Subcellular Localization of Full-Length and Truncated Trk Receptor Isoforms in Polarized Neurons and Epithelial Cells
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The Journal of Neuroscience, July 15, 1999, 19(14):5823–5833 Subcellular Localization of Full-Length and Truncated Trk Receptor Isoforms in Polarized Neurons and Epithelial Cells David Kryl,1 Talene Yacoubian,2 Annakaisa Haapasalo,3 Eero Castren,3 Donald Lo,2 and Philip A. Barker1 1Centre for Neuronal Survival, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada, H3A 2B4, 2Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710, and 3A. I. Virtanen Institute, University of Kuopio, Kuopio, Finland Neurotrophins affect neuronal development and plasticity via both apical and basolateral domains. Full-length TrkB and TrkC spatially localized effects, yet little is known about the subcel- were found throughout transfected primary cultured hippocam- lular distribution of the Trk neurotrophin receptors and the pal neurons and transfected neurons in neocortical brain slices impact of this distribution on neurotrophin action. To address and showed no evidence of vectorial sorting. Truncated forms this, we examined the subcellular location of full-length TrkB of TrkB were also homogeneously distributed in MDCK cells, and TrkC tyrosine kinase receptors and truncated TrkB isoforms dissociated hippocampal neurons, and cortical neurons within after transfection of Madin–Darby canine kidney (MDCK) cells, slice preparations. Levels of full-length and truncated TrkB were dissociated primary hippocampal neurons, and cortical neu- examined in postsynaptic densities; both receptor isoforms rons within intact brain slices. Myc-, herpes virus glycoprotein were present but only moderately enriched in these structures. (HVG)-, or FLAG-derived epitope–tagged receptor isoforms Together, these findings suggest that Trk receptors are uni- were created to allow their unambiguous identification and formly distributed in both axonal and dendritic compartments localization after transfection. All tagged receptors were appro- and that local neurotrophin responses are controlled by other priately synthesized, and full-length myc-TrkB and myc-TrkC mechanisms. mediated appropriate neurotrophin-signaling events. We found that full-length TrkB receptors were excluded from the apical Key words: neurotrophins; MDCK; vectorial sorting; hip- domain of MDCK cells but that TrkC receptors were present in pocampal neurons; Trk; postsynaptic density Neurotrophins play pleiotropic roles in nervous system develop- see Naismith and Sprang, 1998). Ligand binding to Trk receptors ment and plasticity (Davies, 1994; Snider, 1994; Thoenen, 1995; activates several signaling cascades, including phosphatidylino- Lewin and Barde, 1996; McAllister et al., 1999). Some functions, sitol-3-kinase, phospholipase C, SNT, and ras/mitogen-activated such as the promotion of neuronal survival, exert global effects on protein kinase pathways, that mediate growth and survival re- neuronal cell state, whereas others, such as the regulation of sponses of the neurotrophins (for review, see Kaplan and Miller, synaptic plasticity, are more localized and produce subcellular 1997). The predominant types of Trk receptors expressed in the changes in neural function. Neurotrophins may have effects that mammalian CNS are TrkB and TrkC. Both of these are produced are localized to particular neuronal compartments, such as axons primarily as full-length receptor tyrosine kinases early in devel- or dendrites, or even to specific dendritic branches or spines opment with alternatively spliced variants becoming more prev- (Campenot, 1987; Stoop and Poo, 1996; Li et al., 1998; McAllister alent with increasing age (Valenzuela et al., 1993; Allendoerfer et et al., 1999). In regulating neuronal morphology, neurotrophins al., 1994; Escando´n et al., 1994; Knu¨sel et al., 1994; Fryer et al., exhibit differential effects on apical versus basal dendritic com- 1996). TrkB-T1 and -T2 isoforms, which bind neurotrophin but partments of cortical pyramidal neurons (McAllister et al., 1995, lack most of the intracellular domain, are expressed at low levels 1997), suggesting that individual neurons are capable of domain- in the prenatal rodent brain, but their expression levels increase specific neurotrophin responses. postnatally, ultimately exceeding levels of full-length TrkB in Neurotrophins mediate their effects by binding to two distinct adulthood (Valenzuela et al., 1993; Allendoerfer et al., 1994; classes of cell surface receptors. In mammals, the Trk family Escando´n et al., 1994; Knu¨sel et al., 1994; Fryer et al., 1996). contains three highly homologous transmembrane receptor ty- Full-length and truncated TrkB isoforms are coexpressed within rosine kinases, whereas the p75 neurotrophin receptor is a mem- central neurons, whereas central glia produce only truncated ber of the tumor necrosis factor receptor superfamily (for review, TrkB isoforms (Rudge et al., 1994; Armanini et al., 1995; Wet- more and Olson, 1995). The physiological function of the Received Jan. 18, 1999; revised April 14, 1999; accepted April 27, 1999. TrkB-T1 and -T2 isoform receptors remains unclear, but they may This work was supported by grants from the Canadian Neurosciences Network serve as dominant-negative regulators of full-length TrkB recep- Program, the Medical Research Council (MRC) of Canada, the Fond de la Re- tors (Eide et al., 1996; Ninkina et al., 1996), may sequester ligand cherche´ en Sante´ du Quebec (P.A.B.), the McKnight Foundation, the National Institutes of Health (D.L.), and the Academy of Finland and by European Union and limit diffusion (Biffo et al., 1995; Fryer et al., 1997), may Biotechnology Grant PL970259 to E.C. P.A.B. is an MRC Scholar and a Scholar of recruit ligand and regulate cell morphology (Haapasalo et al., the Killam Foundation. 1999), and may even autonomously activate signaling cascades in Correspondence should be addressed to Dr. Philip A. Barker, Centre for Neuro- nal Survival, 3801 University Avenue, Montreal, Quebec, Canada, H3A 2B4. a neurotrophin-dependent manner (Baxter et al., 1997). Copyright © 1999 Society for Neuroscience 0270-6474/99/195823-11$05.00/0 The mechanisms that restrict neurotrophin signaling to specific 5824 J. Neurosci., July 15, 1999, 19(14):5823–5833 Kryl et al. • Subcellular Localization of TrkB and TrkC neuronal subdomains remain unknown, but one possibility is that medium in the first tube. This was repeated several times to collect Trk receptors are specifically concentrated within functionally sufficient numbers of neurons for plating. Cultures were supplemented with B27 (Life Technologies) and then plated on either coverslips or distinct regions within the somatic, dendritic, and axonal com- tissue culture plastic that had been precoated with poly-L-lysine and partments of neurons. Differential localization of multiple alter- collagen. Medium was replaced 48 hr after plating with Neurobasal natively spliced Trk receptor variants could serve as a powerful containing B27 and 0.5 mM glutamine; cultures were subsequently main- mechanism for limiting neurotrophin responsiveness to particular tained in this latter medium. subcellular domains and might explain why specific regions of Visual cortical slices from 2-week-old ferrets were prepared and cul- tured with slight modifications of a previously described protocol (McAl- single neurons show different responses to the same neurotrophin lister et al., 1995). Briefly, 400 mm coronal slices of ferret cortex were (McAllister et al., 1995, 1997; Li et al., 1998). To examine Trk prepared under sterile conditions in artificial CSF bubbled with 5% receptor distribution, we created epitope-tagged forms of Trk CO2 /95% O2 (124 mM NaCl, 5 mM KCl, 0.65 mM MgSO4 ,3mM CaCl2 , receptors, determined their cellular distribution within epithelial 1.2 mM KH2PO4 ,10mM dextrose, 26 mM NaHCO3 ,and1mM kynurenic m cells and primary neurons, and performed biochemical analyses acid) and placed on culture inserts in six-well culture plates (0.4 m pore size; Falcon). Culture medium (1.5 ml; 50% b-mercaptoethanol, 25% to establish whether full-length and truncated TrkB receptors HBSS, and 25% horse serum; HyClone) containing 36 mM dextrose, 26 were enriched in postsynaptic densities isolated from intact brain. mM NaHCO3 ,1mM kynurenic acid, and 100 U/ml penicillin–strepto- Our results indicate that Trk receptor isoforms are uniformly mycin (Life Technologies; bubbled 10 min with 5% CO2 /95% O2 ) was distributed within neurons and that domain-specific receptor lo- added under each insert, and the plates were incubated in 5% CO2 at calization is unlikely to account for specific local effects of 37°C for 36–38 hr before fixation for immunocytochemistry. Transfections. Transfections of MDCK and PC12 cells were performed neurotrophins. using Lipofectamine (Life Technologies) essentially according to the manufacturer’s instructions. PC12 transfections were performed in the MATERIALS AND METHODS absence of serum for 4 hr and then supplemented with PC12 medium for Materials. The anti-myc antibody 9E10 was either produced in ascites 20 hr before initiating neurite extension assays (described below). For fluid in Balb/c mice and purified using Immunopure columns (Pierce, transient MDCK transfections, cells were plated on permeable mem- Rockford, IL) according to the manufacturer’s instructions or purchased brane inserts (Falcon) before transfection. To produce stable MDCK from PharMingen (San Diego, CA). The pan-Trk antibody 203, the ZO-1 sublines overexpressing Trk receptors, we transfected MDCK cells antibody, and the