Involvement of membrane protein GDE2 in retinoic acid-induced
neurite formation in Neuro2A cells
Noriyuki Yanaka*, Yoshihito Nogusa, Yuichiro Fujioka, Yosuke Yamashita, Norihisa Kato
Department of Molecular and Applied Bioscience, Graduate School of Biosphere Science, Hiroshima University, 4-4, Kagamiyama 1-chome,
Higashi-Hiroshima 739-8528, Japan
Received 27 December 2006; revised 15 January 2007; accepted 15 January 2007
Available online 24 January 2007
Edited by Ned Mantei
terase homolog, GDE2, is widely expressed in brain tissues
including primary neurons, and that the expression of GDE2 in
neuroblastoma Neuro2A cells is significantly upregulated during
neuronal differentiation by retinoic acid (RA) treatment. Stable
expression of GDE2 resulted in neurite formation in the absence
of RA, and GDE2 accumulated at the regions of perinuclear and
growth cones in Neuro2A cells. Furthermore, a loss-of-function
of GDE2 in Neuro2A cells by RNAi blocked RA-induced neurite
formation. These results demonstrate that GDE2 expression
during neuronal differentiation plays an important role for grow-
? ? 2007 Published by Elsevier B.V. on behalf of the Federation of
European Biochemical Societies.
We show that a glycerophosphodiester phosphodies-
Keywords: Glycerophosphodiester phosphodiesterase; GDE2;
Retinoic acid; Neuro2A cells
Escherichia coli (E. coli) glycerophosphodiester phosphodies-
terases (GP-PDEs), GlpQ and UgpQ, are periplasmic and cyto-
solic proteins, which play an important role in the hydrolysis of
deacylated glycerophospholipids to glycerol phosphate and
phate in E. coli [1,2]. In contrast, two novel mammalian GP-
PDEs, GDE1/MIR16 and GDE3, were recently identified, and
considered to be involved in several physiological functions
[3,4]. GDE1/MIR16 was identified by yeast two-hybrid screen-
signaling .Arecent study has shown thatGDE1 isanintegral
membrane-bound glycoprotein that selectively hydrolyzes glyc-
cells can be regulated by stimulation of G protein-coupled a/b-
adrenergic andlysophospholipid receptors .Also, GDE3was
recently identified to be differentially expressed during specific
stages of differentiation in mouse osteoblast-like MC3T3-E1
cells using a differential display method . GDE3 encodes a
protein with seven putative transmembrane regions and an
extracellular loop containing a GP-PDE domain. We demon-
strated that GDE3 protein accumulates at the cell periphery,
a spread to a rounded form . Moreover, endogenous GDE3
ing that GDE3 is involved in the morphological change of cells
lian GP-PDEs are considered to be involved in numerous
physiological functions including signal transduction and cyto-
Very recently, six mammalian GP-PDEs have been virtually
cloned by an approach using bioinformatics [5,6]. In particular,
we showed that GDE2 contained 607 amino acids with seven
putative transmembrane regions, and that it was 43.7% identi-
cal to GDE3at the amino acid level. Although GDE3mRNA is
restrictedly expressed in bone tissue and in the spleen, GDE2
mRNA was highly expressed in mouse brain, suggesting that
GDE2 might have distinct roles in the nervous system. The
physiological function of the nervous system is tightly associ-
ated with the highly specific pattern of connections formed be-
tween neurons. The specificity of these connections requires
neurite extension toward their targets guided by the growth
cone [7–9]. Ever since we showed that GDE3 protein was local-
ized at the cell periphery and played a critical role for morpho-
logical change of cells, we have been particularly interested in
the biological functions of mammalian GP-PDEs in neurons.
These observations led us to hypothesize that GDE2 might play
critical roles for neurite formation and/or neurite retraction.
Neuro2A cells are neuroblastoma established from the mouse
spinal cord, and are widely used for studies on neurite growth
that is induced by retinoic acid (RA) treatment. Although sev-
eral molecules have been reported to be involved in retinoid-in-
duced neuronal differentiation of Neuro2A cells [10–12], our
understanding of the mechanisms involved in the control of
neuronal differentiation of Neuro2A cells remains limited. In
this study, we demonstrated that the expression of GDE2 was
dramatically upregulated during neuronal differentiation in re-
sponse to RA, and that GDE2 played a critical role for neurite
outgrowth of Neuro2A cells.
2. Materials and methods
Restriction endonucleases and DNA-modifying enzymes were pur-
chased from Takara Shuzo (Kyoto, Japan). Murine neuroblastoma
Neuro2A cells were obtained from Health Science Research Resources
Abbreviations: GP-PDE, glycerophosphodiester phosphodiesterase;
RA, retinoic acid; GFP, green fluorescent protein; DMEM, Dulbecco’s
modified Eagle’s medium; FCS, fetal calf serum
*Corresponding author. Fax: +81 82 424 7916.
E-mail address: firstname.lastname@example.org (N. Yanaka).
0014-5793/$32.00 ? 2007 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
FEBS Letters 581 (2007) 712–718
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