Gene name - Leukocyte-antigen-related-like
Synonyms - Dlar
Cytological map position - 38A1
Function - receptor protein-tyrosine phosphatase
Keywords - neural - axon guidance
Symbol - Lar
Genetic map position - 38A
Classification - Fibronectin type-III repeat, Immunoglobulin-C2-like domain, phosphatase domain
Cellular location - surface - transmembrane
|Recent literature||Agrawal, P. and Hardin, P. E. (2016). The Drosophila receptor protein tyrosine phosphatase LAR is required for development of circadian pacemaker neuron processes that support rhythmic activity in constant darkness but not during light/dark cycles. J Neurosci 36: 3860-3870. PubMed ID: 27030770
Little is known about phosphatases that control clock protein dephosphorylation in Drosophila. This study screened RNAi knockdowns of Drosophila phosphatases for altered activity rhythms. One phosphatase that was identified, the receptor protein tyrosine phosphatase leukocyte-antigen-related (LAR), abolished activity rhythms in constant darkness (DD) without disrupting the timekeeping mechanism in brain pacemaker neurons. However, expression of the neuropeptide pigment-dispersing factor (PDF), which mediates pacemaker neuron synchrony and output, is eliminated in the dorsal projections from small ventral lateral (sLNv) pacemaker neurons when Lar expression is knocked down during development, but not in adults. Loss of Lar function eliminates sLNv dorsal projections, but PDF expression persists in sLNv and large ventral lateral neuron cell bodies and their remaining projections. In contrast to the defects in lights-on and lights-off anticipatory activity seen in flies that lack PDF, LarRNAi knockdown flies anticipate the lights-on and lights-off transition normally. These results demonstrate that Lar is required for sLNv dorsal projection development and suggest that PDF expression in LNv cell bodies and their remaining projections mediate anticipation of the lights-on and lights-off transitions during a light/dark cycle.
|Barlan, K., Cetera, M. and Horne-Badovinac, S. (2017). Fat2 and Lar define a basally localized planar signaling system controlling collective cell migration. Dev Cell 40(5): 467-477.e465. PubMed ID: 28292425
Collective migration of epithelial cells underlies diverse tissue-remodeling events, but the mechanisms that coordinate individual cell migratory behaviors for collective movement are largely unknown. Studying the Drosophila follicular epithelium, this study shows that the cadherin Fat2 and the receptor tyrosine phosphatase Lar function in a planar signaling system that coordinates leading and trailing edge dynamics between neighboring cells. Fat2 signals from each cell's trailing edge to induce leading edge protrusions in the cell behind, in part by stabilizing Lar's localization in these cells. Conversely, Lar signals from each cell's leading edge to stimulate trailing edge retraction in the cell ahead. Fat2/Lar signaling is similar to planar cell polarity signaling in terms of sub-cellular protein localization; however, Fat2/Lar signaling mediates short-range communication between neighboring cells instead of transmitting long-range information across a tissue. This work defines a key mechanism promoting epithelial migration and establishes a different paradigm for planar cell-cell signaling.
One of the main ways that signals are transduced between proteins is by the addition or removal of a phosphate group from the amino acid tyrosine. The addition of a phosphate group is carried out by kinases while the removal of phosphate is carried out by phosphatases. Leukocyte-antigen-related-like/Dlar influences the process of axon guidance by removal of phosphate groups from phosphotyrosine residues. Examples of receptors that act as kinases include the EGF-R and Sevenless. Each of these interact with an extracellular ligand and transduce the extracellular signal to the inside of the cell resulting in the activation of the kinase associated with the intracellular domain of the receptor.
Receptor tyrosine phosphatases likewise receive extracellular signals (in most cases from unknown ligands), and transduce those signals to the inside of the cell, activating the phosphatase enzymatic function associated with the intracellular domain of the receptor. Phosphatases remove phosphate groups from proteins, instigating a signal that can be passed from protein to protein, ultimately changing the behavior of the cell.
A family of protein receptor tyrosine phosphatases exists in Drosophila as well as in vertebrates that share adhesion-like domains in the extracellular region and phosphatase domains associated with the intracellular region. The receptor tyrosine phosphatases in both insects and vertebrates are found associated with the nervous system. In Drosophila they are involved in axon guidance.
In each segment of the fly, specific motor axons upon leaving the CNS diverge from a common motor pathway and divide into five major peripheral nerve branches that project to different groups of muscles. In Dlar mutant embryos, the common motor pathway appears normal, but two of the nerves that branch from the intersegmental nerve, carrying information between segments, are abnormal. In one of these two nerves, the SNb axons bypass their normal entry point to the ventral muscle region (at a defined choice point) and instead continue to extend distally as a distinct fascicle (bundle), following the intersegmental nerve further along its dorsal trajectory to a more lateral region of muscles. In such mutants, ventral muscles are completely devoid of innervation. In some cases nerves terminate without leaving the dorsal axon pathway, and in other cases inappropriate muscles become innervated (Krueger, 1996).
It is believed that each SNb growth cone may have an independent ability to navigate the choice point; that is, no individual axon is required to pioneer the SNb pathway, with the rest relegated to the status of faithful followers. This conclusion is based on studies in which the aCC motoneuron (whose axon pioneers the intersegmental nerve) is ablated. Later growth cones are capable (with some delay) of independently pioneering the intersegmental nerve (Lin, 1995).
The ability of motoneuron growth cones to both recognize and enter their correct target appears to be dependent on the phosphorylation state of key intracellular proteins. A dynamic balance of kinase and phosphatase activities at the leading edge of the growth cone endows it with the ability to integrate signals and translate them into appropriate directional choices. Two mechanisms for axon guidance are involved. At the choice point, SNb axons defasciculate from the intersegmental nerve axons and form a separate bundle. This bundle of axons is then directed or steered into the ventral muscle region. Genes other than Dlar produce subtly different phenotypes capable of altering defasciculation and/or steering, suggesting a complex genetic basis for axon pathfinding in the fly (Krueger, 1996).
Genetic analysis of growth cone guidance choice points in Drosophila has identified neuronal receptor protein tyrosine phosphatases (RPTPs) as key determinants of axon pathfinding behavior. The Drosophila Abl tyrosine kinase functions in the intersegmental nerve b (ISNb) motor choice point pathway as an antagonist of the RPTP Dlar. The function of Abl in this pathway is dependent on an intact catalytic domain. The Abl phosphoprotein substrate Enabled (Ena) is required for choice point navigation. Both Abl and Ena proteins associate with the Dlar cytoplasmic domain and serve as substrates for Dlar in vitro, suggesting that they play a direct role in the Dlar pathway. These data suggest that Dlar, Abl, and Ena define a phosphorylation state-dependent switch that controls growth cone behavior by transmitting signals at the cell surface to the actin cytoskeleton (Wills, 1999).
The reciprocal catalytic activities of a tyrosine kinase and phosphatase predict that a reduction in kinase activity within the Dlar pathway might suppress the Dlar motor axon phenotype. In Dlar mutant embryos, subsets of axons derived from the intersegmental nerve route (ISN), called ISNb and ISNd, fail to enter adjacent muscle target domains just outside the ventral nerve cord. Instead, Dlar mutant ISNb and ISNd axons follow the ISN toward dorsal targets (the bypass phenotype. Since abl loss of function is known to disrupt the outgrowth of ISNb, the Abl tyrosine kinase is an excellent candidate for a role in Dlar signaling. Therefore, various genetic backgrounds were examined in which homozygous Dlar mutations were combined with mutations in a single allele of abl. Reduction of abl of up to half the normal gene dose has a profound effect on the penetrance of the Dlar motor axon guidance phenotype, suppressing the Dlar phenotype up to 10-fold; for example, ISNb bypass in Dlar mutants is reduced from 38% to 4% in abl heterozygote mutants (Wills, 1999).
Western blot analysis shows that endogenous Abl protein binds specifically to the full-length Dlar cytoplasmic domain (GST-Dlar D1-D2). The association of Dlar and Abl in cell extracts is consistent with a direct functional relationship between the two proteins. However, the binding could depend on other factors present in the crude extract. Therefore, the association of purified recombinant Abl protein with Dlar fusion proteins was examined in the absence of other Drosophila proteins. Recombinant Abl binds to Dlar with somewhat less specificity than does the Abl endogenous to S2 cells. Purified mammalian v-Abl binds to Dlar under the same conditions, with a profile of specificity very similar to that of Drosophila Abl. Since v-Abl represents only the kinase and SH2 domains of Abl, these domains appear sufficient to mediate Dlar binding. As further evidence of direct physical interactions between Abl and the Dlar D2 domain, kinase assays reveal that Drosophila Abl phosphorylates GST-Dlar D2 in vitro. In addition to the Dlar D2 domain, Drosophila Abl can weakly phosphorylate the D2 domain of another receptor tyrosine kinase, DPTP69D; this is interesting, since DPTP69D is tyrosine phosphorylated in S2 cells. The physical interactions between Abl and Dlar support a model whereby both proteins function in the same signaling pathway. Furthermore, the phosphorylation of the D2 domain in vitro raises the intriguing possibility that d-Abl activity regulates Dlar function in vivo (Wills, 1999).
The contrast between the abl and Dlar phenotypes and the suppression of the Dlar phenotype by abl alleles suggest that Abl and Dlar play functionally antagonistic roles in ISNb development. This hypothesis makes a simple prediction: gain of function in Abl should result in a phenotype similar to loss of Dlar. Therefore, the GAL4 expression system was used to target high-level expression of wild-type Abl to postmitotic neurons and then the development of motor axon pathways was examined. With three independent neural specific GAL4 drivers, in combination with an abl cDNA under the control of the GAL4 upstream activator sequence (UAS), GAL4-dependent phenotypes were observed. When wild-type Abl is overexpressed, ISNb axons bypass their ventral target muscles in a manner indistinguishable from that of the ISNb phenotype observed in Dlar mutants. The kinase activity of Abl has been shown to be necessary for its role in ISNb neurons (Wills, 1999).
Since Ena acts as a genetic antagonist of Abl, it was reasoned that loss of Ena should resemble gain of Abl. ISNb bypass phenotypes are seen in all ena mutant combinations. Two types of ISNb phenotypes are observed in ena mutants: (1) failure of ISNb to enter the ventral muscles after a successful defasciculation (characteristic of embryos lacking Dlar alone), and (2) failure of ISNb axons to defasciculate from the ISN pathway (characteristic of embryos lacking multiple phosphatases. In addition, the frequency of ISNb bypass in strong ena mutants is twice that observed in the strongest Dlar alleles. These observations may indicate that Ena acts as a point of convergence for multiple inputs in the ISNb guidance mechanism. Ena family members share a conserved domain structure, including an N-terminal EVH1 domain that mediates binding to Zyxin and Listeria ActA, a proline-rich region that supports associations with Profilin and SH3 domains, and a C-terminal EVH2 domain that promotes multimerization. Mutations are available that specifically disrupt either the EVH1 or the EVH2 domains of Ena. Mutations in either domain display highly penetrant ISNb bypass, demonstrating a requirement for both domains in the guidance mechanism. Although Ena is restricted to axons in the developing nervous system late in embryogenesis, it is expressed broadly prior to germ band retraction. To confirm that neuronal Ena function is necessary for ISNb choice point navigation, wild-type ena cDNA was expressed under neuronal GAL4 control in an ena mutant background. Neural specific ena expression attenuates the ISNb phenotype significantly. If the quantity of Ena protein is rate limiting in wild-type ISNb axons, one might expect Ena overexpression to disrupt ISNb guidance. However, no ISNb phenotypes are observed, even when UAS-ena is combined with the strongest neural driver P[elav-GAL4] (Wills, 1999).
The genetic relationship between Abl and Dlar and the requirement of Ena function for ISNb target entry suggest that Ena might act in the Dlar signaling pathway. To test this model, it was asked whether Ena associates with the cytoplasmic domain of Dlar. Endogenous Ena protein associates with a Dlar full-length cytoplasmic domain (GST-Dlar D1-D2) or with D2 alone but not comparably with wild-type D1. Since Abl is known to associate with Ena, and since binding between Abl and Dlar has been demonstrated, it is possible that Ena binding to Dlar requires Abl or additional proteins. Purified Ena has been shown to bind to the Dlar cytoplasmic domain. In both extract and recombinant protein binding assays, Ena shows only weak association with DPTP10D. However, Ena binds effectively to the D2 domain of DPTP69D. The preferential binding of Ena to the D2 domains of Dlar and DPTP69D, as compared with the D1 domains of the same RPTPs, suggests that these interactions are specific. The parallel between Dlar and DPTP69D binding is interesting, given the published observation that DPTP69D is required for ISNb guidance and can partially substitute for Dlar in vivo. Furthermore, the nature and penetrance of ISNb defects in ena mutants suggest that Ena may function downstream of multiple inputs (Wills, 1999).
The relationships between Abl, Ena, and Dlar in motor axon guidance suggest a model whereby Abl and Dlar compete for shared substrates to regulate growth cone behavior. Although the Dlar cytoplasmic domain was previously shown to encode an active PTP domain, using artificial phospho-peptide substrates in vitro, no physiological substrates have been identified. Since nearly all of the tyrosine phosphatase activity of LAR family RPTPs resides in the D1 domain, the ability of the GST-Dlar D1 fusion protein to dephosphorylate purified Drosophila Abl or Ena proteins after these proteins have been phosphorylated with recombinant d-Abl was examined. Incorporated 32P is rapidly released from both Abl and Ena after addition of wild-type GST-Dlar D1 but not after addition of the catalytically inactive C-to-S mutant GST-Dlar D1 fusion protein. These results suggest that the bacterially expressed GST-Dlar protein is correctly folded and that Drosophila Abl and Ena are both potential Dlar substrates. However, because PTPs are known to be promiscuous in vitro, additional experiments will be necessary to determine whether Abl and/or Ena are targets for Dlar activity in vivo (Wills, 1999).
The stem cell niche provides a supportive microenvironment to maintain adult stem cells in their undifferentiated state. Adhesion between adult stem cells and niche cells or the local basement membrane ensures retention of stem cells in the niche environment. Drosophila male germline stem cells (GSCs) attach to somatic hub cells, a component of their niche, through E-cadherin-mediated adherens junctions, and orient their centrosomes toward these localized junctional complexes to carry out asymmetric divisions. This study shows that the transmembrane receptor tyrosine phosphatase Leukocyte-antigen-related-like (Lar), which is best known for its function in axonal migration and synapse morphogenesis in the nervous system, helps maintain GSCs at the hub by promoting E-cadherin-based adhesion between hub cells and GSCs. Lar is expressed in GSCs and early spermatogonial cells and localizes to the hub-GSC interface. Loss of Lar function resulted in a reduced number of GSCs at the hub. Lar function was required cell-autonomously in germ cells for proper localization of Adenomatous polyposis coli 2 and E-cadherin at the hub-GSC interface and for the proper orientation of centrosomes in GSCs. Ultrastructural analysis revealed that in Lar mutants the adherens junctions between hub cells and GSCs lack the characteristic dense staining seen in wild-type controls. Thus, the Lar receptor tyrosine phosphatase appears to polarize and retain GSCs through maintenance of localized E-cadherin-based adherens junctions (Srinivasan, 2012).
This work identifies a role for the transmembrane receptor tyrosine phosphatase Lar, acting cell-autonomously to maintain attachment of Drosophila male GSCs to the hub. Lar function appears to promote the maintenance of robust adherens junctions between GSCs and hub cells and to localize and/or retain E-cadherin at the hub-GSC interface. Consistent with the recently demonstrated requirement for E-cadherin to polarize GSCs by localizing Apc2 at the hub-GSC interface and to establish centrosome orientation in GSCs (Inaba, 2010), Apc2 was often mislocalized around the GSC cortex and centrosomes were often misoriented in Lar mutant GSCs (Srinivasan, 2012).
Lar may function in parallel with other cell signaling pathways that are important for maintaining attachment of GSCs to the hub. Activation of the JAK-STAT pathway in Drosophila male GSCs maintains GSCs at the hub. However, Stat92E protein levels appeared normal in Lar mutant GSCs, suggesting that Lar function is not required for activation of the JAK-STAT pathway. The Rap1 GTPase/Rap1 guanine nucleotide exchange factor (Rap-GEF) signaling pathway also regulates hub-GSC adhesion. Like Lar mutants, Rap-GEF mutants have impaired adherens junctions at the hub- GSC interface resulting in GSC loss. However, Rap-GEF function is required in hub cells, whereas Lar functions in GSCs to promote hub-GSC adhesion. Interestingly, expression of E-cadherin-GFP in either hub cells or GSCs of Rap-GEF mutants resulted in wild-type numbers of GSCs and restored E-cadherin localization at the hub- GSC interface, whereas expression of Ecadherin-GFP in Lar mutant GSCs did not rescue the loss of GSCs, suggesting that the Rap-GEF and Lar signaling pathways might use different mechanisms to build and/or maintain adherens junctions between the hub cells and GSCs (Srinivasan, 2012).
The ability of some GSCs to persist next to the hub in Lar mutant testes might be due to partial redundancy between Lar and other tyrosine phosphatases such as the type IIA family receptor tyrosine phosphatase Ptp69D, which has overlapping functions with Drosophila Lar in the central nervous system and the visual cortex and shares common signaling mechanisms. Alternatively, weak hub-GSC adhesion in Lar mutant testes might enable CySCs to compete for attachment to the hub, displacing some, but not all, GSCs from the hub. CySCs normally have smaller regions of contact with the hub than do GSCs but can outcompete GSCs from the hub when provided with an advantage. For example, overexpression of components of the integrin-based adhesion system in CySCs resulted in displacement of GSCs from the hub by CySCs (Srinivasan, 2012).
In wild-type testes, Lar localizes to the hub-GSC interface, which is the region of cell cortex where localized adherens junctions anchor GSCs to their niche. Adherens junctions are formed by extended clustering of transmembrane cadherin proteins that form homotypic interactions with cadherins on opposing cell membranes. The highly conserved cytoplasmic tail of E-cadherin acts as an anchor for β-catenin and p120-catenin and indirectly for α- catenin through its interaction with β-catenin. Lar also localizes to adherens junctions in epithelial cells and in neuronal synapses that are enriched in cadherin-catenin complexes. Lar physically associates with the cadherin-catenin complex in cultured cells and with N-cadherin in Drosophila embryos (Srinivasan, 2012).
Adherens junctions are associated with underlying arrays of cortical F-actin, organized by the high local concentration of β-catenin dimers. F-actin filaments, in turn, regulate the stability and strength of adherens junctions. Biochemical and genetic analyses of Lar indicate a role in regulating the actin cytoskeleton. Loss of Lar function in Drosophila oocytes results in defects in follicle formation, egg elongation and anterior-posterior polarity that are correlated with defects in actin filament organization. Lar might help to maintain hub- GSC adhesion by interacting with and modulating the function of regulators of F-actin. Drosophila Lar and its homologs physically and genetically interact with Ena, a member of the Ena/VASP family of actin regulators. Drosophila Ena and its mammalian homologs localize to adherens junctions and have been implicated in the formation and strengthening of adherens junctions in several cell types. However, although Ena localized to the hub-GSC interface, where adherens junctions are present, its function was not absolutely required for GSC maintenance, suggesting that other F-actin regulators in addition to Ena function to maintain hub-GSC adhesion (Srinivasan, 2012).
Lar protein localized to the hub-GSC interface might instead, or in addition, regulate the tyrosine phosphorylation state of components of the adherens junctions to maintain strong adhesion between hub cells and GSCs. Regulation of the tyrosine phosphorylation of components of adherens junctions plays an important role in modulating the adhesive state of cells. Tyrosine phosphorylation of E-cadherin in epithelial cells induces loss of cell-cell contacts and the endocytosis of E-cadherin. A possible role of Lar is to maintain adherens junctions by dephosphorylating E-cadherin. Alternatively, Lar might target E-cadherin to the membrane to build adherens junctions, as has been shown for the mammalian homolog of Lar in cultured hippocampal neurons, where it promotes the accumulation of cadherin-catenin complexes at the synapse to enhance cell adhesion. Alternatively, or in addition, Lar might regulate tyrosine phosphorylation of the catenins associated with E-cadherin at the hub-GSC interface. Tyrosine phosphorylation of β-catenin leads to loss of cadherin-β-catenin interaction and to internalization of Ecadherin, reducing the strength of adherens junctions. Mammalian Lar has been shown to dephosphorylate β-catenin in vitro, suggesting that in vivo Lar might promote cell adhesion by regulating the phosphorylation of β-catenin (Srinivasan, 2012).
In addition to GSCs, Lar protein was also detected in two-, four- and eight-cell transit-amplifying spermatogonial cysts, which have the ability to dedifferentiate and reoccupy the hub to replace lost GSCs. Under conditions that promote dedifferentiation, spermatogonial cells send out dynamic, actin-rich, thin protrusions, suggesting acquisition of motility. An intriguing possibility is that Lar might facilitate the ability of dedifferentiating spermatogonial cells to reorganize their actin cytoskeleton to recognize and build adherens junctions with the hub, similar to the role of Lar in the nervous system, where it promotes axonal migration, possibly by facilitating reorganization of the actin cytoskeleton. One of the ligands of Lar identified in the nervous system, the heparan sulfate proteoglycan Dally-like (Dlp), is expressed by hub cells and helps maintain GSCs in their undifferentiated state. At Drosophila neuromuscular junctions, Dlp interacts with and inhibits the phosphatase activity of Lar to regulate active zone morphology and function at synapses. Similarly, in dedifferentiating spermatogonial cells, interaction of Lar in GSCs with Dlp on hub cells might inhibit cell motility and promote the formation of adherens junctions between hub cells and the dedifferentiating germ cells (Srinivasan, 2012).
Nearly half the protein coding sequence is present in exon 14, a single, large (3.3 kb) exon. Exons 1 through 4, which code for a total of less than 900 bp, are spread over 50 kb of genomic DNA. The screw gene is nested entirely between exons 4 and 5 of Dlar (Krueger, 1996 and Arora, 1994).
Exons - 17
DLAR has an N-terminal signal sequence, an extracellular domain of 1345aa, a transmembrane domain of 25aa and a 627aa cytoplasmic region. The cytoplasmic region of DLAR has 72% identity to vertebrate LAR, with two repeated phosphatase domains. Whereas LAR has eight FNIII repeats, DLAR has nine (Streuli, 1989).
The structure of DLAR is quite different from the structure of its mammalian relatives, LAR and HPTP delta. LAR has a modular gene structure, in which individual exon boundaries correspond to structural domains (such as the Ig-like domains and FNIII repeats). In contrast, the boundaries of exons in the Dlar gene show virtually no correspondence to structural domains of DLAR. The mammalian proteins are alternatively spliced to produce multiple isoforms, while it is unlikely that DLAR possesses multiple isoforms (Krueger, 1996).
DLAR has three N-terminal Ig domains, nine fibronectin III domains, a transmembrane domain and two C-terminal protein tyrosine phosphatase domains. Sequence comparison of the PTP-like domains indicates that DLAR is closely related the the mammalin PTPs, having 77% identity to LAR (Krueger, 1996 and Streuli, 1989).
date revised: 10 May 98
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