Domain Of LAR Research Articles

The receptor protein tyrosine phosphatase LAR promotes R7 photoreceptor axon targeting by a phosphatase-independent signaling mechanism

Receptor protein tyrosine phosphatases (RPTPs) control many aspects of nervous system development. At the Drosophila neuromuscular junction (NMJ), regulation of synapse growth and maturation by the RPTP LAR depends on catalytic phosphatase activity and on the extracellular ligands Syndecan and Dally-like. We show here that the function of LAR in controlling R7 photoreceptor axon targeting in the visual system differs in several respects. The extracellular domain of LAR important for this process is distinct from the domains known to bind Syndecan and Dally-like, suggesting the involvement of a different ligand. R7 targeting does not require LAR phosphatase activity, but instead depends on the phosphatase activity of another RPTP, PTP69D. In addition, a mutation that prevents dimerization of the intracellular domain of LAR interferes with its ability to promote R7 targeting, although it does not disrupt phosphatase activity or neuromuscular synapse growth. We propose that LAR function in R7 is independent of its phosphatase activity, but requires structural features that allow dimerization and may promote the assembly of downstream effectors.

Liprin-α has LAR-independent functions in R7 photoreceptor axon targeting

In the Drosophila visual system, the color-sensing photoreceptors R7 and R8 project their axons to two distinct layers in the medulla. Loss of the receptor tyrosine phosphatase LAR from R7 photoreceptors causes their axons to terminate prematurely in the R8 layer. Here we identify a null mutation in the Liprin-alpha gene based on a similar R7 projection defect. Liprin-alpha physically interacts with the inactive D2 phosphatase domain of LAR, and this domain is also essential for R7 targeting. However, another LAR-dependent function, egg elongation, requires neither Liprin-alpha nor the LAR D2 domain. Although human and Caenorhabditis elegans Liprin-alpha proteins have been reported to control the localization of LAR, we find that LAR localizes to focal adhesions in Drosophila S2R+ cells and to photoreceptor growth cones in vivo independently of Liprin-alpha. In addition, Liprin-alpha overexpression or loss of function can affect R7 targeting in the complete absence of LAR. We conclude that Liprin-alpha does not simply act by regulating LAR localization but also has LAR-independent functions.

Cell-Autonomous and -Nonautonomous Functions of LAR in R7 Photoreceptor Axon Targeting

During Drosophila visual system development, photoreceptors R7 and R8 project axons to targets in distinct layers of the optic lobe. We show here that the LAR receptor tyrosine phosphatase is required in the eye for correct targeting of R7 axons. In LAR mutants, R7 axons initially project to their correct target layer, but then retract to the R8 target layer. This targeting defect can be fully rescued by transgenic expression of LAR in R7, and partially rescued by expression of LAR in R8. The phosphatase domains of LAR are required for its activity in R7, but not in R8. These data suggest that LAR can act both as a receptor in R7, and as a ligand provided by R8. Genetic interactions implicate both Enabled and Trio in LAR signal transduction.

The phosphatase domains of LAR, CD45, and PTP1B: structural correlations with peptide-based inhibitors1

PTP1B is a cytosolic protein tyrosine phosphatase that is a regulator of the kinase activity of the insulin receptor; the two protein tyrosine phosphatases LAR and CD45 are receptor type phosphatases crucially important to cell function. LAR also is involved in regulation of the insulin receptor while CD45 is critical for T-cell activation. Although LAR and CD45 are both transmembrane phosphatases, these enzymes manifest their phosphatase activity through a catalytic cytosolic domain. We have utilized X-ray coordinates of related phosphatases (RPTPalpha and RPTmu) and comparative protein modeling to obtain molecular models of the D1 catalytic domains of CD45 and LAR. The models were tested using established protocols and found to be comparable to low resolution X-ray structures. The structure obtained for LAR was compared with the recently reported X-ray structure. Both the CD45-D1 and LAR-D1 structures were then compared to and contrasted with PTP1B. The active site of pockets of the three enzymes were found to be very uniform in structure and charge distribution. Also, the gross surface topology around the active site was found to be somewhat similar for the 3 phosphatases. However, there were significant differences in surface topology, and, more importantly, large changes in surface charge distribution. The differences between the surface features of these enzymes provide an explanation for the selectivity of inhibition by a number of peptides.

Cellular redistribution of protein tyrosine phosphatases LAR and PTPsigma by inducible proteolytic processing.

Most receptor-like protein tyrosine phosphatases (PTPases) display a high degree of homology with cell adhesion molecules in their extracellular domains. We studied the functional significance of processing for the receptor-like PTPases LAR and PTPsigma. PTPsigma biosynthesis and intracellular processing resembled that of the related PTPase LAR and was expressed on the cell surface as a two-subunit complex. Both LAR and PTPsigma underwent further proteolytical processing upon treatment of cells with either calcium ionophore A23187 or phorbol ester TPA. Induction of LAR processing by TPA in 293 cells did require overexpression of PKCalpha. Induced proteolysis resulted in shedding of the extracellular domains of both PTPases. This was in agreement with the identification of a specific PTPsigma cleavage site between amino acids Pro821 and Ile822. Confocal microscopy studies identified adherens junctions and desmosomes as the preferential subcellular localization for both PTPases matching that of plakoglobin. Consistent with this observation, we found direct association of plakoglobin and beta-catenin with the intracellular domain of LAR in vitro. Taken together, these data suggested an involvement of LAR and PTPsigma in the regulation of cell contacts in concert with cell adhesion molecules of the cadherin/catenin family. After processing and shedding of the extracellular domain, the catalytically active intracellular portions of both PTPases were internalized and redistributed away from the sites of cell-cell contact, suggesting a mechanism that regulates the activity and target specificity of these PTPases. Calcium withdrawal, which led to cell contact disruption, also resulted in internalization but was not associated with prior proteolytic cleavage and shedding of the extracellular domain. We conclude that the subcellular localization of LAR and PTPsigma is regulated by at least two independent mechanisms, one of which requires the presence of their extracellular domains and one of which involves the presence of intact cell-cell contacts.

Molecular cloning and expression of a unique receptor-like protein-tyrosine-phosphatase in the leucocyte-common-antigen-related phosphatase family

Protein-tyrosine-phosphatases (PTPases) have been implicated in the regulation of certain tyrosine kinase growth factor receptors in that they dephosphorylate the activated (autophosphorylated) form of the receptors. In order to identify PTPases that potentially act on receptor targets in liver, we used the human leucocyte common antigen-related PTPase (LAR) cDNA [Streuli, Krueger, Hall, Schlossman and Saito (1988) J. Exp. Med. 168, 1523-1530] and isolated two closely related transmembrane PTPase homologues from a rat hepatic cDNA library. Both PTPases had large extracellular domains that contained three immunoglobulin-like repeats and eight type-III fibronectin repeats. Both enzymes had tandem homologous PTPase domains following a single hydrophobic transmembrane domain. One sequence encoded the rat homologue of LAR. The second PTPase, designated LAR-PTP2, had 79 and 90% identity with rat LAR in the respective cytoplasmic PTPase domains, with only 57% sequence similarity in the extracellular domain. The catalytic domains of LAR and LAR-PTP2 prepared by bacterial expression were active in dephosphorylating a variety of phosphotyrosyl substrates but did not hydrolyse phosphoserine or phosphothreonine residues of labelled casein. Both enzymes exhibited rapid turnover numbers of 4-7 s-1 for myelin basic protein and 78-150 s-1 for derivatized lysozyme. LAR and LAR-PTP2 displayed similar PTPase activity towards the simultaneous dephosphorylation of receptors of intact insulin and epidermal growth factor from liver membranes. These data indicate that there is a family of LAR-related PTPases that may regulate the phosphorylation state of receptor tyrosine kinases in liver and other tissues.

Insulin receptor and epidermal growth factor receptor dephosphorylation by three major rat liver protein-tyrosine phosphatases expressed in a recombinant bacterial system.

Protein-tyrosine phosphatases (PTPases) play an essential role in the regulation of signal transduction mediated by reversible protein-tyrosine phosphorylation. In order to characterize individual rat hepatic PTPases that might have specificity for autophosphorylated receptor tyrosine kinases, we isolated cDNA segments encoding three PTPases (PTPase 1B, LAR and LRP) that are expressed in insulin-sensitive liver and skeletal muscle tissue, and evaluated their catalytic activity in vitro. The intrinsic PTPase activities of the full-length PTPase 1B protein and the cytoplasmic domains of LAR and LRP were studied by expression of recombinant cDNA constructs in the inducible bacterial vector pKK233-2 using extracts of a host strain of Escherichia coli that lacks endogenous PTPase activity. Each of the cloned cDNAs dephosphorylated a cognate phosphopeptide derived from the regulatory region of the insulin receptor. Despite having only 30-39% sequence identity in their catalytic domains, LAR and PTPase 1B had similar relative activities between the peptide substrate and intact insulin receptors, and also displayed similar initial rates of simultaneous dephosphorylation of insulin and epidermal growth factor (EGF) receptors. In contrast, LRP exhibited a higher rate of dephosphorylation of both intact receptors relative to the peptide substrate, and also dephosphorylated EGF receptors more rapidly than insulin receptors. These studies indicate that three PTPases with markedly divergent structures have the catalytic potential to dephosphorylate both insulin and EGF receptors in intact cells and that redundant PTPase activity may occur in vivo. For these PTPases to have specific physiological actions in intact cells, they must be influenced by steric effects of the additional protein segments of the native transmembrane enzymes, cellular compartmentalization and/or interactions with regulatory proteins.

Distinct functional roles of the two intracellular phosphatase like domains of the receptor-linked protein tyrosine phosphatases LCA and LAR.

Protein tyrosine phosphorylation is regulated by both protein tyrosine kinases and protein tyrosine phosphatases (PTPases). Recently, the structures of a family of PTPases have been described. In order to study the structure-function relationships of receptor-linked PTPases, we analyzed the effects of deletion and point mutations within the cytoplasmic region of the receptor-linked PTPases, LCA and LAR. We show that the first of the two domains has enzyme activity by itself, and that one cysteine residue in the first domain of both LCA and LAR is absolutely required for activity. The second PTPase like domains do not have detectable catalytic activity using a variety of substrates, but sequences within the second domains influence substrate specificity. The functional significance of a stretch of 10 highly conserved amino acid residues surrounding the critical cysteine residue located in the first domain of LAR was assessed. At most positions, any substitution severely reduced enzyme activity, while missense mutations at the other positions tested could be tolerated to varying degrees depending on the amino acid substitution. It is suggested that this stretch of amino acids may be part of the catalytic center of PTPases.