SUMMARY The LAR receptor-like protein tyrosine phosphatase is comprised of two intracellular tyrosine phosphate domains and a cell adhesion molecular-like extracellular region containing three immunoglobulin-like domains in combination with eight fibronectin type-III-like repeats. This architecture suggests that LAR may function in cellular signalling by the regulation of tyrosine phosphorylation through cell-cell or cell-matrix interactions. We used gene targeting in mouse embryonic stem cells to generate mice lacking sequences encoding both LAR phosphatase domains. Northern blot analysis of various tissues revealed the presence of a truncated LAR mRNA lacking the cytoplasmic tyrosine phosphatase domains and indicated that this LAR mutation is not accompanied by obvious changes in the expression levels of one of the LAR-like receptor tyrosine phosphatase PTPd or PTPs. LAR÷ mice develop and grow normally and display no appreciable histological tissue abnormalities. However, upon breeding we observed an abnormal neonatal death rate for pups from LAR÷ females. Mammary glands of LAR÷ females were incapable of delivering milk due to an impaired terminal differentiation of alveoli at late pregnancy. As a result, the glands failed to switch to a lactational state and showed a rapid involution postpartum. In wild-type mice, LAR expression is regulated during pregnancy reaching maximum levels around day 16 of gestation. Taken together, these findings suggest an important role for LAR mediated signalling in mammary gland development and function.
Citations Roel Schaapveld, Jan Schepens, Joline Attema, Frank Oerlemans, Jack Fransen, Gertraud Robinson, Lothar Hennighausen, Michel Streuli, Be Wieringa and Wiljan Hendriks (1997) Developmental Biology, in press.
Background Protein tyrosine phosphorylation, a key event in signal transduction, is a post-translational modification controlled by the agonistic or antagonistic action of protein tyrosine kinases (PTKs) and protein tyrosine phosphatase (PTPases). The PTPases comprise a diverse family of receptor-like and cytoplasmic-type enzymes including multiple isoforms resulting from alternative splicing and post-translational modifications.
Receptor-like PTPases (RPTPases) contain on or two homologous PTPase domains, a transmembrane segment, and diverse combinations of domains with possible ligand-binding properties in the extracellular part. For example, the closely related RPTPases RPTPm and PTPk mediate cell-cell interactions in a homophilic, but heterophilic manner. In RPTPß the extracellular moiety binds to a non-related cell-surface protein, the neuronal recognition molecule contactin. It has not been clarified if and how such interactions modulate tyrosine phosphatase activity largely because the intracellular substrates for almost all RPTPases remain to be identified. As a consequence, it is not known whether individual members in the large RPTPase family play a role early in signal transduction or later in adaption or response cessation. Only for the RPTPase CD45, which is expressed exclusively on nucleated cells of haematopoietic origin, an important function in the regulation of activation of haematopoietic cells through antigen receptors has been demonstrated. In addition, studies on mice lacking exon 6 (one of the alternative spliced exon encoding extracellular sequences) provided evidence for a function of CD45 in development of the immune system.
Although LAR was among the first PTPases to be identified and has been studied extensively at the cellular level, its physiological function remains unclear. LAR is composed of two cytoplasmic phosphatase domains in tandem, a transmembrane segment, and an extracellular part which shares homology to immunoglobin (Ig)-like and fibronectin type III (Fn-III) domains, commonly found in cell adhesion molecules. This architecture suggests a physiological function for LAR in cellular signalling by tyrosine dephosphorylation as a response to cell-cell or cell-matrix interactions. Recently, an intracellular LAR-interacting protein (LIP.1) was identified, which binds to the second PTPase domain and colocalizes with LAR to the ends of focal adhesions most proximal to the cell nucleus, suggesting a role for LAR in disassembly of focal adhesions.
LAR has a broad tissue distribution, and is expressed on the cell surface as a complex of two noncovalently associated subunits derived from a pro-protein. A ~200 kDa precursor is intracellularly processed at a paired basic amino acid site by furin or a furin-like endoprotease generating 150 kDa and 85 kDa fragments. The N-linked glycosylated 150 kDa fragment represents the amino terminus of the protein, and is shed during growth. Whether this shedding, which is PMA-inducible has an effect on the intracellular phosphatase activity remains to be established. The 85 kDa fragment contains short ectodomain, the transmembrane segment and the PTPase domains of ~280 amino acid residues each. The highly homologous LAR-like RPTPases PTPd and PTPs show a similar intracellular processing and cell surface expression. Shedding of the cell adhesion molecule-like ectodomain has also been reported for PTPd and may be a common feature of this type of RPTPases.
In vitro site-directed mutagenesis studies suggested that the first, membrane-proximal phosphatase domain of LAR exhibits catalytic activity whereas, the second, membrane distal phosphatase domain may have only a regulatory function in modulating the substrate specificity of the first domain. However, the extracellular ligand(s) and downstream substrate(s) of LAR have not been identified. Therefore, we set out to study the consequences of the loss of LAR PTPase function in vivo by means of gene targeting using homologous recombination in mouse embryonic stem (ES) cells. Characterization of the resulting LAR÷ mice points to a role for LAR PTPase activity in mammary gland development during pregnancy.
Contributed by
Wiljan Hendriks
University of Nijmwegen
The Netherlands
tel. 31-243-614334
Lothar Hennighausen
National Institutes of Health
Bethesda, MD 20892
Phone: 301-435-8907
Fax: 301-496-0839
e-mail: mammary@nih.gov
last update: June, 1998