BIOLOGICAL OVERVIEW

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).

GENE STRUCTURE

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

PROTEIN STRUCTURE

Amino Acids - 1997

Structural Domains

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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