNF)
The gene encoding BDNF is located on chromosome 11p in human. This gene (70 kb) includes eleven exons of which one 3' exon encodes the BDNF protein. Through alternative splicing 10 different transcripts are produced. While transcripts containing exons I, II and IV (exon III according to the old nomenclature) are predominant in the brain, transcript V (exon IV according to the old nomenclature) is expressed more in the lung and heart. BDNF, the same as NGF, is produced as a precursor called proBDNF (36kDa) which can be processed to the mature form (14kDa) either intracellularly by furin or extracellularly in the synaptic cleft by tissue plasminogen activator via plasmin. 
The non-covalent homodimer of mature BDNF binds to TrkB leading to receptor dimerization and autophosphorylation of its intracellular kinase domain. This kinase domain can activate several downstream neuroprotective signaling pathways such as the Ras-MAPK cascade and phosphorylation of cyclic AMP-response element binding protein (CREB). BDNF, like all neurotrophins, binds to p75NTR with a low affinity and can induce apoptosis.
BDNF plays an important role not only in neuronal survival, but also in synaptic plasticity learning, and memory formation. It has been shown that BDNF enhances action potential frequency and excitatory synaptic activities of cultured neurons.

Neurotrophin-3 and neurotrophin-4
Based on the substantial similarities between the sequences of NGF and BDNF, it was possible to search for additional transcripts with similar sequences. Using upon this approach two additional neurotrophins were identified, named neurotrophin-3 (NT3, also initially known as hippocampal-derived neurotrophic factor) and neurotrophin-4 (NT4, also named neurotrophin-5 or neurotrophin-4/5). These proteins both promote neuronal survival and neurite growth in a similar manner to, but with different neuronal specificities than, NGF. Both NT3 and NT4 exist as dimers with similar structures to NGF and BDNF, although a comparison of the sequences of the neurotrophins reveals that several loop regions show substantial variability which allows for specificity in receptor binding. NT3 is produced as a precursor within cells, which is cleaved to yield the mature protein and can be processed by over-expressed pro-protein convertases. 

Other neurotrophins
In addition to the four neurotrophins described above, two others have been identified in fish, named NT-6 and NT-7. Both are able to support the survival of and axon growth from chick neurons and appear to be most closely related to NGF. However, homologues of these neurotrophins have not been identified in mammals.

The neurotrophins and their receptors
TrkA is a receptor for NGF, TrkB for BDNF and NT4/5, and TrkC for NT3. NT-3 also signals through TrkA and TrkB under certain circumstances. In addition, each of the neurotrophins binds to the structurally unrelated neurotrophin receptor p75. Splice variants of Trk receptors are also expressed at high levels in the nervous system especially during later stages of development and in the adult nervous system. Some of these splice variants lack a kinase domain and were thought to sequester signaling of the full-length receptor. It was also discovered that the truncated TrkB receptor TrkB-Tl binds a calcium channel in cis and directly regulates signaling in adult glia cells.

Signaling mechanisms of neurotrophins

PC 12 cells, a cell line derived from rat adrenal pheochromocytoma, have been a valuable tool to elucidate the signaling mechanism of neurotrophins. PC 12 cells express endogenous TrkA and p75, and send out processes called neurites in response to NGF.
Using PC 12 cells, it was shown that upon neurotrophin binding, Trk receptors dimerize and undergo trans-phosphorylation on several tyrosine residues in the C-terminal domain. Two tyrosine residues, Y490 and Y785, which are outside the kinase domain, are major components of TrkA signal transduction. Y490 is the docking site for two adaptor proteins, She and FRS-2. She binding further induces the activation of two signaling pathways: the Ras/MAPK pathway leads to activation of transcription factors CREB and SRF, and the PI3K pathway which activates Akt kinase and increases the survival of host cells. FRS-2, on the other hand, competes for binding to Y490 with She and activates Src kinase whose function in NGF signaling is still not understood. Y785 is a docking site for PLCγ, and binding to this site initiates the PKC signaling pathway. PLCγ binding is also linked to activation of MAPK. 
In contrast to NGF-TrkA signaling which promotes the survival of neurons, neurotrophin-p75 signaling regulates gene expression and is, surprisingly, linked to apoptosis. p75 signaling manifests itself through binding to different sets of adaptor molecules, including TRAF6, RhoA and NRAGE. TRAF6 activates the canonical NFkB pathway which leads to the transcriptional control of gene expression. RhoA, a small GTPase, leads to a reduction in growth cone motility. Adaptor molecules NRAGE, NRIF, SC-1 promote cell cycle arrest and eventually cell death. p75 activation also leads to production of ceramide, a sphingolipid produced from the cell membrane. The presence of ceramide partially inhibits activation of PI3K and Ras signaling pathways. 
Under physiological conditions, neurotrophins signal via retrograde transport from target field to cell body to control neuronal survival and gene expression. This signaling mechanism was shown by local application of NGF directly to axons of sympathetic neurons using a compartmentalized culture system. The requirement of retrograde signaling for the support of sympathetic neuron survival was partially tested in vivo through overexpression of NGF in target fields using a keratin-specific promoter. As before, the number of surviving SCG neurons doubled in this gain of function system further supporting the notion that target-derived NGF is a limiting factor for sympathetic neuron survival. An ideal test for the requirement of retrograde signaling will involve a local inactivation of TrkA signaling directly at the cell body at an appropriate stage of development. During development of the sympathetic nervous system, NGF and NT3 acting through the same receptor TrkA orchestrate target innervation of sympathetic neurons. It is proposed that during initial projections, NT3 supports growth of sympathetic neuron axons while they project along the blood vessels before reaching their final targets, then, as neurons enter the target field, NGF secreted locally leads to an upregulation of p75 which in turn downregulates the neurons’ responsiveness to NT3.

Neurotrophins references
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2. Holland D R, Cousens L S, Meng W, et al. Nerve growth factor in different crystal forms displays structural flexibility and reveals zinc binding sites[J]. Journal of molecular biology, 1994, 239(3): 385-400.
3. Fariñas I, Jones K R, Tessarollo L, et al. Spatial shaping of cochlear innervation by temporally regulated neurotrophin expression[J]. The Journal of Neuroscience, 2001, 21(16): 6170-6180.
4. Reichardt L F. Neurotrophin-regulated signalling pathways[J]. Philosophical Transactions of the Royal Society of London B: Biological Sciences, 2006, 361(1473): 1545-1564.
5. Binder D K, Scharfman H E. Mini review[J]. Growth factors, 2004, 22(3): 123-131.
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7. Butte M J, Hwang P K, Mobley W C, et al. Crystal structure of neurotrophin-3 homodimer shows distinct regions are used to bind its receptors[J]. Biochemistry, 1998, 37(48): 16846-16852.
8. Robinson R C, Radziejewski C, Spraggon G, et al. The structures of the neurotrophin 4 homodimer and the brain-derived neurotrophic factor/neurotrophin 4 heterodimer reveal a common Trk-binding site[J]. Protein Science, 1999, 8(12): 2589-2597.
9. Götz R, Köster R, Winkler C, et al. Neurotrophin-6 is a new member of the nerve growth factor family[J]. Nature, 1994, 372(6503): 266-269. 
10. Lai K O, Fu W Y, Ip F C F, et al. Cloning and expression of a novel neurotrophin, NT-7, from carp[J]. Molecular and Cellular Neuroscience, 1998, 11(1): 64-76.
11. Bamji S X, Majdan M, Pozniak C D, et al. The p75 neurotrophin receptor mediates neuronal apoptosis and is essential for naturally occurring sympathetic neuron death[J]. The Journal of cell biology, 1998, 140(4): 911-923.
12. Albers K M, Wright D E, Davis B M. Overexpression of nerve growth factor in epidermis of transgenic mice causes hypertrophy of the peripheral nervous system[J]. The Journal of neuroscience, 1994, 14(3): 1422-1432.
13. Kuruvilla R, Zweifel L S, Glebova N O, et al. A neurotrophin signaling cascade coordinates sympathetic neuron development through differential control of TrkA trafficking and retrograde signaling[J]. Cell, 2004, 118(2): 243-255.

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