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PubMedDate not supplied

A proposed structure of bacteriophage T4 gene product 22--a major prohead scaffolding core protein.

Gene 22 of bacteriophage T4 encodes a major prohead scaffolding core protein of 269 amino acid residues. From its nucleotide sequence the gene product (gp) 22 has a predicted Mr of 29.9 and a pI of 4.3. The protein is rich in charged residues (glutamic acid and lysine) and contains low amounts of proline and glycine and no cysteine residues. We suggest that gp22 undergoes limited proteolytic processing which eliminates the short C-terminal piece from the molecule during the early steps of prohead assembly. Most amino acid residues of the gp22 polypeptide chain (80%) have an alpha-helical conformation and form seven peculiar alpha-helices. A model suggesting the spatial organization of gp22 is presented. Three long alpha-helices numbered 1 (1A and 1B), 3, and 5 (5A and 5B) are packed in an antiparallel fashion along the major axis of the road-shaped molecule. Two rather short alpha-helices (2 and 4) are located at the distal and proximal ends of the protein molecule, respectively. Helix number 2, which is a proteolytic fragment of gp22 found in mature T4 heads, is packed with helices 1A and 3, similar to a novel element of supersecondary structure, the alpha alpha-corner. Helix number 4 probably interacts with the gp20 connector of the prohead. The implications of the structure of the gp22 molecule for the assembly of the prohead core are discussed.

Amino Acid Sequence↗

PubMed2000

Rifampicin-dependent antibodies bind a similar or identical epitope to glycoprotein IX-specific quinine-dependent antibodies.

The drug-dependent antibody of a patient with rifampicin-induced thrombocytopenia was characterized using the antigen-capture enzyme-linked immunosorbent assay (MAIPA assay), flow cytometry, and immunoprecipitation. The antibody was found to bind glycoprotein (GP) Ib-IX but not GPIIb-IIIa because (1) it immunoprecipitated drug-dependently the former but not the latter glycoprotein complex and (2) the MAIPA assay showed strong rifampicin-dependent antibody binding when anti-GPIb-IX monoclonal antibodies (mAbs) (AK2 and FMC25) but not anti-GPIIb-IIIa mAbs (AP2, SZ21, and SZ22) were used to capture the antigen. The antibody binding site was further localized to the GPIX subunit of the GPIb-IX complex because flow cytometric analysis revealed drug-dependent antibody binding to L cells transfected with human GPIbbeta and GPIX complementary DNA (L betaIX cells) but not with human GPIbalpha and GPIbbeta complementary DNA (L alphabeta cells). Finally, in the MAIPA assay, the rifampicin-dependent antibody almost completely cross-blocked the binding of the anti-GPIX mAb (SZ1) to platelets. Similar cross-blocking of SZ1binding to platelets by the quinine-dependent antibodies was also observed. This finding not only confirms that the epitope of the rifampicin-dependent antibody is on GPIX but it is also identical to or located in close proximity to that of the quinine-dependent antibody and SZ1. Further characterization of the epitopes of these antibodies may have important implications for a general understanding of the mechanism of drug-induced thrombocytopenia. (Blood. 2000;95:1988-1992)

Aged↗

PubMed1995

Sequence diversification and exon inactivation in the glycophorin A gene family from chimpanzee to human.

In humans, the allelic diversity of MNSs glycophorins (GP) occurs mainly through the recombinational modulation of silent exons (pseudoexons) in duplicated genes. To address the origin of such a mechanism, structures of GPA, GPB, and GPE were determined in chimpanzee, the only higher primate known to have achieved a three-gene framework as in humans. Pairwise comparison of the chimpanzee and human genes revealed a high degree of sequence identity and similar exon-intron organization. However, the chimpanzee GPA gene lacks a completely formed M- or N-defining sequence as well as a consensus sequence for the Asn-linked glycosylation. In the case of the GPB gene, exon III is expressed in the chimpanzee but silenced, as a pseudoexon, in the human. Therefore, the protein product in the chimpanzee bears a larger extracellular domain than in the human. For the GPE genes, exon III and exon IV have been inactivated by identical donor splice-site mutations in the two species. Nevertheless, the chimpanzee GPE-like mRNA appeared to be transcribed from a GPB/E composite gene containing no 24-bp insertion sequence in exon V for the transmembrane domain. These results suggest a divergent processing of exonic units from chimpanzee to human in which the inactivation of GPB exon III preserved a limited sequence repertoire for diversification of human glycophorins.

Amino Acid Sequence↗

PubMed2006

Suppressor of cytokine signalling-3 at pathological levels does not regulate lipopolysaccharide or interleukin-10 control of tumour necrosis factor-alpha production by human monocytes.

Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine that suppresses the production of tumour necrosis factor-alpha (TNF-alpha) by monocytes and macrophages. Suppressor of cytokine signalling-3 (SOCS3), a negative regulator of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, is induced following IL-10 exposure but recent studies in mice suggest that SOCS3 only targets gp-130-dependent signal transduction pathways. Understanding the signalling pathways responsible for IL-10-mediated effects in primary human monocytes is relevant to human inflammatory disease and necessary for the identification of potential therapeutic targets. An adenoviral transfection system was used to express different levels of SOCS3 (quantified experimentally with its tag green fluorescent protein (GFP)) with the aim of investigating the role of SOCS3 in LPS-induced and IL-10-mediated suppression of TNF-alpha production by non-transformed human monocytes. SOCS3 over-expression had no effect on TNF-alpha mRNA levels induced by LPS or LPS plus IL-10, or on IL-10 phosphorylation of STAT3, STAT1 and ERK1/2. When data from all donors were combined, adenoviral overexpression of SOCS3 significantly reversed the suppressive effects of IL-10 on LPS-induced TNF-alpha production after 2 hr. However, there was a direct correlation between mean GFP intensity (extent of viral infection) and extent of reversal of IL-10's inhibitory effects. Physiological levels of SOCS3 detected in IL-10-exposed human monocytes had no effect on LPS-induced TNF-alpha production. Although overexpression of SOCS3 to supraphysiological levels transiently antagonized the regulatory properties of IL-10 by a post-transcriptional mechanism, these findings suggest that under pathological conditions SOCS3 does not control LPS-activation or the anti-inflammatory properties of IL-10 in primary human monocytes.

Adenoviridae↗

PubMed1993

Determination of human platelet antigen frequencies in the Dutch population by immunophenotyping and DNA (allele-specific restriction enzyme) analysis.

Platelets from 200 random Dutch blood donors were typed for the human platelet alloantigens HPA-1 to -5 recognized at present and for Naka. Naka is an epitope on glycoprotein IV, not expressed on the platelet of individuals with hereditary GP IV deficiency. Platelet immunofluorescence and monoclonal antibody-specific immobilization of platelet antigens (MAIPA) were applied for this purpose. The observed phenotype frequencies were 97.86% and 28.64% for HPA-1a and -1b, 100% and 13.15% for HPA-2a and -2b, 80.95% and 69.84% for HPA-3a and -3b, 100% and 0% for HPA-4a and -4b, 100% and 19.7% for HPA-5a and HPA-5b, respectively. Platelets from all donors reacted with the anti-Naka antibodies. To determine the gene frequencies for the HPA-1, HPA-2 and HPA-3 systems directly, DNA from 98 of these donors was isolated from peripheral blood mononuclear leucocytes and specific fragments were amplified by polymerase chain reaction (PCR). The fragments were analyzed using allele-specific restriction enzymes (ASRA). In all amplified PCR products an "internal control" for each assay, ie, a restriction site for the applied enzyme independent from the phenotype of the donor was present. In all donors tested, phenotypes, as determined by serological methods and genotypes, directly determined by the ASRA, were identical. Thus, the PCR-ASRA described in this report is a practical and reliable technique for the determination of alleles that code for platelet antigen allotypes, at least in the Dutch population.

Alleles↗

PubMed2003

Glycoprotein IIb/IIIa inhibition attenuates platelet-activating factor-induced platelet activation by reducing protein kinase C activity.

Glycoprotein (GP)IIb/IIIa inhibition may abolish activated leukocyte-induced platelet activation, in which leukocyte-released platelet-activating factor (PAF) is a major mediator. The present study thus investigated if and how GPIIb/IIIa inhibitors interfere with PAF-induced platelet activation. Platelet and leukocyte activation were monitored by flow cytometry and immunoblotting. GPIIb/IIIa inhibitors (c7E3, non-peptide SR121566, and MAb RFGP56) attenuated PAF-induced, but not adenosine diphosphate (ADP)- or thrombin receptor activating peptide (TRAP)-induced platelet P-selectin expression in whole blood. GPIIb/IIIa blockade enhanced ADP- or TRAP-induced leukocyte CD11b expression, but not the response to PAF. GPIIb/IIIa blockade attenuated PAF-induced, but enhanced ADP- or TRAP-induced platelet-leukocyte aggregation. Under the present experimental conditions, thromboxane A2 receptor antagonism did not significantly influence PAF-induced platelet activation, and GPIIb/IIIa inhibition did not interfere with calcium mobilization/influx in platelets. Protein kinase C (PKC) blockade inhibited PAF-induced platelet P-selectin expression, and PAF-induced PKC activity was reduced by GPIIb/IIIa inhibition. PAF (=1 micro m) did not induce MEK 1/2 or ERK 1/2 phosphorylation, whilst thrombin induced marked responses, which were enhanced by GPIIb/IIIa blockade. Thus, GPIIb/IIIa inhibition attenuates PAF-induced platelet activation via inhibiting PKC activity. GPIIb/IIIa blockade enhances thrombin-induced platelet MEK 1/2 and ERK 1/2 activation, and augments ADP- and TRAP-induced leukocyte activation by enhancing platelet-leukocyte aggregation.

Adenosine Diphosphate↗

PubMed1994

Identification of a regulatory domain of the interleukin-6 receptor.

IL-6 signal transduction occurs when the liganded interleukin-6 receptor (IL-6R) interacts with glycoprotein (gp) 130. We hypothesized that synthetic peptides modeled from the extramembranous domain of the IL-6R may interfere with the IL-6-induced reaction between IL-6R and gp130 and may serve to elucidate the initial steps in IL-6 signal transduction. The capacity of such peptides to modulate two different IL-6 functions was evaluated: 1) IL-6-dependent B9 cell mitogenesis, and 2) IL-6-induced acute phase protein synthesis in HepG2 cells. A synthetic peptide, 249Y16T264, corresponding to residues 249-264, inhibited IL-6-dependent B9 proliferation and IL-6-induced acute phase protein up-regulation in HepG2 cells. Other peptides modeled from different regions of the IL-6R were not inhibitory. 249Y16T264 did not inhibit IL-6-independent HepG2 cell proliferation or total cellular protein synthesis. The inhibitory effect was reversible, indicating that the peptide was not cytotoxic. 249Y16T264 did not inhibit 125I-IL-6 binding in U266 cells. Delineation of this domain identified 249Y10R258 as the minimum effective sequence capable of inhibiting fibrinogen synthesis. Amino acid substitutions in 249Y10R258 obliterated the inhibitory effect on fibrinogen synthesis. In conclusion, a region of the extramembranous domain of the IL-6R has been identified that is involved in the regulation of IL-6 signal transmission. A synthetic peptide representing this region inhibits IL-6-dependent B9 cell mitogenesis and IL-6-stimulated acute phase response in HepG2 cells without affecting ligand binding.

Acute-Phase Proteins↗

PubMed2006

Thrombin induces GPIb-IX-mediated fibrin binding to alphaIIbbeta3 in a reconstituted Chinese hamster ovary cell model.

BACKGROUND: The interaction of thrombin with platelet glycoprotein (GP) Ib-IX-V has been recently suggested to induce fibrin-dependent platelet aggregation associated with signaling events. The approaches used to avoid the protease-activated receptor (PAR) thrombin receptors in platelets have provided controversial conclusions regarding the precise mechanism and molecules involved in the response. OBJECTIVES: In the present study, we developed a cellular model to investigate the functional consequences following the binding of thrombin to GPIb-IX. METHODS: We used Chinese hamster ovary (CHO) cells stably expressing human alpha(IIb)beta(3) and/or GPIb-IX complexes (CHO-alpha(IIb)beta(3)-IbIX cells) to analyze the effect of thrombin on the binding of polymerizing fibrin by using fluorescein isothiocyanate-fibrinogen as precursor. RESULTS: Thrombin induces, in a dose-dependent manner, the binding of polymerizing fibrin to CHO-alpha(IIb)beta(3)-IbIX cells. This response is not observed in cells expressing only one of the receptors, and it can be blocked by monoclonal antibodies against alpha(IIb)beta(3) and GPIbalpha. We show that the reaction is not due to simple cell trapping by the fibrin clot, and provide data supporting a role of a signaling pathway in which the 14-3-3zeta adaptor and calcium-calmodulin-dependent events are involved. CONCLUSIONS: The present data support a significant role of GPIb-IX and alpha(IIb)beta(3) receptors in an alternative fibrin-mediated pathway of platelet activation induced by thrombin.

Animals↗

PubMed1994

Remodeling of the transmembrane segment in human glycophorin by aberrant RNA splicing.

This paper describes the identification in S-s-U-erythrocytes of a novel glycophorin (GP), He(P2), with structural variations in both its extracellular and transmembrane domains. In the exon II-intron 2 region, a sequence transfer from GPA to GPB, probably via the mechanism of gene conversion, was associated with the induction of multiple untemplated nucleotide replacements. These changes defined the sequence for the He epitope while concomitantly abolishing GPB-associated N antigenicity. Moreover, the GPHe(P2) gene carries two splice site mutations that coordinately affect the processing of exon V coding for the transmembrane segment. The C-->G transversion at the 3' end of exon V created a cryptic acceptor splice site, whereas the G-->T transversion at the +5 position of intron 5 altered the consensus of the donor splice site. Transcript sequencing revealed that neither site was utilized in the splicing of GPHe(P2) pre-mRNA. Rather, complete skipping of exon V and subsequent joining of exon IV to exon VI caused a shift in the open reading frame, which remodeled GPHe(P2) with an elongated new hydrophobic sequence for membrane anchoring. As a result, GPHe(P2) does not display the S and U epitopes although it still contains an intact linear sequence for the two antigens. These findings illustrate how exon and intron sequences concertedly determine the specificity of in vivo splice site selection. In addition, they pinpoint the conformational dependence of the S, s, and U antigens and the importance of the hinge region for their presentation.

Amino Acid Sequence↗

PubMed1998

Poor expression of MDR1 transgene in HeLa cells by bicistronic Moloney murine leukemia virus-based vector.

Cotransfer of a therapeutic gene together with the human MDR1 gene provides an opportunity to increase the number of transduced marrow cells, expressing the therapeutic gene, by in vivo selection for MDR1. We have used an Lg-MDR1-IRES-neo (LgMIN) retroviral vector, containing MDR1 and neo genes, separated by the EMCV IRES. Human HeLa or canine CTAC cells, transduced with GALV env pseudotyped LgMIN at an MOI of less than 0.01 to ensure 1 proviral copy/genome, were selected with either G418 for neo expression or colchicine for MDR1 expression. The titer determined on HeLa cells with G418 selection was eight-fold higher than that with colchicine selection. In contrast, the same viral supernatant exhibited only a 1.4-fold difference between neo- and MDR1-based viral titer values for CTAC cells. The transduced HeLa cells, with one intact proviral copy per genome, exhibited a 55-fold higher resistance to G418 but only a 4-fold higher resistance to colchicine and a 2-fold higher resistance to Taxol compared with nontransduced cells. About 23% of the transduced cell population did not express vector-derived P-glycoprotein (P-gp) as detected by anti-human P-gp MAb MRK-16. This could explain the difference in viral titers obtained on CTAC cells but not that obtained on HeLa cells. The vector-mediated increase in expression of P-gp was about 20-fold higher in CTAC cells as compared with HeLa cells. These results indicated suppression of expression of vector-derived MDR1 in HeLa cells, in contrast with CTAC cells. To investigate further the possible reasons for this difference, genomic DNA was isolated from the G418-resistant individual colonies of infected cells and analyzed by PCR for full-length proviral MDR1. For transduced CTAC and HeLa cells, selected at a G418 concentration of 1 mg/ml, PCR detected aberrant forms of MDR1 in 17 to 25% of colonies tested. The aberrant forms consisted of MDR1 genes with 2- and 0.7-kb deletions. DNA sequencing across the 2-kb and the 0.7-kb deletion junction suggests cryptic splicing in the producer cell line as the origin of these deletions. The 2-kb deletion corresponds to MDR1 mRNA cryptic splicing via donor (codon 113) and acceptor (codon 773). The 0.7-kb deletion corresponds to splicing via the same donor and a different acceptor (codon 344). When transduced HeLa cells were selected at a higher concentration of G418 (3 mg/ml), the aberrant forms were detected at an increased frequency of about 50% of colonies tested. These results indicate that vector-derived MDR1 is a poor selective marker in HeLa cells but not in CTAC cells and that deletions, which inactivated the MDR1 gene in a bicistronic Mo-MuLV vector, may provide an advantage for expression of the second transgene in HeLa cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2002

Interaction between von Willebrand factor and glycoprotein Ib activates Src kinase in human platelets: role of phosphoinositide 3-kinase.

The binding of von Willebrand factor (VWF) to glycoprotein (GP) Ib-IX-V stimulates transmembrane signaling events that lead to platelet adhesion and aggregation. Recent studies have implied that activation of Src family kinases is involved in GPIb-mediated platelet activation, although the related signal transduction pathway remains poorly defined. This study presents evidence for an important role of Src and GPIb association. In platelet lysates containing Complete, a broad-spectrum protease inhibitor mixture, Src and Lyn dynamically associated with GPIb on VWF-botrocetin stimulation. Cytochalasin D, which inhibits translocation of Src kinases to the cytoskeleton, further increased Src and GPIb association. Similar results were obtained with botrocetin and monomeric A1 domain, instead of intact VWF, with induction of both Src activation and association between GPIb and Src. These findings suggest that ligand binding of GPIb, without receptor clustering, is sufficient to activate Src. Immunoprecipitation studies demonstrated that Src, phosphoinositide 3- kinase (PI 3-kinase), and GPIb form a complex in GPIb-stimulated platelets. When the p85 subunit of PI 3-kinase was immunodepleted, association of Src with GPIb was abrogated. However, wortmannin, a specific PI 3-kinase inhibitor, failed to block complex formation between Src and GPIb. The Src-SH3 domain as a glutathione S-transferase (GST)-fusion protein coprecipitated the p85 subunit of PI 3-kinase and GPIb. These findings taken together suggest that the p85 subunit of PI 3-kinase mediates GPIb-related activation signals and activates Src independently of the enzymatic activity of PI 3- kinase.

Androstadienes↗

PubMed1989

Human platelet glycoprotein IX: an adhesive prototype of leucine-rich glycoproteins with flank-center-flank structures.

The glycoprotein (GP) Ib-IX complex on the surface of human platelets functions as the von Willebrand factor receptor and mediates von Willebrand factor-dependent platelet adhesion to blood vessels. GPIX is a relatively small (Mr, 17,000) protein that may provide for membrane insertion and orientation of the larger component of the complex, GPIb (Mr, 165,000). Using antibody screening, we cloned a cDNA encoding GPIX from a human erythroleukemia cell cDNA library constructed in phage lambda gt11. Lacking a 5' untranslated region and start codon, the cDNA sequence includes 604 nucleotides, beginning with 495 bases at the 5' end coding for 165 amino acids, followed by a stop codon and 106 noncoding bases at the 3' end. By Northern blot analysis, the GPIX cDNA hybridizes with a single 1.0-kilobase species of platelet poly(A)+ RNA. Translation of the cDNA sequence gives a predicted protein sequence beginning with a truncated putative signal sequence of 5 amino acid followed by a sequence of 17 amino acids matching that determined directly by Edman degradation of intact GPIX. The predicted amino acid sequence of mature GPIX includes an NH2-terminal extracytoplasmic domain of 134 residues, a transmembrane domain of 20 residues, 6 intracytoplasmic residues, and 1 N-linked glycosylation site. GPIX contains a leucine-rich glycoprotein (LRG) sequence of 24 amino acids similar to conserved LRG sequences in GPIb and other proteins from humans, Drosophila, and yeast. "Flanking" sequences of approximately 22 amino acids are present at the NH2 and/or COOH sides of the "central" LRG sequence(s) in GPIX, GPIb, and the other human and Drosophila members of the LRG family. The role of the flank-LRG center-flank structure in the evolution and function of the LRG proteins remains to be defined.

Amino Acid Sequence↗

PubMed1990

The genomic organization of platelet glycoprotein IIIa.

The platelet membrane glycoprotein (GP) IIb/IIIa complex, a member of the integrin family of adhesive receptors involved in cell-cell and cell-matrix interactions, contains binding sites for fibrinogen, von Willebrand factor, fibronectin, and vitronectin. Absence or defects of this receptor result in the platelet bleeding disorder Glanzmann's thrombasthenia. In this report, we describe the isolation of genomic DNA coding for the entire mature GPIIIa protein. Mature GPIIIa is encoded by 14 exons which range in length from 90 to 3618 base pairs, which are contained within an approximately 46-kilobase (kb) stretch of genomic DNA on chromosome 17. All of the exon/intron junctions were found to conform to the consensus splice donor and acceptor sequences. The coding region of the GPIIIa gene is identical with the previously described cDNA sequence except for three silent substitutions. One substitution creates a TaqI site which may be the site of a known GPIIIa polymorphism. A second substitution eliminates a SmaI site. Aside from the start of the first exon described, which begins at the second base of the first codon of the mature protein, there is no correlation between the organization of the exons in this gene and proposed functional domains of the protein based on analysis of the primary amino acid sequence. The less frequently used polyadenylation signal AAATTAAA was present at the 3'-end of the major RNA transcript. Recently, an alternatively processed GPIIIa transcript has been described. We demonstrate that this transcript results from nonsplicing of the final intron. The description of the GPIIIa gene organization should be of importance in understanding the evolution of the integrin family of receptors and should be useful in the molecular biology analysis of thrombasthenic patients who have a defect in the GPIIIa gene.

Amino Acid Sequence↗

PubMed2003

Role of the intracellular domains of GPIb in controlling the adhesive properties of the platelet GPIb/V/IX complex.

Glycoprotein (GP) Ib/V/IX complex-dependent platelet adhesion to von Willebrand factor (VWF) is supported by the 45-kd N-terminal extracellular domain of the GPIb alpha subunit. Recent results with an adhesion blocking antibody (RAM.1) against GPIb beta, which is disulfide linked to GPIb alpha, have suggested a novel function of this subunit in regulating VWF-mediated platelet adhesion, possibly involving its intracellular face. A putative cooperation between the GPIb alpha and GPIb beta cytoplasmic domains was investigated by measuring the adhesion under flow to immobilized VWF of K562 and Chinese hamster ovary (CHO) cells transfected with GPIb/(V)/IX containing mutations in this region. Adhesion of cells carrying a glycine substitution of the GPIb beta Ser166 phosphorylation site was 50% lower than normal and became insensitive to inhibition by RAM.1. In contrast, forskolin or PGE(1) treatment increased both the phosphorylation of GPIb beta and adhesion of control cells, both effects being reversed by RAM.1, but had no influence on cells expressing the Ser166Gly mutation. A role of the GPIb alpha intracellular domain was also apparent as the VWF-dependent adhesion of cells containing deletions of the entire (Delta 518-610) or portions (Delta 535-568, Delta 569-610) of the GPIb alpha cytoplasmic tail was insensitive to RAM.1 inhibition. Cells carrying progressive 11 amino acid deletions spanning the GPIb alpha 535-590 region were equally unresponsive to RAM.1, with the exception of those containing GPIb alpha Delta 569-579, which behaved like control cells. These findings support a role of the GPIb beta intracellular domain in controlling the adhesive properties of the GPIb/V/IX complex through phosphorylation of GPIb beta Ser166 and point to the existence of cross-talk between the GPIb beta and GPIb alpha intracellular domains.

Alprostadil↗

PubMed1997

Gene cloning of rat and mouse platelet glycoprotein V: identification of megakaryocyte-specific promoters and demonstration of functional thrombin cleavage.

Platelet glycoprotein (GP) V is a major surface protein cleaved during thrombin-induced platelet activation. GPV associates noncovalently with the GPIb-IX complex to form GPIb-V-IX, a receptor for von Willebrand factor and thrombin. We describe the cloning of the genes coding for rat and mouse GPV and compare them with the human gene. The two rodent genes have a similar structure and resemble the human GPV gene with a coding sequence (approximately 1,700 nucleotides) entirely contained in one exon and a single intron (approximately 900 nucleotides) in the 5' untranslated region. Both genes have megakaryocyte-type promoters with conserved tandem Ets and GATA recognition motifs and lack a TATA box. The mature rat and mouse proteins comprise 551 amino acids, have 70% sequence identity, and contain an additional 8-amino acid intracellular segment as compared with the human protein. As in human GPV, there is an NH2-terminal leucine-rich region of 15 repeats and a thrombin cleavage recognition sequence. Whereas the rat and human thrombin cleavage sites are similar, the mouse cleavage site resembles that of the human thrombin receptor. Functionality of these sites was demonstrated by thrombin cleavage of synthetic peptides and analysis by high-performance liquid chromatography (HPLC) or mass spectrometry. Cleavage of native rat GPV was confirmed by means of a polyclonal antibody directed against the new NH2-terminal peptide exposed after thrombin cleavage. This antibody specifically recognized thrombin-activated rat platelets by fluorescence-activated cell sorting (FACS) analysis. In addition, we raised monoclonal antibodies specific for rat GPV (88 kD), which recognized the NH2-terminal soluble fragment (70 kD) liberated after thrombin cleavage. Knowledge of these rodent GPV genes and availability of species-specific peptides and antibodies will be essential to further studies aiming to define the exact in vivo function of platelet GPV using animal models of thrombosis and gene inactivation experiments.

Amino Acid Sequence↗

PubMed1985

Primate globus pallidus and subthalamic nucleus: functional organization.

Neuronal relations to active movements of individual body parts and neuronal responses to somatosensory stimulation were studied in the external (GPe) and internal (GPi) segments of the globus pallidus (GP) and the subthalamic nucleus (STN) of awake monkeys. In GPe (n = 249), GPi (n = 151), and STN (n = 153), 47, 29, and 28% of the cells, respectively, discharged in relation to active arm movements, 10, 11, and 15% to leg movements, and 22, 22, and 18% to orofacial movements. Of the neurons whose activity was related to arm movements, 26, 16, and 21% in GPe, GPi, and STN, respectively, discharged in relation to movements of distal parts of the limb. Of cells whose discharge was related to active limb movements, 37, 22, and 20% in GPe, GPi, and STN, respectively, also responded to passive joint rotation, which was usually specific in terms of joint and direction of movement. Only a small percentage of cells responded to muscle or joint palpation, tendon taps, or cutaneous stimulation. Short-latency, direction-specific neuronal responses to load perturbations confirmed the existence of proprioceptive driving. In both GPe and GPi, leg movement-related neurons were centrally located in the rostrocaudal and dorsoventral dimensions. In contrast, arm movement-related cells were found throughout the entire rostrocaudal extent of both segments, although in greater numbers caudally. In the central portions they were situated largely inferior and lateral to leg movement-related neurons. Neurons related to orofacial movements were largely confined to the caudal halves of both segments, where they were located largely ventral to arm movement-related cells. The STN cells whose activity was related to leg movements were observed largely in the central portions of the nucleus in the rostrocaudal and mediolateral dimensions. Cells whose activity was related to arm movements were found throughout the rostrocaudal extent of the nucleus, but were most numerous at the rostral and caudal poles. Neurons related to movements of the facial musculature and to licking and chewing movements were distributed over the entire rostrocaudal extent of the nucleus, where they generally occupied the ventrolateral regions. In all three nuclei, neurons with similar functional properties were sometimes clustered together. Within the arm and leg areas, however, there was no clear evidence for a simple organization of clusters related to different parts of the limb. These studies provide further evidence for a role of the basal ganglia in the control of limb movements.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗