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.     

Expression of the receptor-linked protein tyrosine phosphatase LAR: proteolytic cleavage and shedding of the CAM-like extracellular region.

The human transmembrane molecule LAR is a protein tyrosine phosphatase (PTPase) with a cell adhesion molecule-like extracellular receptor region. The structure of LAR hinted at its involvement in the regulation of tyrosine phosphorylation through cell-cell or cell-matrix interactions. We show here that LAR is expressed on the cell surface as a complex of two non-covalently associated subunits derived from a proprotein. The LAR E-subunit contains the cell adhesion molecule-like receptor region, while the LAR P-subunit contains a short segment of the extracellular region, the transmembrane peptide and the cytoplasmic PTPase domains. Proprotein processing occurs intracellularly. Analysis of LAR mutants suggested that cleavage occurs in the LAR extracellular region at a paired basic amino acid site by a subtilisin-like endoprotease. A single amino acid substitution at this site blocked LAR proprotein cleavage. The LAR E-subunit is shed during cell growth, suggesting that LAR receptor shedding may be a mechanism for regulating PTPase function. The use of immunohistochemistry techniques on human tissues demonstrated the expression of LAR by various cell lineages, including epithelial cells, smooth muscle cells and cardiac myocytes. The LAR gene is mapped to chromosome 1, region p32-33, which contains candidate tumor suppressor genes.     

Mutational analysis of human immunodeficiency virus type 1 protease suggests functional homology with aspartic proteinases.

Processing of the retroviral gag and pol gene products is mediated by a viral protease. Bacterial expression systems have been developed which permit genetic analysis of the human immunodeficiency virus type 1 protease as measured by cleavage of the pol protein precursor. Deletion analysis of the pol reading frame locates the sequences required to encode a protein with appropriate proteolytic activity near the left end of the pol reading frame but largely outside the gag-pol overlap region, which is at the extreme left end of pol. Most missense mutations within an 11-amino-acid domain highly conserved among retroviral proteases and with sequence similarity to the active site of aspartic proteinases abolish appropriate processing, suggesting that the retrovirus proteases share a catalytic mechanism with aspartic proteinases. Substitution of the amino acids flanking the scissile bond at three of the processing sites encoded by pol demonstrates distinct sequence requirements for cleavage at these different sites. The inclusion of a charged amino acid at the processing site blocks cleavage. A subset of these substitutions also inhibits processing at the nonmutated sites.     

Molecular cloning and characterization of a protein tyrosine phosphatase enriched in testis, a putative murine homologue of human PTPMEG

Protein tyrosine phosphorylation is regulated by protein tyrosine kinase and protein tyrosine phosphatase activities. These two counteracting proteins are implicated in cell growth and transformation. Using polymerase chain reaction with degenerate primers, we have identified a novel mouse protein tyrosine phosphatase (PTP). This cDNA contains a single open reading frame of the predicted 926 amino acids. Those predicted amino acids showed significant identity with human megakaryocyte protein-tyrosine phosphatase by 91% in nucleotide sequences and 94% in amino acid sequences. We have identified that expression of this PTP is highly enriched in the testis in mouse and human and has been termed here as a 'testis-enriched phosphatase' (TEP). Northern analysis detected two mRNA species of 3.7 and 3.2kb for this PTP in mouse testis and the expression of TEP is regulated during development. The recombinant phosphatase domain possesses protein tyrosine phosphatase activity when expressed in Escherichia coli. Immunohistochemical analysis of the cellular localization of TEP on mouse testis sections showed that this PTP is specifically expressed in spermatocytes and spermatids within seminiferous tubules, suggesting an important role in spermatogenesis.     

Site-specific mutations in the COOH-terminus of placental alkaline phosphatase: a single amino acid change converts a phosphatidylinositol- glycan-anchored protein to a secreted protein

Placental alkaline phosphatase (PLAP) is anchored in the plasma membrane by a phosphatidylinositol-glycan moiety (PI-glycan). PI-glycan is added posttranslationally to the nascent peptide chain after the removal of 29 amino acids from the COOH-terminus. The contribution of selected COOH-terminal amino acids to the signal for PI-glycan addition was tested by creating a fusion protein with the COOH-terminus of PLAP and a secreted protein and by mutagenesis of specific PLAP COOH-terminal amino acids. The cDNA encoding the COOH-terminus of PLAP was fused in frame to the cDNA for human clotting Factor X and expressed in transfected COS-1 cells. Fusion proteins containing 32 amino acids of the PLAP COOH-terminus were modified by PI-glycan addition. Thus, the signal for PI-glycan modification must reside in these amino acids. Next, the region between the hydrophobic domain and the cleavage site was examined for additional determinants. Mutations of the hydrophilic residues in the spacer region demonstrated that these amino acids do not contribute to the signal for PI-glycan addition. Deletion of amino acids in the spacer region prevented the addition of PI-glycan suggesting that the length of the spacer domain or the amino acids around the cleavage site are important determinants. Finally, we demonstrated that interruption of the hydrophobic domain by a charged residue prevents PI-glycan addition and results in a protein that is secreted into the medium. The finding that a single Leu to Arg substitution in the hydrophobic domain converts a PI-glycan anchored, membrane protein to a secreted protein suggests that an essential signal for the correct sorting of PI-glycan anchored proteins versus secreted proteins resides in the hydrophobic domain. Substitution of a charged amino acid for a hydrophobic amino acid may be a mechanism for producing membrane bound and secreted forms of the same protein.     

WARP is a new member of the von Willebrand factor A-domain superfamily of extracellular matrix proteins

We report a new member of the von Willebrand factor A-domain protein superfamily, WARP (for von Willebrand factor A-domain-related protein). The full-length mouse WARP cDNA is 2.3 kb in size and predicts a protein of 415 amino acids which contains a signal sequence, a VA-like domain, two fibronectin type III-like repeats, and a short proline- and arginine-rich segment. WARP mRNA was expressed predominantly in chondrocytes and in vitro expression experiments in transfected 293 cells indicated that WARP is a secreted glycoprotein that forms disulphide-bonded oligomers. We conclude that WARP is a new member of the von Willebrand factor A-domain (VA-domain) superfamily of extracellular matrix proteins which may play a role in cartilage structure and function.     

The protein tyrosine phosphatase alpha modifies insulin secretion in INS-1E cells

Increasing evidence indicates a role of insulin signalling for insulin secretion from the pancreatic beta-cells. Therefore, regulators of insulin signalling, like protein tyrosine phosphatases, could also have an impact on insulin secretion. Here, we investigated a possible role of the negative regulator protein tyrosine phosphatase alpha (PTP alpha) for insulin secretion. RT-PCR analysis confirmed that both splice variants of the extracellular domain of PTP alpha that vary by an insert of 9 amino acids are expressed in human islets and insulinoma cells (INS-1E, RIN1046-38). Overexpression of the wild type PTP alpha splice variant containing the 9 amino acids reduced insulin secretion, as did a mutant form unable to bind Grb2 (Tyr798Phe). By contrast, overexpression of a phosphatase inactive mutant improved insulin secretion. These data reveal a functional relevance of PTP alpha for insulin secretion.     

Cleavage of p75 neurotrophin receptor by alpha-secretase and gamma-secretase requires specific receptor domains

The p75 neurotrophin receptor (p75(NTR)), a member of the tumor necrosis factor superfamily of receptors, undergoes multiple proteolytic cleavage events. These events are initiated by an alpha-secretase-mediated release of the extracellular domain followed by a gamma-secretase-mediated intramembrane cleavage. However, the specific determinants of p75(NTR) cleavage events are unknown. Many other substrates of gamma-secretase cleavage have been identified, including Notch, amyloid precursor protein, and ErbB4, indicating there is broad substrate recognition by gamma-secretase. Using a series of deletion mutations and chimeric receptors of p75(NTR) and the related Fas receptor, we have identified domains that are essential for p75(NTR) proteolysis. The initial alpha-secretase cleavage was extracellular to the transmembrane domain. Unfortunately, deletion mutants were not capable of defining the requirements of ectodomain shedding. Although this cleavage is promiscuous with respect to amino acid sequence, its position with respect to the transmembrane domain is invariant. The generation of chimeric receptors exchanging different domains of noncleavable Fas receptor with p75(NTR), however, revealed that a discrete domain above the membrane is sufficient for efficient cleavage of p75(NTR). Mass spectrometric analysis confirmed the cleavage can occur with a truncated p75(NTR) displaying only 15 extracellular amino acids in the stalk region.     

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.     

Multiple variants of receptor-type protein tyrosine phosphatase beta are expressed in the central nervous system of Xenopus

We have isolated cDNA clones encoding the Xenopus homologue of receptor-type protein tyrosine phosphatase beta (RPTPbeta), and identified 13 forms of the mRNA provably generated by alternative splicing. All the conceptual translates have a carbonic anhydrase-like domain, a fibronectin type III-like repeat and a spacer in the extracellular segment. Eleven of them (designated XRPTPbeta.1-XRPTPbeta.11) also have highly conserved two intracellular PTP domains, whereas the other two variants (sXRPTPbeta.1 and sXRPTPbeta.2) have neither transmembrane nor cytoplasmic segment. There are five peptides that can be inserted in various combinations into the spacer region. Northern and Western blot analyses show central nervous system-specific expression of the XRPTPbeta mRNAs and proteins. Chondroitinase ABC treatment of the brain and spinal cord extracts results in separation of six protein bands on the Western blot, in association with a decrease in the size of major bands, indicating that the major XRPTPbeta variants are chondroitin sulfate proteoglycans. The results of these as well as reverse-transcribed polymerase chain reaction analyses suggest that the amounts of different XRPTPbeta variants are regulated in tissue- and developmental stage-specific manners.     

MPTP delta, a putative murine homolog of HPTP delta, is expressed in specialized regions of the brain and in the B-cell lineage.

Protein tyrosine phosphatases (PTPs), together with protein tyrosine kinases (PTKs), are involved in the regulation of cell activation, growth, and differentiation. To further elucidate the fine tuning of cell growth and differentiation through tyrosine phosphorylation, we tried to isolate mouse receptor-type PTP (RPTP) cDNA clones by screening mouse brain cDNA libraries with mouse CD45 PTP domain probes under reduced-stringency conditions. Characterization of isolated cDNA clones for RPTP showed that the cytoplasmic region contains two tandem repeats of PTP domain of about 230 amino acids with intrinsic phosphatase activity. The extracellular region was composed of immunoglobulin (Ig)-like domains and fibronectin type III (FN-III)-like domains. The gene was highly homologous to human PTP delta (HPTP delta) and thus was named MPTP delta (murine counterpart of HPTP delta). The MPTP delta gene appeared to generate at least three species of mRNA, which differ in the composition of the extracellular domain: type A, one Ig-like and four FN-III-like domains; type B, one Ig-like and eight FN-III-like domains; and type C, three Ig-like and eight FN-III-like domains. Interestingly, the 5' untranslated region and the leader peptide of types A and B were completely different from those of type C. Northern (RNA) blot analysis demonstrated that brain, kidney, and heart cells express three mRNA species of about 7 kb. Antibody directed against part of the extracellular domain of type A MPTP delta recognized a 210-kDa protein in brain and kidney lysates. In situ hybridization of brain samples revealed that MPTP delta mRNA is present in the hippocampus, thalamic reticular nucleus, and piriform cortex, where some Src family PTKs have been also demonstrated to exist. Although MPTP delta mRNA was not detected in lymphoid tissues, all of the pre-B-cell lines tested and one of three B-cell lines tested expressed MPTP delta mRNA, whereas antibody-producing B-cell hybridomas and T-cell and macrophage lines did not. Finally, the MPTP delta locus was tightly linked to the brown (b) locus on mouse chromosome 4.     

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.     

The MUC1 SEA module is a self-cleaving domain

MUC1, a glycoprotein overexpressed by a variety of human adenocarcinomas, is a type I transmembrane protein (MUC1/TM) that soon after its synthesis undergoes proteolytic cleavage in its extracellular domain. This cleavage generates two subunits, alpha and beta, that specifically recognize each other and bind together in a strong noncovalent interaction. Proteolysis occurs within the SEA module, a 120-amino acid domain that is highly conserved in a number of heavily glycosylated mucin-like proteins. Post-translational cleavage of the SEA module occurs at a site similar to that in MUC1 in the glycoproteins IgHepta and MUC3. However, as in the case of other proteins containing the cleaved SEA module, the mechanism of MUC1 proteolysis has not been elucidated. Alternative splicing generates two transmembrane MUC1 isoforms, designated MUC1/Y and MUC1/X. We demonstrated here that MUC1/X, whose extracellular domain is comprised solely of the SEA module in addition to 30 MUC1 N-terminal amino acids, undergoes proteolytic cleavage at the same site as the MUC1/TM protein. In contrast, the MUC1/Y isoform, composed of an N-terminally truncated SEA module, is not cleaved. Cysteine or threonine mutations of the MUC1/X serine residue (Ser-63) immediately C-terminal to the cleavage site generated cleaved proteins, whereas mutation of the Ser-63 residue of MUC1/X to any other of 17 amino acids did not result in cleavage. In vitro incubation of highly purified precursor MUC1/X protein resulted in self-cleavage. Furthermore, addition of hydroxylamine, a strong nucleophile, markedly enhanced cleavage. Both these features are signature characteristics of self-cleaving proteins, and we concluded that MUC1 undergoes autoproteolysis mediated by an N --> O-acyl rearrangement at the cleavage site followed by hydrolytic resolution of the unstable ester and concomitant cleavage. It is likely that all cleaved SEA module-containing proteins follow a similar route.     

The primary structure of NG2, a novel membrane-spanning proteoglycan

The complete primary structure of the core protein of rat NG2, a large, chondroitin sulfate proteoglycan expressed on O2A progenitor cells, has been determined from cDNA clones. These cDNAs hybridize to an mRNA species of 8.9 kbp from rat neural cell lines. The total contiguous cDNA spans 8,071 nucleotides and contains an open reading frame for 2,325 amino acids. The predicted protein is an integral membrane protein with a large extracellular domain (2,224 amino acids), a single transmembrane domain (25 amino acids), and a short cytoplasmic tail (76 amino acids). Based on the deduced amino acid sequence and immunochemical analysis of proteolytic fragments of NG2, the extracellular region can be divided into three domains: an amino terminal cysteine-containing domain which is stabilized by intrachain disulfide bonds, a serine-glycine-containing domain to which chondroitin sulfate chains are attached, and another cysteine-containing domain. Four internal repeats, each consisting of 200 amino acids, are found in the extracellular domain of NG2. These repeats contain a short sequence that resembles the putative Ca(++)-binding region of the cadherins. The sequence of NG2 does not show significant homology with any other known proteins, suggesting that NG2 is a novel species of integral membrane proteoglycan.     

Formation of the flavivirus envelope: role of the viral NS2B-NS3 protease.

One of the late processing events in the flavivirus replication cycle involves cleavage of the intracellular form of the flavivirus capsid protein (Cint) to the mature virion form (Cvir) lacking the carboxy-terminal stretch of hydrophobic amino acids which serves as a signal peptide for the downstream prM protein. This cleavage event was hypothesized to be effected by a viral protease and to be associated with virion formation. We have proposed a model of flavivirus virion formation in which processing of the C-prM precursor at the upstream signalase site is upregulated by interaction of the NS2B part of the protease with the prM signal peptide or with an adjacent carboxy-terminal region of the capsid protein in the precursor, and processing of Cint by the NS2B-NS3 protease follows the signalase cleavage. Recently, an alternative hypothesis was proposed which suggests a reverse order of these two cleavage events, namely, that cleavage of the C-prM precursor by the NS2B-NS3 protease at the Cint-->Cvir dibasic cleavage site is a prerequisite for the subsequent signalase cleavage of the prM signal peptide. To distinguish between these alternative models, we prepared a series of expression cassettes carrying mutations at the Cint-->Cvir dibasic cleavage site and investigated the effects of these mutations on signalase processing of C-prM and on formation and secretion of prM-E heterodimers. For certain mutated C-prM precursors, namely, for those with Lys-->Gly disruption of the dibasic site, efficient formation of prM was observed upon expression from larger cassettes encoding the viral protease, despite the absence of processing at the Cint-->Cvir cleavage site. Surprisingly, formation and secretion of prM-E heterodimers accompanied by late cleavage of prM was also observed for these cassettes, with an efficiency comparable to that of the wild-type expression cassette. These observations contradict the model in which cleavage of the C-prM precursor at the Cint-->Cvir dibasic site is a prerequisite for signalase cleavage.