Arcadlin Interacts with N-Cadherin
Arcadlin is an activity-regulated cell-adhesion molecule whose expression level is very low in the resting brain but is vigorously induced by neural stimulation and recruited to the dendritic spine (Yamagata et al., 1999
). Upon neural stimulation, such as maximal electroconvulsive seizure (MECS), arcadlin immunoreactivity increased and displayed a punctate distribution in the stratum lucidum of the hippocampal CA3 region, in which N-cadherin is also found (; Fannon and Colman, 1996
Arcadlin Is Associated with N-Cadherin at Hippocampal Synaptic Puncta
In cultured hippocampal neurons, spontaneous synaptic activity causes detectable expression of arcadlin, which was increased by brief treatment with glutamate or elevation in cAMP with isobutyl methylxanthine (IBMX) and forskolin ( and data not shown). Although N-cadherin is also known to be induced by elevating cAMP level in acute hippocampal slices (Bozdagi et al., 2000
), there was no significant induction of N-cadherin in our culture (). This discrepancy is presumably due to the lack of glial support and the higher spontaneous activity of neurons in dispersed culture. The expression of arcadlin in cultures treated with IBMX and forskolin was confined to glutamate decarboxylase 65 (GAD6)-negative, non-GABAergic neurons (, arrow). In such neurons, arcadlin showed punctate distribution in dendrites (, arrow). The arcadlin puncta were not colocalized or apposed to the GAD6-puncta, which correspond to inhibitory axonal termini (, arrowhead). In contrast, the arcadlin-puncta colocalized with postsynaptic markers for excitatory synapses, such as PSD-95 and the NMDA receptor subunit NR1, as well as N-cadherin (). Arcadlin was expressed in developing axonal growth cones of young neurons, showing that arcadlin is also present in developing presynaptic membranes (, arrow).
We discovered the association of arcadlin with N-cadherin fortuitously during the course of immunoprecipitation studies of N-cadherin in hippocampal lysates. Arcadlin was coimmunoprecipitated with N-cadherin in dissected rat hippocampi 4 hr after electroconvulsions, but very little was found in unstimulated hippocampi (; note that the amount of β-catenin bound to N-cadherin is not affected). Coimmunoprecipitation was detectable in resting cultured neurons, which was increased by neural stimulation, such as depolarization by KCl (). Another synaptic classical cadherin, cadherin-11, was also found to be associated with the induced arcadlin in the brain (). The fact that arcadlin is targeted to multiple classical cadherins is reminiscent of the notion that Xenopus
arcadlin/PAPC downregulates the adhesion activity of C-cadherin (Chen and Gumbiner, 2006
). In this study, we focused on N-cadherin as the most abundant example of these cadherins. Coimmnoprecipitation experiments from cocultured cell lines transfected with N-cadherin
independently (single transfections) or simultaneously (cotransfection) revealed that they associated laterally in the same membrane (). Consistently, we were able to localize their interaction domains to their transmembrane segments by deletion mutant analyses (). The affinity of N-cadherin to arcadlin was significantly reduced by a point mutation L561P or L561P/M562G in the middle of the transmembrane α-helix of N-cadherin (). The corresponding amino acid of E-cadherin plays a pivotal role in homophilic cis
dimerization (Huber et al., 1999
Arcadlin Induces the Internalization of N-Cadherin
To test whether arcadlin had any effect on the adhesive activity of N-cadherin, we performed cell-aggregation assays using L929 cells (Takeichi, 1977
). Although arcadlin itself has a homophilic adhesive activity, it is too weak to be detected in the aggregation assay optimized for classical cadherins (Yamagata et al., 1999
). We found that arcadlin downregulated the homophilic adhesiveness of N-cadherin (). The inhibitory effect was not attributable to either the expression level of N-cadherin () or the intracellular molecules associated with N-cadherin, such as catenins (). We therefore hypothesized that the downregulation of N-cadherin activity could involve the internalization of N-cadherin by analogy to the case of apCAM (Bailey et al., 1992
) and L1 (Kamiguchi and Lemmon, 2000
). Recently, a key role for endocytosis in the disassembly of E-cadherin cell-cell adhesion has been reported (Troyanovsky et al., 2006
Arcadlin Inhibits the Adhesive Activity of N-Cadherin
To examine whether arcadlin enhances the internalization of N-cadherin, we first quantified surface N-cadherin level by labeling neuronal proteins on the extracellular surface with biotin. The biotin-labeled surface proteins were isolated with avidin-sepharose beads and immunoblotted for N-cadherin (). Membrane depolarization with KCl and elevating cAMP level, both of which induced endogenous arcadlin (), resulted in significant reduction in surface N-cadherin levels (), whereas surface levels of neuroligin, as a control, did not change ().
Depolarization or Elevated cAMP Level Increases N-Cadherin Internalization through an Arcadlin-Dependent Pathway
Next, we quantified the amount of surface-associated N-cadherin microscopically. We generated a new polyclonal antibody, MT79, which recognizes the extracellular domain of N-cadherin (see Figure S1 in the Supplemental Data
available with this article online). Mouse hippocampal neurons at age 14–17 days were incubated in medium containing anti-N-cadherin MT79 antibody at 4°C for 30 min. Surface-associated N-cadherins were then labeled with the secondary antibody without permeabilization of plasma membrane (, green). To analyze the synaptic population, we examined the N-cadherin signal that overlapped with synaptophysin (; synaptic versus extrasynaptic). A 33.4% ± 5.1% decrease in the mean intensity of total surface N-cadherin in the IBMX + forskolin treatment group relative to control was observed (). (The mean intensity of synaptophysin did not differ between groups.) The decrease in the surface N-cadherin mean intensity was observed in both the synaptic (synaptophysin-overlapping fraction) and extrasynaptic (synaptophysin-nonoverlapping fraction) populations.
To show that the cAMP (IBMX + forskolin)-induced internalization of N-cadherin was indeed mediated by arcadlin, we utilized arcadlin (acad/papc)−/−
mice (Yamamoto et al., 2000
). The acad−/−
mice were apparently normal, and the gross expression level of N-cadherin appeared the same as wild-type in brain sections and cultured neurons (data not shown). Neurons cultured from acad−/−
mice extended dendrites and axons normally. The surface N-cadherin intensity of acad−/−
neurons was slightly higher than that of acad+/+
neurons (9.8% ± 3.6% increase in biotinylation assay, n = 5) and of acad+/−
neurons (37.9 ± 1.3 [n = 100] versus 33.1 ± 0.9 [n = 100], arbitrary fluorescence units in microscopic analysis, data collected from ten independent experiments). In these acad−/−
neurons, there was no significant change in the mean intensity of total surface N-cadherin in the IBMX + forskolin treatment group relative to control (). The data indicate that the IBMX + forskolin treatment-induced N-cadherin internalization is mediated by arcadlin.
Identification of an Isoform of TAO2 Kinase as a Signal Transducer of Arcadlin
To dissect the molecular mechanism of endocytosis of N-cadherin by arcadlin, we searched for an intracellular binding partner of arcadlin. A splice form of TAO2 kinase was cloned in a yeast two-hybrid screen using a cDNA library prepared from electroconvulsed rat hippocampi and the cytoplasmic domain of arcadlin as bait ( and Figure S2
). This isoform of TAO2 kinase (named TAO2β) of 1056 amino acids shares the common serine/threonine protein kinase catalytic domain and the MEK binding domain with the original TAO2 kinase (renamed as TAO2α hereafter) but has a unique carboxy-terminal regulatory domain that shows no apparent homology to any known protein motif ( and Figure S2B
). The mRNA portion corresponding to the C-terminal domain of TAO2α is transcribed from only one exon, whereas that encoding the TAO2β C terminus is derived partly from the same exon, and mostly from three downstream exons, suggesting that tao2
α and -β mRNAs are alternative splicing products from the same gene (Figure S2A
TAO2β Binds to the Intracellular Domain of Arcadlin
A recombinant TAO2β tagged at its carboxyl terminus with enhanced cyan fluorescent protein (TAO2β-ECFP) colocalized with arcadlin-EYFP (enhanced yellow fluorescent protein) in HEK293T cells (). The molecular interaction between these proteins was confirmed by coimmunoprecipitation (). The formation of a trimeric complex of TAO2β, arcadlin, and N-cadherin was confirmed in triple-transfected HEK293T cells (). The association of EGFP-TAO2β (398–751), the central domain common to both α and β isoforms, suggested that TAO2α can also bind to arcadlin (). In order to examine in vivo interactions of TAO2β, we produced and purified a polyclonal antibody recognizing the carboxy-terminal domain specific for the β isoform of TAO2 kinase (). Coimmunoprecipitation of endogenous arcadlin protein with the anti-TAO2β antibody () and, reciprocally, of TAO2β with anti-arcadlin antibody () confirmed that these molecules associate in vivo in MECS-treated rat hippocampi. Immunolocalization of TAO2β showed puncta in dendrites (). Arcadlin-EYFP and TAO2β-ECFP transfected into cultured hippocampal neurons colocalized in dendrites (). We conclude that the arcadlin protocadherin and the TAO2β MAPKKK interact in hippocampal neurons.
Arcadlin Homophilic Interaction Triggers Activation of p38 MAPK and Internalization
The arcadlin/PAPC extracellular domain mediates homophilic binding (Chen and Gumbiner, 2006
; Kim et al., 1998
; Yamagata et al., 1999
). In addition, arcadlin is a transiently expressed protein in hippocampal neurons, whose protein level peaks at 4 hr after the synaptic stimulation and largely disappears within 8 hr (Yamagata et al., 1999
). During this period, the arcadlin protein is transported to pre- and postsynaptic membranes and rapidly turned over (Yamagata et al., 1999
). In HEK293T cells cotransfected with arcadlin-EGFP
, arcadlin-arcadlin lateral interactions in the same membrane were readily detectable (, lane3). To analyze trans
interactions specifically, we utilized arcadlin-L, a splice variant of arcadlin containing a 98 amino acid insertion in its cytoplasmic region. Arcadlin-L-EGFP-expressing cells and arcadlin-L-FLAG-expressing cells were cocultured so that these two types of cells attached to each other. Immunoprecipitation of arcadlin-L-EGFP with anti-flag antibody indicated that there is significant binding activity in trans
(, lane 1). Application of a soluble extracellular fragment of recombinant arcadlin protein (Acad-EC, purified via a His-tag) into the culture medium competed trans
-association, indicating that Acad-EC binds to the extracellular domain of arcadlin in trans
(, lane 2). Although cis
interaction may be also involved, Acad-EC at this concentration was not sufficient to replace the lateral oligomerization (data not shown).
Homophilic Interaction of Arcadlin Causes Internalization via the Activation of Arcadlin-TAO2β-p38 MAPK Signaling Pathway
Arcadlin molecules expressed in HEK293T cells abundantly localized to the cell surface. There was also detectable fraction of arcadlin in intracellular vesicles (, 0 min). Homophilic interaction of arcadlin on the cell surface with Acad-EC added to the culture medium triggered the rapid translocation of arcadlin from the periphery to the center of HEK293T cells cotransfected as described below (). This shift is mediated by endocytosis, because the moved arcadlin colocalized with EGFP-rab5 as a marker for endosomes and because the internalization of arcadlin was blocked by a coexpression of a dominant-negative form of dynamin, as shown in . A similar endocytic response was observed upon the application of the antibody against the extracellular region of arcadlin (data not shown). Therefore, a binding of the extracellular domain is sufficient to enhance the endocytosis of arcadlin. We next used this Acad-EC reagent to investigate the signal transduction mechanism that triggers endocytosis. It should be noted that a detectable level of background arcadlin endocytosis before the addition of Acad-EC may be triggered by the cis homophilic interaction of the transfected arcadlin (, 0 min).
Because TAO2β forms a molecular complex with the arcadlin intracellular domain, we asked whether p38 MAPK, a main target kinase of the TAO2α-MAPKKK pathway bridged by MEK3 (MAPKK-3) (Chen et al., 2003
), was activated by the arcadlin signal. HEK293T cells cotransfected with arcadlin
, and p38 MAPK
were treated by addition of purified Acad-EC (10 μg/ml) to the culture medium. Immunostaining and immunoblot of the phosphorylated forms of p38 MAPK and MEK3 revealed that the phosphorylation levels of both kinases were enhanced within 30 min of the application of Acad-EC (). The p38 MAPK phosphorylation is mediated by arcadlin and TAO2β, because Acad-EC did not exert any response in HEK293T cells lacking either arcadlin
transfection (). Importantly, addition of Acad-EC protein triggered the activation of endogenous p38 MAPK in the dendritic shaft of primary cultures of rat hippocampal neurons (). Taken together, the results suggest that the arcadlin extracellular domain activates p38 MAPK via TAO2β. In cultured neurons, activation of p38 was detected specifically in the dendritic shaft after addition of protocadherin extracellular domain.
TAO2β Is Required for p38 Activation and Endocytosis of Arcadlin
We then addressed whether TAO2β is necessary for the phosphorylation of p38 MAPK and the endocytosis of arcadlin. We reconstituted the arcadlin-TAO2β-MEK3-p38 MAPK signaling pathway in HEK293T cells by cotransfecting arcadlin
(catalytically defective TAO2 kinase; Chen et al., 2003
), or tao2α
. After the treatment with Acad-EC for 30 min, cells were analyzed for the phosphorylation of p38 MAPK () and endocytosis of arcadlin by the surface biotinylation assay (). The phosphorylation of p38 MAPK was significantly increased, and surface arcadlin levels were significantly reduced in cells expressing wild-type TAO2β (). In mock
-transfected cells, neither the endocytosis of arcadlin nor the phosphorylation of p38 MAPK was observed (). Cells expressing TAO2α displayed full activation of p38 MAPK but were deficient in the internalization of arcadlin (). We conclude from these data that the kinase activity of TAO2α and -β that resides in their common catalytic domains is sufficient for the phosphorylation and activation of p38 MAPK. The endocytosis of arcadlin, however, depends exclusively on TAO2β, suggesting that the unique carboxy-terminal domain of TAO2β is required for the endocytosis of the arcadlin protocadherin.
A TAO2β-p38 MAPK Feed-Back Loop Mediates the Endocytosis of Arcadlin
We next investigated the requirement of p38 MAPK for the endocytosis of arcadlin. We found that SB203580, a p38 MAPK inhibitor, blocked the endocytosis of arcadlin (, compare lanes 2 and 3). This suggested that a feed-back loop, in which p38 MAPK regulates the endocytosis of arcadlin, might exist. We first postulated that arcadlin itself was a direct substrate of p38 MAPK. However, arcadlin was not phosphorylated by p38 MAPK (data not shown). We then tested whether p38 MAPK phosphorylates TAO2β. Because the experiments above indicated that the function of TAO2β responsible for the endocytosis of arcadlin resided in the carboxy-terminal domain, the unique carboxy-terminal region (751–1056) of TAO2β was fused to glutathione S-transferase (GST) and subjected to an in vitro kinase reaction with purified p38 MAPK. GST-TAO2β (751–1056) was indeed phosphorylated by activated p38 MAPK (, lane 2). Within this region, there were two sites encoding a serine preceded by a proline in positions 951 and 1010 as possible substrate sites for MAPK family members (; Kyriakis and Avruch, 2001
), but their mutation into phosphorylation-resistant alanines did not affect the incorporation of 32
P (, lane 3 and data not shown). A detailed deletion mutant analysis (data not shown) then revealed that the main target of p38 MAPK was localized within the region between positions 1036 and 1056 (). We generated Ser to Ala point mutations on positions 1038, 1040, 1042, and 1045 and found that incorporation of 32
P diminished in TAO2βS1038A, indicating that Ser1038 is the main target of p38 MAPK (, lane 4).
Feed-Back Phosphorylation of TAO2β at Ser1038 by p38 MAPK Is Essential for the Endocytosis of Arcadlin
We next investigated whether phosphorylation of Ser1038 was required for the endocytosis of arcadlin. Cells expressing TAO2βS951A, used here as a control, exhibited normal endocytosis of arcadlin, whereas the phosphorylation-resistant TAO2βS1038A remained in the surface after addition of Acad-EC protein (, lanes 1–4; see for quantification). Time-lapse images of HEK293T cells expressing arcadlin-EYFP and TAO2βS1038A-ECFP or control TAO2βS951A-ECFP confirmed that TAO2βS1038A fails to induce the endocytosis of arcadlin in living cells ().
These results indicate that the p38 MAPK that is activated by the arcadlin-TAO2β-MEK3 signaling pathway in turn phosphorylates TAO2β on Ser1038, resulting in the formation of a feed-back signaling loop. Phosphorylation of Ser1038 of TAO2β seems to be essential to activate the endocytic machinery following homophilic interaction of arcadlin. Thus, it appears we have identified a molecular pathway for protocadherin-mediated endocytosis.
The Arcadlin-TAO2β-p38 MAPK Pathway Regulates N-Cadherin Endocytosis
Finally, we tested whether the arcadlin-induced internalization of N-cadherin is mediated by the arcadlin-TAO2β-p38 MAPK molecular pathway defined above. The quantification of neuronal surface proteins by biotinylation showed that treatment with Acad-EC caused the reduction in surface arcadlin/N-cadherin levels in neurons (; see for quantification). This reduction was inhibited by SB203580, suggesting that the internalization was mediated by the p38 MAPK pathway (). Although arcadlin also binds to cadherin-11 (see ), there was no significant endocytosis of cadherin-11 upon the treatment with Acad-EC (). It seems that arcadlin is targeted to multiple species of classical cadherins, but not all of them undergo the endocytosis through this pathway. Individual classical cadherins might code for distinct subsets of synapses.
The Arcadlin-TAO2β-p38 MAPK Pathway Mediates the Internalization of N-Cadherin
Similar results were obtained in the microscopic quantification of N-cadherin internalization. A 28.3% ± 2.7% decrease in the mean intensity of total surface N-cadherin in the Acad-EC treatment group (30 min) relative to control was observed. The decrease in the surface N-cadherin mean intensity was observed in both the synaptic and extrasynaptic populations (). In acad−/− neurons, there was no significant change in the mean intensity of total surface N-cadherin in the Acad-EC treatment group relative to control ().
In HEK293T cells cotransfected with arcadlin
, and tao2β
, addition of Acad-EC protein also triggered endocytosis (Figure S3
). Arcadlin and N-cadherin were cointernalized in the presence of TAO2β but were retained on the plasma membrane in its absence (Figure S3
). Taken together, these data indicate that the internalization of N-cadherin is mediated by the arcadlin-TAO2β-p38 MAPK molecular pathway and triggered by homophilic interactions between arcadlin protocadherin extracellular domains.
Arcadlin Mutation Increases Dendritic Spine Density
What is the consequence of the arcadlin-induced internalization of N-cadherin in the dendritic spine membrane? To address this question, we examined the morphology and the number of spines of hippocampal neurons. Cultured hippocampal neurons derived from acad−/−
mice protruded a significantly larger number of spines than wild-type neurons (). This phenotype was rescued by the transfection of arcadlin
cDNA ( and Figure S4A
). The rescuing effect was more prominent where the axons of transfected neurons were attached to the transfected dendrites (, square bracket). The other splice variant arcadlin-L
did not recover the spine number ( and Figure S4A
), indicating that only arcadlin, not arcadlin-L, regulates spine density.
The Effects of Arcadlin on Dendritic Spine Density
We then asked whether the N-cadherin endocytosis caused the arcadlin-induced change in spine density. There are several studies showing the relationship between N-cadherin activity and spine number. The spine number of N-cadherin
KO neurons is maintained (Jungling et al., 2006
; Kadowaki et al., 2007
); a sustained period of N-cadherin loss in these neurons might allow other synaptic cell adhesion molecules to compensate for N-cadherin. In contrast, spine number is suppressed in neurons whose N-cadherin is knocked down by RNAi techniques (Saglietti et al., 2007
). Consistently, an expression of a dominant-negative form of N-cadherin reduces the number of synaptic puncta (Togashi et al., 2002
). In the present study, the spine density of acad−/−
neurons was reduced by siRNA knockdown of N-cadherin ( and Figure S4B
). Moreover, a similar effect was observed by the expression of a dominant-negative form of N-cadherin (NcadΔE) (). The data suggest that the arcadlin-induced change in spine number is due at least in part to the N-cadherin endocytosis.