Leukocyte Common Antigen-related Research Articles

Two Receptor Tyrosine Phosphatases of the LAR Family Are Expressed in the Developing Leech by Specific Central Neurons as well as Select Peripheral Neurons, Muscles, and Other Cells

Receptor protein tyrosine phosphatases (rPTPs) are thought to play a crucial role in neuronal development, particularly in pathfinding by growing processes. We have cloned and sequenced two Hirudo medicinalis rPTPs that are homologous to the Drosophila and vertebrate rPTPs of the Leukocyte common antigen-related (LAR) subfamily. These Hirudo rPTPs, HmLAR1 and HmLAR2, are products of different, homologous genes, both containing two tandem intracellular phosphatase domains and ectodomains with three tandem Ig domains and different numbers of tandem fibronectin type III (FIII) domains. They are expressed in distinct patterns during embryogenesis. HmLAR1 mRNA is expressed by a subset of central and peripheral neurons and by several peripheral muscular structures, whereas HmLAR2 mRNA is expressed by a different subset of central neurons and by the peripheral, neuron-like Comb cells. HmLAR1 and HmLAR2 proteins are located on the neurites of central neurons. In addition, HmLAR2 is expressed on the cell body, processes, and growth cones of the Comb cells. Because of their CAM-like ectodomains and homology to proteins known to be involved in pathfinding and because they are expressed by different subsets of neurons, we hypothesize that HmLAR1 and HmLAR2 participate in navigational decisions that distinguish the sets of neurons that express them. Furthermore, we hypothesize that HmLAR2 is also involved in setting up the highly regular array of parallel processes established by the Comb cells. Lastly, we propose that the HmLAR1 ectodomain on peripheral muscle cells plays a role in target recognition via interactions with neuronal receptors, which might include HmLAR1 or HmLAR2.

LAR Tyrosine Phosphatase Receptor: A Developmental Isoform Is Present in Neurites and Growth Cones and Its Expression Is Regional- and Cell-Specific

Transgenic mice and Drosophila mutant studies demonstrate that the leukocyte common antigen-related (LAR) protein tyrosine phosphatase (PTPase) receptor is required for formation of neural networks. We assessed the hypothesis that alternative splicing of the LAR extracellular region contributes to this function by establishing temporospatial expression patterns of LAR isoforms containing an alternatively spliced extracellular nine amino acid segment (LAR alternatively spliced element-c; LASE-c). LASE-c was present in multiple alternatively spliced and truncated LAR transcripts. In contrast to LAR isoforms without LASE-c, levels of LAR transcripts and protein isoforms containing LASE-c were primarily present during development, suggesting a mechanism for developmental regulation of LAR function.In situanalysis demonstrated increasingly region- and cell-specific expression of LASE-c during maturation. Immunostaining revealed LASE-c-containing LAR protein along neurites and in growth cones. The discovery of highly regulated, temporospatial extracellular domain alternative splicing of LAR-type PTPase receptors points to a novel mechanism by which these receptors might influence network formation.

Overexpression of the transmembrane tyrosine phosphatase LAR activates the caspase pathway and induces apoptosis.

The protein tyrosine phosphatase family comprises transmembrane receptor-like and cytosolic forms. Although the exact biological functions of these enzymes are largely unknown, they are believed to counter-balance the effects of protein tyrosine kinases. We have previously identified and characterized a mammalian transmembrane protein tyrosine phosphatase, called LAR (leukocyte common antigen related gene), whose expression is often associated with proliferating epithelial cells or epithelial progenitor cells. This study investigates the potential role of LAR in the regulation of cell growth and death in mammals. We overexpressed in mammalian cells in culture either the full-length wild-type LAR or a truncation mutant containing only the extracellular domain of the molecule, and found that whereas the truncated LAR could be readily overexpressed in various cell lines, cells overexpressing the wild-type LAR were negatively selected. Using an inducible expression system, we demonstrated that overexpression of the wild-type LAR, but not the truncated LAR, activated the caspase pathway directly and induced p53-independent apoptosis. Our data suggest that LAR might regulate cellular signals essential for cell survival. Overproduction of LAR may tilt the balance between the tyrosine phosphorylation and dephosphorylation of proteins whose activities are critical for cell survival, and therefore lead to cell death. In addition, our observations that overexpression of LAR induces cell death without affecting cell adhesion suggest that LAR may activate the caspase pathway and induce cell death directly. This work is the first example of the involvement of a receptor-like protein tyrosine phosphatase in cell-death control and provides the basis for searching for molecules and mechanisms linking signal transduction by protein tyrosine phosphorylation to the caspase-mediated cell-death pathway.

Cloning of Novel Trinucleotide-Repeat (CAG) Containing Genes in Mouse Brain

CAG trinucleotide repeat (CTR) sequence often appears in mammalian genome including transcription-regulatory protein and homeobox genes. Its expansion is associated with six genetic disorders in human. To identify novel CTR-containing genes expressed in mouse brain, a brain cDNA library was screened using an oligonucleotide, (CTG)10. Eight clones were novel mouse genes and they were sequenced on both strands. The size of the cloned DNA ranged from 0.5 to 2.1 kb. The number of the CAG repeats in the clones ranged from 6 to 25. The inserts of the clones were analyzed for open reading frames and the peptide sequences were used for a GenBank homology search. Of the clones, one (CAG-6) shared 13 consecutive identical amino acid residues with the OB-cadherin gene, a member of cadherin family. CAG-14 showed high homology (657 nucleotides identity in 1022 nucleotides; 64%) with the 3′-untranslated region of rat leukocyte common antigen-related (LAR) tyrosine phosphatase receptor. All the 8 clones were originated from mouse DNA as judged by Southern blot analysis of mouse genomic DNA. The expression of the clones in mouse brain was addressed by RT-PCR and 4 clones showed specific expression.

Deficient LAR expression decreases basal forebrain cholinergic neuronal size and hippocampal cholinergic innervation.

A role in neural development for protein tyrosine phosphatase (PTPase) receptors has been suggested by the finding of aberrant neurite outgrowth in Drosophila mutants lacking functional leukocyte common antigen-related (LAR) PTPase receptors; however, PTPase functions in the mammalian nervous system remain to be established. In transgenic mice containing a gene trap in the LAR gene, only trace expression of full-length LAR transcripts was found. In these mice, the size of basal forebrain cholinergic neurons was significantly reduced and cholinergic innervation of the dentate gyrus was markedly decreased. These findings constitute the first demonstration of an aberrant neuronal phenotype in a mammalian PTPase mutant and support the hypothesis that LAR-type PTPase receptors function to establish and/or maintain neuronal networks.

Functional Association between the Insulin Receptor and the Transmembrane Protein-tyrosine Phosphatase LAR in Intact Cells

The receptor-type protein-tyrosine phosphatase LAR (for leukocyte common antigen-related) has been implicated as a physiological regulator of the insulin receptor. To demonstrate a functional interaction between LAR and the insulin receptor, we incubated CHO cells overexpressing the human insulin receptor with an antibody to the extracellular domain of LAR and found a 47% decrease in insulin receptor autophosphorylation and kinase activity. A physical association between LAR and the insulin receptor was then shown by immunoprecipitation of LAR from cell lysates and immunoblotting with antibody to the insulin receptor, or vice versa. Up to 11.8% of the LAR protein in the lysates of CHO cells overexpressing both the insulin receptor and LAR co-immunoprecipitated with the insulin receptor. The LAR/insulin receptor association was related to the level of LAR or insulin receptor overexpression and was increased 6.5-fold by chemical cross-linking and 3.9-fold by treatment with insulin, suggesting that receptor activation influences the affinity of LAR for the insulin receptor. In insulin-stimulated rat liver, LAR was temporally enriched in endosomes with the insulin receptor, and incubation of endosomes with neutralizing LAR antibodies decreased insulin receptor dephosphorylation in situ by 28% (p = 0.01 versus control). These data provide more direct evidence of a role for LAR in the physiological regulation of insulin action at the receptor level.

LAR tyrosine phosphatase receptor: alternative splicing is preferential to the nervous system, coordinated with cell growth and generates novel isoforms containing extensive CAG repeats.

Receptor-linked tyrosine phosphatases regulate cell growth by dephosphorylating proteins involved in tyrosine kinase signal transduction. The leukocyte common antigen-related (LAR) tyrosine phosphatase receptor has sequence similarity to the neural cell adhesion molecule N-CAM and is located in a chromosomal region (1p32-33) frequently altered in neuroectodermal tumors. To understand the function of receptor-linked tyrosine phosphatases in neural development, we sought to identify LAR isoforms preferentially expressed in the nervous system and cellular processes regulating LAR alternative splicing. We report here the isolation of a series of rat LAR cDNA clones arising from complex combinatorial alternative splicing, not previously demonstrated for the tyrosine phosphatase-receptor gene family in general. Isoforms included: (a) deletions of the fourth, sixth and seventh fibronectin type III-like domains; (b) an alternatively spliced novel cassette exon in the fifth fibronectin type III-like domain; (c) two alternatively spliced novel cassette exons in the juxtamembrane region; (d) a retained intron in the extracellular region with in-frame stop codons predicting a secreted LAR isoform; and (e) an LAR transcript including an alternative 3' untranslated region containing multiple stretches of tandem CAG repeats up to 21 repeats in length. This number of repeats was in the range found in normal alleles of genes in which expansions of repeats are associated with neurodegenerative disease and the genetic phenomenon of anticipation. RT-PCR and Northern analysis demonstrated that LAR alternative splicing occurred preferentially in neuromuscular tissue in vivo and in neurons compared to astrocytes in vitro and was developmentally regulated. Alternative splicing was also regulated in PC12 cells by NGF, in 3T3 fibroblasts by cell confluence and in sciatic nerve and muscle subsequent to nerve transection. Western blot analysis demonstrated that alternatively spliced cassette exons result in the presence of corresponding amino acid segments of LAR protein in vivo. These studies suggest specialized functions of LAR isoforms in the nervous system and support our hypothesis that LAR-like tyrosine phosphatase receptors play a role in neural development and regeneration.

Leukocyte common antigen-related receptor-linked tyrosine phosphatase. Regulation of mRNA expression.

Receptor-linked tyrosine phosphatases regulate cell growth by dephosphorylating proteins involved in tyrosine kinase signal transduction. Within this gene family, the leukocyte common antigen-related (LAR) gene is of particular interest with respect to the nervous system because it has sequence similarity to the neural cell adhesion molecule N-CAM and is located in a chromosomal region (1p32-33) frequently deleted in neuroectodermal tumors. However, immunostaining has detected LAR in non-neural tissues, but not in the central nervous system, peripheral neurons, or adrenal medulla. In this study, rat brain cDNA library LAR clones corresponding to cytoplasmic and 3'-untranslated regions of human LAR were identified. Using probes derived from these clones, high stringency Northern blots revealed approximately 8 kilobase and variable length tissue- and cell-specific LAR transcripts in cortex, brainstem, cerebellum, spinal cord, peripheral tissues, and cultured neural, glial, and pheochromocytoma cells. In situ hybridization showed expression by brain and dorsal root ganglion neurons. LAR expression was developmentally regulated in a region-dependent manner. Changes in LAR expression were also found during nerve growth factor-induced PC12 pheochromocytoma cell differentiation and with contact-mediated inhibition of fibroblast growth. These observations and studies demonstrating neurotrophins functioning via tyrosine kinase receptors suggest that LAR represents an additional mechanism regulating neural development.