Donor T-cell chimerism and early post-transplant cytomegalovirus viremia in patients treated with myeloablative allogeneic hematopoietic stem cell transplant.

BACKGROUND: Cytomegalovirus (CMV) is a common infection after myeloablative allogeneic hematopoietic stem cell transplant (M-alloHSCT). Achievement of complete donor T-cell chimerism (CDC-T) post transplant is a measure of immune reconstitution. We investigated the association between CDC-T post M-alloHSCT and the incidence of CMV viremia. METHODS: We retrospectively reviewed all CMV and chimerism results of 47 patients for the first 6 months post M-alloHSCT. CDC-T was analyzed as a time-varying covariate for association with post M-alloHSCT CMV viremia. RESULTS: CMV viremia occurred in 15 (32%) and CDC-T was achieved in 38 (81%) recipients within the first 6 months post M-alloHSCT. On univariable analysis, increased CMV viremia was seen among patients with CDC-T (hazard ratio 2.81 [P = 0.07, 95% confidence interval = 0.93-8.52]). A 30-day landmark analysis showed that the incidence of CMV viremia at 6 months (regardless of recipient CMV serostatus) was 50% among those who had achieved CDC-T by day 30, and 23% among those who had not (P = 0.06). CONCLUSION: We conclude that shorter time to CDC-T may be associated with higher risk of CMV viremia. If confirmed in a larger cohort, this might be a marker for risk stratification in the management of CMV in this population.

Effect of substrate size on immunoinhibition of amylase activity.

Immunoinhibition assays are hypothesized to work by antibodies blocking substrate access to enzyme active sites. To test this hypothesis, the inhibition of amylase isoenzymes by monoclonal and polyclonal antisera was assessed using substrates of varying sizes: chromogenic sustrates 3, 5, or 7 glucose units in length, novel synthetic macromolecular substrates, and starch. The synthetic macromolecular substrates consisted of small oligosaccharide substrates linked to an inert polymer that conferred a large size to substrate molecules as determined by gel filtration chromatography. When substrate size increased, amylase activity could be inhibited equivalently by antibody concentrations that are 10-fold lower. Progressively less polyclonal serum was required to inhibit amylase activity as substrate length increased from 3 to 5 to 7 glucose units and as size was increased by linkage to a polymer. Different effects of substrate size were observed with two monoclonal antibodies. One monoclonal antibody blocked amylase activity independent of substrate size, while another monoclonal antibody had little inhibitory effect except using starch as substrate. We conclude that use of larger substrates can expand the repertoire of inhibitory epitopes on enzymes and convert a noninhibitory antibody into an inhibitory one.

Macromolecular chromogenic substrates for measuring proteinase activity.

BACKGROUND: Proteinase activities are often measured using chromogenic substrates that are much smaller than physiological substrates. METHODS: The hydrodynamic size of macromolecular substrates (macrosubstrates) prepared by linking small chromogenic substrates to polyethylene glycol was determined by gel filtration. Efficiency of macrosubstrate cleavage by proteinases and alpha(2)-macroglobulin-proteinase complexes was monitored spectrophotometrically. RESULTS: Macrosubstrates had hydrodynamic radii of approximately 20 A, similar to proteins with a molecular weight of 18,000. Different macrosubstrates served as efficient substrates for chymotrypsin, trypsin, and thrombin. Linking small substrates to a polymer variably affected substrate efficiency, with the impact on activity ranging from a 60-fold decrease to a 30-fold increase. Proteinases complexed with alpha(2)-macroglobulin had approximately 10-fold lower activity vs macrosubstrates than small substrates. CONCLUSIONS: Macrosubstrates are efficient substrates that allow decreased measurement of sterically hindered proteinase molecules such as alpha(2)-macroglobulin-proteinase complexes. Thus, macrosubstrates may provide more accurate functional assays of proteinases such as coagulation factors.

Glypican-3 is a binding protein on the HepG2 cell surface for tissue factor pathway inhibitor.

Tissue factor pathway inhibitor (TFPI) is a primary regulator of the initiation of blood coagulation. TFPI is internalized and degraded by HepG2 cells through the low-density-lipoprotein receptor-related protein (LRP) but also binds another molecule present on the cell surface at approx. 10-fold the abundance of LRP [Warshawsky, Broze and Schwartz (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 6664-6668]. When HepG2 cells are washed with heparin or dextran sulphate, a substance that binds TFPI is removed from the cell surface and can be detected in a slot-blot assay. Preincubation with trypsin destroys the reactivity of the TFPI-binding component in the slot-blot assay, suggesting that it is a protein. In addition, when the sulphation of glycosaminoglycans (GAGs) is prevented by growing the HepG2 cells in the presence of 30 mM sodium chlorate, TFPI binding is unaffected, whereas the binding of bovine lipoprotein lipase, a protein known to associate with cell-surface GAGs, falls to 50% of control levels. Dextran sulphate washes of HepG2 cells grown in sodium chlorate have an equal reactivity in slot-blot experiments to that of non-treated cells, suggesting that GAGs are not totally responsible for the binding activity observed. By using the slot blot to follow binding activity and conventional protein purification techniques, a protein species that migrates at 40 kDa after reduction was identified in the HepG2 cell wash. The binding of this protein to TFPI was confirmed with immobilized TFPI. Amino acid sequence analysis identified this protein species as a proteolytic fragment of glypican-3 (also called OCI-5), a member of the glypican family of glycosylphosphatidylinositol-anchored proteoglycans.

Differential functions of triplicated repeats suggest two independent roles for the receptor-associated protein as a molecular chaperone.

The 39-kDa receptor-associated protein (RAP) is a molecular chaperone for the low density lipoprotein receptor-related protein (LRP), a large endocytic receptor that binds multiple ligands. The primary function of RAP has been defined as promotion of the correct folding of LRP, and prevention of premature interaction of ligands with LRP within the early secretory pathway. Previous examination of the RAP sequence revealed an internal triplication. However, the functional implication of the triplicated repeats was unknown. In the current study using various RAP and LRP domain constructs, we found that the carboxyl-terminal repeat of RAP possesses high affinities to each of the three ligand-binding domains on LRP, whereas the amino-terminal and central repeats of RAP exhibit only low affinity to the second and the fourth ligand-binding domains of LRP, respectively. Using truncated soluble minireceptors of LRP, we identified five independent RAP-binding sites, two on each of the second and fourth, and one on the third ligand-binding domain of LRP. By coexpressing soluble LRP minireceptors and RAP repeat constructs, we found that only the carboxyl-terminal repeat of RAP was able to promote the folding and subsequent secretion of the soluble LRP minireceptors. However, when the ability of each RAP repeat to inhibit ligand interactions with LRP was examined, differential effects were observed for individual LRP ligands. Most striking, both the amino-terminal and central repeats, but not the carboxyl-terminal repeat, of RAP inhibited the interaction of alpha2-macroglobulin with LRP. These differential functions of the RAP repeats suggest that the roles of RAP in the folding of LRP and in the prevention of premature interaction of ligand with the receptor are independent.

The low density lipoprotein receptor-related protein can function independently from heparan sulfate proteoglycans in tissue factor pathway inhibitor endocytosis.

Tissue factor pathway inhibitor (TFPI) is a plasma serine protease inhibitor that directly inhibits coagulation factor Xa and regulates blood coagulation via inhibition of factor VIIa-tissue factor enzymatic activity. We previously demonstrated that >90% of TFPI bound to a single population of low affinity binding sites on hepatoma cells (2 x 10(6) sites/cell, Kd = 30 nM), and, that following binding, the low density lipoprotein receptor-related protein (LRP) mediated TFPI uptake and degradation. We subsequently reported heparan sulfate proteoglycans (HSPGs) constitute a second receptor system involved in TFPI catabolism. In the present study, mouse embryonic fibroblasts heterozygous and homozygous-negative for disruption of the LRP gene were used to further examine the roles of LRP and HSPGs in TFPI endocytosis. We demonstrate that LRP is absolutely required for degrading 125I-TFPI. LRP heterozygous and homozygous-negative cells bind 125I-TFPI similarly, and the 39-kDa protein, an inhibitor of all known ligand interactions with LRP, does not alter 125I-TFPI binding to these cells. TFPI can be cross-linked to LRP on [35S]cysteine-labeled hepatoma and LRP-heterozygous cells but not LRP-negative cells. When HSPGs are blocked with protamine, 125I-TFPI binds in a 39-kDa protein-inhibitable manner to 41,000 high affinity sites/hepatoma cell (Kd = 2.3 nM). Blockade of HSPGs with protamine results in significantly more 125I-TFPI degradation by LRP-positive cells. TFPI can be cross-linked to LRP in the absence and presence of protamine. However, in the presence of protamine, relative to the total pool of cross-linked proteins, 5-fold more TFPI is cross-linked to LRP. Finally, we show TFPI inhibits 125I-alpha2-macroglobulin-methylamine binding to hepatoma cells and that carboxyl-terminal residues 115-319 of the 39-kDa protein inhibit both 125I-TFPI degradation and binding when binding conditions contain protamine. Together, our results suggest that while the majority of TFPI binds to cell surface HSPGs, LRP can function independently from HSPGs in the binding and uptake of TFPI.

The 39-kDa protein regulates LRP activity in cultured endothelial and smooth muscle cells.

The low density lipoprotein receptor-related protein (LRP) has been proposed to function as an endocytosis receptor for chylomicron remnants and protease-inhibitor complexes so that these particles can be cleared from the plasma or extracellular fluid. The kidney glycoprotein 330 (gp330) may have an analogous role to LRP in the kidney. A 39-kDa protein which copurifies with LRP and gp330 inhibits the binding and/or cellular uptake of ligands to these receptors and may regulate LRP and gp330 activity in vivo. Recently, LRP has been immunochemically localized to endothelial and vascular smooth muscle cells. In the present study, the biology of the 39-kDa protein was studied in cultured endothelial cells and vascular smooth muscle cells. The 39-kDa protein is synthesized by both cell types and has an average half-life of 15 hours. Immunofluorescence shows the major part of the 39-kDa protein has an intracellular localization with enrichment in the perinuclear region. Tissue-type plasminogen activator (t-PA), a plasma serine protease that binds specifically and with high affinity to LRP on hepatoma cells, also binds to endothelial cells and vascular smooth muscle cells. 125I-t-PA binding to both cell types is inhibited by the 39-kDa protein. However, only the endothelial cells are capable of rapidly internalizing and degrading 125I-t-PA. These data thus suggest that LRP may function as a clearance receptor for t-PA on endothelial cells.

Analysis of ligand binding to the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein. Evidence that lipoprotein lipase and the carboxyl-terminal domain of the receptor-associated protein bind to the same site.

The endocytic alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) binds several classes of extracellular ligands at independent sites. In addition, alpha 2MR/LRP can bind multiple copies of the 39-40-kDa receptor-associated protein (RAP). Both amino-terminal and carboxyl-terminal fragments of RAP exhibit affinity, and the fragments apparently bind to different sites on the receptor. RAP completely inhibits the binding of all presently known extracellular ligands, whereas several ligands such as alpha 2-macroglobulin and tissue-type plasminogen activator are poor inhibitors of RAP binding. Since RAP is largely an intracellular molecule that normally does not occupy alpha 2MR/LRP at the cell surface, we hypothesized that an established extracellular ligand might bind to those sites on the receptor capable of binding the RAP fragments. We found complete cross-competition between carboxyl-terminal RAP fragments and fragments of lipoprotein lipase containing the recently identified binding domain for alpha 2MR/LRP (Nykjaer, A., Nielsen, M., Lookene, A., Meyer, N., Røigaard, H., Etzerodt, M., Beisiegel, U., Olivecrona, G., and Gliemann, J. (1994) J. Biol. Chem. 269, 31747-31755). Moreover, the lipoprotein lipase fragment completely inhibited the binding of several alpha 2MR/LRP ligands in a pattern similar to that of carboxyl-terminal RAP fragments. On the other hand, the amino-terminal RAP fragment was a poor competitor of binding of the lipoprotein lipase fragment, whereas it competed effectively with pro-uPA for binding to the receptor. The results provide evidence that lipoprotein lipase binds to the site on alpha2MR/LRP also available for binding of the carboxyl-terminal domain of RAP and suggest that pro-uPA may bind to or overlap the site available for the amino-terminal domain of RAP.

The carboxy terminus of tissue factor pathway inhibitor is required for interacting with hepatoma cells in vitro and in vivo.

Tissue factor pathway inhibitor (TFPI) is a plasma Kunitz-type serine protease inhibitor that directly inhibits coagulation Factor Xa and also inhibits tissue factor-initiated coagulation. Normal human plasma TFPI exists both as the full-length molecule and as variably carboxy-terminal truncated forms. We reported recently that the low density lipoprotein receptor-related protein mediates the cellular degradation of TFPI after TFPI binding to the hepatoma cell surface. To examine whether the carboxy terminus of TFPI was required for interacting with hepatoma cells, a mutant of TFPI lacking the third Kunitz-type domain and basic carboxy terminus was generated. We found that this mutant, TFPI-160, did not compete with full-length 125I-TFPI-160 for binding to hepatoma cells. We were also unable to demonstrate specific binding of 125I-TFPI-160 to hepatoma cells at 4 degrees C. At 37 degrees C, significantly less 125I-TFPI-160 was internalized and degraded via low density lipoprotein receptor-related protein than full-length 125I-TFPI. Full-length 125I-TFPI binding to hepatoma cells could be inhibited > 90% by heparin and other highly charged molecules. Since TFPI, but not TFPI-160, was capable of effectively binding to cultured hepatoma cells, the fates of TFPI and TFPI-160 in vivo were examined. Both 125I-TFPI and 125I-TFPI-160 disappeared rapidly from the circulation after their intravenous administration into rats. The initial plasma half-life of 125I-TFPI was approximately 30 s whereas the half-life of 125I-TFPI-160 was approximately 4 min. 125I-TFPI was cleared predominantly by the liver. In contrast, 125I-TFPI-160 accumulated in the outer cortex of the kidney. Using microscopic autoradiography, we demonstrate that 125I-TFPI clearance is largely hepatocellular, whereas 125I-TFPI-160 accumulates mainly in the cells of the kidney proximal tubules. Together our findings demonstrate that the carboxy-terminal region(s) distal to amino acid 160 of TFPI mediates TFPI binding to hepatoma cells both in vitro and in vivo.

Sites within the 39-kDa protein important for regulating ligand binding to the low-density lipoprotein receptor-related protein.

A 39-kDa protein copurifies with the low-density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) and inhibits the binding and/or cellular uptake of ligands by this receptor. We recently utilized glutathione S-transferase (GST)-39-kDa fusion protein constructs to demonstrate that constructs encoding amino-terminal residues 1-114 and carboxy-terminal residues 115-319 of the 39-kDa protein independently bind to purified LRP and to LRP on hepatoma cells with similar affinities as the full-length GST-39-kDa protein (Kd approximately 8-10 nM). These regions, however, inhibit ligand binding to LRP differently: GST/1-114 inhibits both tissue-type plasminogen activator (t-PA) and alpha 2-macroglobulin-methylamine (alpha 2M*) binding whereas GST/115-319 only potently inhibits t-PA binding. Four domains, containing residues 18-24 and 100-107 within amino-terminal constructs and residues 200-225 and 311-319 within carboxy-terminal constructs, are required for inhibition of ligand binding. In the present study, we generated additional 39-kDa protein constructs to precisely define residues within each domain required for inhibition of t-PA and alpha 2M* binding to LRP. The potential importance of these residues in mediating direct binding both to purified LRP and to LRP on hepatoma cells was examined. Within amino-terminal residues 1-114, alanine 103 and leucine 104 are required for inhibition of t-PA and alpha 2M* binding. These residues, however, are not required for binding either to purified LRP or to LRP on hepatoma cells. Within domain 18-24, arginine 21 is required for inhibition of t-PA and alpha 2M* binding as well as for the direct binding of amino-terminal constructs to LRP. Within carboxy-terminal domains 200-225 and 311-319, leucine 222 and leucine 319 are both required for inhibition of t-PA binding. Deletion of leucine 319 changes the ligand specificity from inhibition of t-PA binding to inhibition of alpha 2M* binding. Thus, leucine 319 is not required for direct binding to LRP whereas leucine 222 is required for high-affinity binding to LRP.

The low density lipoprotein receptor-related protein mediates the cellular degradation of tissue factor pathway inhibitor.

The low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) is a cell-surface glycoprotein of 4525 amino acids that functions as a hepatic endocytosis receptor for several plasma proteins. These include alpha 2-macroglobulin-protease complexes, free plasminogen activators as well as plasminogen activators complexed with their inhibitors, and beta-migrating very low density lipoproteins complexed with either apolipoprotein E or lipoprotein lipase. In the current study we used human and rat hepatoma cell lines to demonstrate that LRP can mediate the degradation of tissue factor pathway inhibitor (TFPI), a Kunitz-type plasma serine protease inhibitor that regulates tissue factor-induced blood coagulation. The cellular degradation of 125I-labeled TFPI (125I-TFPI) was inhibited more than 80% both by antibodies directed against LRP and by the LRP-associated 39-kDa protein, a protein that inhibits the binding and/or cell-mediated degradation of all ligands by LRP. Using rat hepatoma cells, we report that at 4 degrees C, 125I-TFPI binds to approximately 2 x 10(6) sites per cell with an equilibrium dissociation constant of approximately 30 nM. 125I-TFPI binding to the cell surface is not inhibited by the 39-kDa protein. Taken together, our results suggest that TFPI binds to an as-yet-unidentified cell surface molecule. After binding, LRP mediates the cellular degradation of TFPI.

Binding analysis of amino-terminal and carboxyl-terminal regions of the 39-kDa protein to the low density lipoprotein receptor-related protein.

A 39-kDa protein binds to the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP/alpha 2MR) and inhibits the binding of ligands to this receptor. We recently reported that inhibition of tissue-type plasminogen activator binding to LRP/alpha 2MR is mediated by both amino-terminal and carboxyl-terminal regions of the 39-kDa protein, whereas inhibition of alpha 2-macroglobulin-proteinase binding is mediated only by amino-terminal regions. In this report we show that amino-terminal and carboxyl-terminal regions of the 39-kDa protein bind specifically and with high affinity to LRP/alpha 2MR on rat hepatoma MH1C1 cells. Following binding, these amino-terminal and carboxyl-terminal regions of the 39-kDa protein are each rapidly endocytosed and degraded with kinetics identical to the full-length 39-kDa protein. Competition binding experiments with these constructs demonstrate that amino-terminal and carboxyl-terminal regions of the 39-kDa protein compete with one another for binding to LRP/alpha 2MR. A model is proposed in which amino-terminal and carboxyl-terminal regions of the 39-kDa protein bind to different sites on LRP/alpha 2MR in order to inhibit ligand binding.

Identification of domains on the 39-kDa protein that inhibit the binding of ligands to the low density lipoprotein receptor-related protein.

The low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP/alpha 2MR) binds and internalizes several plasma proteins including tissue-type plasminogen activator (t-PA) and alpha 2-macroglobulin-protease complexes (alpha 2M*). A 39-kDa protein that copurifies with LRP/alpha 2MR inhibits the binding and uptake of ligands by LRP/alpha 2MR, including t-PA and alpha 2M*. To define domains on the 39-kDa protein which are essential for inhibition of t-PA and alpha 2M* binding to LRP/alpha 2MR, we have generated bacterial expression constructs encoding discrete regions of the 39-kDa protein as fusion proteins with glutathione S-transferase. Inhibition of t-PA and alpha 2M* binding to LRP/alpha 2MR on rat hepatoma MH1C1 cells are shown to require amino acid residues 18-24 and 100-107 on the 39-kDa protein. Inhibition of t-PA but not alpha 2M* binding to LRP/alpha 2MR is also mediated by residues 200-225 and 311-319. The same 39-kDa protein constructs that inhibit alpha 2M* and t-PA binding to MH1C1 cells are able to bind directly to purified LRP/alpha 2MR immobilized on nitrocellulose. Thus, our studies demonstrate several specific regions on the 39-kDa protein which are required for the inhibition of t-PA and alpha 2M* binding to LRP/alpha 2MR.

39-kD protein inhibits tissue-type plasminogen activator clearance in vivo.

Tissue-type plasminogen activator (t-PA) is a plasma serine protease that catalyzes the initial and rate-limiting step in the fibrinolytic cascade. t-PA is widely used as a thrombolytic agent in the treatment of acute myocardial infarction. However, its use has been impaired by its rapid hepatic clearance from the circulation following intravenous administration. Studies with both rat hepatoma MH1C1 cells (G. Bu, S. Williams, D. K. Strickland, and A. L. Schwartz, 1992. Proc. Natl. Acad. Sci. USA. 89:7427-7431) and human hepatoma HepG2 cells (G. Bu, E. A. Maksymovitch, and A. L. Schwartz. 1993. J. Biol. Chem. 28:13002-13009) have shown that binding of t-PA to its clearance receptor, the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor, is inhibited by a 39-kD protein that copurifies with this receptor. Herein we investigated whether administration of purified recombinant 39-kD protein would alter t-PA clearance in vivo. We found that intravenous administration of purified 39-kD protein to rats prolonged the plasma half-life of 125I-t-PA from 1 min to approximately 5-6 min. The plasma half-life of t-PA enzymatic activity was similarly prolonged following intravenous administration of purified 39-kD protein. In addition we found that the 39-kD protein itself was rapidly cleared from the circulation in vivo. Clearance of 125I-39-kD protein was a biphasic process with half-lives of 30 s and 9 min and the liver was the primary organ of clearance. Preadministration of excess unlabeled 39-kD protein slowed 125I-39-kD protein clearance in rats in a dose-dependent manner, suggesting that specific clearance receptors were responsible for this process. Administration of increasing doses of unlabeled 39-kD protein along with labeled 39-kD protein resulted in a decrease in the amount of labeled 39-kD protein associating with the liver and a concomitant increase in the amount of labeled 39-kD protein associating with the kidneys, indicating two clearance mechanisms exist for the 39-kD protein.

Cloning and expression of a cytosolic megakaryocyte protein-tyrosine-phosphatase with sequence homology to retinaldehyde-binding protein and yeast SEC14p.

Protein tyrosine phosphorylation is important in the regulation of cell growth, the cell cycle, and malignant transformation. We have cloned a cDNA that encodes a cytosolic protein-tyrosine-phosphatase (PTPase), MEG2, from MEG-01 cell and human umbilical vein endothelial cell cDNA libraries. The 4-kilobase cDNA sequence of PTPase MEG2 corresponds in length to the mRNA transcript detected by Northern blotting. The predicted open reading frame encodes a 68-kDa protein composed of 593 amino acids and has no apparent signal or transmembrane sequences, suggesting that it is a cytosolic protein. The C-terminal region has a PTPase catalytic domain that has 30-40% amino acid identity to other known PTPases. The N-terminal region has 254 amino acids that are 28% identical to cellular retinaldehyde-binding protein and 24% identical to yeast SEC14p, a protein that has phosphatidylinositol transfer activity and is required for protein secretion through the Golgi complex in yeast. Recombinant PTPase MEG2 expressed in Escherichia coli possesses PTPase activity. PTPase MEG2 mRNA was detected in 12 cell lines tested, which suggests that this phosphatase is widely expressed. The structure of PTPase MEG2 implies that a tyrosine phosphatase could participate in the transfer of hydrophobic ligands or in functions of the Golgi apparatus.

Identification, cloning, and expression of a cytosolic megakaryocyte protein-tyrosine-phosphatase with sequence homology to cytoskeletal protein 4.1.

We have isolated a cDNA encoding a third type of protein-tyrosine-phosphatase. We screened human megakaryoblastic cell line (MEG-01) an umbilical vein endothelial cell cDNA libraries to obtain a 3.7-kilobase cDNA designated PTPase MEG. Northern blot analysis of MEG-01 RNA detected a 3.7-kilobase transcript, suggesting that a full-length cDNA has been identified. PTPase MEG cDNA contains an open reading frame of 926 amino acids. The cDNA has a G+C-rich 5' untranslated region of 771 nucleotides that has the potential to form stable stem-loop structures and has two upstream ATG codons. The predicted protein (Mr = 105,910) has no apparent membrane-spanning region and contains a single protein-tyrosine-phosphatase domain (amino acids 659-909) that is 35-40% identical to previously described tyrosine-phosphatase domains. The recombinant phosphatase domain possesses protein-tyrosine-phosphatase activity when expressed in Escherichia coli. The amino-terminal region (amino acids 31-367) is 45% identical to the amino terminus of human erythrocyte protein 4.1, a cytoskeletal protein. The identification of a protein-tyrosine-phosphatase that is related to cytoskeletal proteins implies that cell signaling activities reside not only in transmembrane receptors but in cytoskeletal elements as well.