Expression of the naturally occurring truncated trkB neurotrophin receptor induces outgrowth of ®lopodia and processes in neuroblastoma cells Annakaisa Haapasalo 1 , Tommi Saarelainen 1 , Maxim Moshnyakov 3 , Urmas ArumaÈe 3 , Tiila-Riikka Kiema 1,4 , Mart Saarma 3 , Garry Wong 1 , and Eero CastreÂn 1,2 1 Laboratory of Molecular Pharmacology, A.I. Virtanen Institute and 2 Department of Psychiatry, University of Kuopio, PO Box 1627, 70100 Kuopio, Finland and 3 Institute for Biotechnology, University of Helsinki, PO Box 56, 00014, Helsinki, Finland We have investigated the eects of the truncated trkB receptor isoform T1 (trkB.T1) by transient transfection into mouse N2a neuroblastoma cells. We observed that expression of trkB.T1 leads to a striking change in cell morphology characterized by outgrowth of ®lopodia and processes. A similar morphological response was also observed in SH-SY5Y human neuroblastoma cells and NIH3T3 ®broblasts transfected with trkB.T1. N2a cells lack endogenous expression of trkB isoforms, but express barely detectable amounts of its ligands, brain- derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4). The morphological change was ligand-indepen- dent, since addition of exogenous BDNF or NT-4 or blockade of endogenous trkB ligands did not in¯uence this response. Filopodia and process outgrowth was signi®cantly suppressed when full-length trkB.TK+ was cotransfected together with trkB.T1 and this inhibitory eect was blocked by tyrosine kinase inhibitor K252a. Transfection of trkB.T1 deletion mutants showed that the morphological response is dependent on the extra- cellular, but not the intracellular domain of the receptor. Our results suggest a novel ligand-independent role for truncated trkB in the regulation of cellular morphology. Keywords: trkB; BDNF; NT-4; N2a; neuroblastoma Introduction Neuroblastomas, which are derived from the embry- onal neural crest cells, are among the most common tumors in children. These tumors harbor various stages of dierentiation, and in some children, spontaneous or therapy-induced tumor maturation to benign gang- lioneuromas can occur. Unfortunately, tumors in the majority of children over one year of age often lead to death (Vaughan III et al., 1979). Neurotrophins function through trk family of receptor tyrosine kinases (Barbacid, 1994; Lewin and Barde, 1996). Endogenous trk receptors are expressed in many human neuroblastoma cells and their expression appears to correlate with the differentiation stage. In particular, expression of trkA (Martin-Zanca et al., 1989), the signal transducing receptor for nerve growth factor (NGF) (Kaplan et al., 1991a,b; Klein et al., 1991a; Barbacid, 1994) or trkC, the receptor for neurotrophin-3 (NT-3) (Lamballe et al., 1991) is associated with a higher dierentiation stage and more favorable outcome (Nakagawara et al., 1993; Yamashiro et al., 1996). In contrast, expression of trkB (Klein et al., 1989; Middlemas et al., 1991), the receptor for brain-derived neurotrophic factor (BDNF) (Klein et al., 1991b; Soppet et al., 1991; Squinto et al., 1991) and neurotrophin 4/5 (NT-4) (Berkemeier et al., 1991; HallboÈoÈk et al., 1991; Ip et al., 1992; Klein et al., 1992) is correlated with N-myc expression and malignant phenotype (Nakagawara et al., 1994). Dierentiation of several human neuroblas- toma cells with retinoic acid induces trkB expression (Kaplan et al., 1993; Lucarelli et al., 1994, 1995). Retinoic acid dierentiated SH-SY5Y human neuro- blastoma cells, which express trkB display character- istics of metastatic cells, such as increased survival, disaggregation, and invasiveness in response to BDNF treatment (Matsumoto et al., 1995). TrkB is alternatively spliced into several dierent isoforms (Klein et al., 1990; Middlemas et al., 1991). Most notably, alternative splicing produces, in addition to the full-length, tyrosine kinase-containing isoform (trkB.TK+) also truncated tyrosine kinase-lacking isoforms. The truncated trkB.TK7 isoforms share with the trkB.TK+ form identical extracellular and transmembrane domains and a 12-amino acid intracel- lular juxta-membrane region, but diverge thereafter and contain only a short intracellular tail region (Klein et al., 1990; Middlemas et al., 1991). Rodents have two dierent trkB.TK7 isoforms: trkB.T1, with an 11 amino acid tail; and trkB.T2, with a unique 9 amino acid tail (Middlemas et al., 1991). Only one trkB.TK7 isoform corresponding to the T1 form has been reported in humans, and this isoform is highly conserved in evolution with humans, rats and chick, sharing an identical intracellular region (Bio et al., 1995; Shelton et al., 1995). The physiological functions of trkB.TK7 isoforms are unclear. TrkB.TK7 isoforms, in particular T1, are expressed in glial cells and in the ependymal lining of brain ventricles, whereas trkB.TK+ forms are restricted to neurons. It has therefore been proposed that trkB.TK7 forms act as `sponges' to restrict BDNF diusion within brain parenchyma (Klein et al., 1990; Beck et al., 1993; FriseÂn et al., 1993; Bio et al., 1995). Indeed, trkB.T1 is internalized into leptomeningeal cells and its expression pattern in developing chick nervous system is consistent with a compartmentalization function (Bio et al., 1995). TrkB.T1-mediated internalization may clear extracel- lular BDNF and shorten the time that it is available to Correspondence: E CastreÂn 4 Current address: Department of Biochemistry, University of Oulu, Finland Received 30 March 1998; revised 7 September 1998; accepted 7 September 1998 Oncogene (1999) 18, 1285 ± 1296 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00 http://www.stockton-press.co.uk/onc