ABSTRACT
Talin is a major cytosolic protein that links the intracellular domains of beta1 and beta3 integrins to the cytoskeleton. It is required for focal adhesion assembly. However, its downregulation not only slows down cell spreading and organization of focal adhesions but also impairs the maturation of some beta1 integrins, including the fibronectin receptor alpha5beta1. To investigate this, we characterized the beta1 integrin synthesized in cells expressing talin anti-sense RNA (AT22 cells). We identified a large intracellular pool of beta1 integrins that is abnormally accumulated in an earlier compartment of the secretory pathway. In this report, we show that in talin-deficient AT22 cells, the aberrant glycosylation of integrin receptors is accompanied by a delay in the export of the integrin alpha5beta1. In normal cells, talin was found associated with beta1 integrins in an enriched membrane fraction containing Golgi and endoplasmic reticulum. Finally, microinjection of anti-talin antibodies resulted in accumulation of the integrins within the cells. These data strongly suggest that talin plays a specific role in the export of newly synthesized integrins. We propose that talin binding to the integrin may disclose a diphenylalanine export signal, which is present in the membrane-proximal GFFKR motif conserved in all integrin alpha chains.
Subject(s)
Cell Compartmentation/physiology , Cytoplasmic Granules/physiology , Receptors, Fibronectin/metabolism , Talin/metabolism , Antisense Elements (Genetics) , Biological Transport/physiology , Cell Membrane/metabolism , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , HeLa Cells , Humans , Microinjections , Microsomes/metabolism , Talin/genetics , Talin/immunologyABSTRACT
With the exception of the divergent beta4 and beta8 chains, the integrin beta subunit cytoplasmic domains are short and highly conserved sequences. Consensus motifs are found among the different cytoplasmic beta chains. Experiments using chimeric receptors demonstrated that the 47 amino acids of the beta1 subunit cytoplasmic domain contain sufficient information to target integrins to adhesion plaques. Three clusters of amino acids, named cyto-1, cyto-2 and cyto-3, seem to contribute to this localization. Cyto-2 and cyto-3 exhibit NPXY motifs. At present, the exact function of these motifs remains unknown but it is likely that these sequences are involved in protein-protein interactions. Although NPXY motifs often act as internalization signals at the cytoplasmic tail of membrane receptors, our previous results showed that the two NPXY motifs are not responsible for the alpha5beta1 integrin endocytosis. Herein, we address the question of the role of the two highly conserved NPXY motifs found in the beta1 cytoplasmic domain, and which correspond to the conserved domains cyto-2 and cyto-3. We demonstrate that, within the integrin beta1 cytoplasmic tail, the two NPXY motifs are required for the recruitment of the integrin in focal adhesions. In addition, our results indicate that these two motifs control but do not belong to the talin-binding sites. Finally, the analysis of the phenotypes of NPXY mutants reveals that the interaction of talin with the beta1 cytosolic domain is not sufficient to target the integrins to focal adhesions.
Subject(s)
Cell Adhesion/physiology , Integrin beta1/metabolism , Receptors, Fibronectin/metabolism , Talin/metabolism , Amino Acid Sequence , Animals , CHO Cells , Cricetinae , Fibronectins/metabolism , Fluorescent Antibody Technique, Indirect , Humans , Integrin beta1/genetics , Microscopy, Electron , Molecular Sequence Data , Mutation , Talin/ultrastructure , TransfectionABSTRACT
The role of talin was addressed by down regulating its expression using an antisense RNA strategy. HeLa cells were transfected with a talin 5' cDNA fragment under the control of the inducible human metallothionein promotor. Isolated clones displayed a decrease in talin level down to 10% of control. The reduction in talin expression dramatically slowed down the kinetics of cell spreading. Mock-transfected cells, spread out onto fibronectin, exhibited large peripheral adhesion plaques. In contrast, cells with reduced talin expression showed smaller focal contacts localized all over the ventral face, and displayed a marked decrease in the number of stress fibers. Immunoprecipitation experiments carried out with a polyclonal antibody on surface-labeled receptor indicated a shift in the mobility for both alpha 5 and beta 1 subunits. Surprisingly, beta 1 integrin chains could not be detected by indirect immunofluorescence using monoclonal antibodies in talin deficient clones. Western blot analysis indicated the presence of two forms of beta 1. We analyzed the processing of beta 1 in normal and talin deficient cells using pulse chase experiments. Normal cells required a minimum of 5 hours for the processing of mature beta 1, while the talin deficient AT22 clone showed that the beta 1 precursor was slowly converted into a very low molecular mass product. Our data demonstrate that talin plays a central role in the establishment of cell-matrix contacts. In addition, down regulation of talin impairs the folding and processing of beta 1 integrins.
Subject(s)
Cell Adhesion , Receptors, Fibronectin/metabolism , Talin/metabolism , Animals , Base Sequence , CHO Cells , Cricetinae , Down-Regulation , Gene Transfer Techniques , HeLa Cells , Humans , Molecular Sequence Data , RNA, Antisense/metabolism , Talin/geneticsABSTRACT
Using Chinese hamster ovary cell lysate, an in vitro assay has been developed to study the interaction of fibronectin with the alpha 5 beta 1 integrin in a cytosolic environment. In our solid phase assay, 96-well microtiter plates were coated with fibronectin in which cell lysate was incubated. A dose-dependent binding of the fibronectin receptor onto the coated plastic was immunodetected by specific polyclonal antibodies raised against the alpha 5 beta 1 integrin. Both soluble fibronectin and PB1, a monoclonal antibody raised against the fibronectin receptor, competed with the alpha 5 beta 1 integrin for binding to the fibronectin-coated plastic. General phosphatase inhibitors used during cell lysis completely abolished the fibronectin/integrin interaction in the assay, indicating that the affinity of the fibronectin receptor might be modulated by a protein phosphatase activity. Furthermore, in this assay, the interaction between the fibronectin receptor and its substrate in a cytosolic environment required intracellular calcium. Additionally, the action of more specific phosphatase inhibitors and the inhibition of the integrin/fibronectin interaction by a monoclonal antibody raised against the calcium/calmodulin-dependent protein phosphatase calcineurin suggested that calcineurin allowed the interaction between the alpha 5 beta 1 integrin and fibronectin. Metabolical labeling experiments showed that alpha 5 beta 1 itself was not the target of phosphorylation/dephosphorylation cascades involving calcineurin and leading to the modulation of integrin affinity. Taken together, these results showed that in vitro one substrate of the serine/threonine protein phosphatase calcineurin regulates the alpha 5 beta 1 integrin affinity by interacting with a yet unidentified effector.
Subject(s)
Calmodulin-Binding Proteins/metabolism , Fibronectins/metabolism , Integrins/metabolism , Phosphoprotein Phosphatases/metabolism , Receptors, Fibronectin/metabolism , Animals , Antibodies, Monoclonal , CHO Cells , Calcineurin , Calcium/metabolism , Cricetinae , Cricetulus , Precipitin Tests , Receptors, Fibronectin/immunologyABSTRACT
Integrins are alpha beta heterodimers that play a major role in cell-cell contacts and in interactions between cells and extracellular matrices. Identification of structural domains that are critical for the expression of such receptors at the cell surface in a functional conformation is one of the major issues that has not yet been resolved. In the present study, the role of the cytoplasmic and transmembrane domains of each of the subunits has been examined using platelet GPIIb/IIIa as a prototypic integrin. GPIIb/IIIa (alpha IIb/beta 3) is a member of the integrin family and functions as a receptor for fibrinogen, fibronectin, von Willebrand factor, and vitronectin at the surface of activated platelets. Human megakaryocyte GPIIb and GPIIIa cDNAs were used to create a GPIIb mutant coding for the extracellular GPIIb heavy chain alone (GPIIb delta 1) and a GPIIIa mutant lacking the transmembrane and cytoplasmic domains (GPIIIa delta m). Full length and mutant cDNAs were subcloned into the expression vector pECE and used to transfect COS cells. The formation of heterodimers and their cellular localization was analyzed by immunoprecipitation and immunofluorescence labeling using anti-platelet GPIIb/IIIa antibodies. We show here that the extracellular domains of alpha and beta subunits are able to form a heterodimer, although with a lower efficiency, in the absence of the transmembrane and cytoplasmic domains. The presence of the cytoplasmic and transmembrane domains in the alpha subunit is, however, necessary for expression at the surface of the cell whereas the corresponding domains of the beta subunit are not required.
Subject(s)
Platelet Membrane Glycoproteins/physiology , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Cell Membrane/chemistry , Chlorocebus aethiops , Cytoplasm/chemistry , Cytoplasm/metabolism , Gene Expression , Humans , Molecular Sequence Data , Mutation , Plasmids , Platelet Membrane Glycoproteins/chemistry , Platelet Membrane Glycoproteins/genetics , Precipitin Tests , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/physiology , TransfectionABSTRACT
The CD9 antigen was described originally as a 24-kDa surface protein of non-T acute lymphoblastic leukemia cells and developing B-lymphocytes. It is also strongly expressed on platelets, among other cells, where it shows the property of mediating platelet activation and aggregation upon binding with mAbs. The primary structure has been elucidated by cloning the cDNA from a lambda gt11 expression vector library constructed with megakaryocytic mRNA. Monoclonal antibodies were used as probes with an APAAP amplification of the signal. The 5' region was further cloned in a lambda gt10 randomly primed cDNA library. The initiation codon was immediately followed by a sequence coding for the tetrapeptide corresponding to the NH2-terminal sequence identified in a microsequencing procedure. Only one species of mRNA was found with an estimated size of 1.4 kilobase. CD9 antigen appears to be a 227-amino acid molecule with four hydrophobic domains and one N-glycosylation site. Sequence and structural comparisons showed extensive similarity of the CD9 antigen with a 237-amino acid molecule described previously as the human melanoma-associated antigen ME491 and a Schistosoma mansoni membrane protein of 218 amino acids. These three proteins identify a new family of cell-surface proteins.
Subject(s)
Antigens, CD/genetics , Antigens, Differentiation/genetics , Blood Platelets/immunology , Amino Acid Sequence , Antibodies, Monoclonal , Antigens, CD/isolation & purification , Antigens, Differentiation/isolation & purification , Base Sequence , Cell Membrane/immunology , Cloning, Molecular , Humans , Megakaryocytes/immunology , Membrane Glycoproteins/genetics , Molecular Sequence Data , Protein Conformation , Sequence Homology, Nucleic Acid , Tetraspanin 29Subject(s)
Antigens, CD/genetics , Antigens, Differentiation/genetics , Chromosomes, Human, Pair 12 , Membrane Glycoproteins , Animals , Blotting, Southern , Cell Line , Chromosome Banding , Chromosome Mapping , Cricetinae , DNA/genetics , DNA/isolation & purification , DNA Probes , Deoxyribonuclease HindIII , Humans , Hybrid Cells/cytology , Karyotyping , Mice , Restriction Mapping , Tetraspanin 29ABSTRACT
Platelet GPIIbIIIa is only synthesized in megakaryocyte or in cell lines with megakaryocytic features. The sequence for GPIIb and GPIIIa have recently been derived from cDNAs obtained from HEL cells. The sequence of these proteins produced by the megakaryocyte, has however, not been determined yet. This study describes full length cDNAs for GPIIb and GPIIIa isolated from megakaryocyte cDNA libraries. The cDNA sequences indicate the presence of nucleotide differences, between the sequence of the GPIIIa cDNAs from HEL cells, endothelial cells and megakaryocytes. One difference was also observed between HEL and megakaryocyte GPIIb at position 633 where a cysteine in the megakaryocyte GPIIb, is replaced by a serine in the HEL sequence. The mRNA species for GPIIb (3.4 kb) and GPIIIa (6.1 kb) were of the same size in HEL cells and human megakaryocytes.
Subject(s)
Megakaryocytes/analysis , Platelet Membrane Glycoproteins/genetics , Amino Acid Sequence , Base Sequence , Cells, Cultured , DNA/genetics , Endothelium, Vascular/cytology , Genes , Humans , Leukemia, Erythroblastic, Acute/pathology , Leukemia, Myeloid, Chronic-Phase/pathology , Molecular Sequence Data , RNA, Messenger/genetics , RNA, Messenger/isolation & purification , RNA, Neoplasm/genetics , RNA, Neoplasm/isolation & purification , Sequence Homology, Nucleic Acid , Tumor Cells, CulturedSubject(s)
Hematopoiesis , Megakaryocytes/metabolism , Platelet Membrane Glycoproteins/metabolism , Amino Acid Sequence , Base Sequence , Binding Sites , Biomarkers , DNA/genetics , DNA/metabolism , Humans , Megakaryocytes/cytology , Molecular Sequence Data , Platelet Membrane Glycoproteins/chemistry , Platelet Membrane Glycoproteins/geneticsABSTRACT
The platelet membrane glycoproteins GPIIb and GPIIIa form a calcium-dependent heterodimer that functions as a receptor for adhesive proteins on stimulated platelets. In this study, we have investigated the kinetics of the assembly reaction that result in GPIIb-IIIa dimerization. Pulse-chase experiments analysis performed on human megakaryocytes obtained from liquid cultures of chronic myelogenous leukemic patients with antibodies specific for GPIIIa or GPIIb demonstrated the existence of a pro-GPIIb-GPIIIa complex and of a large pool (60%) of unassociated GPIIIa; nearly all the GPIIb and the pro-GPIIb molecules were found associated with GPIIIa. This free GPIIIa was not exposed on the cell surface. Pulse-chase experiments on a subclone of the human megakaryocytic cell line LAMA-84 revealed that the cells from this subclone produced only the pro-GPIIb, which was neither processed into mature GPIIb nor expressed on the cell surface. The expression of GPIIIa in PMA treated cells resulted in the production of the mature GPIIb form and the expression of the GPIIb-IIIa complex on the cell surface. These results indicate that assembly between the early forms of pro-GPIIb and GPIIIa is an obligatory step for the maturation of the heterodimer and its expression on the cell surface.
Subject(s)
Integrin beta3 , Megakaryocytes/metabolism , Platelet Membrane Glycoprotein IIb , Platelet Membrane Glycoproteins/biosynthesis , Protein Precursors/biosynthesis , RNA, Messenger/genetics , Antibodies, Monoclonal , Cell Membrane/metabolism , Cells, Cultured , DNA/genetics , Fluorescent Antibody Technique , Humans , Immunoblotting , Megakaryocytes/drug effects , Molecular Weight , Nucleic Acid Hybridization , Platelet Membrane Glycoproteins/genetics , Platelet Membrane Glycoproteins/isolation & purification , Protein Precursors/genetics , Protein Processing, Post-Translational , RNA/genetics , RNA, Messenger/isolation & purification , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
The platelet GPIIb-IIIa complex functions as a receptor for fibrinogen, fibronectin, and von Willebrand factor on activated platelets. This glycoprotein is a member of a broadly distributed family of structurally and immunologically related membrane receptors involved in cell-cell contact and cell-matrices interactions. GPIIb-IIIa is a heterodimer complex composed of GPIIb (the alpha subunit), which consists of two disulfide-linked heavy and light chains, and GPIIIa (the beta subunit), which is a single polypeptide chain. Congenital absence of platelet GPIIb-IIIa in Glanzmann's thrombasthenia results in a severe bleeding disorder characterized by defective platelet aggregation and failure of fibrinogen to bind to platelets. The gene coding for GPIIb was located on 17q21.1-17q21.3 as determined by in situ hybridization with a 2650-bp GP2B (GPIIb) cDNA probe prepared from human megakaryocytes.
Subject(s)
Blood Platelets/analysis , Chromosomes, Human, Pair 17 , Membrane Glycoproteins/genetics , Platelet Glycoprotein GPIb-IX Complex , Platelet Membrane Glycoproteins , Receptors, Immunologic/genetics , Chromosome Banding , Chromosome Mapping , DNA Probes , Humans , Integrin alpha2 , Karyotyping , Nucleic Acid HybridizationABSTRACT
Platelet membrane glycoprotein (GP) IIbIIIa complex functions as a receptor for fibrinogen, von Willebrand factor and fibronectin, and mediates adhesive reactions of platelets. The gene for the GPIIb subunit is only active in megakaryocytic cell type. We have isolated this gene from a genomic library. The GPIIb gene was characterized by restriction mapping and sequencing of the 5' and 3' regions containing the first and the last exons. The transcription start site and the polyadenylation signal were identified. From these data we deduced that the gene spans a region of 22 kb and that the mRNA contains a leader sequence of 32 nucleotides. At the 3' end the last exon encodes the 19 amino acids corresponding to the cytoplasmic domain of the GPIIb light chain. Upstream the transcription start site, two sequences are homologous to consensus binding sites of the nuclear factors SP1 and CP2. Two inverted repeats were also identified in this region.
Subject(s)
Exons , Genes , Platelet Membrane Glycoproteins/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA/blood , DNA/genetics , DNA Restriction Enzymes , Humans , Megakaryocytes/metabolism , Molecular Sequence Data , Transcription, GeneticABSTRACT
Platelet glycoprotein (GP) IIb is one of the two subunits of the common platelet adhesion receptor, GPIIb-IIIa. The isolation, characterization and sequencing of cDNA clones encoding for the two polypeptide chains of GPIIb are described. A number of clones were isolated from lambda gt11 libraries constructed with mRNA from an erythroleukemic cell line, HEL, and human megakaryocytes. Two of these clones, lambda IIb1, from HEL cells, and lambda IIb2, from megakaryocytes, cross-hybridized and were selected for detailed analysis. The identification of these as authentic GPIIb clones was based on immunological criteria and confirmed by the presence of nucleotide sequences in each insert encoding for known protein sequences of platelet GPIIb. These clones contained inserts of 1.54 kb and 1.39 kb, respectively, with an overlapping sequence of 801 bp. The nucleotide sequence of the overlapping region was identical indicating that HEL cells produce a protein closely related, if not identical, to platelet GPIIb. The determined nucleotide sequence of two inserts included a coding sequence for 648 amino acid residues, a TAG stop codon and 185 nucleotides of 3' non-coding sequence followed by a poly(A) tail. The coding sequence contained a portion of the heavy chain, the junction between the heavy and light chains and the entire light chain including a potential transmembrane-spanning domain and a short cytoplasmic tail. When these cDNA were used to probe for GPIIb mRNA, a single mRNA species of 3.9 kb was identified in both HEL cells and human megakaryocytes. A comparison of the deduced amino acid sequence for GPIIb with those of the alpha subunit of the vitronectin and the fibronectin receptors revealed extensive homologies. These homologies further establish that GPIIb-IIIa from platelets, together with the vitronectin and the fibronectin receptors, are members of a supergene family of adhesion receptors with a recognition specificity for Arg-Gly-Asp amino acid sequences.