Distinct functions of the two protein tyrosine phosphatase domains of LAR (leukocyte common antigen-related) on tyrosine dephosphorylation of insulin receptor

Most receptor-like, transmembrane protein tyrosine phosphatases (PTPases), such as CD45 and the leukocyte common antigen-related (LAR) molecule, have two tandemly repeated PTPase domains in the cytoplasmic segment. The role of each PTPase domain in mediating PTPase activity remains unclear; however, it has been proposed that PTPase activity is associated with only the first of the two domains, PTPase domain 1, and the membrane-distal PTPase domain 2, which has no catalytic activity, would regulate substrate specificity. In this paper, we examine the function of each PTPase domain of LAR in vivo using a potential physiological substrate, namely insulin receptor, and LAR mutant proteins in which the conserved cysteine residue was changed to a serine residue in the active site of either or both PTPase domains. LAR associated with and preferentially dephosphorylated the insulin receptor that was tyrosine phosphorylated by insulin stimulation. Its association was mediated by PTPase domain 2, because the mutation of Cys-1813 to Ser in domain 2 resulted in weakening of the association. The Cys-1522 to Ser mutant protein, which is defective in the LAR PTPase domain 1 catalytic site, was tightly associated with tyrosine-phosphorylated insulin receptor, but failed to dephosphorylate it, indicating that LAR PTPase domain 1 is critical for dephosphorylation of tyrosine-phosphorylated insulin receptor. This hypothesis was further confirmed by using LAR mutants in which either PTPase domain 1 or domain 2 was deleted. Moreover, the association of the extracellular domains of both LAR and insulin receptor was supported by using the LAR mutant protein without the two PTPase domains. LAR was phosphorylated by insulin receptor tyrosine kinase and autodephosphorylated by the catalytic activity of the PTPase domain 1. These results indicate that each domain of LAR plays distinct functional roles through phosphorylation and dephosphorylation in vivo.     

Isolation and characterization of temperature-sensitive and thermostable mutants of the human receptor-like protein tyrosine phosphatase LAR.

Human LAR is a transmembrane receptor-like protein whose cytoplasmic region contains two tandemly duplicated domains homologous to protein tyrosine phosphatases (PTPases). Whereas the membrane-proximal domain I has enzymatic activity, the membrane-distal domain II has no apparent catalytic activity but seems to have a regulatory function. In order to study structure-function relationships of the LAR PTPase, LAR domain I was expressed in Escherichia coli, and mutants that have reduced catalytic activity or reduced thermostability were isolated and characterized. We isolated 18 unique hydroxylamine-induced missense mutations in the LAR domain I segment, of which three were temperature-sensitive. Five additional temperature-sensitive mutations were isolated using N-methyl-N'-nitro-N-nitrosoguanidine. All eight temperature-sensitive mutations are confined within a short segment of the LAR domain I sequence between amino acid positions 1329 and 1407. To examine whether this region is particularly prone to temperature-sensitive mutations, tyrosine at amino acid position 1379 was changed to a phenylalanine by oligonucleotide-directed mutagenesis. This mutant, Y1379-F, was indeed temperature-sensitive. We also isolated a revertant of a temperature-sensitive mutant. The revertant contained a second-site mutation (C1446-Y) that suppresses several temperature-sensitive mutations and also enhances the folding of LAR protein produced in E. coli.     

Cellular Redistribution of Protein Tyrosine Phosphatases LAR and PTPσ 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 PTPσ. PTPσ 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 PTPσ 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 PKCα. Induced proteolysis resulted in shedding of the extracellular domains of both PTPases. This was in agreement with the identification of a specific PTPσ 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 β-catenin with the intracellular domain of LAR in vitro. Taken together, these data suggested an involvement of LAR and PTPσ 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 PTPσ 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. A key element in the regulation of cell–cell and cell– matrix contacts is the tyrosine phosphorylation of proteins that are localized in focal adhesions and at intercellular junctions (for reviews see Kemler, 1993; Clark and Brugge, 1995). While much is known about the protein tyrosine kinases involved in the phosphorylation of cell adhesion components, very little information exists about the identity of protein tyrosine phosphatases (PTPases),¹ which are responsible for the dephosphorylation and thereby regulation of these structural complexes. Probable candidates are those receptor-like PTPases that contain cell adhesion molecule-like extracellular domains and could therefore regulate their intrinsic phosphatase activity in response to cell contact. Recent reports suggest that some PTPases do, in fact, possess properties that resemble those of classical cell adhesion molecules (for review see Brady-Kalnay and Tonks, 1995). A direct involvement in cell–cell contact has so far been demonstrated for PTPμ (Brady-Kalnay et al., 1993; Gebbink et al., 1993) and PTPκ (Sap et al., 1994), for which a homophilic interaction between their extracellular domains was found. The localization of PTPμ (Brady-Kalnay et al., 1995; Gebbink et al., 1995), PTPκ (Fuchs et al., 1996), and PCP-2 (Wang et al., 1996) was restricted to sites of cell–cell contact and surface expression of PTPμ (Gebbink et al., 1995), and PTPκ (Fuchs et al., 1996) was increased in a cell density-dependent manner. Moreover, a direct association of PTPκ (Fuchs et al., 1996) and PTPμ (Brady-Kalnay et al., 1995) with members of the cadherin/catenin family suggests that proteins of the cell adhesion complex represent physiological substrates for these PTPases. A possible regulatory function in cell–matrix adhesion has been proposed for LAR, another receptor-like PTPase, which associated with focal cell–substratum adhesions via the newly identified LAR interacting protein 1, LIP-1 (Serra-Pages et al., 1995).PTPμ (Gebbink et al., 1991), PTPκ (Jiang et al., 1993; Fuchs et al., 1996), PTPδ (Krueger et al., 1990; Mizuno et al., 1993, Pulido et al., 1995a), PCP-2 (Wang et al., 1996), and LAR (Streuli et al., 1988, Pot et al., 1991) are members of the so-called type II receptor-like PTPases. The extracellular domains of these PTPases contain a variable number of Ig-like and fibronectin type III-like (FNIII) domains (for review see Charbonneau and Tonks, 1992). With the exception of PCP-2 (Wang et al., 1996), these PTPases also share characteristics in their biosynthesis. They all underwent proteolytic processing by a furin-like endoprotease and were expressed at the cell surface in two subunits which were not covalently linked (Streuli et al., 1992; Yu et al., 1992; Jiang et al., 1993; Brady-Kalnay and Tonks, 1994; Gebbink et al., 1995; Pulido et al., 1995a; Fuchs et al., 1996). It was shown for LAR that the E subunit, which contains the cell adhesion molecule-like extracellular domain, was shed from the cell surface when cells were grown to a high density (Streuli et al., 1992). This shedding of the E subunit of LAR was the result of an additional proteolytic processing step that could also be induced by treatment of the cells with the phorbol ester TPA (Serra-Pages et al., 1995). An accumulation of E subunits in the supernatant of cells was also observed for PTPμ (Gebbink et al., 1995) and PTPδ (Pulido et al., 1995a), and this suggests a common mechanism in the regulation of type II PTPases. However, the effect of proteolytic processing on either the catalytic activity, the substrate specificity, or the cellular localization of these PTPases has not yet been determined. We report here that PTPσ, a recently identified new member of the family of receptor-like type II PTPases (Pan et al., 1993; Walton et al., 1993; Yan et al., 1993; Ogata et al., 1994; Zhang et al., 1994), underwent biosynthesis and proteolytic processing in a manner that resembled that of the most closely related PTPase LAR. Moreover, further proteolytic processing of PTPσ as well as of LAR could be induced by treatment of the cells with TPA or the calcium ionophore A23187. Transient expression studies indicated that TPA-induced processing of LAR, but not PTPσ, was dependent on the coexpression of PKCα. Inducible processing of both PTPases took place in the extracellular segment of the P subunit in a juxtamembrane position and led to the shedding of the E subunit. Both LAR and PTPσ were predominantly localized in regions of cell–cell contact and accumulated in dot-like structures that could be identified as adherens junctions and desmosomes by colocalization with plakoglobin (Cowin et al., 1986). Moreover, plakoglobin and β-catenin, another component of E-cadherin–containing cell adhesion complexes in adherens junctions, associated directly with the intracellular domain of LAR in vitro. The inducible shedding of the E subunit of LAR and PTPσ was followed by a redistribution of the PTPases within the cell membrane and by an internalization of the cleaved P subunits. It therefore represents a mechanism through which the phosphatase activity of these PTPases could be regulated in response to cell–cell contact. The cell adhesion molecule-like character of LAR and PTPσ was further supported by the fact that the internalization of LAR and PTPσ occurred independently of the proteolytic processing if cells were grown in calcium-depleted growth medium. The analogies in specific localization as well as internalization behavior of PTPσ and LAR, with molecules of the cadherin/catenin family, strongly suggest a direct involvement of PTPσ and LAR in the formation or maintenance of intercellular contacts.     

Reduced proteolysis of rabbit growth hormone (GH) receptor substituted with mouse GH receptor cleavage site

GH binding protein (GHBP) is a circulating form of the GH receptor (GHR) extracellular domain, which derives by alternative splicing of the GHR gene (in mice and rats) and by metalloprotease-mediated GHR proteolysis with shedding of the extracellular domain as GHBP (in rabbits, humans, and other species). Inducible proteolysis of either mouse (m) or rabbit (rb) GHR is detected in cell culture in response to phorbol ester and other stimuli, yielding a cell-associated GHR remnant (comprised of the cytoplasmic and transmembrane domains and a small portion of the proximal extracellular domain) and down-regulating GH signaling. In this report, we map the mGHR cleavage site by adenoviral overexpression of a membrane-anchored mGHR mutant lacking its cytoplasmic domain and purification and N-terminal sequencing of the phorbol 12-myristate 13-acetate-induced remnant protein. The sequence obtained was LEACEEDI, which matches the mGHR extracellular domain stem region sequence L265EACEEDI272, indicating that mGHR cleavage occurs in the extracellular domain nine residues outside of the transmembrane domain, in the same region (but at different residues) as the rbGHR cleavage site we recently mapped. We studied the effects on receptor proteolysis and GHBP shedding of replacing rbGHR cleavage site residues with those corresponding to the mGHR cleavage site. We analyzed five separate rodentized rbGHR mutants incorporating mGHR amino acids either at or surrounding the cleavage site. Each mutant was normally processed, displayed at the cell surface, and responded to GH stimulation by undergoing tyrosine phosphorylation. Only the mutants replaced with mGHR cleavage site residues, rather than surrounding residues, exhibited deficient inducible proteolysis and GHBP shedding. These findings suggested that the GHR cleavage sites in the two species differ in their susceptibility to cleavage. This difference may underlie interspecies variation in utilization of proteolysis to generate GHBP.     

Structural diversity and evolution of human receptor-like protein tyrosine phosphatases.

Protein tyrosine phosphatases (PTPases), together with protein tyrosine kinases, regulate the tyrosine phosphorylation that controls cell activities and proliferation. Previously, it has been recognized that both cytosolic PTPases and membrane associated, receptor-like PTPases exist. In order to examine the structural diversity of receptor-like PTPases, we isolated human cDNA clones that cross-hybridized to a Drosophila PTPase cDNA clone, DPTP12, under non-stringent hybridization conditions. The cDNA clones thus isolated included LCA and six other novel receptor-like PTPases, named HPTP alpha, beta, gamma, delta, epsilon, and zeta. The cytoplasmic regions of HPTP alpha and epsilon are highly homologous, and are composed of two tandemly duplicated PTPase-like domains. The extracellular regions of HPTP alpha and epsilon are, respectively, 123 amino acids and 27 amino acids, and do not have obvious similarity to any known protein. The cytoplasmic region of HPTP beta contains only one PTPase domain. The extracellular region of HPTP beta, which is 1599 amino acids, is composed of 16 fibronectin type-III repeats. HPTP delta is very similar to leukocyte common antigen related molecule (LAR), in both the extracellular and cytoplasmic regions. Partial sequences of HPTP gamma and zeta indicate that they are highly homologous and contain two PTPase-like domains. The PTPase-like domains of HPTP alpha, beta and delta expressed in Escherichia coli had tyrosine phosphatase activities.     

Purification and characterization of a soluble catalytic fragment of the human transmembrane leukocyte antigen related (LAR) protein tyrosine phosphatase from an Escherichia coli expression system.

A 350 amino acid soluble fragment of the intracellular catalytic domain of the human transmembrane leukocyte antigen related (LAR) protein tyrosine phosphatase has been purified 17-fold to greater than 90% purity from an Escherichia coli expression vector in quantities sufficient for kinetic and structural characterization. To assess substrate specificity, phosphotyrosine peptides corresponding to autophosphorylation sites of the two major classes of tyrosine kinases have been synthesized. Thus 6-12-residue phosphotyrosine peptides of the insulin receptor and epidermal growth factor receptor kinase domains and of the autophosphorylation and C-terminal regulatory sites of p60src and p56lck have been analyzed for kcat and KM by using a nonradioactive chromogenic assay for Pi release. The catalytic domain of LAR PTPase shows kcat values of 20-70 s-1 for phosphotyrosine peptides and affinities that vary 150-fold from 27 microM to 4.1 mM.     

Insulin receptor protein-tyrosine phosphatases. Leukocyte common antigen-related phosphatase rapidly deactivates the insulin receptor kinase by preferential dephosphorylation of the receptor regulatory domain.

A number of protein-tyrosine phosphatase(s) (PTPases) have been shown to dephosphorylate the insulin receptor in vitro; however, it is not known whether any individual PTPase has specificity for certain phosphotyrosine residues of the receptor that regulate its intrinsic tyrosine kinase activity. We evaluated the deactivation of the insulin receptor kinase by three candidate enzymes that are expressed in insulin-sensitive rat tissues, including the receptor-like PTPases LAR and LRP, and the intracellular enzyme, PTPase1B. Purified insulin receptors were activated by insulin and receptor dephosphorylation, and kinase activity was quantitated after incubation with recombinant PTPases from an Escherichia coli expression system. When related to the level of overall receptor dephosphorylation, LAR deactivated the receptor kinase 3.1 and 2.1 times more rapidly than either PTPase1B or LRP, respectively (p less than 0.03). To assess whether these effects were associated with preferential dephosphorylation of the regulatory (Tyr-1150) domain of the receptor beta-subunit, we performed tryptic mapping of the insulin receptor beta-subunit after dephosphorylation by PTPases. Relative to the rate of initial loss of 32P from receptor C-terminal sites, LAR dephosphorylated the Tris-phosphorylated Tyr-1150 domain 3.5 and 3.7 times more rapidly than either PTPase1B or LRP, respectively (p less than 0.01). The accelerated deactivation of the insulin receptor kinase by LAR and its relative preference for regulatory phosphotyrosine residues further support a potential role for this transmembrane PTPase in the physiological regulation of insulin receptors in intact cells.     

Regulation of Lck and Fyn tyrosine kinase activities by transmembrane protein tyrosine phosphatase leukocyte common antigen-related molecule

Leukocyte common antigen-related molecule (LAR) is a receptor-like protein tyrosine phosphatase (PTPase) with two PTPase domains. In the present study, we detected the expression of LAR in the brain, kidney, and thymus of mice using anti-LAR PTPase domain subunit monoclonal antibody (mAb) YU1. In the thymus, LAR was expressed on CD4(-)CD8(-) and CD4(-)CD8(low) thymocytes. The development of thymocytes in CD45 knockout mice is blocked partially in the maturation of CD4(-)CD8(-) to CD4(+)CD8(+). We postulated that LAR regulates Lck and Fyn in the immature thymocytes. Transfection of wild-type LAR activated extracellular signal-regulated kinase signal transduction pathway in CD45-deficient Jurkat cells stimulated with anti-CD3 mAb. LAR mutants, with Cys to Ser mutation in the catalytic center of PTPase D1, bound to tyrosine-phosphorylated Lck and Fyn, and LAR PTPase domain 2 was tyrosine phosphorylated by Fyn tyrosine kinase. The phosphorylated LAR was associated with Fyn Src homology 2 domain. Moreover, LAR dephosphorylated phosphorylated tyrosine residues in both the COOH terminus and kinase domain of Fyn in vitro. Our results indicate that Lck and Fyn would be substrates of LAR in immature thymocytes and that each LAR PTPase domain plays distinct functional roles in phosphorylation and dephosphorylation.     

Nuclear translocation of intracellular domain of Protogenin by proteolytic cleavage

Protogenin (PRTG) is a transmembrane protein of immunoglobulin superfamily, which has multiple roles in embryogenesis as a receptor or an adhesion molecule. In this study, we present sequential proteolytic cleavage of PRTG. The cleavage first occurs at the extracellular domain, then at the interface of the transmembrane and the intracellular domain by γ-secretase, which results in the release of the intracellular domain of PRTG (PRTG-ICD). PRTG-ICD contains putative nuclear localization signal (NLS) at its N-terminal, and translocates to the nucleus in cultured cells and in the neuroepithelial cells of chick embryos. We propose that the PRTG-ICD is cleaved by γ-secretase and translocates to the nucleus, which is potentially implicated in signaling for neural differentiation and in cell adhesion mediated by PRTG.     

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.     

Biochemical comparisons of the normal and oncogenic forms of insect cell-expressed neu tyrosine kinases.

The rat neu oncogene product is a member of the epidermal growth factor (EGF) receptor subgroup of the superfamily of growth factor receptor tyrosine kinases. The oncogenic activation of the neu protein occurs by a point mutation within its transmembrane region which results in an increase in its tyrosine kinase activity. Using three different forms of neu expressed in insect cells via baculovirus infection, we have examined the biochemical differences between the normal and transforming forms of neu and investigated the role of the transmembrane domain in its tyrosine kinase activity. One form of neu which was expressed in insect cells consisted of the complete tyrosine kinase domain but lacked the extracellular and transmembrane regions (designated NTK). The other two forms consisted of the tyrosine kinase domain, the transmembrane domain, and 40 amino acids of the extracellular domain. One of these transmembrane forms of neu contained the normal valine residue at position 664 within the transmembrane region (MS-N), while the other contained the oncogenic glutamic acid residue at this position (MS-T). Direct comparisons of NTK, MS-N, and MS-T have shown that the NTK protein is capable of the highest extents of both autophosphorylation activity and the tyrosine phosphorylation of exogenous substrate, suggesting that the presence of the transmembrane region of neu suppresses the tyrosine kinase activity of this receptor. In addition, we have found that the oncogenic point mutation within the transmembrane region stimulates the tyrosine kinase activity of the neu protein by allowing it to more effectively utilize Mg2+. Overall, the results of these studies suggest that the valine to glutamic acid substitution at position 664 may at least partially relieve a negative constraint imparted by the membrane-spanning domain on the tyrosine kinase activity of neu and enables a more effective use of Mg2+ in the catalysis of tyrosine phosphorylation of exogenous substrates.     

Active site labeling of a receptor-like protein tyrosine phosphatase.

The inactivation of the cytoplasmic domain of rat LAR, a receptor-like protein tyrosine phosphatase (PTPase), by iodoacetate and not by iodoacetamide suggested that iodoacetate interacts in a highly selective manner with the enzyme. The data indicate that iodoacetate binds at the active site of the enzyme with a stoichiometry of 0.8 mol of iodoacetate bound per mol of rat LAR. A single [14C]iodoacetate-labeled peptide was isolated following endoproteinase Lys-C digestion of the radiolabeled PTPase. Sequence analysis of the active site labeled peptide demonstrates that Cys-1522 contains the radiolabel. This residue has been shown by site-directed mutagenesis to be essential for rat LAR activity (Pot, D. A., Woodford, T. A., Remboutsika, E., Haun, R. S., and Dixon, J. E. (1991) J. Biol. Chem. 266, 19688-19696). Iodoacetate reacts only with the first domain of this double domain PTPase. These results, for the first time, directly identify the highly reactive cysteine residue at the active site of a PTPase and highlight the ability of this residue to participate as a nucleophile in the hydrolysis of phosphate from tyrosine.     

The catalytic activity of the CD45 membrane-proximal phosphatase domain is required for TCR signaling and regulation.

Cell surface expression of CD45, a receptor-like protein tyrosine phosphatase (PTPase), is required for T cell antigen receptor (TCR)-mediated signal transduction. Like the majority of transmembrane PTPases, CD45 contains two cytoplasmic phosphatase domains, whose relative in vivo function is not known. Site-directed mutagenesis of the individual catalytic residues of the two CD45 phosphatase domains indicates that the catalytic activity of the membrane-proximal domain is both necessary and sufficient for restoration of TCR signal transduction in a CD45-deficient cell. The putative catalytic activity of the distal phosphatase domain is not required for proximal TCR-mediated signaling events. Moreover, in the context of a chimeric PTPase receptor, the putative catalytic activity of the distal phosphatase domain is not required for ligand-induced negative regulation of PTPase function. We also demonstrate that the phosphorylation of the C-terminal tyrosine of Lck, a site of negative regulation, is reduced only when CD45 mutants with demonstrable in vitro phosphatase activity are introduced into the CD45-deficient cells. These results demonstrate that the phosphatase activity of CD45 is critical for TCR signaling, and for regulating the levels of C-terminal phosphorylated Lck molecules.     

Cloning and characterization of R-PTP-kappa, a new member of the receptor protein tyrosine phosphatase family with a proteolytically cleaved cellular adhesion molecule-like extracellular region.

We describe a new member of the receptor protein tyrosine phosphatase family, R-PTP-kappa, cDNA cloning predicts that R-PTP-kappa is synthesized from a precursor protein of 1,457 amino acids. Its intracellular domain displays the classical tandemly repeated protein tyrosine phosphatase homology, separated from the transmembrane segment by an uncharacteristically large juxta-membrane region. The extracellular domain of the R-PTP-kappa precursor protein contains an immunoglobulin-like domain and four fibronectin type III-like repeats, preceded by a signal peptide and a region of about 150 amino acids with similarity to the Xenopus A5 antigen, a putative neuronal recognition molecule (S. Takagi, T. Hsrata, K. Agata, M. Mochii, G. Eguchi, and H. Fujisawa, Neuron 7:295-307, 1991). Antibodies directed against the intra- and extracellular domains reveal that the R-PTP-kappa precursor protein undergoes proteolytic processing, following which both cleavage products remain associated. By site-directed mutagenesis, the likely cleavage site was shown to be a consensus sequence for cleavage by the processing endopeptidase furin, located in the fourth fibronectin type III-like repeat. In situ hybridization analysis indicates that expression of R-PTP-kappa in the central nervous system is developmentally regulated, with highest expression seen in actively developing areas and, in the adult, in areas capable of developmental plasticity such as the hippocampal formation and cerebral cortex. The mouse R-PTP-kappa gene maps to chromosome 10, at approximately 21 centimorgans from the centromere.     

Receptor tyrosine phosphatase R-PTP-kappa mediates homophilic binding.

Receptor tyrosine phosphatases (R-PTPases) feature PTPase domains in the context of a receptor-like transmembrane topology. The R-PTPase R-PTP-kappa displays an extracellular domain composed of fibronectin type III motifs, a single immunoglobulin domain, as well as a recently defined MAM domain (Y.-P. Jiang, H. Wang, P. D'Eustachio, J.M. Musacchio, J. Schlessinger, and J. Sap, Mol. Cell. Biol. 13:2942-2951, 1993). We report here that R-PTP-kappa can mediate homophilic intercellular interaction. Inducible expression of the R-PTP-kappa protein in heterologous cells results in formation of stable cellular aggregates strictly consisting of R-PTP-kappa-expressing cells. Moreover, the purified extracellular domain of R-PTP-kappa functions as a substrate for adhesion by cells expressing R-PTP-kappa and induces aggregation of coated synthetic beads. R-PTP-kappa-mediated intercellular adhesion does not require PTPase activity or posttranslational proteolytic cleavage of the R-PTP-kappa protein and is calcium independent. The results suggest that R-PTPases may provide a link between cell-cell contact and cellular signaling events involving tyrosine phosphorylation.     

The human insulin receptor cDNA: the structural basis for hormone-activated transmembrane signalling

A cloned approximately 5 kb cDNA (human placenta) contains the coding sequences for the insulin receptor. The nucleotide sequence predicts a 1382 amino acid precursor. The alpha subunit comprises the N-terminal portion of the precursor and contains a striking cysteine-rich "cross-linking" domain. The beta-subunit (the C-terminal portion of the precursor) contains a transmembrane domain and, in the intracellular region, the elements of a tyrosine phosphokinase: an ATP-binding site and a possible tyrosine autophosphorylation site or sites. The overall structure is reminiscent of the EGF receptor; the cross-linking domain of the alpha subunit and several regions of the beta subunit exhibit sequence homology with the EGF receptor. The phosphokinase domain also exhibits homology with some oncogenic proteins that have tyrosine phosphokinase activity, in particular, a striking homology with v-ros. Southern blotting experiments suggest that the coding region spans more than 45 kb. The insulin receptor gene is located on chromosome 19.     

Identification of a functional proprotein convertase cleavage site in microfibril-associated glycoprotein 2

Microfibril-associated glycoprotein 2 (MAGP2) is a secreted protein associated with multiple cellular activities including the organization of elastic fibers in the extracellular matrix (ECM), angiogenesis, as well as regulating Notch and integrin signaling. Importantly, increases in MAGP2 positively correlate with poor prognosis for some ovarian cancers. It has been assumed that full-length MAGP2 is responsible for all reported effects; however, here we show MAGP2 is a substrate for the proprotein convertase (PC) family of endoproteases. Proteolytic processing of MAGP2 by PC cleavage could serve to regulate secretion and thus, activity and function as reported for other extracellular and cell-surface proteins. In support of this idea, MAGP2 contains an evolutionarily conserved PC consensus cleavage site, and amino acid sequencing of a newly identified MAGP2 C-terminal cleavage product confirmed functional PC cleavage. Additionally, mutagenesis of the MAGP2 PC consensus cleavage site or treatment with PC inhibitors prevented MAGP2 proteolytic processing. Finally, both cleaved and uncleaved MAGP2 were detected extracellularly and MAGP2 secretion appeared independent of PC cleavage, suggesting that PC processing occurs mainly outside the cell. Our characterization of alternative forms of MAGP2 present in the extracellular space not only enhances diversity of this ECM protein but also provides a previously unrecognized molecular mechanism for regulation of MAGP2 biological activity.     

Post-translational proteolytic processing of the calcium-independent receptor of alpha-latrotoxin (CIRL), a natural chimera of the cell adhesion protein and the G protein-coupled receptor. Role of the G protein-coupled receptor proteolysis site (GPS) motif

The calcium-independent receptor of alpha-latrotoxin (CIRL), a neuronal cell surface receptor implicated in the regulation of exocytosis, is a natural chimera of the cell adhesion protein and the G protein-coupled receptor (GPCR). In contrast with canonic GPCRs, CIRL consists of two heterologous non-covalently bound subunits, p120 and p85, due to endogenous proteolytic processing of the receptor precursor in the endoplasmic reticulum. Extracellularly oriented p120 contains hydrophilic cell adhesion domains, whereas p85 resembles a generic GPCR. We determined that the site of the CIRL cleavage is located within a juxtamembrane Cys- and Trp-rich domain of the N-terminal extracellular region of CIRL. Mutations in this domain make CIRL resistant to the cleavage and impair its trafficking. Therefore, we have named it GPS for G protein-coupled receptor proteolysis site. The GPS motif is found in homologous adhesion GPCRs and thus defines a novel receptor family. We postulate that the proteolytic processing and two-subunit structure is a common characteristic feature in the family of GPS-containing adhesion GPCRs.     

Effect of tumor necrosis factor-α on the phosphorylation of tyrosine kinase receptors is associated with dynamic alterations in specific protein-tyrosine phosphatases

Tumor necrosis factor-α (TNF-α) can modulate the signalling capacity of tyrosine kinase receptors; in particular, TNF-α has been shown to mediate the insulin resistance associated with animal models of obesity and noninsulin-dependent diabetes mellitus. In order to determine whether the effects of TNF-α might involve alterations in the expression of specific protein-tyrosine phosphatases (PTPases) that have been implicated in the regulation of growth factor receptor signalling, KRC-7 rat hepatoma cells were treated with TNF-α, and changes in overall tissue PTPase activity and the abundance of three major hepatic PTPases (LAR, PTP1B, and SH-PTP2) were measured in addition to effects of TNF-α on ligand-stimulated autophosphorylation of insulin and epidermal growth factor (EGF) receptors and insulin-stimulated insulin receptor substrate-1 (IRS-1) phosphorylation. TNF-α caused a dose-dependent decrease in insulin-stimulated IRS-1 phosphorylation and EGF-stimulated receptor autophosphorylation to 47–50% of control. Overall PTPase activity in the cytosol fraction did not change with TNF-α treatment, and PTPase activity in the particulate fraction was decreased by 55–66%, demonstrating that increases in total cellular PTPase activity did not account for the observed alterations in receptor signalling. However, immunoblot analysis showed that TNF-α treatment resulted in a 2.5-fold increase in the abundance of SH-PTP2, a 49% decrease in the transmembrane PTPase LAR, and no evident change in the expression of PTP1B. These data suggest that at least part of the TNF-α effect on pathways of reversible tyrosine phosphorylation may be exerted through the dynamic modulation of the expression of specific PTPases. Since SH-PTP2 has been shown to interact directly with both the EGF receptor and IRS-1, increased abundance of this PTPase may mediate the TNF-α effect to inhibit signalling through these proteins. Furthermore, decreased abundance of the LAR PTPase, which has been implicated in the regulation of insulin receptor phosphorylation, may account for the less marked effect of TNF-α on the autophosphorylation state of the insulin receptor while postreceptor actions of insulin are inhibited. J. Cell. Biochem. 64:117–127. © 1997 Wiley-Liss, Inc.