The upper limb of Paranthropus boisei from Ileret, Kenya.

Paranthropus boisei was first described in 1959 based on fossils from the Olduvai Gorge and now includes many fossils from Ethiopia to Malawi. Knowledge about its postcranial anatomy has remained elusive because, until recently, no postcranial remains could be reliably attributed to this taxon. Here, we report the first associated hand and upper limb skeleton (KNM-ER 47000) of P. boisei from 1.51 to 1.53 Ma sediments at Ileret, Kenya. While the fossils show a combination of primitive and derived traits, the overall anatomy is characterized by primitive traits that resemble those found in Australopithecus, including an oblique scapular spine, relatively long and curved ulna, lack of third metacarpal styloid process, gracile thumb metacarpal, and curved manual phalanges. Very thick cortical bone throughout the upper limb shows that P. boisei had great upper limb strength, supporting hypotheses that this species spent time climbing trees, although probably to a lesser extent than earlier australopiths. Hand anatomy shows that P. boisei, like earlier australopiths, was capable of the manual dexterity needed to create and use stone tools, but lacked the robust thumb of Homo erectus, which arguably reflects adaptations to the intensification of precision grips and tool use. KNM-ER 47000 provides conclusive evidence that early Pleistocene hominins diverged in postcranial and craniodental anatomy, supporting hypotheses of competitive displacement among these contemporaneous hominins.

Landscape scale heterogeneity in the East Turkana ecosystem during the Okote Member (1.56-1.38 Ma).

Placing the biological adaptations of Pleistocene hominins within a well-resolved ecological framework has been a longstanding goal of paleoanthropology. This effort, however, has been challenging due to the discontinuous nature of paleoecological data spanning many important periods in hominin evolution. Sediments from the Upper Burgi (1.98-1.87 Ma), KBS (1.87-1.56 Ma) and Okote (1.56-1.38 Ma) members of the Koobi Fora Formation at East Turkana in northern Kenya document an important time interval in the evolutionary history of the hominin genera Homo and Paranthropus. Although much attention has been paid to Upper Burgi and KBS member deposits, far less is known regarding the East Turkana paleoecosystem during Okote Member times. This study pairs spatially-resolved faunal abundance data with stable isotope geochemistry from mammalian enamel to investigate landscape-scale ecosystem variability during Okote Member times. We find that during this period 1) taxa within the East Turkana large mammal community were distributed heterogeneously across space, 2) the abundance of C3 and C4 vegetation varied between East Turkana subregions, and 3) the Karari subregion, an area with abundant evidence of hominin stone tool manufacture, had significantly more C3 vegetation than regions closer to the central axis of the Turkana Basin (i.e., Ileret and Koobi Fora). These findings indicate that the East Turkana paleoecosystem during the Okote Member was highly variable across space and provided a complex adaptive landscape for Pleistocene hominins.

Platelet PMCA- and SERCA-type Ca2+ -ATPase expression in diabetes: a novel signature of abnormal megakaryocytopoiesis.

BACKGROUND: Previous studies have shown platelet Ca(2+) abnormalities in diabetes mellitus and some reports suggest abnormal platelet production. Platelet Ca(2+) homeostasis is controlled by a multi-Ca(2+)-ATPase system that includes two plasma membrane Ca(2+)-ATPase (PMCA) and seven sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) isoforms. In addition, we recently found that the expression of PMCA4b and SERCA3 isoforms may serve as new markers of abnormal megakaryocytopoiesis [Nurden P et al. Impaired megakaryocytopoiesis in type 2B von Willebrand disease with severe thrombocytopenia. Blood 2006; 108: 2587-95]. AIM: To analyze the expression of major platelet Ca(2+)-ATPases in 27 patients with type 1 or type 2 diabetes (T1D or T2D) compared with normal donors. METHODS: Investigation of protein and mRNA expressions of PMCA1b and PMCA4b, and SERCA2b, SERCA3a and SERCA3b, using specific Western blotting and reverse transcriptase-polymerase chain reaction, respectively. RESULTS: Remarkably, all patients with T1D were found to present a higher expression of PMCA4b protein (212% +/- 28%; n = 10) and PMCA4b mRNA (155% +/- 16%; n = 17), coupled with a higher expression of SERCA3b mRNA (165% +/- 9%) in some cases. Patients with T2D (n = 10) were also studied for protein expression and were found to present similar major upregulation of the expression of PMCA4b protein (180% +/- 28%; n = 10). Lastly, five of 10 patients with T1D were studied for PMCA4b expression after insulin treatment, with four of five recovering normal expression (96% +/- 15%; n = 5). CONCLUSIONS: Compared with the expression of PMCA4b upon platelet maturation, platelets from diabetic patients exhibit similarities with immature megakaryocytes. Thus, this study reinforces the idea that abnormal megakaryocytopoiesis can provide additional insights into diabetes and could represent a novel therapeutic target for antithrombotic drugs.

How many Ca(2)+ATPase isoforms are expressed in a cell type? A growing family of membrane proteins illustrated by studies in platelets.

Ca(2+) signaling plays a key role in normal and abnormal platelet functions. Understanding platelet Ca(2+) signaling requires the knowledge of proteins involved in this process. Among these proteins are Ca(2+)ATPases or Ca(2+) pumps that deplete the cytosol of Ca(2+) ions. Here, we will particularly focus on two Ca(2+) pump families: the plasma membrane Ca(2+)ATPases (PMCAs) that extrude cytosolic Ca(2+) towards the extracellular medium and the sarco/endoplasmic reticulum Ca(2+)ATPases (SERCAs) that pump Ca(2+) into the endoplasmic reticulum (ER). In the present review, we will summarize data on platelet Ca(2+)ATPases including their identification and biogenesis. First of all, we will present the Ca(2+)ATPase genes and their isoforms expressed in platelets. We will especially focus on a member of the SERCA family, SERCA3, recently found to give rise to a number of species-specific isoforms. Next, we will describe the differences in Ca(2+)ATPase patterns observed in human and rat platelets. Last, we will analyze how the expression of Ca(2+)ATPase isoforms changes during megakaryocytic maturation and show that megakaryocytopoiesis is associated with a profound reorganization of the expression and/or activity of Ca(2+)ATPases. Taken together, these data provide new aspects of investigations to better understand normal and abnormal platelet Ca(2+) signaling.

The role of ITAM- and ITIM-coupled receptors in platelet activation by collagen.

The major activation-inducing collagen receptor glycoprotein VI (GPVI) has been cloned within the last two years. It is a member of the Ig superfamily of proteins and is constitutively associated with the ITAM-bearing Fc receptor gamma-chain (FcR gamma-chain). GPVI signals through a pathway that involves several of the proteins used by Fc, B- and T-lymphocyte receptors and which takes place in glycolipid-enriched membrane domains in the plasma membrane known as GEMs. Responses to GPVI are regulated by PECAM-1 (CD31) and possibly other ITIM-bearing receptors. Despite a pivotal role for GPVI, there are important differences between signalling events to collagen and GPVI-specific ligands. This may reflect a role for co-receptors in the response to collagen.

Phosphatidylinositol 3-kinase-dependent translocation of phospholipase Cgamma2 in mouse megakaryocytes is independent of Bruton tyrosine kinase translocation.

Activation of the collagen receptor glycoprotein VI (GPVI) by a collagen-related peptide (CRP) induces stimulation of platelets and megakaryocytes through the phosphatidylinositol (PI) 3-kinase-dependent pathway leading to activation of Bruton tyrosine kinase (Btk) and phospholipase Cgamma2 (PLCgamma2). Here, we present evidence that both proteins undergo PI 3-kinase-dependent translocation to the plasma membrane on CRP stimulation that is markedly inhibited by wortmannin and LY294002. Translocation of PLCgamma2 but not Btk is also seen in megakaryocytes from X-linked immunodeficiency mice, which have a mutation that reduces the affinity of the pleckstrin homology (PH) domain of Btk for PI 3,4,5-trisphosphate (PI 3,4,5-P3). Activation of PC12 cells by epidermal growth factor (EGF) results in increased PI 3-kinase activity and high PI 3,4,5-P3 levels that trigger translocation of the green fluorescent protein (GFP)-labeled PH of Btk, but not the GFP-labeled PH and tandem Src homology 2 (SH2) domains of PLCgamma2. In contrast to the results with CRP, the G protein-coupled receptor agonist thrombin stimulates PI 3-kinase-independent translocation of Btk but not PLCgamma2. In conclusion, these results demonstrate that in mouse megakaryocytes, CRP leads to PI 3-kinase-dependent translocation of PLCgamma2 and Btk that are independent of one another, whereas thrombin only induces translocation of Btk through a pathway that is independent of PI 3-kinase activity.

Expression of the collagen receptor glycoprotein VI during megakaryocyte differentiation.

This study examined the expression of the platelet collagen receptor glycoprotein VI (GPVI) in megakaryocyte cell lines and primary megakaryocytes by reverse transcriptase-polymerase chain reaction and by flow cytometry and ligand blotting using the snake venom toxin convulxin. Expression of GPVI is increased in the megakaryoblastic cell lines HEL and CMK on differentiation with the phorbol ester phorbol 12-myristate 13-acetate (PMA), along with the Fc receptor gamma-chain (FcR gamma-chain). The increase in GPVI expression is associated with marked potentiation of tyrosine phosphorylation and Ca(++) elevation in response to convulxin. Syk, linker for activated T cells, and phospholipase C gamma 2 (PLC gamma 2) are among the proteins tyrosine phosphorylated on convulxin stimulation in PMA-differentiated HEL cells. Studies on primary murine megakaryocytes grown in vitro confirmed that GPVI is up-regulated in parallel with functional activation, assessed by measurement of [Ca(++)](i), during differentiation. The results demonstrate that expression of GPVI is up-regulated along with the FcR gamma-chain during differentiation of megakaryocytes. (Blood. 2000;96:2740-2745)

Phosphatidylinositol 3,4,5-trisphosphate regulates Ca(2+) entry via btk in platelets and megakaryocytes without increasing phospholipase C activity.

The role of phosphatidylinositol 3,4,5-trisphosphate (PI3,4,5P(3)) and Btk in signalling by the collagen receptor glycoprotein VI was investigated. PI3,4,5P(3) was increased in platelets from mice deficient in the SH2 domain-containing inositol 5-phosphatase (SHIP), in response to collagen related peptide (CRP). Tyrosine phosphorylation and activation of phospholipase Cgamma2 (PLCgamma2) were unaltered in SHIP(-/-) platelets, whereas Btk was heavily tyrosine phosphorylated under basal conditions and maximally phosphorylated by low concentrations of CRP. There was an increase in basal Ca(2+), maximal expression of P-selectin, and potentiation of Ca(2+) and aminophospholipid exposure to CRP in SHIP(-/-) platelets in the presence of Ca(2+) (1 mM). Microinjection of PI3,4, 5P(3) into megakaryocytes caused a 3-fold increase in Ca(2+) in response to CRP, which was absent in X-linked immunodeficiency (Xid) mice, which have a mutation in the PH domain of Btk. There was a corresponding partial reduction in the sustained level of intracellular Ca(2+) in response to CRP in Xid mice but no change in PLC activity. These results demonstrate a novel pathway of Ca(2+) entry that involves PI3,4,5P(3) and Btk, and which is independent of increased PLC activity.

A collagen-related peptide regulates phospholipase Cgamma2 via phosphatidylinositol 3-kinase in human platelets.

The collagen receptor glycoprotein VI (GPVI) induces platelet activation through a similar pathway to that used by immune receptors. In the present study we have investigated the role of phosphatidylinositol 3-kinase (PI 3-kinase) in GPVI signalling. Our results show that collagen-related peptide {CRP: [GCP*(GPP*)(10)GCP*G](n); P*=hydroxyproline}, which is selective to GPVI, induces formation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] and phosphatidylinositol 3,4-bisphosphate [PI(3, 4)P(2)] in platelets. The increase in the two 3-phosphorylated lipids is inhibited completely by wortmannin and by LY294002, two structurally unrelated inhibitors of PI 3-kinase. The formation of inositol phosphates and phosphatidic acid (PA), two markers of phospholipase C (PLC) activation, by CRP are inhibited by between 50 and 85% in the presence of wortmannin and LY294002. This is associated with inhibition of elevation of intracellular Ca(2+) ([Ca(2+)](i)) and aggregation. Wortmannin and LY294002 also partially inhibit elevation of Ca(2+) by CRP in murine megakaryocytes. Microinjection of the pleckstrin-homology PH domain of Bruton's tyrosine kinase, which binds selectively to PI(3,4, 5)P(3), but not the R28C (Arg(28)-->Cys) mutant which binds to PI(3, 4,5)P(3) with low affinity, also inhibits elevation of [Ca(2+)](i) in megakaryocytes, suggesting that it is this lipid species which mediates the action of the PI 3-kinase pathway. Studies in platelets show that the action of wortmannin and LY294002 is not mediated through an alteration in tyrosine phosphorylation of PLCgamma2. These results demonstrate that PI 3-kinase is required for full activation of PLCgamma2 by GPVI in platelets and megakaryocytes.

Characterization of the 3' end of the mouse SERCA 3 gene and tissue distribution of mRNA spliced variants.

The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) type 1 and 2 genes are alternatively spliced at their 3' end. We hypothesized that similar mechanism may occur for SERCA 3. Two spliced variants were identified by RNase protection analysis. We then isolated and sequenced the 3' end portion of the mouse SERCA 3 gene, and confirmed the presence of an alternative mRNA transcript by sequencing a cDNA fragment obtained by RT-PCR. Tissue distribution of the alternatively spliced mRNAs was studied by RT-PCR: SERCA 3b was the only isoform expressed in endothelial cells from aorta and heart and also was the major isoform in lung and kidney whereas SERCA 3a and 3b were coexpressed in trachea, intestine, thymus, spleen, and fetal liver.

Platelet sarco/endoplasmic reticulum Ca2+ATPase isoform 3b and Rap 1b: interrelation and regulation in physiopathology.

Platelet Ca2+ signalling involves intracellular Ca2+ pools, whose content is controlled by sarco/endoplasmic reticulum Ca2+ATPases (SERCAs). Among these, a key role is played by the inositol trisphosphate-sensitive Ca2+ pool, associated with the SERCA 3b isoform. We have investigated the control of this Ca2+ pool through the cAMP-dependent phosphorylation of the GTP-binding protein, Rap (Ras-proximate) 1b. We first looked for this Ca2+ pool target of regulation by studying the expression of the different SERCA and Rap 1 proteins in human platelets and various cell lines, by Western blotting and reverse transcription-PCR. Since co-expression of Rap 1b and SERCA 3b was obtained, we looked for their protein-protein interaction as a function of the cAMP-dependent phosphorylation of Rap 1b. Co-immunoprecipitations of SERCA 3b and Rap 1b proteins were found in the absence of phosphorylation, induced by the catalytic subunit of the cAMP-dependent protein kinase (csPKA). In contrast, upon pre-treatment of platelet membranes with csPKA, the SERCA 3b dissociated from the Rap 1b protein, in agreement with a role of its phosphorylated state in their interaction. Finally, we looked for adaptation of this complex in a platelet pathological model of hypertension. We investigated the expression of both proteins, as well as the cAMP-dependent phosphorylation of Rap 1b and SERCA 3b activity in platelets from control normotensive Wistar-Kyoto rats and from spontaneously hypertensive rats (SHRs). A decrease in SERCA 3b activity was associated with a decrease in Rap 1b endogenous phosphorylation in SHR platelets, consistent with a functional role in the regulation of the SERCA 3b-associated Ca2+ pool.

Expression of two isoforms of the third sarco/endoplasmic reticulum Ca2+ ATPase (SERCA3) in platelets. Possible recognition of the SERCA3b isoform by the PL/IM430 monoclonal antibody.

Human platelets express several sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) isoenzymes: SERCA2b of 100 kDa apparent molecular mass and two distinct enzymes of 97 kDa, one of them identified as being the SERCA3a isoform. The molecular identity of the third enzyme specifically recognized by the PL/IM430 monoclonal antibody has remained elusive. First, the study of the 3'-end part of platelet SERCA3 mRNA, by means of RT-PCR amplification using sets of primers covering the N-3 to N (ultimate) exons of the human SERCA3 sequence, revealed the presence of two distinct mRNA sequences, SERCA3a and a longer variant. Second, this additional sequence was identified as SERCA3b and found to refer to the insertion of a new exon of 73 bp, located at bp 349 from the beginning of the intronic sequence, linking the penultimate (N-1) exon to the last exon (N) of the human SERCA3 gene. Third, a relationship between the expression of this SERCA3b mRNA and the PL/ IM430 recognizable SERCA protein was observed. SERCA3b mRNA was found to be absent in epithelial HeLa cells not recognized by the PL/IM430 antibody and the expression of this SERCA3b RNA species correlated with that of the SERCA protein recognized by PL/IM430 which was down-modulated in the platelet precursor megakaryocytic CHRF 288-11 cell line as well as upon in vitro lymphocyte activation. Taken together, these results strongly support the notion of the presence of the SERCA3b protein in human cells by showing SERCA3b mRNA in platelets and the fact that the protein corresponding to this mRNA species is very likely the 97 kDa protein recognized by the PL/IM430 antibody.

Modulation of endoplasmic reticulum calcium pump expression during T lymphocyte activation.

Calcium mobilization from intracellular storage organelles is a key component of the second messenger system inducing cell activation. Calcium transport ATPases associated with intracellular calcium storage organelles play a major role in controlling this process by accumulating calcium from the cytosol into intracellular calcium pools. In this study the modulation of the expression of the sarco-endoplasmic reticulum calcium transport ATPase (SERCA) isoenzymes has been studied in lymphocytes undergoing phorbol myristate acetate and ionomycin-induced activation. In several T lymphocyte cell lines a combined treatment by the two drugs resulted in an approximately 90% decrease of the expression of the calcium pump isoform recognized by the PLIM430 isoform-specific antibody, whereas the expression of the SERCA 2b isoform was increased approximately 2-fold. Phorbol ester or ionomycin applied separately was ineffective. In Jurkat T cells the down-modulation of expression of the SERCA isoform recognized by the PLIM430 antibody appeared concomitantly with the induction of interleukin-2 expression and could be inhibited by the immunosuppressant drug cyclosporine-A. These data indicate that T cell activation induces a selective and cyclosporine-A-sensitive modulation of the expression of the SERCA calcium pump isoforms. This reflects a profound reorganization of the calcium homeostasis of T cells undergoing activation and may open new avenues in the understanding of the plasticity of the calcium homeostasis of differentiating cells and in the pharmacological modulation of lymphocyte function.

Immunolocalization of the multi-sarco/endoplasmic reticulum Ca2+ ATPase system in human platelets.

We recently identified a multi-SERCA (sarco/endoplasmic reticulum Ca2+ ATPase) system in haemopoietic cells comprising the SERCA 2b, SERCA 3 and a new monoclonal anti-Ca2+ ATPase antibody (PL/IM 430) recognizable SERCA isoforms. We have now investigated the subcellular localization of these enzymes in human platelets by Western blotting of subcellular membrane fractions and by immunoelectron microscopy. We precisely defined the recognition specificity of the polyclonal anti-SERCA 2b, anti-SERCA 3, anti-SERCA 1 antibodies as well as of the monoclonal antibody PL/IM 430 by testing their recognition of the tryptic fragments of the SERCA isoforms. The analysis of fragmented membranes enriched in plasma membrane and intracellular membrane components by Western blotting showed that the SERCA 2b and the SERCA 3 isoforms were found in both the plasma membrane and the intracellular membrane fractions, whereas the PL/IM 430 recognizable SERCA isoform was restricted to membranes associated with the plasma membrane fraction. The immunoelectron microscopical study of the SERCA isoforms in resting platelets showed that: (i) the SERCA 2b isoform was expressed in membranes associated with the plasma membrane and open canalicular system, some alpha-granules and in unidentified membranes; (ii) the SERCA 3 isoform was found associated with plasma and intracellular membranes; and (iii) the PL/IM 430 recognizable SERCA isoform was observed only in structures associated with the cytoplasmic face of the plasma membranes, as confirmed by flow cytometry. Finally, since the PL/IM 430 antibody was raised against intracellular membranes, we looked for a potential membrane redistribution during the isolation procedure used for the preparation of the immunizing membranes. Neuraminidase treatment indeed induced a translocation of the PL/IM 430 recognizable SERCA isoform from plasma to intracellular membranes. Thus, the multi-SERCA system in platelets: (i) is distributed over different platelet membranes, (ii) presents a sub-compartmental organization with some overlapping, and (iii) is partly associated with motile membranes, reflecting an unrecognized level of complexity of Ca2+ stores in these cells.

The platelet Ca2+ transport ATPase system.

The Ca2+ signal accompanying cell function involves the activities of plasma membrane Ca2+ transport ATPases (PMCA) which transport Ca2+ ions out of the cell and those of sarco/endoplasmic reticulum Ca2+ transport ATPases (SERCA), which pump Ca2+ ions into intracellular Ca2+ pools. Although a platelet Ca2+ transport ATPase was described three decades ago, for a long time it remained poorly understood in terms of its cellular localization and identity. By integrating data obtained during recent years, including newly available information in the literature for the PMCAs and aspects of our work concerning the SERCAs, the present review will show how the overall view of the platelet Ca2+ATPase system has to be modified due to the presence of a number of Ca2+ATPases in these cells. These Ca2+ATPases include a typical 144 kDa PMCA protein, although its molecular identity still remains to be established, expressed together with a multi-SERCA system constituted by the ubiquitous 100 kDa SERCA 2b isoform, the 97 kDa SERCA 3 isoform and a new 97 kDa SERCA isoform recognized by the monoclonal antibody termed PL/IM 430 which also remains to be identified. The new paradigm of the platelet multi-Ca2+ATPase system will be discussed including: (i) the problems solved, as it has now become possible to reconciliate previous contradictory observations and (ii) those which still remain due to the fact that the platelet Ca2+ATPase system is more complex than previously assumed. Finally, to put this complexity of the platelet Ca2+ transport ATPase system into perspective, the biological significance of the multi-SERCA system in the context of Ca2+ signalling will be tentatively discussed in an attempt to produce a model of the organization of the intracellular Ca2+ pools in platelets.

Differential up-regulation of Rap1a and Rap1b proteins during smooth muscle cell cycle.

The relationship between Rap1 proteins and cell proliferation was assessed by investigating the effect of isoforms AA and BB of platelet-derived growth factor (PDGF) on Rap1 protein and mRNA expression throughout the smooth muscle cell cycle. Firstly, PDGF BB-induced cell cycle traverse was studied, thus demonstrating entry into S phase at 18 to 20 h. Western blotting carried out on total Rap1 proteins showed that 5 ng/ml of PDGF BB instigated a biphasic induction of total Rap1 proteins during the cell cycle. This involved a 2.1 +/- 0.4-fold increase at 6 h (early G1) and a 2.8 +/- 0.6-fold increase at 20 to 24 h (G1/S transition). Such an up-regulation was abolished by addition of 1 ng/ml of transforming growth factor-beta 1 (TGF-beta 1), which inhibited up to 80% of the PDGF BB-induced entry into S phase. Comparative RT-PCR of both rap1a and rap1b mRNAs throughout the cell cycle allowed us to differentiate between the two rap1a and rap1b species. PDGF BB induced a 1.9 +/- 0.3-fold increase at 4 h and a 2.4 +/- 0.2-fold relative increase at 16 h for rap1b mRNA, whereas a unique 1.9 +/- 0.5-fold increase in rap1a mRNA was observed at 14 h. Again, this induction of rap1a and rap1b mRNAs by PDGF BB was totally abolished by TGF-beta 1. We conclude that the differential up-regulation of Rap1a and Rap1b proteins during the smooth muscle cell cycle is directly linked to cell proliferation.

A sarco/endoplasmic reticulum Ca(2+)-ATPase 3-type Ca2+ pump is expressed in platelets, in lymphoid cells, and in mast cells.

An organellar-type of Ca2+ pump formerly detected by means of its phosphoprotein intermediate in platelets and in lymphoid cells, and which runs in acid gels at 97 kDa, is now characterized as sarco/endoplasmic reticulum Ca2+ATPase 3 (SERCA3). SERCA3 is co-expressed in these cells along with the housekeeping SERCA2b. This conclusion is based on the following observations. 1) Tryptic digestion the phosphoprotein intermediate of SERCA3 expressed in COS cells yields a phosphorylated fragment of about 80 kDa, which can be clearly distinguished from the 57-kDa fragments formed in the SERCA1 and SERCA2 pumps. This 80-kDa fragment comigrates with a similar phosphoprotein fragment previously observed in human platelets (Papp, B., Enyedi, A., Pászty, K., Kovács, T., Sarkadi, B., Gárdos, G., Wuytack, F., and Enouf, J. (1992) Biochem. J. 288, 297-302). 2) An antiserum directed against an NH2-terminal SERCA3-specific peptide (N89) reacts with SERCA3 expressed in COS cells and with the 97-kDa protein in rat platelets and the corresponding protein in human platelets. Likewise an antiserum against the rat SERCA3 terminus (C90) binds to SERCA3 expressed in COS cells and to the 97-kDa band in rat platelets, but it does not recognize the human platelet pump. In conformity with the predicted absence of the T1 tryptic cleavage site in SERCA3, the autophosphorylated aspartyl residue and the COOH-terminal epitope were co-localized on the 80-kDa fragment. 3) The co-expression of nearly equal levels of SERCA3 and SERCA2b messengers in human lymphoblastoid Jurkat cells and in proliferating rat mucosal mast cells was also demonstrated by reverse transcriptase polymerase chain reaction.

The rat platelet 97-kDa Ca2+ATPase isoform is the sarcoendoplasmic reticulum Ca2+ATPase 3 protein.

We recently showed that human and rat platelets express two types of SERCAs (Sarco Endoplasmic Reticulum Ca2+ATPases): a 100-kDa SERCA2b isoform and a 97-kDa SERCA isoform. Here, we explored the possibility that the rat 97-kDa isoform is identical to the SERCA3 protein. For this purpose, we first attempted to detect SERCA3 mRNA in rat platelet total RNA by reverse transcription-polymerase chain reaction using SERCA3-specific primers, and demonstrated the presence of this mRNA species by sequencing the amplification product. We then searched for a relationship between the expression of the SERCA3 mRNA and of the 97-kDa protein using either rat aortic smooth muscle cells, previously found not to express the 97-kDa SERCA isoform (negative model), or platelets of spontaneously hypertensive rats (SHR), which overexpress this isoform (overexpression model) but express the 100-kDa SERCA2b isoform normally. No expression of SERCA3 mRNA was detectable by analysis of smooth muscle cell RNA, but comparison by reverse transcription-polymerase chain reaction of the SERCA2b and SERCA3 mRNAs from the platelets of normotensive (Wistar-Kyoto, WKY) rats and SHR clearly demonstrated a 238 +/- 43% increase in the expression of the SERCA3 mRNA in SHR platelets only. Last, by comparative Western blotting of WKY rat and SHR platelet membranes using a recently developed polyclonal anti-SERCA3 antibody, we established that the 97-kDa SERCA and the SERCA3 protein are identical, as immunostaining of the 97-kDa protein revealed a 230 +/- 25% increase in the expression of this protein in SHR versus WKY rat platelets. It is concluded that the 97-kDa platelet SERCA isoform, which is up-regulated in SHR, is the SERCA3 protein. As far as we know, this constitutes the first demonstration of the actual presence of this Ca2+ATPase isoform in normal cells, in addition to the artificial transfection systems.