Role of the transmembrane and cytoplasmic domains in the assembly and surface exposure of the platelet integrin GPIIb/IIIa.

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.     

GPIIb and GPIIIa amino acid sequences deduced from human megakaryocyte cDNAs

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 cystein in the megakaryocyte GPIIb, is replaced by a serine in the HEL sequence. The mRNA species for GPIIb (3.4kb) and GPIIIa (6.1 kb) were of the same size in HEL cells and human megakaryocytes.     

Activation of the fibrinogen receptor on human platelets exposed to alpha chymotrypsin. Relationship with a major proteolytic cleavage at the carboxyterminus of the membrane glycoprotein IIb heavy chain

The serine proteinase alpha chymotrypsin from bovine pancreas (CT) is known to expose fibrinogen binding sites on the surface of human platelets in the absence of cell activation and granular secretion. This is accompanied by the appearance of membrane-bound chymotryptic fragments of both glycoprotein (GP) IIb and GPIIIa, the two subunits of the platelet fibrinogen receptor, the GPIIb-IIIa complex. However, no clear relationship between discrete proteolytic event(s) within GPIIb-IIIa and fibrinogen-binding-site expression has yet been established. We have now evaluated the proteolysis of GPIIb-IIIa by CT by Western blot analyses using a panel of polyclonal and monoclonal antibodies against GPIIb or GPIIIa. The different proteolytic events were then correlated with the kinetics of the expression of active fibrinogen binding sites on platelets, as measured through the binding of 125I-labelled purified fibrinogen and to the capacity of CT-treated platelets to aggregate. Treatment of platelets with CT at 22 degrees C resulted in the expression of fibrinogen binding sites prior to cleavage of GPIIIa (Mr approximately 90,000) into a previously described, major membrane-bound fragment with Mr 60,000. In contrast, fibrinogen receptor expression closely paralleled a proteolytic cleavage at the carboxy terminus of the GPIIb heavy chain (Mr approximately 120,000), which was converted into a faster migrating species with Mr approximately 115,000). This proteolysis resulted in the release of a soluble peptide with an expected molecular mass of less than 3.7 kDa. Quantitation of this peptide using a competitive immunoenzymatic assay, confirmed that its release from the platelet surface correlated with the expression of fibrinogen binding sites and aggregability. When platelets were exposed to CT at 37 degrees C, a prompt increase in fibrinogen binding sites and platelet aggregability was observed, whereas the GPIIb heavy chain was rapidly converted into the carboxy-terminal-cleaved form. However, incubation at 37 degrees C for longer than 10 min resulted in extensive and simultaneous degradation of both the GPIIb heavy and light chains and of GPIIIa, with the latter being converted into the 60-kDa fragment. These later events were associated with a sharp decline of platelet aggregability and a reduction in the number of fibrinogen binding sites. These data allow us to propose that an early and limited proteolytic processing of the GPIIb component of the platelet fibrinogen receptor is associated with a shift of this receptor complex into a state which expresses specific binding sites for fibrinogen. Further cleavage of GPIIIa to generate the 60-kDa fragment results in loss of receptor activity.     

A highly conserved sequence of the Arg-Gly-Asp-binding domain of the integrin beta 3 subunit is sensitive to stimulation

The Arg-Gly-Asp (RGD)-binding domain of GPIIb-IIIa has been localized in a fragment of the GPIIIa subunit that includes the sequence between amino acids 109 and 171. To examine, in a platelet membrane environment, the activated versus nonactivated status of this domain, we have produced a monoclonal antibody against a synthetic peptide (residues 109-128) located within the RGD-binding region on GPIIIa. This kappa-IgM, named AC7, was specific for GPIIIa peptide 109-128 and interacted only with activated platelets. Fibrinogen, RGDF peptide, and the fibrinogen phi chain decapeptide LGGAKQAGDV inhibited the binding of AC7 to ADP-stimulated platelets. AC7 IgM and "small fragments" inhibited fibrinogen binding and platelet aggregation in a dose-dependent fashion. Induction of AC7 binding by D33C, a monoclonal antibody recognizing the GPIIb 426-437 sequence and stimulating fibrinogen binding, indicated that the GPIIb 426-437 and the GPIIIa 109-128 sequences were both involved in a stimulation-dependent conformational modification of the receptor. AC7 was able to recognize beta subunits other than GPIIIa on leucocyte surfaces but only after cell fixation with glutaraldehyde. The results are consistent with the implication of the RGD-binding domain in receptor ligand interaction on the platelet surface and its conformational modification and exposure upon receptor induction.     

Processing and assembly of the integrin, glycoprotein IIb-IIIa, in HEL cells

We examined the biosynthetic processing and assembly of the platelet glycoprotein (GP) IIb-IIIa complex in [35S]methionine-labeled HEL cells, a human cell line with features of megakaryocytes. Both GPIIb and GPIIIa were synthesized as single-chain precursors to which high mannose N-linked oligosaccharides were added in the endoplasmic reticulum (ER). A 5-fold excess of the major IIb precursor, preIIb, was synthesized relative to GPIIIa. Two smaller proteins immunologically related to GPIIb were synthesized in smaller amounts. Assembly of the GPIIb and GPIIIa precursors required 4-6 h for completion. All GPIIIa molecules were eventually assembled; the excess GPIIb precursors were degraded without reaching the cell surface. Following assembly, preIIb-IIIa complexes were rapidly transported to the Golgi apparatus where preIIb underwent modification of high mannose chains into complex oligosaccharides and proteolytic cleavage to yield disulfide-linked heavy and light chains. Pretreating cells with the ionophore monensin blocked cleavage of preIIb but not its carbohydrate modification or its assembly with GPIIIa. These studies suggest that 1) assembly of the precursors of GPIIb and GPIIIa in the ER is a slow process requiring conformational maturation of one or both subunits, and 2) only heterodimers assembled in the ER are transported to the Golgi apparatus for additional processing and, ultimately, expression on the cell surface.     

Biosynthesis and processing of platelet GPIIb-IIIa in human megakaryocytes

Platelet membrane glycoprotein IIb-IIIa forms a calcium-dependent heterodimer and constitutes the fibrinogen receptor on stimulated platelets. GPIIb is a two-chain protein containing disulfide-linked alpha and beta subunits. GPIIIa is a single chain protein. These proteins are synthesized in the bone marrow by megakaryocytes, but the study of their synthesis has been hampered by the difficulty in obtaining enriched population of megakaryocytes in large numbers. To examine the biosynthesis and processing of GPIIb-IIIa, purified human megakaryocytes were isolated from liquid cultures of cryopreserved leukocytes stem cell concentrates from patients with chronic myelogenous leukemia. Immunoprecipitation of [35S]methionine pulse-chase-labeled cell extracts by antibodies specific for the alpha or beta subunits of GPIIb indicated that GPIIb was derived from a precursor of Mr 130,000 that contains the alpha and beta subunits. This precursor was converted to GPIIb with a half-life of 4-5 h. No precursor form of GPIIIa was detected. The glycosylation of GPIIb-IIIa was examined in megakaryocytes by metabolic labeling in the presence of tunicamycin, monensin, or treatment with endoglycosidase H. The polypeptide backbones of the GPIIb and the GPIIIa have molecular masses of 120 and 90 kD, respectively. High-mannose oligosaccharides are added to these polypeptide backbones co-translationally. The GPIIb precursor is then processed with conversion of high-mannose to complex type carbohydrates yielding the mature subunits GPIIb alpha (Mr 116,000) and GPIIb beta (Mr 25,000). No posttranslational processing of GPIIIa was detected.     

C-terminal amino acid determination of the transmembrane subunits of the human platelet fibrinogen receptor, the GPIIb/IIIa complex

Glycoproteins IIb (GPIIb) and IIIa (GPIIIa) form the Ca2(+)-dependent GPIIb/IIIa complex, which acts as the fibrinogen receptor on activated platelets. GPIIb and GPIIIa are synthesized as single peptide chains. The GPIIb precursor is processed proteolytically to yield two disulphide-bonded chains, GPIIb alpha and GPIIb beta. The GPIIb/IIIa complex has two membrane attachment sites located at the C-termini of GPIIb beta and GPIIIa. The short cytoplasmic tails of GPIIb beta and/or GPIIIa become most likely associated to the cytoskeleton of activated platelets. In the present work the C-terminal amino acid residues of platelet GPIIb beta and GPIIIa have been analyzed by protein-chemical methods and compared with those predicted from cDNA analysis. We were able to confirm the positions of the C-termini in both glycoproteins and the identity of the C-terminus predicted for GPIIIa, i.e. threonine. However, glutamine, not glutamic acid as predicted for GPIIb beta from the human erythroleukemic cell line and megakaryocyte cells, was found to be the C-terminal amino acid of GPIIb beta. This indicates that the glutamic acid in the GPIIb precursor is posttranslationally modified to glutamine.     

Structure of the platelet membrane glycoprotein IIb. Homology to the alpha subunits of the vitronectin and fibronectin membrane receptors

The platelet membrane glycoprotein IIb X IIIa heterodimer complex (GPIIb X IIIa) is the platelet receptor for adhesive proteins, containing binding sites for fibrinogen, von Willebrand factor, and fibronectin on activated platelets. GPIIb X IIIa also appears to be a member of a family of membrane adhesive protein receptors that plays a major role in cell-cell and cell-matrix interactions. GPIb is the larger component of this platelet receptor and is composed of two disulfide-linked subunits. In this report we describe the analysis of cDNA clones for human GPIIb that were isolated from a lambda gt11 expression library prepared using RNA from HEL cells. A total of 3.3 kilobases of cDNA was sequence, revealing a continuous open reading frame encoding both GPIIb subunits. The cDNA encodes 1039 amino acids: 137 constituting the smaller subunit, 871 constituting the larger subunit, and 30 constituting an NH2-terminal signal peptide. No homology was found between the larger and smaller subunits. The smaller subunit contains a 26-residue hydrophobic sequence near its COOH terminus that represents a potential transmembrane domain. Four stretches of 12 amino acids present in the larger subunit are homologous to the calcium binding sites of calmodulin and troponin C. Northern blot analysis using HEL cell RNA indicated that the mature mRNA coding for GPIIb is 4.1 kilobases in size. A comparison of the GPIIb coding region with available cDNA sequences of the alpha-chains of the vitronectin and fibronectin receptors revealed 41% DNA homology and 74% and 63% amino acid homology, respectively. Our data establish the amino acid sequence for the human platelet glycoprotein IIb and provide additional evidence for the existence of a family of cellular adhesion protein receptors.     

Structural and functional characterization of major platelet membrane components derived by limited proteolysis of glycoprotein IIIa

The authors isolated a product of proteolytic degradation of glycoprotein IIIa (GPIIIa) which is formed on the surface of human platelets during incubation with chymotrypsin and which was previously described as the 66 kDa platelet membrane component. This component migrated with an apparent Mr 62,400 in a non-reduced system of sodium dodecyl sulfate polyacrylamide gel electrophoresis. In a reduced system it yielded two major subunits migrating with apparent Mr 14,000-17,000 and 65,000. The low-molecular weight component began with the NH2-terminal sequence of GPIIIa (GPNICTTR...) and the larger component with residue 348 of GPIIIa (GKIRSKKA...) as deduced from a cDNA clone of this glycoprotein. The two subunits appeared to be linked by one or more S-S bridges supporting the contention that GPIIIa is a highly folded molecule on the platelet membrane. In contrast to GPIIIa, the '66 kDa component' did not bind to GRGDSPK-agarose, to fibrinogen-agarose nor to insolubilized monoclonal antibody recognizing the GPIIb/IIIa complex. The exposure of fibrinogen receptors during the course of incubation of platelets with chymotrypsin preceded the formation of the '66 kDa component' characterized in this study. An intermediate product of GPIIIa proteolysis migrating with an apparent Mr 120,000 in a non-reduced system and Mr 80,000 in a reduced system was identified as a precursor of the '66 kDa component'. The '120 kDa component' was not retained on GRGDSPK-agarose or on fibrinogen-agarose but it was retained on insolubilized antibody recognizing the GPIIb/IIIa complex. Incubation of platelets with porcine pancreatic elastase or human granulocytic elastase resulted in the formation of similar proteolytic degradation fragments.     

Localization of the cross-linking sites of RGD and KQAGDV peptides to the isolated fibrinogen receptor, the human platelet integrin glycoprotein IIb/IIIa. Influence of peptide length.

The non-covalent and Ca(2+)-dependent heterodimer GPIIb/IIIa, formed by platelet glycoproteins IIb (GPIIb) and IIIa (GPIIIa), also known as the integrin alpha IIb beta 3, is the inducible receptor for fibrinogen and other adhesive proteins on the surface of activated platelets. A fraction of the isolated GPIIb/IIIa in solution binds RGD or KQAGDV inhibitory peptides and, upon peptide removal, apparently acquires the capacity to bind fibrinogen ('activated' GPIIb/IIIa) [Du, X., Plow, E. F., Frelinger, A. L., III, O'Toole, T. E., Loftus, J. C. & Ginsberg, M. H. (1991) Cell 65, 409-416]. Photoaffinity labelling was used here to study the ligand binding site(s) of GPIIb/IIIa in solution, for which the peptides CKRKRKRKRRGDV (alpha 1), CGRGDF (alpha 2), CYHHLGGAKQAGDV (gamma 1) and CGAKQAGDV (gamma 2) were synthesized with a photoactivable cross-linker group and a fluorescent reporter group attached to the N-terminal cysteine residue. Contrary to the situation in activated platelets, both GPIIb and GPIIIa were equally labelled by the four peptides and the cross-linking sites were localized by protein chemical analyses of the fluorescently labelled tryptic peptides of both subunits. Thus, the localization of the cross-linking sites in GPIIb varies considerably with the peptide length and is very different from that localization observed in activated platelets: alpha 2 and gamma 2 were found cross-linked to the N-terminal of both the heavy (GPIIbH 42-73) and the light (GPIIbL2 30-75) chains of GPIIb; while the longer peptides alpha 1 and gamma 1 were cross-linked to the C-terminal of GPIIbH within the 696-724 and 752-768 peptide stretches, respectively. On the other hand, the cross-linking sites of the four inhibitory peptides in GPIIIa were found mainly within the proteolysis susceptible region, between the N-terminal (GPIIIa 1-52) and the core (GPIIb 423-622) highly disulphide-bonded domains, observing that the longer the peptide the closer the cross-linking site is to the N-terminal of GPIIIa: alpha 1 at GPIIIa 63-87 and 303-350; gamma 1 at GPIIIa 9-37; alpha 2 at GPIIIa 151-191; and gamma 2 at GPIIIa 303-350. These results led us to the following conclusions. (a) The GPIIIa 100-400 region contributes to the ligand-binding domain in GPIIb/IIIa both in solution and in activated platelets.(ABSTRACT TRUNCATED AT 400 WORDS)     

Assignment of human platelet GP2B (GPIIb) gene to chromosome 17, region q21.1-q21.3

Summary 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 subunit), which consists of two disulfide-linked heavy and light chains, and GPIIIa (the 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-pb GP2B (GPIIb) cDNA probe prepared from human megakaryocytes.     

Engagement of αIIbβ3 (GPIIb/IIIa) with ανβ3 integrin mediates interaction of melanoma cells with platelets: a connection to hematogenous metastasis

A mutual relationship exists between metastasizing tumor cells and components of the coagulation cascade. The exact mechanisms as to how platelets influence blood-borne metastasis, however, remain poorly understood. Here, we used murine B16 melanoma cells to observe functional aspects of how platelets contribute to the process of hematogenous metastasis. We found that platelets interfere with a distinct step of the metastasis cascade, as they promote adhesion of melanoma cells to the endothelium in vitro under shear conditions. Constitutively active platelet receptor GPIIb/IIIa (integrin αIIbβ3) expressed on Chinese hamster ovary cells promoted melanoma cell adhesion in the presence of fibrinogen, whereas blocking antibodies to aνβ3 integrin on melanoma cells or to GPIIb/IIIa significantly reduced melanoma cell adhesion to platelets. Furthermore, using intravital microscopy, we observed functional platelet-melanoma cell interactions, as platelet depletion resulted in significantly reduced melanoma cell adhesion to the injured vascular wall in vivo. Using a mouse model of hematogenous metastasis to the lung, we observed decreased metastasis of B16 melanoma cells to the lung by treatment with a mAb blocking the aν subunit of aνβ3 integrin. This effect was significantly reduced when platelets were depleted in vivo. Thus, the engagement of GPIIb/IIIa with aνβ3 integrin interaction mediates tumor cell-platelet interactions and highlights how this interaction is involved in hematogenous tumor metastasis.     

A novel fibronectin receptor with an unexpected subunit composition (alpha v beta 1)

The integrins are a family of heterodimeric cell surface receptors for extracellular matrix molecules. An analysis of integrin subunits expressed by a number of cell lines identified a novel heterodimer. The alpha subunit of this integrin was immunologically and electrophoretically indistinguishable from the vitronectin receptor alpha subunit (alpha v) and the beta subunit was indistinguishable from beta 1. Affinity chromatography experiments and cell adhesion assays indicated that this receptor complex is a new fibronectin receptor. Its unexpected subunit composition demonstrates the importance of the beta subunit in determining the ligand specificity of integrins and suggests that the current integrin classification scheme needs revision.     

Binding of glycoprotein IIIa-derived peptide 217-231 to fibrinogen and von Willebrand factors and its inhibition by platelet glycoprotein IIb/IIIa complex.

Based on previous reports in the literature and the high homology between platelet glycoprotein (GP) IIIa 217-231 and similar portions of other beta subunits of integrin receptors, we hypothesized that this region may participate in ligand binding. Using a polyclonal antibody against GPIIIa 217-231(YC), we tested the interaction of a synthetic peptide representing this region with fibrinogen (Fg), in the enzyme-linked immunosorbent assay (ELISA) system. Results show a calcium-independent, dose-related, direct interaction between GPIIIa 217-231(Y) and immobilized Fg. This peptide also bound to von Willebrand Factor (vWF) and fibronectin (Fn), but did not attach to a 50 kDa Fn fragment which is deficient in the cell attachment site. In addition, purified GPIIb/IIIa displaced GPIIIa 217-231(Y) from Fg and vWF. Binding of 125I-GPIIIa 217-231(Y) to Fg coated tubes was inhibited by soluble Fg and by the GPIIb/IIIa complex. We synthesized this peptide with several alterations; similar peptides with Pro-219 replaced with an Ala showed significantly reduced binding to Fg and vWF. The decreased binding of the peptides with Pro-219 substitutes suggests that the confirmation of GPIIIa 217-230 is important for its ability to bind to adhesive ligands. In conclusion, the amino acid residues between 217 and 231 of GPIIIa appear to be involved in ligand binding and Pro-219 probably plays a significant role in this interaction.     

Endothelial cells use alpha 2 beta 1 integrin as a laminin receptor

Human umbilical vein endothelial cells attach and spread on laminin-coated substrates. Affinity chromatography was used to identify the attachment receptor. Fractionation of extracts from surface-iodinated endothelial cells on human laminin-Sepharose yielded a heterodimeric complex, the subunits of which migrated with molecular sizes corresponding to 160/120 kD and 160/140 kD under nonreducing and reducing conditions, respectively. The purified receptor bound to laminin and slightly less to fibronectin and type IV collagen in a radioreceptor assay. This endothelial cell laminin receptor was classified as an alpha 2 beta 1 integrin because monoclonal and polyclonal antibodies directed against the alpha 2 and bet 1 subunits immunoprecipitated the receptor. Cytofluorometric analysis and immunoprecipitation showed that the alpha 2 subunit is an abundant integrin alpha subunit in the endothelial cells and that the alpha subunits associated with laminin binding in other types of cells are expressed in these cells only at low levels. The alpha 2 beta 1 integrin appears to be a major receptor for laminin in the endothelial cells, because an anti-alpha 2 monoclonal antibody inhibited the attachment of the endothelial cells to human laminin. These results define a new role for the alpha 2 subunit in laminin binding and suggest that the ligand specificity of the alpha 2 beta 1 integrin, which is known as a collagen receptor in other types of cells, can be modulated by cell type-specific factors to include laminin binding.     

Cloning and expression of a divergent integrin subunit beta 8

Rabbit and human cDNA clones have been identified that encode a novel integrin beta subunit. The sequences that encode this subunit, which has been designated as beta 8, were isolated initially from rabbit placental cDNA libraries using an oligonucleotide probe derived from a highly conserved region of integrin beta subunit sequences. The rabbit clone was used to isolate human beta 8 cDNA clones from human placental and MG-63 osteosarcoma cell libraries. The putative beta 8 polypeptides, which comprise 769 and 768 residues in human and rabbit, respectively, show a high degree of inter-species conservation (approximately 90% identity). In contrast, beta 8 is distinct from the other integrin beta subunits. At the amino acid level human beta 8 ranges from 31 to 37% identity with human beta 1-7. The domain structure of beta 8 is typical of the integrin beta subunits. Human beta 8 has a 42-residue N-terminal signal peptide, a large extracellular domain (approximately 639 residues) that contains four cysteine-rich repeats, a transmembrane domain (approximately 30 residues), and a C-terminal cytoplasmic domain (approximately 58 residues). There are several structural features that are unique to the beta 8 polypeptide, as compared with the other integrin beta subunits. Six of the 56 cysteine residues that are conserved within the extracellular domains of beta 1, beta 2, beta 3, beta 5, beta 6, and the beta subunit from Drosophila are absent in the beta 8 polypeptide. Also, the cytoplasmic domain of the beta 8 subunit shares no homology with the cytoplasmic regions of any of the other integrin beta subunits. Northern analysis demonstrated an approximately 8-kilobase beta 8 mRNA in rabbit placenta, kidney, brain, ovary, and uterus. PCR analysis revealed that beta 8 mRNA is also present in several transformed human cell lines. The beta 8 polypeptide has been transiently expressed in 293 human embryonic kidney cells. A polyclonal antipeptide antibody specific for beta 8 and a polyclonal antibody that recognizes alpha v epitopes were used to show that beta 8 can complex with the endogenous alpha v subunit in 293 cells and that the resulting integrin is expressed as a cell surface complex.     

High-efficiency utilization of the bovine integrin alpha(v)beta(3) as a receptor for foot-and-mouth disease virus is dependent on the bovine beta(3) subunit

We have previously reported that Foot-and-mouth disease virus (FMDV), which is virulent for cattle and swine, can utilize the integrin alpha(v)beta(3) as a receptor on cultured cells. Since those studies were performed with the human integrin, we have molecularly cloned the bovine homolog of the integrin alpha(v)beta(3) and have compared the two receptors for utilization by FMDV. Both the alpha(v) and beta(3) subunits of the bovine integrin have high degrees of amino acid sequence similarity to their corresponding human subunits in the ectodomains (96%) and essentially identical transmembrane and cytoplasmic domains. Within the putative ligand-binding domains, the bovine and human alpha(v) subunits have a 98.8% amino acid sequence similarity while there is only a 93% similarity between the beta(3) subunits of these two species. COS cell cultures, which are not susceptible to FMDV infection, become susceptible if cotransfected with alpha(v) and beta(3) subunit cDNAs from a bovine or human source. Cultures cotransfected with the bovine alpha(v)beta(3) subunit cDNAs and infected with FMDV synthesize greater amounts of viral proteins than do infected cultures cotransfected with the human integrin subunits. Cells cotransfected with a bovine alpha(v) subunit and a human beta(3) subunit synthesize viral proteins at levels equivalent to those in cells expressing both human subunits. However, cells cotransfected with the human alpha(v) and the bovine beta(3) subunits synthesize amounts of viral proteins equivalent to those in cells expressing both bovine subunits, indicating that the bovine beta(3) subunit is responsible for the increased effectiveness of this receptor. By engineering chimeric bovine-human beta(3) subunits, we have shown that this increase in receptor efficiency is due to sequences encoding the C-terminal one-third of the subunit ectodomain, which contains a highly structured cysteine-rich repeat region. We postulate that amino acid sequence differences within this region may be responsible for structural differences between the human and bovine beta(3) subunit, leading to more efficient utilization of the bovine receptor by this bovine pathogen.     

Single subunit chimeric integrins as mimics and inhibitors of endogenous integrin functions in receptor localization, cell spreading and migration, and matrix assembly

The ability of single subunit chimeric receptors containing various integrin beta intracellular domains to mimic and/or inhibit endogenous integrin function was examined. Chimeric receptors consisting of the extracellular and transmembrane domains of the small subunit of the human interleukin-2 receptor connected to either the beta 1, beta 3, beta 3B, or beta 5 intracellular domain were transiently expressed in normal human fibroblasts. When expressed at relatively low levels, the beta 3 and beta 5 chimeras mimicked endogenous ligand-occupied integrins and, like the beta 1 chimera (LaFlamme, S. E., S. K. Akiyama, and K. M. Yamada. 1992. J. Cell Biol. 117:437), concentrated with endogenous integrins in focal adhesions and sites of fibronectin fibril formation. In contrast, the chimeric receptor containing the beta 3B intracellular domain (a beta 3 intracellular domain modified by alternative splicing) was expressed diffusely on the cell surface, indicating that alternative splicing can regulate integrin receptor distribution by an intracellular mechanism. Furthermore, when expressed at higher levels, the beta 1 and beta 3 chimeric receptors functioned as dominant negative mutants and inhibited endogenous integrin function in localization to fibronectin fibrils, fibronectin matrix assembly, cell spreading, and cell migration. The beta 5 chimera was a less effective inhibitor, and the beta 3B chimera and the reporter lacking an intracellular domain did not inhibit endogenous integrin function. Comparison of the relative levels of expression of the transfected beta 1 chimera and the endogenous beta 1 subunit indicated that in 10 to 15 h assays, the beta 1 chimera can inhibit cell spreading when expressed at levels approximately equal to the endogenous beta 1 subunit. Levels of chimeric receptor expression that inhibited cell spreading also inhibited cell migration, whereas lower levels were able to inhibit alpha 5 beta 1 localization to fibrils and matrix assembly. Our results indicate that single subunit chimeric integrins can mimic and/or inhibit endogenous integrin receptor function, presumably by interacting with cytoplasmic components critical for endogenous integrin function. Our results also demonstrate that beta intracellular domains, expressed in this context, display specificity in their abilities to mimic and inhibit endogenous integrin function. Furthermore, the approach that we have used permits the analysis of intracellular domain function in the processes of cell spreading, migration and extracellular matrix assembly independent of effects due to the rest of integrin dimers. This approach should prove valuable in the further analysis of integrin intracellular domain function in these and other integrin-mediated processes requiring the interaction of integrins with cytoplasmic components.     

Complex formation of platelet membrane glycoproteins IIb and IIIa with the fibrinogen D domain

Glycoprotein IIb (GPIIb) and glycoprotein IIIa (GPIIIa) form a macromolecular complex on the activated platelet surface which contains the fibrinogen-binding site necessary for normal platelet aggregation. To identify the specific region of the fibrinogen molecule responsible for its interaction with the GPIIb-GPIIIa complex, purified fragment D1 (Mr = 100,000) and fragment E (Mr = 50,000) were prepared from plasmin digests of purified human fibrinogen. In addition, the polypeptide chain subunits A alpha, B beta, and gamma of fibrinogen were prepared. Using an enzyme-linked immunosorbent assay we have demonstrated that isolated fragment D1 in a solid phase system forms a complex with a mixture of GPIIb and GPIIIa. The binding of the GPIIb-GPIIIa mixture to fragment D1-coated plates reached saturation at 8 nM and to fibrinogen-coated plates at 24 nM. Isolated A alpha, B beta, and gamma chains were not reactive with added glycoproteins. Fragment E coated directly on plastic plates or immobilized on antibody-coated plastic plates did not form a complex with GPIIb-GPIIIa. Only fluid phase fibrinogen and fragment D1 but not fragment E were inhibitory toward formation of a complex between solid phase fibrinogen and GPIIb-GPIIIa. Isolated A alpha, B beta, and gamma chains at concentrations equivalent to fluid phase fibrinogen were inactive. Binding of fragment D1 but not fragment E to the GPIIb-GPIIIa complex was also demonstrated by rocket immunoelectrophoresis of the membrane glycoprotein mixture through a gel containing the individual fragments and subsequent autoradiography of the complex following exposure to 125I-anti-fibrinogen. These observations with isolated platelet membrane glycoproteins provide strong evidence that each of the D domains of the fibrinogen molecule interacts directly with the GPIIb-GPIIIa complex on the activated platelet surface, thus allowing formation of a tertiary molecular "bridge" across the surface of two adjacent activated platelets.