A mouse homolog of Drosophila Disabled (Dab), mDab1, is an adaptor molecule that functions in neural development. mDab1 is expressed in certain neuronal and hematopoietic cells, and is localized to the growing nerves of embryonic mice. mDab1 expression is observed in the head in neural tracts corresponding to the developing cranial nerves, such as the oculomotor and trochlear nerves. In the body, mDab1 expression is apparent in the spinal accessory nerve and dorsal root ganglia. At embryonic day 13, mDab1 expression is observed in sensory nerves that innervate the vibrissae, and in the extremities of developing bone. All nerves identified at these times by neurofilament antibody also express mDab1. During embryogenesis, mDab1 is tyrosine phosphorylated when the nervous system is undergoing dramatic expansion. However, once nerve tracts are established, mDab1 lacks detectable phosphotyrosine. Tyrosine-phosphorylated mDab1 associates with the SH2 domains of Src, Fyn and Abl. An interaction between mDab1 and Src is observed when embryonal carcinoma cells undergo differentiation into neuronal cell types. mDab1 can also form complexes with cellular phosphotyrosyl proteins through a domain that is related to the phosphotyrosine binding (PTB) domains of the Shc family of adaptor proteins. The mDab1 PTB domain binds to phosphotyrosine-containing proteins of 200, 120, and 40 kDa from extracts of embryonic mouse heads. The properties of mDab1 and genetic analysis of Dab in Drosophila suggest that these molecules function in key signal transduction pathways involved in the formation of neural networks (Howell, 1997a).
During mammalian brain development, immature neurons migrate radially from the neuroectoderm to defined locations, giving rise to characteristic cell layers. Targeted disruption of the mouse disabled1 (mdab1) gene disturbs neuronal layering in the cerebral cortex, hippocampus and cerebellum. The gene encodes a cytoplasmic protein, mDab1 p80, which is expressed and tyrosine-phosphorylated in the developing nervous system. It is likely to be an adaptor protein, docking to others through its phosphotyrosine residues and protein-interacting domain. The mdab1 mutant phenotype is very similar to that of the reeler mouse mutant. The product of the reeler gene, Reelin, is a secreted protein that has been proposed to act as an extracellular signpost for migrating neurons. Because mDab1 is expressed in wild-type cortical neurons, and Reelin expression is normal in mdab1 mutants, mDab1 may be part of a Reelin-regulated or parallel pathway that controls the final positioning of neurons (Howell, 1997b).
The reelin (reln) and disabled 1 (dab1) genes both ensure correct neuronal positioning during brain development. The intracellular Dab1 protein receives a tyrosine phosphorylation signal from extracellular Reln protein. Genetic analysis shows that reln function depends on dab1, and vice versa, as expected if both genes are in the same pathway. To investigate whether there is a worsening of phenotype in dab1::reln double mutants, double heterozygous dab1/+ reln/+ animals were generated and interbred. Double homozygous mutant progeny were obtained at expected frequency and resemble the single homozygotes. The cerebral cortex loses lamination; the hippocampal scrolls are split, and the granule cells of the dentate gyrus are intermingled. One characteristic of the neocortex in dab1 and reln homozygotes is invasion of the marginal zone by neurons that are normally found deep in the cortex. Increased neuron numbers are also detected in the marginal zone of compound homozygotes, but there is no significant quantitative difference between the marginal zone population in single and double homozygotes. The cerebella of the double mutant mice are also indistinguishable from the single mutants. Mutant cerebella are unfoliated and small. A molecular layer is present but the granule cells are reduced in number. Furthermore, the Purkinje cells, which normally express Dab1, are found clustered in central regions. In contrast, the Purkinje cells in a wild-type cerebellum form a monolayer above a dense granule cell layer. The compound mutants are also indistinguishable behaviorally from the single mutants. Thus it seems unlikely that either Reln or Dab1 has residual function in the absence of the other protein. Dab1 is expressed at a higher level, yet phosphorylated at a lower level, in reln mutant embryo brains. In primary neuronal cultures, Dab1 tyrosine phosphorylation is stimulated by exogenous Reln. These results show that Dab1 and Reln act on the same pathway to control neuronal positioning in the developing brain. The kinetics of tyrosine phosphorylation suggest that Reln interacts directly with a receptor on neurons that express Dab1, and the effects of a generic phosphotyrosine inhibitor suggest that the Reln receptor is linked to a tyrosine kinase. Reln may also stimulate other signaling pathways that may act in parallel to Dab1 tyrosine phosphorylation, but the genetics suggest that Dab1 tyrosine phosphorylation is one step in an essential, nonredundant pathway that regulates neuron position. Because Reln is also involved in the ingrowth of entorhinal afferents into the hippocampus, and Dab1 is in growth cones, a Reln-Dab1 pathway may also regulate growth cone migration. Increased tyrosine phosphorylation of Dab1 is expected to potentiate interactions with proteins that contain SH2 domains or other phosphotyrosine-dependent domains. These interactions may arrest neuronal migration by modulating the actin cytoskeleton, membrane flow, or adhesion complexes. (Howell, 1999a).
Disabled gene products are important for nervous system development in Drosophila and mammals. During neuronal positioning in mice, the Dab1 protein is thought to function downstream of the extracellular protein Reelin. The structures of Dab proteins suggest that they mediate protein-protein or protein-membrane docking functions. The amino-terminal phosphotyrosine-binding (PTB) domain of Dab1 binds to the transmembrane glycoproteins of the amyloid precursor protein (APP) and low-density lipoprotein receptor families and the cytoplasmic signaling protein Ship. Dab1 associates with the APP cytoplasmic domain in transfected cells and is coexpressed with APP in hippocampal neurons. Screening of a set of altered peptide sequences has shown that the sequence GYXNPXY present in APP family members is an optimal binding sequence, with approximately 0.5 µM affinity. Unlike other PTB domains, the Dab1 PTB does not bind to tyrosine-phosphorylated peptide ligands. The PTB domain also binds specifically to phospholipid bilayers containing phosphatidylinositol 4P (PtdIns4P) or PtdIns4,5P2 in a manner that does not interfere with protein binding. It is proposed that the PTB domain permits Dab1 to bind specifically to transmembrane proteins containing an NPXY internalization signal (Howell, 1999b).
The function of Dab1 binding to LDL receptor-related protein (LRP)-alpha-2 macroglobulin receptor, APP, and their relatives could be to regulate trafficking or processing. The internalization signals of LRP and APP contain the NPXY sequence, which is bound by the Dab1 PTB domain. Nonetheless, two observations make it unlikely that Dab1 functions in the internalization process per se. (1) Dab1 is absent from many cell types that successfully internalize LDL receptors or APP, and (2) the LDL receptor is thought to be clustered into coated pits by direct binding to clathrin. However, Dab1 may compete for internalization signals. By altering protein sorting into coated pits, Dab1 may affect membrane flow from the surface to intracellular membrane systems and hence influence membrane recycling, which is important for cell movement. APP is best known for its increased cleavage and the accumulation of a degradation product, beta amyloid, in Alzheimer's disease. Interestingly, the NPXY motif appears to be important for proteolysis of APP to produce beta amyloid. Overexpression of brain proteins X11 and FE65 affects APP processing in an opposing manner. While X11 overexpression leads to an increased half-life for APP, possibly by slowing the sorting of this protein into the endosomal compartment, overexpression of FE65 leads to increased translocation of APP to the cell surface and increased production of the proteolytic fragments. It remains to be determined what effect Dab1 might have on these processes (Howell, 1999b and references).
It is also possible that Dab1 PTB domain signaling is mediated by proteins other than APP and LDL receptor family proteins. The high-affinity binding sequence found in APP family members, GYXNPXY, is found in approximately 10 other eukaryotic proteins listed in current (November 1998) databases, including the APP orthologs from Drosophila and C. elegans. The high degree of conservation of this motif, 100% over the seven residues, suggests that selective pressures, possibly exerted by binding partners, act on it. The binding studies do not exclude the possibility that the Dab1 PTB domain has additional ligands with distinct sequences. Five cDNA clones were isolated that encode apparent Dab1 PTB domain binding partners that lack NPXY motifs in the interacting regions. They also lack other common sequence patterns. They may, therefore, bind to the PTB domain through novel interacting sequences (Howell, 1999b).
PTB domains and PH domains are similar in structure. Many PH domains bind with 10-5 to 10-7 M affinity to phosphoinositides with characteristic stereospecificity. Making use of a triple-charge mutation of Shc that prevents lipid binding without preventing phosphopeptide binding evidence has shown that lipid binding is important for Shc function. A model has been suggested in which weak interaction between the Shc PTB domain and membrane phospholipids is a prerequisite for recruitment of Shc to activated growth factor receptors and Shc is released from phospholipids as it binds to the receptor. Similarly, the Shc PTB domain binds to PtdIns4P, PtdIns4,5P2, and PtdIns3,4,5P3 with affinities in the 10-4 to 10-5 M range. Making use of a triple-charge mutation of Shc that prevents lipid binding without preventing phosphopeptide binding, the Dab1 PTB domain binds to PtdIns4P and PtdIns4,5P2 but less to PtdIns or PtdIns3,4,5P3. Since PtdIns4,5P2 is more abundant than PtdIns3,4,5P3, it is likely to be the major lipid bound to the Dab1 PTB domain in the cell. Unlike the Shc PTB domain, the Dab1 PTB domain can bind simultaneously to synthetic peptides and phosphoinositides. Thus, binding to membrane phospholipids could reinforce binding to PhiXNPXY motifs in the cytoplasmic domains of transmembrane proteins. Unlike Shc, recruitment of Dab1 to a protein ligand would therefore not occur at the expense of binding to phospholipids (Howell, 1999 and references).
The Dab1 protein acts within embryonic neurons and is required for appropriate neuronal placement within the brain. Recent findings suggest that it functions downstream of the extracellular matrix protein Reelin, which may define targets for the migrating neurons. The current findings show that the Dab1 PTB domain binds with high affinity to unphosphorylated targets and that binding is dramatically reduced by tyrosine phosphorylation. One of the targets identified in this study, Ship, is regulated by phosphorylation, while APP and LRP are not known to be tyrosine phosphorylated. However, Reelin increases tyrosine phosphorylation of Dab1 itself, and Dab1 PTB domain function may be regulated as a consequence of this. Exposure of binding surfaces or changes in subcellular distribution could alter Dab1 PTB domain activity. It will be interesting to determine the Dab1 PTB domain functions required for Reelin signaling and how the PTB domain ligands are involved in neuronal placement (Howell, 1999b and references).
Formation of the mammalian brain requires choreographed migration of neurons to generate highly ordered laminar structures, such as those in the cortices of the forebrain and the cerebellum. These processes are severely disrupted by mutations in Reelin protein, which cause widespread misplacement of neurons and associated ataxia in reeler mutant mice. Reelin is a large extracellular protein secreted by pioneer neurons that coordinates cell positioning during neurodevelopment. Two new autosomal recessive mouse mutations, scramble and yotari have been described that exhibit a phenotype identical to reeler. scrambler and yotari arise from mutations in mdab1, a mouse gene related to the Drosophila gene disabled (dab). Both scrambler and yotari mice express mutated forms of mdab1 messenger RNA and little or no mDab1 protein. mDab1 is a phosphoprotein that appears to function as an intracellular adaptor in protein kinase pathways. mdab1 is expressed in neuronal populations exposed to Reelin. The similar phenotypes of reeler, scrambler, yotari and mdab1 null mice indicate that Reelin and mDab1 function as signaling molecules that regulate cell positioning in the developing brain (Sheldon, 1997).
Two novel mouse mutations, scrambler and yotari exhibit remarkable behavioral and histopathological similarities to reeler. Molecular genetic studies reveal that scrambler and yotari arose from independent mutations in the disabled-1 (Dab1) gene coding for a phosphotyrosine-interacting/phosphotyrosine-binding domain protein that exhibits some degree of similarity to the Drosophila disabled gene. It is an intracellular protein that becomes phosphorylated on tyrosine residues during embryonic development and it can bind to the protein tyrosine kinases Src, Abl and Fyn. Based on its biochemical properties, Dab1 is thought to function as an adapter molecule in the transduction of protein kinase signals. Mutation of either reelin (Reln) or Dab1 results in widespread abnormalities in laminar structures throughout the brain and ataxia in reeler and scrambler mice. Both exhibit the same neuroanatomical defects, including cerebellar hypoplasia with Purkinje cell ectopia and disruption of neuronal layers in the cerebral cortex and hippocampus. Despite these phenotypic similarities, Reln and Dab1 have distinct molecular properties. Reln is a large extracellular protein secreted by Cajal-Retzius cells in the forebrain and by granule neurons in the cerebellum. In contrast, Dab1 is a cytoplasmic protein that has properties of an adapter protein which functions in phosphorylation-dependent intracellular signal transduction. Dab1 is shown to participate in the same developmental process as Reln. In scrambler mice, neuronal precursors are unable to invade the preplate of the cerebral cortex and consequently, they do not align within the cortical plate. During development, cells expressing Dab1 are located next to those secreting Reln at critical stages of formation of the cerebral cortex, cerebellum and hippocampus, before the first abnormalities in cell position become apparent in either reeler or scrambler. In reeler, the major populations of displaced neurons contain elevated levels of Dab1 protein, although they express normal levels of Dab1 mRNA. This suggests that Dab1 accumulates in the absence of a Reln-evoked signal. Taken together, these results indicate that Dab1 functions downstream of Reln in a signaling pathway that controls cell positioning in the developing brain (Rice, 1998).
Layering of neurons in the cerebral cortex and cerebellum requires Reelin, an extracellular matrix protein, and mammalian Disabled (mDab1), a cytosolic protein that activates tyrosine kinases. A molecular pathway that regulates the migration of neurons along the radial glial fiber network involves the large extracellular protein Reelin. In the cortex, this modular protein is either associated with the extracellular matrix or with the surface of a special class of neurons that produce it in the outermost layer just beneath the pial surface. These so-called Cajal-Retzius neurons are formed during the early stages of neural development. In the reeler strain of mice, the gene encoding Reelin is defective. As a result, migratory neurons in these mice apparently do not receive a critical cue that informs them of their position, leading to an inversion of the cortical layers. These layers normally form from the inside out, with later-born neurons migrating past older ones to form progressively more superficial, and thus younger, layers of the neocortex. In the cerebellum, Reelin is required for the Purkinje cells to migrate outward, where they form a well-defined cortical plate through which postmitotic granule cells migrate inward to form the internal granular layer. Both of these laminated structures do not form in the reeler mouse. The cytoplasmic adaptor protein mDab1 is related to the Drosophila disabled gene product. It is predominantly expressed in neurons and has been shown to function downstream of Reelin. mDab1-deficient mice (identified as a naturally occurring strain called scrambler and also generated by gene knockout) develop a phenotype indistinguishable from reeler. Furthermore, in reeler mice, mDab1 protein expression is greatly increased even after the end of the migratory period, indicating a failure of neurons to adjust mDab1 expression due to lack of Reelin signal input. mDab1 contains a protein interaction domain that binds to NPxY motifs in the cytoplasmic tails of receptors. mDab1 can undergo tyrosine phosphorylation and subsequently interact with nonreceptor tyrosine kinases of the Abl and Src family, suggesting that the Reelin signaling pathway involves coupling of the signal, via mDab1, to intracellular kinase pathways. However, the cell surface receptors that mediate transmission of the signal across the neuronal plasma membrane are unknown. The requirement for two other proteins has been documented. These are cell surface receptors termed very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2). The LDL receptor gene family comprises a group of structurally related multifunctional cell surface receptors that mediate endocytosis of extracellular ligands. The five known mammalian members of the family are the LDL receptor, LRP, Megalin, VLDLR, and ApoER2. The role of the LDL receptor in the regulation of cholesterol homeostasis is well understood. LRP and Megalin are both multifunctional and bind a diverse spectrum of ligands, including lipoproteins, proteases and their inhibitors, peptide hormones, and carrier proteins for vitamins. The VLDLR and ApoER2, like all members of the family, can bind ApoE, but the relevance of this interaction is unclear and their true physiological functions are unknown. Both receptors can bind mDab1 on their cytoplasmic tails and are expressed in cortical and cerebellar layers adjacent to layers that express Reelin. mDab1 expression is upregulated in knockout mice that lack both VLDLR and ApoER2. Inversion of cortical layers and absence of cerebellar foliation in these animals precisely mimic the phenotype of mice lacking Reelin or mDab1. These findings suggest that VLDLR and ApoER2 participate in transmitting the extracellular Reelin signal to intracellular signaling processes initiated by mDab1 (Trommsdorff, 1999).
Three models have been proposed to explain the phenotype of Reelin- and mDab1-deficient mice. One model suggests that Reelin acts as a repellent that induces the cortical preplate to split into marginal zone and subplate. The failure of the preplate to separate in reeler causes the cortical plate to develop ectopically underneath the subplate neurons. In another model, a Reelin gradient may selectively induce cortical plate neurons to migrate past the subplate neurons. In a third model, Reelin is thought to act as a stop signal that instructs migrating neurons to detach from their glial guidance fiber. The present findings are consistent with all these models, but they also indicate that neuronal migration along radial glia is more complex than these current models suggest. VLDLR and ApoER2 are both required for normal neuronal migration. Both receptors coordinate the development of the cortex and of the cerebellum; however, the phenotype of the VLDLR defect manifests itself mainly in the cerebellum, whereas deficiency in ApoER2 predominantly affects the development of the neocortex. Only the absence of both receptors causes an almost exact neuroanatomical phenocopy of the reeler and scrambler (mDab1-deficient) mutation. In the cortex, VLDLR and mDab1 are selectively expressed in the migrating neurons that are about to make contact with Reelin in the marginal zone, consistent with a role of VLDLR as a receptor for a migratory stop signal. Also consistent with such a model is the finding that in VLDLR-deficient cerebellum, Purkinje cells are ectopically located, apparently due to their inability to properly respond to the Reelin signal that emanates from the granule cells in the external granular layer. In contrast, ApoER2 is more ubiquitously expressed throughout the developing brain. Thus, other non-cell autonomous functions for this receptor cannot be ruled out. Absence of ApoER2 alone prevents a large portion of neurons from completing their migration and causes a partial inversion of the layers in the neocortex. As a result, the large and easily identifiable pyramidal neurons end up in a relatively ordered, but more superficial layer than the layer 5 in which they normally reside (Trommsdorff, 1999).
Furthermore, in VLDLR-deficient cortex, neurons are strictly radially aligned, and although they apparently have reached their normal assigned layer, they have failed to distribute within that layer. In contrast, in ApoER2-deficient cortex, there is no recognizable radial pattern; instead, neurons are packed into tight consecutive horizontal layers. In the absence of either VLDLR or ApoER2, neurons do not invade layer 1, in contrast to the double knockout and to reeler and mDab1 mutants (Trommsdorff, 1999 and references).
Taken together, these findings suggest that VLDLR and ApoER2 function in a coordinated and partially overlapping fashion. Either receptor is apparently capable of interpreting the stop signal conferred by Reelin, thus allowing migrating neurons to detach from the radial guidance fiber and preventing them from invading layer 1. Consistent with this interpretation is the finding that mDab1 expression is only slightly increased in either of the single knockouts. The distinct neuroanatomical phenotypes of vldlr and apoER2 knockouts may indicate that both receptors further interact with specific sets of other matrix or glial surface components. Other adaptor proteins besides mDab1 may also be involved and control different stages of neuronal migration (Trommsdorff, 1999).
Disruption of the disabled-1 gene (Dab1) results in aberrant migration of neurons during development and disorganization of laminar structures throughout the brain. Dab1 is thought to function as an adapter molecule in signal transduction processes. It contains a protein-interaction (PI) domain similar to the phosphotyrosine-binding domain of the Shc oncoprotein; it is phosphorylated by the Src protein tyrosine kinase, and it binds to SH2 domains in a phosphotyrosine-dependent manner. To investigate the function of Dab1, binding proteins were sought using the yeast two-hybrid system. The PI domain of Dab1 interacts with the amyloid precursor-like protein 1 (APLP1), a member of the family of proteins including APP. The association of Dab1 with APLP1 was confirmed in biochemical assays, and the site of interaction was localized to a cytoplasmic region of APLP1 containing the amino acid sequence motif Asn-Pro-x-Tyr (NPxY). NPxY motifs are involved in clathrin-mediated endocytosis, and they have been shown to bind to PI domains present in several proteins. This region of APLP1 is conserved among all members of the amyloid precursor family of proteins. Indeed, it was found that Dab1 also interacts with amyloid precursor protein (APP) and APLP2 in biochemical association experiments. In transiently transfected cells, Dab1 and APLP1 colocalized in membrane ruffles and vesicular structures. Cotransfection assays in cultured cells indicate that APP family members increase serine phosphorylation of Dab1. Dab1 and APLP1 are expressed in similar cell populations in developing and adult brain tissue. These results suggest that Dab1 may function, at least in part, through association with APLP1 in the brain (Homayouni, 1999).
Dab1-deficient mice show abnormalities in neuronal migration and positioning of neurons in the brain. The observation that members of theAPP family of proteins interact with Dab1 suggests APP proteins may play a role in neuronal migration during brain development. However, targeted disruption of APP, APLP1, and APLP2 genes in mice does not result in altered lamination in the brain. The lack of a major phenotype in these mice may be attributable, in part, to compensation or functional redundancy among closely related APP family members. Importantly, mice in which two of the genes have been disrupted, for example APP and APLP2 or APLP1 and APLP2, die before birth. Thus, it seems that the overlapping function of APP family members is required for normal development. Several studies have suggested developmental roles for APP family genes. For example, their level of gene expression is regulated during development of the nervous system. Also, induction of neuronal differentiation in cultured cells increases expression of all three family members. Furthermore, both APP and APLP2 are present in elongating axons. Other studies have shown that APP is expressed on radial fibers, which are present transiently in the developing cortex and provide a substrate for neuronal migration. Thus, there is some circumstantial evidence supporting an interaction between the Reelin-Dab1 pathway and APP family proteins. Mutations in APP have been linked to autosomal dominant familial Alzheimer's disease, the most common form of late-onset dementia. Alzheimer's disease is characterized pathologically by the appearance of neuritic plaques containing Abeta peptide derived from APP and neurofibrillary tangles containing hyperphosphorylated tau protein. Thus far, no direct link has been established between the appearance of amyloid plaques and tau phosphorylation. One of the kinases responsible for the phosphorylation of tau is Cdk5, and Cdk5 immunoreactivity increases in neurons that exhibit early-stage neurofibrillary tangles. It is intriguing that disruption of either Cdk5 or its activating subunit p35 in mice causes a neuronal migration defect similar to that seen in mice lacking Reelin or Dab1. These findings, in combination with the data presented here, suggest that Cdk5-p35 and Dab1 may provide a link between APP and tau metabolism in the adult brain. Numerous functions have been suggested for APP. It has been implicated in differentiation, attachment, survival, and outgrowth of neurons. Different regions of the extracellular domain of APP have been shown to inhibit proteases and to modulate synaptic activity. However, the normal function of APP and the consequences of the interaction of Dab1 with APP family proteins in the adult brain are unclear at present. The results presented here suggest that Dab1 may influence processes involving the APP family of proteins that are important in the developing, as well as the adult, brain (Homayouni, 1999 and references).
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