Neurotrophins and their receptors in breast cancer

Growth factors of the neurotrophin family and their receptors have been mainly studied in the nervous system, but they are also expressed in carcinomas, and in breast cancer they significantly impact tumor cell growth and metastasis through various signaling pathways. Nerve growth factor and its precursor proNGF, brain-derived neurotrophic factor and neurotrophin-4/5 stimulate breast cancer cells through an autocrine loop involving the tyrosine kinase receptors TrkA, TrkB as well as its truncated form TrkB.T1 and the p75^NTR death receptor. Preclinical studies have shown that targeting neurotrophins and their receptors induce an inhibition of breast cancer cell survival, proliferation and invasion. Furthermore, targeting neurotrophins may also decrease tumor-induced cancer pain and this additional effect further strengthens their clinical relevance.     

Cdk5 activity is required for Purkinje cell dendritic growth in cell‐autonomous and non‐cell‐autonomous manners

Cyclin‐dependent kinase 5 (Cdk5) is recognized as a unique member among other Cdks due to its versatile roles in many biochemical processes in the nervous system. The proper development of neuronal dendrites is required for the formation of complex neural networks providing the physiological basis of various neuronal functions. We previously reported that sparse dendrites were observed on cultured Cdk5‐null Purkinje cells and Purkinje cells in Wnt1^cre‐mediated Cdk5 conditional knockout (KO) mice. In the present study, we generated L7^cre‐mediated p35; p39 double KO (L7^cre‐p35^f/f; p39^–/–) mice whose Cdk5 activity was eliminated specifically in Purkinje cells of the developing cerebellum. Consequently, these mice exhibited defective Purkinje cell migration, motor coordination deficiency and a Purkinje dendritic abnormality similar to what we have observed before, suggesting that dendritic growth of Purkinje cells was cell‐autonomous in vivo . We found that mixed and overlay cultures of WT cerebellar cells rescued the dendritic deficits in Cdk5‐null Purkinje cells, however, indicating that Purkinje cell dendritic development was also supported by non‐cell‐autonomous factors. We then again rescued these abnormalities in vitro by applying exogenous brain‐derived neurotrophic factor (BDNF). Based on the results from culture experiments, we attempted to rescue the developmental defects of Purkinje cells in L7^cre‐p35^f/f; p39^–/– mice by using a TrkB agonist. We observed partial rescue of morphological defects of dendritic structures of Purkinje cells. These results suggest that Cdk5 activity is required for Purkinje cell dendritic growth in cell‐autonomous and non‐cell‐autonomous manners. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1175–1187, 2017     

p75 reduces TrkB tyrosine autophosphorylation in response to brain-derived neurotrophic factor and neurotrophin 4/5

Neurotrophins mediate their signals through two different receptors: the family of receptor tyrosine kinases, Trks, and the low affinity pan-neurotrophin receptor p75. Trk receptors show more restricted ligand specificity, whereas all neurotrophins are able to bind to p75. One important function of p75 is the enhancement of nerve growth factor signaling via TrkA by increasing TrkA tyrosine autophosphorylation. Here, we have examined the importance of p75 on TrkB- and TrkC-mediated neurotrophin signaling in an MG87 fibroblast cell line stably transfected with either p75 and TrkB or p75 and TrkC, as well as in PC12 cells stably transfected with TrkB. In contrast to TrkA signaling, p75 had a negative effect on TrkB tyrosine autophosphorylation in response to its cognate neurotrophins, brain-derived neurotrophic factor and neurotrophin 4/5. On the other hand, p75 had no effect on TrkB or TrkC activation in neurotrophin 3 treatment. p75 did not effect extracellular signal-regulated kinase 2 tyrosine phosphorylation in response to brain-derived neurotrophic factor, neurotrophin 3, or neurotrophin 4/5. These results suggest that the observed reduction in TrkB tyrosine autophosphorylation caused by p75 does not influence Ras/mitogen-activated protein kinase signaling pathway in neurotrophin treatments.     

Trypanosome trans-sialidase mediates neuroprotection against oxidative stress, serum/glucose deprivation, and hypoxia-induced neurite retraction in Trk-expressing PC12 cells

Trypanosome trans-sialidase (TS) is a sialic acid-transferring enzyme and a novel ligand of tyrosine kinase (TrkA) receptors but not of neurotrophin receptor p75NTR. Here, we show that TS targets TrkB receptors on TrkB-expressing pheochromocytoma PC12 cells and colocalizes with TrkB receptor internalization and phosphorylation (pTrkB). Wild-type TS but not the catalytically inactive mutant TSDeltaAsp98-Glu induces pTrkB and mediates cell survival responses against death caused by oxidative stress in TrkA- and TrkB-expressing cells like those seen with nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). These same effects are not observed in Trk deficient PC12(nnr5) cells, but are re-established in PC12(nnr5) cells stably transfected with TrkA or TrkB, are partially blocked by inhibitors of tyrosine kinase (K-252a), mitogen-activated protein/mitogen-activated kinase (PD98059) and completely blocked by LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K). Both TrkA- and TrkB-expressing cells pretreated with TS or their natural ligands are protected against cell death caused by serum/glucose deprivation or from hypoxia-induced neurite retraction. The cell survival effects of NGF and BDNF against oxidative stress are significantly inhibited by the neuraminidase inhibitor, Tamiflu. Together, these observations suggest that trypanosome TS mimics neurotrophic factors in cell survival responses against oxidative stress, hypoxia-induced neurite retraction and serum/glucose deprivation.     

TrkB receptors are required for follicular growth and oocyte survival in the mammalian ovary

Although it is well established that both follicular assembly and the initiation of follicle growth in the mammalian ovary occur independently of pituitary hormone support, the factors controlling these processes remain poorly understood. We now report that neurotrophins (NTs) signaling via TrkB receptors are required for the growth of newly formed follicles. Both neurotrophin-4/5 (NT-4) and brain-derived neurotrophic factor (BDNF), the preferred TrkB ligands, are expressed in the infantile mouse ovary. Initially, they are present in oocytes, but this site of expression switches to granulosa cells after the newly assembled primordial follicles develop into growing primary follicles. Full-length kinase domain-containing TrkB receptors are expressed at low and seemingly unchanging levels in the oocytes and granulosa cells of both primordial and growing follicles. In contrast, a truncated TrkB isoform lacking the intracellular domain of the receptor is selectively expressed in oocytes, where it is targeted to the cell membrane as primary follicles initiate growth. Using gene-targeted mice lacking all TrkB isoforms, we show that the ovaries of these mice or those lacking both NT-4 and BDNF suffer a stage-selective deficiency in early follicular development that compromises the ability of follicles to grow beyond the primary stage. Proliferation of granulosa cells-required for this transition-and expression of FSH receptors (FSHR), which reflects the degree of biochemical differentiation of growing follicles, are reduced in trkB-null mice. Ovaries from these animals grafted under the kidney capsule of wild-type mice fail to sustain follicular growth and show a striking loss of follicular organization, preceded by massive oocyte death. These results indicate that TrkB receptors are required for the early growth of ovarian follicles and that they exert this function by primarily supporting oocyte development as well as providing granulosa cells with a proliferative signal that requires oocyte-somatic cell bidirectional communication. The predominance of truncated TrkB receptors in oocytes and their developmental pattern of subcellular expression suggest that a significant number of NT-4/BDNF actions in the developing mammalian ovary are mediated by these receptors.     

Trkb Signaling in Pericytes Is Required for Cardiac Microvessel Stabilization

Pericyte and vascular smooth muscle cell (SMC) recruitment to the developing vasculature is an important step in blood vessel maturation. Brain-derived neurotrophic factor (BDNF), expressed by endothelial cells, activates the receptor tyrosine kinase TrkB to stabilize the cardiac microvasculature in the perinatal period. However, the effects of the BDNF/TrkB signaling on pericytes/SMCs and the mechanisms downstream of TrkB that promote vessel maturation are unknown. To confirm the involvement of TrkB in vessel maturation, we evaluated TrkB deficient (trkb ^−/−) embryos and observed severe cardiac vascular abnormalities leading to lethality in late gestation to early prenatal life. Ultrastructural analysis demonstrates that trkb^−/− embryos exhibit defects in endothelial cell integrity and perivascular edema. As TrkB is selectively expressed by pericytes and SMCs in the developing cardiac vasculature, we generated mice deficient in TrkB in these cells. Mice with TrkB deficiency in perivascular cells exhibit reduced pericyte/SMC coverage of the cardiac microvasculature, abnormal endothelial cell ultrastructure, and increased vascular permeability. To dissect biological actions and the signaling pathways downstream of TrkB in pericytes/SMCs, human umbilical SMCs were treated with BDNF. This induced membranous protrusions and cell migration, events dependent on myosin light chain phosphorylation. Moreover, inhibition of Rho GTPase and the Rho-associated protein kinase (ROCK) prevented membrane protrusion and myosin light chain phosphorylation in response to BDNF. These results suggest an important role for BDNF in regulating migration of TrkB-expressing pericytes/SMCs to promote cardiac blood vessel ensheathment and functional integrity during development.     

BDNF and NT4/5 promote survival and neurite outgrowth of pontocerebellar mossy fiber neurons

The neurotrophins nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT3), and NT4/5 are all found in the developing cerebellum. Granule cells, the major target neurons of mossy fibers, express BDNF during mossy fiber synaptogenesis. To determine whether neurotrophins contribute to the development of cerebellar afferent axons, we characterized the effects of neurotrophins on the growth of mossy fiber neurons from mice and rats in vitro. For a mossy fiber source, we used the basilar pontine nuclei (BPN), the major source of cerebellar mossy fibers in mammals. BDNF and NT4/5 increased BPN neuron survival, neurite outgrowth, growth cone size, and elongation rate, while neither NT3 nor NGF increased survival or outgrowth. In addition, BDNF and NT4/5 reduced the size of neurite bundles. Consistent with these effects, in situ hybridization on cultured basilar pontine neurons revealed the presence of mRNA encoding the TrkB receptor which binds both BDNF and NT4/5 with high affinity. We detected little or no message encoding the TrkC receptor which preferentially binds NT3. BDNF and NT4/5 also increased TrkB mRNA levels in BPN neurons. In addition to previously established functions as an autocrine/paracrine trophic factor for granule cells, the present results indicate that cerebellar BDNF may also act as a target‐derived trophic factor for basilar pontine mossy fibers. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 254–269, 1999     

Tottering and leaner mutations perturb transient developmental expression of tyrosine hydroxylase in embryologically distinct Purkinje cells.

The mouse mutants tottering and leaner exhibit neurologic disorders associated, in part, with global noradrenergic hyperinnervation. Therefore, the expression of tyrosine hydroxylase (TH) mRNA and protein was examined in mutant and control mice. TH expression was normal in the major catecholaminergic nuclei. However, TH was expressed in vermal Purkinje cells of adult mutant but not control mice. TH expression in the Purkinje cells of both mutants was first observed on P21 and persisted throughout adulthood; in contrast, Purkinje cells of normal mice expressed TH transiently during development from P21 to P35. Thus, tottering and leaner mice are deficient in suppressing the normal transient expression of TH in developing Purkinje cells, suggesting that the protein encoded by the tg locus may play a crucial role in neuronal development.     

Essential Role of Hrs in Endocytic Recycling of Full-length TrkB Receptor but Not Its Isoform TrkB.T1

Brain-derived neurotrophic factor (BDNF) signaling through its receptor, TrkB, modulates survival, differentiation, and synaptic activity of neurons. Both full-length TrkB (TrkB-FL) and its isoform T1 (TrkB.T1) receptors are expressed in neurons; however, whether they follow the same endocytic pathway after BDNF treatment is not known. In this study we report that TrkB-FL and TrkB.T1 receptors traverse divergent endocytic pathways after binding to BDNF. We provide evidence that in neurons TrkB.T1 receptors predominantly recycle back to the cell surface by a “default” mechanism. However, endocytosed TrkB-FL receptors recycle to a lesser extent in a hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)-dependent manner which relies on its tyrosine kinase activity. The distinct role of Hrs in promoting recycling of internalized TrkB-FL receptors is independent of its ubiquitin-interacting motif. Moreover, Hrs-sensitive TrkB-FL recycling plays a role in BDNF-induced prolonged mitogen-activated protein kinase (MAPK) activation. These observations provide evidence for differential postendocytic sorting of TrkB-FL and TrkB.T1 receptors to alternate intracellular pathways.Brain-derived neurotrophic factor (BDNF)³ has been shown to play critical roles in vertebrate nervous system development and function (1–3). The actions of BDNF are dictated by two classes of cell surface receptors, the TrkB receptor and the p75 neurotrophin receptor. BDNF binding to TrkB receptors activates several signaling cascades, including phosphatidylinositol 3-kinase, phospholipase C, and Ras/mitogen-activated protein kinase (MAPK) pathways, that mediate growth and survival responses to BDNF (1, 4, 5). It has been established that upon binding neurotrophins, Trk receptors are rapidly endocytosed in a clathrin-dependent manner (6, 7). Postendocytic sorting of Trk receptors to diverse pathways after ligand binding has a significant impact on the physiological responses to neurotrophins because they also determine the strength and duration of intracellular signaling cascades initiated by activated Trk receptors (8). Three alternate endocytic pathways that Trk receptors can follow are trafficking to lysosomes for degradation, recycling back to the plasma membrane, or being retrogradely transported (9–13). The degradative pathway to lysosomes is characterized by down-regulation of the total number of receptors at the cell surface and a decreased response to ligand. Conversely, recycling of receptors back to the plasma membrane can lead to functional resensitization and prolongation of cell surface-specific signaling events. A recent study has shown that recycled and re-secreted BDNF plays an important role in mediating the maintenance of long term potentiation in hippocampal slices, which suggests a potential role of TrkB recycling in long term potentiation regulation (14).Different TrkB isoforms, including the full-length TrkB (TrkB-FL) and three truncated isoforms named TrkB.T1, TrkB.T2, and TrkB.T-Shc, exist in the mammalian central nervous system because of alternative splicing (15–17). Truncated TrkB.T1 receptor lacks the kinase domain but contains short isoform-specific cytoplasmic domain in its place (15, 16). Many neuronal populations, including hippocampal and cortical neurons, express both full-length and truncated TrkB receptors (18, 19). TrkB.T1 is expressed at low levels in the prenatal rodent brain, but its expression increases postnatally, ultimately exceeding the level of full-length TrkB in adulthood (19–22). The physiological function of the TrkB.T1 receptor remains unclear, but it may serve as dominant-negative regulator of full-length TrkB receptors (23–25), may sequester ligand and limit diffusion (26, 27), may regulate cell morphology and dendritic growth (28, 29), and may even autonomously activate signaling cascades in a neurotrophin-dependent manner (30). TrkB-FL and TrkB.T1 are localized to both somatodendritic and axonal compartments in neurons (31); however, little is known about TrkB.T1 endocytic trafficking fate upon BDNF treatment.In this study we conducted an analysis of the postendocytic fates (degradation and recycling) of TrkB-FL and TrkB.T1 receptors in PC12 cells and neurons. We have determined that, unlike TrkB-FL, TrkB.T1 receptors recycle more efficiently in a default pathway to plasma surface after internalization, which is independent of hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs). Conversely, Hrs could bind with TrkB-FL in a kinase activity-dependent manner and regulate TrkB-FL receptors postendocytic recycling. Hrs was identified as a tyrosine-phosphorylated protein in cells stimulated with growth factors and cytokines (32). Hrs is expressed in the cytoplasm of all cells and is predominantly localized to endosomes (33). Hrs has also been proposed to play a role in regulating cell surface receptor postendocytic trafficking (34). These observations provide evidence for differential postendocytic sorting to alternate intracellular pathways between TrkB-FL and TrkB.T1 receptors after internalization.     

The responsiveness of TrkB to BDNF and antidepressant drugs is differentially regulated during mouse development

Background

Previous studies suggest that the responsiveness of TrkB receptor to BDNF is developmentally regulated in rats. Antidepressant drugs (AD) have been shown to increase TrkB signalling in the adult rodent brain, and recent findings implicate a BDNF-independent mechanism behind this phenomenon. When administered during early postnatal life, ADs produce long-lasting biochemical and behavioural alterations that are observed in adult animals.

Methodology

We have here examined the responsiveness of brain TrkB receptors to BDNF and ADs during early postnatal life of mouse, measured as autophosphorylation of TrkB (pTrkB).

Principal Findings

We found that ADs fail to induce TrkB signalling before postnatal day 12 (P12) after which an adult response of TrkB to ADs was observed. Interestingly, there was a temporally inverse correlation between the appearance of the responsiveness of TrkB to systemic ADs and the marked developmental reduction of BDNF-induced TrkB in brain microslices ex vivo. Basal p-TrkB status in the brain of BDNF deficient mice was significantly reduced only during early postnatal period. Enhancing cAMP (cyclic adenosine monophosphate) signalling failed to facilitate TrkB responsiveness to BDNF. Reduced responsiveness of TrkB to BDNF was not produced by the developmental increase in the expression of dominant-negative truncated TrkB.T1 because this reduction was similarly observed in the brain microslices of trkB.T1 ^−/− mice. Moreover, postnatal AD administration produced long-lasting behavioural alterations observable in adult mice, but the responses were different when mice were treated during the time when ADs did not (P4-9) or did (P16-21) activate TrkB.

Conclusions

We have found that ADs induce the activation of TrkB only in mice older than 2 weeks and that responsiveness of brain microslices to BDNF is reduced during the same time period. Exposure to ADs before and after the age when ADs activate TrkB produces differential long-term behavioural responses in adult mice.     

The Trkb Receptor Tyrosine Kinase Regulates Cellular Proliferation Via Signal Transduction Pathways Involving Shc,Plcγ, and Cbl

Abstract

The TrkB protein tyrosine kinase is a high affinity receptor for brain derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4). TrkB autophosphorylation occurs on five cytoplasmic tyrosines: Y484, Y670, Y674, Y675, and Y785. Using site directed mutagenesis, we have assessed the importance of TrkB tyrosines 484 and 785 in affecting TrkB-mediated signaling events leading to NIH 3T3 cell mitogenesis and survival. Mutation of TrkB tyrosine 484, while having no affect on BDNF-inducible PLCγ and Cbl tyrosine phosphorylation, is essential for the phosphorylation of Shc, the complete activation of extracellular regulated kinase 1/2 (ERK1/2) and the induction of c-fos protein synthesis. In contrast, mutation of Y785 does not significantly affect BDNF-inducible Shc phosphorylation, ERK1/2 activation, or c-fos protein synthesis, but completely inhibits the tyrosine phosphorylation of PLCγ and Cbl. These data indicate that both ERK-dependent and ERK-independent signaling pathways lead to BDNF-inducible mitogenesis and survival.     

Cdk5 is involved in BDNF-stimulated dendritic growth in hippocampal neurons

Neurotrophins are key regulators of neuronal survival and differentiation during development. Activation of their cognate receptors, Trk receptors, a family of receptor tyrosine kinases (RTKs), is pivotal for mediating the downstream functions of neurotrophins. Recent studies reveal that cyclin-dependent kinase 5 (Cdk5), a serine/threonine kinase, may modulate RTK signaling through phosphorylation of the receptor. Given the abundant expression of both Cdk5 and Trk receptors in the nervous system, and their mutual involvement in the regulation of neuronal architecture and synaptic functions, it is of interest to investigate if Cdk5 may also modulate Trk signaling. In the current study, we report the identification of TrkB as a Cdk5 substrate. Cdk5 phosphorylates TrkB at Ser478 at the intracellular juxtamembrane region of TrkB. Interestingly, attenuation of Cdk5 activity or overexpression of a TrkB mutant lacking the Cdk5 phosphorylation site essentially abolishes brain-derived neurotrophic factor (BDNF)–triggered dendritic growth in primary hippocampal neurons. In addition, we found that Cdk5 is involved in BDNF-induced activation of Rho GTPase Cdc42, which is essential for BDNF-triggered dendritic growth. Our observations therefore reveal an unanticipated role of Cdk5 in TrkB-mediated regulation of dendritic growth through modulation of BDNF-induced Cdc42 activation.     

Benzodiazepine Receptor Binding in Cerebellar Cortex: Observations in Olivopontocerebellar Atrophy

Benzodiazepine receptor binding was measured in cerebellar cortex of 15 patients with dominantly inherited olivopontocerebellar atrophy (OPCA). The majority of these patients had a moderate to marked Purkinje cell loss, as judged by the lowered levels of dentate nucleus gamma-aminobutyric acid (GABA), a marker of Purkinje cells. Despite the reduction in Purkinje cell number cerebellar cortical benzodiazepine receptor density was either normal or slightly elevated in the OPCA patients. These results are in contrast to the findings in a mutant strain of mice deficient in Purkinje cells in which the concentration of benzodiazepine receptors in cerebellum is greatly reduced. Our data indicate that in the human, cerebellar cortical benzodiazepine receptors are either not significantly associated with Purkinje cells or that in OPCA Purkinje cell loss triggers a de novo synthesis of extra benzodiazepine binding sites. It is concluded that, in contrast with the rodent, in the human benzodiazepine receptor binding may not serve as a marker for cerebellar Purkinje cells.     

A TrkB/insulin receptor-related receptor chimeric receptor induces PC12 cell differentiation and exhibits prolonged activation of mitogen-activated protein kinase.

Insulin receptor-related receptor (IRR), an orphan receptor in the insulin receptor (IR) family of receptor tyrosine kinases, is primarily localized to neural crest-derived sensory neurons during embryonic development. Expression of IRR closely resembles that of the nerve growth factor receptor, TrkA. To analyze the signaling properties and function of IRR in PC12 cells, a TrkB/IRR hybrid receptor was used. In contrast to IR activation, brain-derived neurotrophic growth factor-mediated activation of the TrkB/IRR receptor resulted in differentiation rather than proliferation. Analysis of cytoplasmic substrates activated by the TrkB/IRR receptor indicates a signaling pathway similar to that of the IR. Mutagenesis studies further show that only TrkB/IRR receptors able to phosphorylate mitogen-activated protein kinase elicit a differentiation response. Our analysis indicates that prolonged kinetics of mitogen-activated protein kinase activation mediated by the TrkB/IRR chimeric receptor correlates with induction to differentiate.     

Differential heat stress stability of epidermal growth factor receptor and erbB-2 receptor tyrosine kinase activities

The epidermal growth factor (EGF) and erbB-2 receptors are structurally related membrane-bound tyrosine kinases. While these proteins exhibit close sequence homology, 50% overall and 80% in the tyrosine kinase domains, they respond very differently to heat stress. In NIH-3T3 or NR6 cells transfected with wild-type EGF-R and incubated at 37°C or heat shocked at 46°C, EGF binds to its receptor and stimulates receptor autophosphorylation to equivalent extents. At 46°C, however, the basal tyrosine kinase activity of the wild-type erbB-2 receptor is rapidly lost. When cells containing chimeric receptors composed of the EGF-R extracellular domain and intracellular domain of erbB-2 were heat stressed, ¹²⁵I-EGF bound to the receptors, but did not stimulate receptor autophosphorylation. The decline in EGF-stimulated chimeric erbB-2 receptor autophosphorylation is dependent on the length of heat shock, with nearly 100% of the kinase activity lost after 60 min at 46°C. The loss of chimeric receptor erbB-2 kinase activity is not due to degradation of receptor protein, nor is it attributable to a specific transmembrane domain from either the EGF or erbB-2 receptors. Sensitivity of erbB-2 to heat stress is also not a result of denaturation of this receptor's carboxy-terminal domain. Insertion of the erbB-2 tyrosine kinase domain into the EGF-R confers heat stress sensitivity to the resultant chimeric receptor. Thus, although the EGF-R and erbB-2 kinase domains show a high degree of homology, the secondary/tertiary structures of these domains would seem to be stabilized in distinct manners. © 1993 Wiley-Liss, Inc.     

Different regulation of Purkinje cell dendritic development in cerebellar slice cultures by protein kinase Cα and ‐β

Activity of protein kinase C (PKC), and in particular the PKCγ‐isoform, has been shown to strongly affect and regulate Purkinje cell dendritic development, suggesting an important role for PKC in activity‐dependent Purkinje cell maturation. In this study we have analyzed the role of two additional Ca²⁺‐dependent PKC isoforms, PKCα and ‐β, in Purkinje cell survival and dendritic morphology in slice cultures using mice deficient in the respective enzymes. Pharmacological PKC activation strongly reduced basal Purkinje cell dendritic growth in wild‐type mice whereas PKC inhibition promoted branching. Purkinje cells from mice deficient in PKCβ, which is expressed in two splice forms by granule but not Purkinje cells, did not yield measurable morphological differences compared to respective wild‐type cells under either experimental condition. In contrast, Purkinje cell dendrites in cultures from PKCα‐deficient mice were clearly protected from the negative effects on dendritic growth of pharmacological PKC activation and showed an increased branching response to PKC inhibition as compared to wild‐type cells. Together with our previous work on the role of PKCγ, these data support a model predicting that normal Purkinje cell dendritic growth is mainly regulated by the PKCγ‐isoform, which is highly activated by developmental processes. The PKCα isoform in this model forms a reserve pool, which only becomes activated upon strong stimulation and then contributes to the limitation of dendritic growth. The PKCβ isoform appears to not be involved in the signaling cascades regulating Purkinje cell dendritic maturation in cerebellar slice cultures. © 2003 Wiley Periodicals, Inc. J Neurobiol 57: 95–109, 2003     

Down-regulation of the neurotrophin receptor TrkB following ligand binding. Evidence for an involvement of the proteasome and differential regulation of TrkA and TrkB

This study examines the mechanisms by which the tyrosine kinase receptor TrkB is down-regulated following binding of brain-derived neurotrophic factor (BDNF). In primary cultures of cerebellar granule neurons, BDNF-induced reduction of TrkB receptors was largely prevented by the addition of specific proteasome inhibitors. HN10 cells, a neuronal cell line that can be readily transfected, also showed a marked down-regulation of cell surface TrkB following BDNF exposure. In addition, we observed that prolonged exposure to nerve growth factor of TrkA-transfected cells did not lead to the down-regulation seen with BDNF and TrkB. TrkA and TrkB chimeric molecules were therefore expressed in HN10 cells and tested for ligand-induced regulation. These experiments led to the conclusion that the motives responsible for down-regulation are contained in the cytoplasmic domain of TrkB, and a short sequence in the juxtamembrane domain of TrkB was identified that confers nerve growth factor-induced down-regulation when inserted into TrkA.     

The bovine papillomavirus type 1 E5 transforming protein specifically binds and activates the beta-type receptor for the platelet-derived growth factor but not other related tyrosine kinase-containing receptors to induce cellular transformation.

The 44-amino-acid E5 protein of bovine papillomavirus type 1 is a highly hydrophobic protein which appears to transform cells through the activation of growth factor receptors. To investigate the specificity of E5-growth factor receptor interactions required for mitogenic signaling, we utilized a nontumorigenic, murine myeloid cell line (32D) which is strictly dependent on interleukin-3 (IL-3) for sustained proliferation in culture. This IL-3 dependence can be functionally substituted by the expression of a variety of surrogate growth factor receptors and the addition of the corresponding ligand. Several receptor cDNAs for the alpha- and beta-type platelet-derived growth factor receptors [alpha PDGFR and beta PDGFR], the epidermal growth factor receptor, and the colony-stimulating factor 1 receptor) were transfected into 32D cells constitutively expressing the E5 protein to test for IL-3-independent growth. Only beta PDGFR was capable of abrogating the IL-3 dependence of 32D cells. The proliferative signal induced by the coexpression of beta PDGFR and E5 was accompanied by stable complex formation between these proteins, constitutive tyrosine phosphorylation of the receptor, and tumorigenicity in nude mice. The lack of cooperative interaction between E5 and the epidermal growth factor receptor, the colony-stimulating factor 1 receptor, and the highly related alpha PDGFR was paralleled by the inability of E5 to bind to these receptors and failure to increase receptor tyrosine phosphorylation. Thus, these data indicate that the ability of E5 to induce sustained proliferation and transformation of 32D cells is a direct consequence of specific interaction between the E5 protein and the beta PDGFR signaling complex and the subsequent stimulation of receptor tyrosine phosphorylation.