Research reportLAR tyrosine phosphatase receptor: proximal membrane alternative splicing is coordinated with regional expression and intraneuronal localization
Introduction
Tyrosine phosphorylation by receptor and intracellular protein tyrosine kinases (PTKs) constitutes a fundamental process regulating neuronal survival and development. PTK signaling is counteracted or in some cases augmented by the action of protein tyrosine phosphatases (PTPs). Elucidation of mechanisms regulating PTP function will provide novel fundamental insights into neuronal survival and development. In the case of most PTK cell surface receptors, activity is induced by growth factor ligand activation or the formation of homodimeric or heterodimeric complexes [26]. In contrast, the mechanisms regulating PTP receptor activity remain to be identified 2, 7, 25. The observation that recombinantly expressed PTP intracytoplasmic catalytic domains demonstrate high activity in the absence of extracellular domains indicates that ligand–extracellular domain interactions or other protein–protein interactions mediated by heretofore unidentified domains might down-regulate constitutive activity.
The proximal membrane domain of PTP and other cell surface receptors constitutes an important candidate site regulating receptor activity. Crystal studies of the PTPα receptor identified a segment in the proximal membrane domain adjacent to the N-terminus of the first PTP catalytic domain, designated as the `N-terminal wedge', that upon receptor dimerization is predicted to insert into the active site of the dyad-related monomer [3]. The observation that this inhibitory wedge is conserved in many receptor PTPs suggested that dimerization and associated active-site blockage might constitute a general mechanism for down regulating PTP activity. Mutagenesis studies of this wedge region in the CD45 PTP receptor have confirmed that this domain is required for ligand-induced inhibition of enzyme activity [17]. A second possible mechanism regulating PTP receptor activity via the proximal membrane domain involves phosphorylation of specific Ser residues in this region leading to decreased catalytic activity 9, 24. It has also been proposed that certain PTP functions are dependent upon interaction with other membrane-associated proteins. In this regard it is of interest to note that the proximal membrane domain of PTPλ is required for association with β-catenin, a membrane associated protein undergoing tyrosine dephosphorylation and involved in cadherin mediated cell adhesion [5].
An important opportunity for assessing the possibility that the PTP receptor proximal membrane domain plays a critical role in PTP function consists of the discovery that the proximal membrane region of the Leukocyte Common Antigen-Related (LAR) PTP receptor undergoes alternative splicing [28]. LAR is the prototype member of the subgroup of PTP receptors that contain Ig-like and fibronectin type III-like (FNIII) cell adhesion domains 4, 23. LAR modulates signaling by the insulin, epidermal growth factor and hepatocyte growth factor tyrosine kinase receptors [14]. Studies in our laboratory have shown that LAR modulates NGF and BDNF Trk-mediated neurotrophin signaling (Tisi and Longo; Yang and Longo, unpublished data). The discoveries that LAR is expressed by mammalian neurons and that its expression is regulated during neural development and by NGF 16, 19, 28, 29suggested that LAR might modulate mammalian neuronal survival and/or neurite outgrowth. This hypothesis was supported by studies showing reduced size of basal forebrain cholinergic neurons and loss of cholinergic innervation of the dentate gyrus in LAR-deficient transgenic mice [27]and aberrant motor neuron pathfinding in Drosophila LAR loss-of-function mutants [13]. LAR-deficient transgenic mice have also been found to have impaired outgrowth of sensory fibers during sciatic nerve regeneration (Xie and Longo, unpublished data). As is the case with most PTP receptors, the extracellular ligands interacting with LAR have not been identified and the mode by which LAR is regulated is unknown.
The proximal membrane region of LAR contains an alternatively spliced 33 bp exon encoding an eleven amino acid (SSAPSCPNISS) segment designated as LAR Alternatively Spliced Element-a (LASE-a; [28]; Fig. 1A). Measurement of the ratio of LAR RT-PCR products with and without the LASE-a insert in cortical, cerebellar and non-neuronal tissue showed that LASE-a splicing occurs preferentially in the nervous system and that the proportion of LAR transcripts containing LASE-a decreases during development. Given the potential importance of the proximal membrane region in regulating LAR-type PTP receptor function and protein–protein interaction, the present study established whether LASE-a splicing occurs in a context in which it might function during neural development. Four fundamental questions essential for the subsequent design of direct functional studies of LASE-a were addressed: (i) Is the LASE-a insert conserved across rat, human and mouse LAR? (ii) How is the expression of LASE-a containing isoforms spatially and developmentally regulated? (iii) Given that LAR is expressed by multiple cell types, are LASE-a containing isoforms expressed selectively by neurons? and (iv) As shown with other LAR protein isoforms, are LASE-a-containing protein isoforms present in neurites?
Section snippets
Isolation and sequencing of mouse and human LASE-a containing cDNA clones
A 33-mer oligonucleotide probe corresponding to the sequence of the rat LASE-a insert was used to screen a human hippocampus cDNA library using standard protocols (2-year-old female, Lambda ZAPII; Stratagene). cDNA was sequenced by the chain termination method using double stranded DNA template with successive oligonucleotide primers (Sequenase 2.0 kit protocol and reagents from United States Biochemical). Mouse LAR cDNA sequence flanking and including the LASE-a insert was obtained by
The LASE-a insert is present in human and mouse LAR
The originally determined human LAR sequence was derived from cDNA clones isolated from a lymphocyte library and did not contain the LASE-a insert [23]. While this finding was consistent with subsequent studies of rat LAR transcripts in multiple organ tissues showing that LASE-a is preferentially spliced in nervous system tissue [28], we first determined whether LASE-a is present in LAR transcripts in the human nervous system. A human brain cDNA library was screened using a probe corresponding
Discussion
This study demonstrates that splicing of the LASE-a insert is conserved across human, rat and mouse LAR transcripts. The predicted amino acid sequence and its conservation across species raise important novel possibilities regarding potential mechanisms for the regulation of LAR function. The introduction of Ser residues at the first and tenth position of the LASE-a insert result in the presence of Ser moieties in one-to-one alignment with Ser-195 and Ser-204 of PTPα. Ser-204 of PTPα is
Acknowledgements
Supported by NIA R01 AG09873 (F.L.), the Veterans Administration (F.L.), and the Finnish Neurology Foundation and the Finnish Academy of Sciences (J.H.). We thank Dr. Frank Sharp in the UCSF/VAMC Department of Neurology for reviewing histological findings.
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