Generation of specific antibodies against the rap1A, rap1B and rap2 small GTP-binding proteins. Analysis of rap and ras proteins in membranes from mammalian cells.

Specific antibodies against rap1A and rap1B small GTP-binding proteins were generated by immunization of rabbits with peptides derived from the C-terminus of the processed proteins. Immunoblot analysis of membranes from several mammalian cell lines and human thrombocytes with affinity-purified antibodies against rap1A or rap1B demonstrated the presence of multiple immunoreactive proteins in the 22-23 kDa range, although at strongly varying levels. Whereas both proteins were present in substantial amounts in membranes from myelocytic HL-60, K-562 and HEL cells, they were hardly detectable in membranes from lymphoma U-937 and S49.1 cyc- cells. Membranes from human thrombocytes and 3T3-Swiss Albino fibroblasts showed strong rap1B immunoreactivity, whereas rap1A protein was present in much lower amounts. In the cytosol of HL-60 cells, only small amounts of rap1A and rap1B proteins were detected, unless the cells were treated with lovastatin, an inhibitor of hydroxymethylglutaryl-coenzyme A reductase, suggesting that both proteins are isoprenylated. By comparison with recombinant proteins, the ratio of rap1A/ras proteins in membranes from HL-60 cells was estimated to be about 4:1. An antiserum directed against the C-terminus of rap2 reacted strongly with recombinant rap2, but not with membranes from tested mammalian cells. In conclusion, rap1A and rap1B proteins are distributed differentially among membranes from various mammalian cell types and are isoprenylated in HL-60 cells.     

Isoprenylation of rap2 proteins in platelets and human erythroleukemia cells

The covalent modification of proteins by isoprenoid derivatives of mevalonic acid was investigated in human platelets, cells that lack the ability to synthesize endogenous cholesterol, and human erythroleukemia (HEL) cells, cholesterol-producing cultured cells derived from megakaryocytes. When washed platelets or HEL cells were incubated with [3H]mevalonic acid, the radiolabel was incorporated into a distinct group of proteins with molecular masses between 21,000 and 28,000. We have identified one of these proteins as a ras-related rap2 protein based on its immunoreactivity with a polyclonal antiserum raised against purified recombinant rap2b. This anti-rap2 antiserum was used for two-dimensional immunoblotting analysis and immunoprecipitation of mevalonate-labeled rap2 from platelets and HEL cells. These results suggest that rap2 may undergo a series of carboxyl-terminal modifications similar to the p21ras proteins. In addition, it is shown that non-cholesterol-producing cells are capable of incorporating isoprenyl groups into specific proteins.     

The cAMP-dependent protein kinase phosphorylates the rap1 protein in vitro as well as in intact fibroblasts, but not the closely related rap2 protein

The products of rap genes (rap1A, rap1B and rap2) are small molecular weight GTP-binding proteins that share approximately 50% homology with ras-p21s. It had previously been shown that a rap1 protein (also named Krev-1 or smg p21) could be phosphorylated on serine residues by the cAMP-dependent protein kinase (PKA) in vitro as well as in intact platelets stimulated by prostaglandin E1. We show here that the rap1A protein purified from recombinant bacteria is phosphorylated in vitro by the catalytic subunit of PKA and that the deletion of the 17 C-terminal amino acids leads to the loss of this phosphorylation. This suggests that the serine residue at position 180 constitutes the site of phosphorylation of the rap1A protein by PKA. The rap1 protein can also be phosphorylated by PKA in intact fibroblasts; this phenomenon is independent of their proliferative state. In contrast, protein kinase C (PKC) does not phosphorylate the rap1 proteins, neither in vitro nor in vivo. Finally, the 60% homologous rap2 protein is neither phosphorylated in vitro nor in vivo by PKA or PKC.     

Regulation of the GTPase activity of the ras-related rap2 protein.

The small GTP-binding protein rap2A exhibits a high level of identity with rap1 and ras proteins (60% and 46%, respectively). Nevertheless, its intrinsic GTPase activity is not stimulated by ras-GAP, and unlike the rap1A protein, it cannot compete with ras proteins for their interaction with ras-GAP. In addition, rap1-GAPm that is highly active on the GTPase activity of the rap1A product, also stimulates the GTPase activity of the rap2A protein but with a 30-40-fold lower efficiency. An activity that greatly stimulated the GTPase activity of the rap2 protein (rap2-GAP) was found in bovine brain cytosol and purified. However, it copurified with the cytosolic form of rap1-GAP and was more efficient at stimulating the GTPase activity of the rap1 protein; this 55 kD polypeptide, that is recognized by an antibody raised against rap1-GAPm, likely represents a degraded and soluble form of the full size 89 kD molecule. In bovine brain membranes, a weak GAP activity toward the rap2A protein was also detected; however, it was also attributable to the membrane-associated rap1-GAPm. Thus, it appears that a single rap-GAP protein, complete or degraded, is able to stimulate the GTPase activity of both rap1 and rap2 proteins.     

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae.

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.     

Association of rap1 and rap2 proteins with the specific granules of human neutrophils. Translocation to the plasma membrane during cell activation.

Activation of human neutrophils involves the degranulation of specific and azurophil granules. This process is GTP-dependent and the presence of small GTP-binding proteins (SGBPs) has been detected in the two granule populations. At present, none of these SGBPs has been definitely identified. In order to characterize some of these proteins and obtain further insights as to their potential role in degranulation processes, we have used specific antibodies directed against the ras-related rap1 and rap2 proteins. By immunoblot analysis, we observed that rap2p is predominantly located in specific granules, whereas rap1p is detected both in specific granules and a fraction enriched in plasma membranes. Neither rap1p nor rap2p was found in the cytosol or in azurophil granules. Similarly, by indirect immunofluorescence, we observed that cytoplasmic granules were stained with anti-rap1p antibodies and anti-rap2p antibodies, and the plasma membrane was labeled with both antibodies but more distinctly with anti-rap1p than with anti-rap2p antibodies. rap1p and rap2p are tightly bound to the membrane of specific granules since they cannot be extracted by high salt or alkaline buffers. In addition, treatment of intact specific granules with pronase induced the degradation of rap proteins suggesting that they are exposed to the cytoplasmic face of the granules. Degranulation of neutrophils consists of the translocation and subsequent fusion of granules with the plasma membrane. Activation of this process induced the accumulation of rap proteins in the plasma membrane as observed by subcellular fractionation and indirect immunofluorescence experiments; this was not associated with the appearance of a soluble form of these proteins, showing that they remain membrane-bound during this process. The identification and subcellular localization of rap1p and rap2p at the surface of specific granules and the observation that they translocate to the plasma membrane upon cell stimulation without appearance of soluble forms constitute an important step toward the understanding of their physiological functions in human neutrophils.     

Effect of protein kinase A on inositide metabolism and rap 1 G-protein in human erythroleukemia cells.

Human erythroleukemia (HEL) cells phosphorylate [3H]inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate; they also contain all the enzymes to sequentially dephosphorylate [3H]inositol 1,4,5-trisphosphate and [3H]inositol 1,3,4,5-tetrakisphosphate to inositol. alpha-Thrombin, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, and sodium fluoride caused the formation of [3H]inositol phosphates in HEL cells that were previously labeled with [3H]inositol. This indicates agonist-induced activation of phospholipase C and hydrolysis of the inositol phospholipids. Pretreatment of the HEL cells with iloprost, a prostacyclin analog that increases cellular cyclic AMP levels, dramatically reduced the formation of inositol phosphates and the increase of [3H]phosphatidylinositol 4,5-bisphosphate. The inhibitory effects of iloprost were associated with the phosphorylation of a 24-kDa protein, which was detected with an antiserum obtained against the rap 1 protein. The catalytic subunit of protein kinase A inhibited formation of polyphosphoinositides during phosphorylation of the rap 1 protein in membranes. This rap 1 protein might have functional relevance in the inhibition of agonist-induced inositide metabolism.     

Rap1-B is phosphorylated by protein kinase A in intact human platelets.

Agonists that increase cAMP levels in platelets promote the phosphorylation of a 24 kDa GTP-binding protein that is immunoreactive with a monoclonal antibody (M90) to the H-ras p21 protein. Evidence is presented which indicates that this protein is rap-1b, not rap1-a as previously suggested (Ohmori, T., Kikuchi, A., Yamamoto, K., Kawata, M., Kondo, J. and Takai, Y. (1988) Biochem. Biophys. Res. Commun. 157, 670-676). The amino acid sequence of labeled peptides obtained by proteolytic cleavage of the purified phosphorylated protein was identical with that of rap-1b. Furthermore, a comparison of the kinetics of phosphorylation of synthetic peptides corresponding to the C-terminal region of rap-1a and rap-1b proteins indicated that rap-1b is the preferred substrate for phosphorylation by cAMP-dependent protein kinase.     

Interaction of the low-molecular-weight GTP-binding protein rap2 with the platelet cytoskeleton is mediated by direct binding to the actin filaments

The interaction of the low-molecular-weight GTP-binding protein rap2 with the cytoskeleton from thrombin-aggregated platelets was investigated by inducing depolymerization of the actin filaments, followed by in vitro-promoted repolymerization. We found that the association of rap2 with the cytoskeleton was spontaneously restored after one cycle of actin depolymerization and repolymerization. Exogenous rap2, but not unrelated proteins, added to depolymerized actin and solubilized actin-binding proteins, was also specifically incorporated into the in vitro reconstituted cytoskeleton. The incorporation of exogenous rap2 was also observed when the cytoskeleton from resting or thrombin-activated platelets was subjected to actin depolymerization-repolymerization. Moreover, such interaction occurred equally well when exogenous rap2 was loaded with either GDP or GTPgammaS. We also found that polyhistidine-tagged rap2 immobilized on Ni(2+)-Sepharose and loaded with either GDP or GTPgammaS, could specifically bind to cytoskeletal actin. Moreover, when purified monomeric actin was induced to polymerize in vitro in the presence of rap2, the small G-protein specifically associated with the actin filaments. Finally, rap2 loaded with either GDP or GTPgammaS was able to bind to purified F-actin immobilized on a plastic surface. These results demonstrate that rap2 interacts with the platelet cytoskeleton by direct binding to the actin filaments and that this interaction is not regulated by the activation state of the protein.     

Partial purification of a GTPase-activating protein for rap2b from bovine brain membranes.

Rap2b is a ras-related GTP-binding protein isolated from a human platelet cDNA library. It shares 90% similarity to the previously described rap2a and is closely related to rap1a (Krev-1, smgp21), which has been shown to possess reversion of transformation activity in Kirsten ras transformed 3T3 cells. In this study we have partially purified a protein from bovine brain membranes which stimulates the GTPase activity of rap2b. This rap2b GTPase-activating protein (GAP) activity is not immunoreactive with antibodies specific for rap1 GAP or ras GAP, yet displays limited GTPase stimulatory activity toward rap1. This result differs from the previously described rap1 GAP which is highly specific for rap1. When the rap2 GAP activity is analyzed by coomassie staining, an enrichment of a approximately 55 kDa protein is observed providing further evidence of a distinct rap2 GAP.     

Purification, identification, and characterization of two GTP-binding proteins with molecular weights of 25,000 and 21,000 in human platelet cytosol. One is the rap1/smg21/Krev-1 protein and the other is a novel GTP-binding protein

We have purified, characterized, and identified two GTP-binding proteins with Mr of 25,000 (c25KG) and 21,000 (c21KG) from the cytosol fraction of human platelets. These two proteins were not copurified with the beta gamma subunits of heterotrimeric GTP-binding proteins. Amino acid sequences of tryptic fragments of c21KG completely matched with those of rap1 protein (Pizon, V., Chardin, P., Lerosey, I., Olofsson, B., and Tavitian, A. (1988) Oncogene 3, 201-204), smg p21 (Kawata, M., Matsui, Y., Kondo, J., Hishida, T., Teranishi, Y., and Takai, Y. (1988) J. Biol. Chem. 263, 18965-18971), and Krev-1 protein (Kitayama, H., Sugimoto, Y., Matsuzaki, T., Ikawa, Y., and Noda, M. (1989) Cell 56, 77-84). The partial amino acid sequence analysis of c25KG revealed that this protein was different from any low Mr GTP-binding proteins already reported. c25KG bound about 1 mol of [35S] guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)/mol of protein, with a Kd value of about 45 nM. [35S]GTP gamma S-binding to c25KG was specifically inhibited by guanine nucleotides, GTP and GDP, but not by adenine nucleotides such as ATP and adenyl-5'-yl beta, gamma-imidodiphosphate. The binding activity was not inhibited by pretreatment with N-ethylmaleimide. c25KG hydrolyzed GTP to librate Pi with the specific activity of 1.8 mmol of Pi/mol of protein/min, which are different from the activities of the already purified low Mr GTP-binding proteins. We conclude that c25KG is a novel GTP-binding protein and c21KG is a rap1/smg p21/Krev-1 product.     

Possible involvement of different GTP-binding proteins in noradrenaline- and thrombin-stimulated release of arachidonic acid in rabbit platelets.

Noradrenaline (NA) stimulated the release of arachidonic acid (AA) from the [3H]AA-labelled rabbit platelets via alpha 2-adrenergic receptors, since the effect of NA was inhibited by yohimbine. The stimulatory effect of NA in digitonin-permeabilized platelets was completely dependent on the simultaneous presence of GTP and Ca2+. The NA- and thrombin-stimulated releases of AA were markedly decreased by the prior ADP-ribosylation of the permeabilized platelets with pertussis toxin. Antiserum directed against the pig brain Go (a GTP-binding protein of unknown function), recognizing both alpha 39 and beta 35,36 subunits, but not alpha 41, of pig brain, reacted with 41 kDa and 40 kDa bands, with not one of 39 kDa, in rabbit platelet membranes. Anti-Go antiserum inhibited guanosine 5'-[gamma-thio]triphosphate-, A1F4(-)-, NA- and thrombin-stimulated AA releases in the membranes. Although the effect of thrombin was inhibited by low concentrations of anti-Go antiserum, high concentrations of the antiserum was needed for inhibition of the NA effect. Antiserum directed against the pig brain G1 (inhibitory G-protein), recognizing both alpha 41 and beta 35,36 subunits, but not alpha 39, of pig brain, reacted with the 41 kDa band in platelets. Anti-G1 antiserum inhibited only the effect of NA. Reconstitution of the platelet membranes ADP-ribosylated by pertussis toxin with Go, not Gi, purified from pig brain restored the thrombin-stimulated release of AA. In contrast, reconstitution of those membranes with Gi, not Go, restored the NA-stimulated release of AA. These results indicate that different GTP-binding proteins, Gi- and Go-like proteins, may be involved in the mechanism of signal transduction from alpha 2-adrenergic receptors and thrombin receptors to phospholipase A2 in rabbit platelets.     

Monoclonal antibodies recognizing different epitopes of the 27-kDa gap junction protein from rat liver

Monoclonal antibodies (2-3E2, 6-3G11, and 7-3H6) against gap junction plaques purified from rat liver were prepared and characterized. Immunoblot analysis of liver gap junctions revealed that all three antibodies reacted with the 27-kDa protein, but not with the 22-kDa one. The 2-3E2 and 6-3G11 antibodies both reacted with the 27-kDa protein in gap junctions purified from livers of the rat, mouse, rabbit, and guinea pig; the 7-3H6 antibody, however, failed to react with the 27-kDa protein from guinea pig liver. The 7-3H6 antibody reacted strongly with the 24- to 26-kDa degradation products of the 27-kDa protein. Indirect immunofluorescence showed that the 6-3G11 and 7-3H6 antibodies both gave the same specific fluorescence labeling on rat liver cryosections, suggesting that these two antibodies recognized the cytoplasmic sites of the 27-kDa protein. Immunoblot analysis of protease-digested fragments from the 27-kDa protein revealed that the 7-3H6 antibody reacted with the 24- and 17-kDa fragments (including portions of the carboxyl-terminal domain of the 27-kDa protein) produced with endoproteinases Arg-C and Lys-C, respectively. Immunoblot analysis of CNBr fragments of the 27-kDa protein revealed that all three antibodies reacted with the 10-kDa fragment, which is thought to be the carboxyl-terminal domain of the 27-kDa protein. These results demonstrate that three monoclonal antibodies recognize different epitopes of the cytoplasmic sites (probably the carboxyl-terminal domain) of the 27-kDa liver gap junction protein.     

rap1B, a cAMP-dependent protein kinase substrate, associates with the platelet cytoskeleton

rap1B is a member of the ras superfamily of low molecular weight GTP binding proteins which constitutes a focal point of GTP and cAMP signal transduction systems. Like other members of this superfamily, rap1B is membrane-associated in resting platelets, presumably through polyisoprenylation. The studies presented here were undertaken to determine the subcellular changes in rap1B localization during cell activation. Activated and unactivated platelets were fractionated by Triton X-100 lysis followed by differential centrifugation to obtain a 10,000 x g cytoskeleton fraction, a 100,000 x g membrane skeleton fraction, and a 100,000 x g supernatant fraction containing solubilized proteins. In unactivated platelets, rap1B was present in the 100,000 x g supernatant fraction. In contrast, in platelets activated with 1 unit/ml alpha-thrombin or with the calcium ionophore, A23187, rap1B was quantitatively recovered in the 10,000 x g cytoskeleton fraction. rap1B was absent from the 100,000 x g fraction containing the membrane skeleton and could not be detected in the 100,000 x g supernatant containing cytosolic proteins and solubilized membrane components. These results indicate that rap1B associates with the cytoskeleton during cell activation.     

Phosphorylation of rap1GAP in vivo and by cAMP-dependent kinase and the cell cycle p34cdc2 kinase in vitro.

rap1GAP is a GTPase activating protein that specifically stimulates the GTP hydrolytic rate of the ras-related protein p21rap1.rap1GAP undergoes post-translational modification that causes a substantial change in its mobility on sodium dodecyl sulfate-polyacrylamide gels. At least part of this modification is due to the phosphorylation. Expression of a rap1GAP cDNA in insect cells labeled with 32Pi resulted in high level incorporation of radioactivity into serine residues of the expressed protein. Purified rap1GAP was phosphorylated in vitro by cAMP-dependent kinase and the cell cycle p34cdc2 kinase. The molar ratio of incorporated phosphate/rap1GAP was approximately 3 by cAMP-dependent kinase and 2 by p34cdc2. The sites of phosphorylation by both kinases were localized to a 100-residue segment contained in the carboxyl-terminal region of the predicted primary structure of rap1GAP. Highly favorable recognition sequences for the two kinases are contained within this fragment and are proposed as the sites of phosphorylation. Treatment of SK-MEL-3 cells with dibutyryl cAMP promoted phosphorylation of rap1GAP in vivo. Based on the results of comparative phosphopeptide mapping the sites of phosphorylation in vivo and in vitro are identical.     

A novel 39-kilodalton membrane protein binds GTP in polyomavirus-transformed cells.

To investigate the possible involvement of GTP-binding proteins in transformation by the DNA tumor virus polyomavirus, the GTP-binding activities of Ras-like proteins and G protein alpha subunit proteins were examined in polyomavirus-transformed cells. No differences in the degrees or patterns of expression of Ras-like proteins were observed. However, a 39-kDa protein specifically bound GTP in membranes from polyomavirus-transformed cells. This protein was not seen in nontransformed or lytically infected cells or in phenotypically normal revertants of polyomavirus-transformed cells. It reappeared, however, in spontaneous retransformants derived from the revertants. The 39-kDa protein was not found stably associated with polyomavirus T antigens, nor was it phosphorylated on tyrosine. The 39-kDa protein was not recognized by an antiserum specific for members of the Gi alpha subfamily of G proteins or by antisera against all other known GTP-binding proteins of similar molecular mass. These results suggest that this novel 39-kDa GTP-binding membrane protein is observed as part of a long-term response that accompanies stable transformation by the virus.     

Identification of multiple ral gene products in human platelets that account for some but not all of the platelet Gn-proteins

Polyclonal antibodies raised against specific recombinant low molecular mass GTP-binding proteins were tested for their ability to recognize partially purified human platelet membrane Gn-proteins (i.e. proteins that bind [alpha-32P]GTP on nitrocellulose blots of SDS/polyacrylamide gels). An antiserum against simian ralA protein recognized a 27 kDa human platelet protein with the same apparent molecular mass as the major platelet Gn-protein (Gn27). In further analysis by two-dimensional polyacrylamide gel electrophoresis, the isoelectric focusing step permitted resolution of 12 major Gn-protein forms, seven of 27 kDa (Gn27a-g), one of 26 kDa (Gn26) and four of 24 kDa (Gn24a-d). The ralA antibody reacted strongly with the five most basic Gn27 species (a-e), weakly with Gn26 and not at all with Gn27f, Gn27g or Gn24a-d. We conclude that ral gene products account for some but probably not for all of the platelet Gn-proteins.     

Identification of two minor subunits in the pilus of Haemophilus influenzae.

Haemophilus influenzae type b (Hib) organisms produce pili, which mediate attachment to human cells and are multimeric structures composed of a 24-kDa subunit called pilin or HifA. Although pili from other organisms contain additional proteins accessory to pilin, no structural components other than pilin have been identified in Hib pili. Previous analysis of a Hib pilus gene cluster, however, suggested that two genes, hifD and hifE, may encode additional pilus subunits. To determine if hifD and hifE encode pilus components, the genes were overexpressed in Escherichia coli and the resulting proteins were purified and used to raise polyclonal antisera. Antisera raised against C-terminal HifD and HifE fragments reacted with H. influenzae HifD and HifE proteins, respectively, on Western immunoblots. Western immunoblot analysis of immunoprecipitated Hib pili demonstrated that HifD and HifE copurified with pili. In enzyme-linked immunosorbent assays, antisera raised against a recombinant HifE protein that contained most of the mature protein reacted more to piliated Hib than to nonpiliated Hib or to a mutant containing a hifE gene insertion. Immunoelectron microscopy confirmed that the HifE antiserum bound to pili and demonstrated that the antiserum bound predominantly to the pilus tips. These data indicate that HifD and HifE are pilus subunits. Adherence inhibition studies demonstrated that the HifE antiserum completely blocked pilus-mediated hemagglutination, suggesting that HifE mediates pilus adherence.     

Demonstration of two forms of calcium pumps by thapsigargin inhibition and radioimmunoblotting in platelet membrane vesicles

In mixed membrane vesicles prepared from human platelets, the presence of two distinct calcium pump enzymes (molecular mass 100 and 97 kDa) was demonstrated by 32P autoradiography, immunoblotting, and thapsigargin inhibition. Both the 100- and 97-kDa membrane proteins showed calcium-dependent phosphoenzyme formation and reacted with a polyclonal anti-sarcoplasmic reticulum calcium pump antiserum, while only the 100-kDa protein reacted with the antiserum specific for the sarco-endoplasmic reticulum-type calcium transport ATPase 2b isoform. Thapsigargin, inhibiting active calcium transport in platelet membrane vesicles, predominantly blocked the phosphoenzyme formation of the 100-kDa isoform and of the tryptic calcium pump fragments of 55 and 35 kDa, while lanthanum specifically increased the phosphoenzyme formation of the 97-kDa enzyme and of the tryptic fragment of 80 kDa. These results indicate the presence of the sarco-endoplasmic reticulum-type calcium transport ATPase 2b isoform and of a yet unidentified, 97-kDa calcium pump protein in human platelet membranes.     

Cloning and expression of human 75-kDa inositol polyphosphate-5-phosphatase.

Inositol polyphosphate-5-phosphatase (5-phosphatase) hydrolyzes inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate and thereby functions as a signal terminating enzyme in cellular calcium ion mobilization. A cDNA encoding human platelet 5-phosphatase has been isolated by screening for beta-galactosidase fusion proteins that bind to inositol 1,3,4,5-tetrakisphosphate. The sensitivity of the screening procedure was enhanced 50- to 100-fold by amplification of "sublibraries" prior to carrying out binding assays. The sequences derived from the "expression clone" were used to screen human erythroleukemia cell line and human megakaryocytic cell line cDNA libraries. We obtained two additional clones which together consist of 2381 base pairs. The amino-terminal amino acid sequence from the 75-kDa 5-phosphatase purified from platelets is identical to amino acids 38-56 predicted from the cDNA. This suggests that the platelet 5-phosphatase is formed by proteolytic processing of a larger precursor. The cDNA predicts that the mature enzyme contains 635 amino acids (Mr 72, 891). Antibodies directed against recombinant TrpE fusion proteins of either an amino-terminal region or a carboxyl-terminal region immunoprecipitate the enzyme activity from a preparation of the 75-kDa form of platelet 5-phosphatase (Type II) but do not precipitate the distinct 47-kDa 5-phosphatase (Type I) also found in platelets. In addition, the recombinant protein expressed in Cos-7 cells has the same 5-phosphatase activity as the platelet 5-phosphatase.