High affinity binding of Dab1 to Reelin receptors promotes normal positioning of upper layer cortical plate neurons

doi:10.1016/j.molbrainres.2004.03.022

Molecular Brain Research

Volume 126, Issue 2, 26 July 2004, Pages 121-128

https://doi.org/10.1016/j.molbrainres.2004.03.022 Get rights and content

Abstract

The positions of neurons in the neocortex, hippocampus, cerebellum and various other laminated brain regions are regulated by a signaling pathway initiated by the secreted protein Reelin and requiring the intracellular adaptor protein Dab1. Dab1 and the Reelin receptors VLDLR and ApoER2 are expressed by neurons whose migrations are coordinated by Reelin. In vitro, Dab1 binds with high affinity to the cytoplasmic tails of VLDLR and ApoER2 via its PTB domain. To test the importance of Dab1 binding to VLDLR and ApoER2, we replaced the Dab1 gene with a cDNA cassette encoding a point mutant allele, Dab1^F158V. This mutation strongly decreases Dab1 binding in vitro to peptides containing the ApoER2 or VLDLR cytoplasmic regions. Surprisingly, Dab1^F158V/F158V homozygotes have no discernable phenotype. However, Dab1^F158V/− hemizygous animals have a subtle phenotype in which late-generated cortical plate neurons migrate excessively into the marginal zone. Early cortical plate neurons, subplate neurons, hippocampal pyramidal cells and cerebellar Purkinje cells are positioned normally. Thus Dab^F158V is a weak loss-of-function (hypomorphic) allele that has no detectable effect when homozygous. The phenotype of Dab1^F158V/− hemizygotes shows that late cortical plate neurons of layers 2–3 require efficient Reelin–Dab1 signaling to prevent them entering the marginal zone. The Dab1^F158V allele adds to a series of Dab1 alleles that demonstrates cell type-specific variation in the Reelin–Dab1 pathway.

Section snippets

Dab1^F158V mutant mice

The Dab1 gene was targeted as described [22]. The targeting vector contained an upstream intron, the second coding exon, part of a F158V mutant Dab1 cDNA [21] a strong polyadenylylation signal, a neomycin phosphotransferase selectable marker flanked by loxP sites, and a downstream intron, as described for the Dab1 WT, 5F and p45 alleles [22], [14]. Mutant mice were obtained as described and mated to Meox2^Cre/+ mice to delete the selection cassette [22]. All studies in this paper were done with

Characterization of F158V mutant Dab1 protein in vitro

We previously showed that a Dab1 F158V mutation, made in a conserved residue in the PTB/PID domain that was predicted to contact the backbone of a bound NPXY ligand [34], reduces binding of an NPXY peptide from APP approximately 25-fold in vitro, and reduces formation of a complex between Dab1 and the cytoplasmic domain of APP when the proteins are co-expressed in cultured 293 cells [21]. Recent structural studies of the Dab1 PTB domain have shown that the F158 side chain contacts the bound

Discussion

We have found that Dab1^F158V is a hypomorphic (weak loss of function) allele, which when homozygous (two copies) has no phenotype but when hemizygous (one copy) causes a subset of late CP neurons to enter the marginal zone and slight disorganization of the hippocampus. The absence of a strong phenotype is surprising, given the reduced affinity of Dab1^F158V for the cytoplasmic tails of APP, VLDLR and ApoER2 in vitro [21] (Fig. 1). There are several possible reasons for the mild effects in vivo.

Acknowledgements

We thank Priscilla Kronstad for expert technical assistance, Lionel Arnaud and Bryan Ballif for advice and discussions, Nanyang Zhang for ES cell electroporation and blastocyst injections, Michelle Tallquist and Phil Soriano for mice, and Robert Hevner and Robert McEvilly for antibodies and protocols for immunofluorescence. This work was supported by grant R37-CA41072 from the National Cancer Institute.

References (34)

L Arnaud et al.
Fyn tyrosine kinase is a critical regulator of Disabled-1 during brain development
Curr. Biol.
(2003)
D Benhayon et al.
Binding of purified Reelin to ApoER2 and VLDLR mediates tyrosine phosphorylation of Disabled-1
Mol. Brain Res.
(2003)
H.H Bock et al.
Reelin activates SRC family tyrosine kinases in neurons
Curr. Biol.
(2003)
G D'Arcangelo et al.
Reelin is a ligand for lipoprotein receptors
Neuron
(1999)
L Dulabon et al.
Reelin binds alpha3beta1 integrin and inhibits neuronal migration
Neuron
(2000)
M Gotthardt et al.
Interactions of the low density lipoprotein receptor gene family with cytosolic adaptor and scaffold proteins suggest diverse biological functions in cellular communication and signal transduction
J. Biol. Chem.
(2000)
R.F Hevner et al.
Tbr1 regulates differentiation of the preplate and layer 6
Neuron
(2001)
T Hiesberger et al.
Direct binding of Reelin to VLDL receptor and ApoE receptor 2 induces tyrosine phosphorylation of disabled-1 and modulates tau phosphorylation
Neuron
(1999)
B.W Howell et al.
Dab1 tyrosine phosphorylation sites relay positional signals during mouse brain development
Curr. Biol.
(2000)
L Keshvara et al.
Identification of reelin-induced sites of tyrosyl phosphorylation on disabled 1
J. Biol. Chem.
(2001)

M Ogawa et al.

The reeler gene-associated antigen on Cajal–Retzius neurons is a crucial molecule for laminar organization of cortical neurons

Neuron

(1995)

P.C Stolt et al.

Origins of peptide selectivity and phosphoinositide binding revealed by structures of Disabled-1 PTB domain complexes

Structure (Camb.)

(2003)

M Trommsdorff et al.

Interaction of cytosolic adaptor proteins with neuronal apolipoprotein E receptors and the amyloid precursor protein

J. Biol. Chem.

(1998)

M.L Ware et al.

Aberrant splicing of a mouse disabled homolog, mdab1, in the scrambler mouse

Neuron

(1997)

M Yun et al.

Crystal structures of the dab homology domains of mouse disabled 1 and 2

J. Biol. Chem.

(2003)

S Alcantara et al.

Regional and cellular patterns of reelin mRNA expression in the forebrain of the developing and adult mouse

J. Neurosci.

(1998)

J.B Angevine et al.

Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse

Nature

(1961)

Cited by (21)

Regulation of neural migration by the CREB/CREM transcription factors and altered Dab1 levels in CREB/CREM mutants
2008, Molecular and Cellular Neuroscience
The family of CREB transcription factors is involved in a variety of biological processes including the development and plasticity of the nervous system. To gain further insight into the roles of CREB family members in the development of the embryonic brain, we examined the migratory phenotype of CREB1^NescreCREM^−/− mutants. We found that the lack of CREB/CREM genes is accompanied by anatomical defects in specific layers of the olfactory bulb, hippocampus and cerebral cortex. These changes are associated with decreased Dab1 expression in CREB1^NescreCREM^−/− mutants. Our results indicate that the lack of CREB/CREM genes, specifically in neural and glial progenitors, leads to migration abnormalities during brain development, suggesting that unidentified age-dependent factors modulate the role of CREB/CREM genes in neural development.
Hippocampal dendritic arbor growth in vitro: Regulation by Reelin-Disabled-1 signaling
2007, Brain Research
Citation Excerpt :
In addition, our results suggest that the majority of hippocampal pyramidal neurons have the same signaling threshold when it comes to Reelin's role in dendritic maturation. In contrast, recent findings suggest that some neurons are less tolerant than others to reductions in the Reelin–Dab1 signal when it comes to being correctly positioned during development in the stratum pyramidale (Herrick and Cooper, 2002; Herrick and Cooper, 2004). Taken together, these findings further support the hypothesis that the roles Reelin plays in neuronal positioning and dendrite maturation are different.
The cytoplasmic adaptor protein Disabled-1 (Dab1), which is a key component of the Reelin-signaling pathway, has been suggested to be required for neuronal dendritic development. However, only data from studies on immature cultures [≤ 6 days in vitro (DIV)] and cytoarchitectural analyses of mutant mice have been used to formulate this hypothesis. Therefore, to determine if Reelin–Dab1 signaling is specifically required for neurons to develop mature dendrites in respect to length and complexity, we analyzed dendritic development in mature cultures derived from Dab1 knockout (ko) embryos. No significant differences in dendritic length or complexity between Dab1 ko and wt cultures were found at 20 DIV. An examination of dendritic development in maturing cultures found significant differences in dendritic length between mutant and wt cultures at 4 DIV, but detected no differences in complexity. In addition, by 7 DIV, all measures were statistically the same between cultures. Therefore, although Reelin–Dab1 signaling promotes hippocampal dendrite development, Dab1 is not required for neurons to reach maturity with respect to dendritic length and complexity. Furthermore, analyses of 4 DIV cultures derived from Dab1 heterozygotes or mice that express only the natural splice form of Dab1 (p45) found that Dab1^p45/− hemizygote, but not Dab1^p45/p45 and Dab1 heterozygote cultures had significantly shorter dendrites than those in wt cultures. Thus, a substantial attenuation of the Reelin–Dab1 signal is required before dendrite elongation is significantly decreased at 4 DIV. Moreover, experiments that incorporated a Reelin-neutralizing antibody support the hypothesis that the role(s) Reelin-signaling plays in dendritic maturation is different than the one it has in neuronal positioning.
DAB1 and reelin effects on amyloid precursor protein and ApoE receptor 2 trafficking and processing
2006, Journal of Biological Chemistry
Citation Excerpt :
The PTB domain of Dab1 expression also slightly increased cell apoEr2 levels (Fig. 1D). Because Dab1 interacts with apoEr2 through its PTB domain (19), we hypothesized that constructs of Dab1 lacking the PTB domain would not affect apoEr2 processing. We found that PTB deletion construct of Dab1 did not affect sapoEr2 or apoEr2 CTF levels (Fig. 1E).
Numerous cytoplasmic adaptor proteins, including JIP1, FE65, and X11α, affect amyloid precursor protein (APP) processing and Aβ production. Dab1 is another adaptor protein that interacts with APP as well as with members of the apoE receptor family. We examined the effect of Dab1 on APP and apoEr2 processing in transfected cells and primary neurons. Dab1 interacted with APP and apoEr2 and increased levels of their secreted extracellular domains and their cytoplasmic C-terminal fragments. These effects depended on the NPXY domains of APP and apoEr2 and on the phosphotyrosine binding domain of Dab1 but did not depend on phosphorylation of Dab1. Dab1 decreased the levels of APP β-C-terminal fragment and secreted Aβ. Full-length Dab1 or its phosphotyrosine binding domain alone increased surface levels of APP, as determined by surface protein biotinylation and live cell staining. A ligand for apoEr2, the extracellular matrix protein Reelin, significantly increased the interaction of apoEr2 with Dab1. Surprisingly, we also found that Reelin treatment significantly increased the interaction of APP and Dab1. Moreover, Reelin treatment increased cleavage of APP and apoEr2 and decreased production of the β-C-terminal fragment of APP and Aβ. Together, these data suggest that Dab1 alters trafficking and processing of APP and apoEr2, and this effect is influenced by extracellular ligands.
Both the phosphoinositide and receptor binding activities of Dab1 are required for Reelin-stimulated Dab1 tyrosine phosphorylation
2005, Molecular Brain Research
Reelin-stimulated tyrosine phosphorylation of the Dab1 adaptor protein is required during brain development for Reelin-dependent neuron positioning in the cerebral cortex and various other laminated regions. Dab1 contains an amino-terminal PTB/PI domain through which it can bind to Reelin receptors and membrane phosphoinositides. The relative contributions of these binding activities were unknown. Here, we identify a mutation in the PTB domain of Dab1 that inhibits membrane localization without inhibiting receptor binding. In neurons, this mutation reduces both basal and Reelin-stimulated Dab1 tyrosine phosphorylation. In contrast, a mutation that inhibits receptor binding reduces Reelin-stimulated but not basal tyrosine phosphorylation. These results support a model in which phospholipids recruit Dab1 to membranes but do not play a direct role in relaying the Reelin signal, while direct Dab1–receptor interaction is responsible for relaying the Reelin signal but not for membrane recruitment.
Signaling through Disabled 1 requires phosphoinositide binding
2005, Biochemical and Biophysical Research Communications
Citation Excerpt :
Since PtdIns-4,5-P2 is the most abundant PI in the plasma membrane [27], PI binding could play an important role in enriching Dab1 at the membrane and consequently influencing the kinetics of Dab1-receptor binding. For example, a point mutation within the peptide-binding pocket of Dab1 PTB domain abolished receptor binding in vitro, but mice hemizygous for this allele have subtle defects and homozygous are normal [30], suggesting that PI binding may partly compensate for the weak affinity of the Dab1 mutant for the receptor in vivo. It is thought that the components of the Reelin pathway, including SFKs, are not diffused throughout the cell but rather exist in lipid-enriched microdomains [31,32].
The Reelin signaling pathway plays a critical role in the correct positioning of neurons within the developing brain. Within this pathway, Disabled 1 (Dab1) serves as an intracellular adaptor that is tyrosine phosphorylated when Reelin, a secreted glycoprotein, binds to the lipoprotein receptors VLDLR and ApoER2 on the surface of neurons. The phosphotyrosine-binding (PTB) domain within its amino terminus enables Dab1 to recognize and bind to a conserved sequence motif within the cytoplasmic tails of the receptors. In addition, the PTB contains a Pleckstrin Homology-like subdomain that binds to phosphoinositides. Here, we show that the phosphoinositide-binding region within Dab1 PTB domain is required for membrane localization and basal tyrosine phosphorylation of Dab1 independently of VLDLR and ApoER2. Furthermore, receptor-independent membrane targeting of Dab1 is required for its interaction with Src and Crk, and disruption of phosphoinositide binding also blocks subsequent Reelin-induced tyrosine phosphorylation of Dab1.
Phosphoinositide binding by the disabled-1 PTB domain is necessary for membrane localization and reelin signal transduction
2005, Journal of Biological Chemistry
Citation Excerpt :
A recent study by Herrick and Cooper (36) has used this technique to investigate the effect of the single F158V NPXY binding mutation on brain development. Mice hemizygous for this mutation present as a hypomorphic phenocopy of the Dab1 knockout (36). This milder phenotype is most likely because of the fact that the F158V mutation alone, without the accompanying S114T mutation we have used in our studies, results only in an ∼10-fold reduction of affinity of the PTB domain for the NPXY peptide in vitro (20), which may still allow sufficient Reelin signal propagation.
Disabled-1 (Dab1) is an essential adaptor protein that functions in the Reelin signaling pathway and is required for the regulation of neuronal migration during embryonic development. Dab1 interacts with NPXY motifs in the cytoplasmic tails of the lipoprotein receptors ApoER2 and very low density lipoprotein receptor through an amino-terminal phosphotyrosine binding (PTB) domain. Binding of Reelin to these receptors leads to tyrosine phosphorylation of Dab1 and the initiation of a signaling cascade that results in remodeling of the cytoskeleton. Structural and biochemical studies of the Dab1 PTB domain have demonstrated that this domain binds to both the NPXY peptide motif in the lipoprotein receptor tails as well as to the head group of phosphoinositide 4,5-P₂ through energetically independent mechanisms. Here we have investigated how phosphoinositide binding by the Dab1 PTB domain influences Reelin signal transduction. Our findings in cultured primary neurons that have been transduced with lentiviral constructs expressing mutant Dab1 forms reveal that phosphoinositide binding by the Dab1 PTB domain is necessary for proper membrane localization of Dab1 and for effective transduction of a Reelin signal.

View all citing articles on Scopus

¹: Present address: School of International and Public Affairs, Columbia University, New York, USA.

View full text

High affinity binding of Dab1 to Reelin receptors promotes normal positioning of upper layer cortical plate neurons

Abstract

Section snippets

Dab1F158V mutant mice

Characterization of F158V mutant Dab1 protein in vitro

Discussion

Acknowledgements

Curr. Biol.

Mol. Brain Res.

Curr. Biol.

Neuron

Neuron

J. Biol. Chem.

Neuron

Neuron

Curr. Biol.

J. Biol. Chem.

Neuron

Structure (Camb.)

J. Biol. Chem.

Neuron

J. Biol. Chem.

Regional and cellular patterns of reelin mRNA expression in the forebrain of the developing and adult mouse

J. Neurosci.

Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse

Nature

Dab1^F158V mutant mice