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PubMed2004

Structural basis for integrin alphaIIbbeta3 clustering.

We have expressed two proteins that correspond to the transmembrane and cytoplasmic domains of integrin alphaIIb and beta3 subunits. Characterization of these proteins, dispersed in anionic and zwitterionic micelles, revealed that, rather than interacting with each other, the two proteins associated into homodimers and homotrimers respectively. Moreover, studies using the TOXCAT assay system confirmed that the alphaIIb and beta3 transmembrane domains can self-associate in biological cell membranes. Transmembrane domain-mediated homo-oligomerization provides a plausible structural basis for integrin clustering and could promote integrin activation as well. Indeed, replacing specific residues in the transmembrane helix of either alphaIIb or beta3 with an asparagine residue resulted in a facilitated homo-oligomerization of the mutated transmembrane helix, promoted the formation of integrin clusters on the cell surface and shifted alphaIIbbeta3 to its activated state. Thus these studies support the hypothesis that the transmembrane domains play a vital role in the function and regulation of alphaIIbbeta3.

Amino Acid Motifs↗

PubMed2005

[GFP fused to the cytoplasmic tail of integrin alphaIIb allows the normal expression of alphaIIb beta3 compound in CHO cells].

To investigate the effect of GFP fused to C terminal of integrin alpha(IIb) on the biosynthesis and expression of alpha(IIb) beta(3) compound, the alpha(IIb) GFP expression plamid, named palpha(IIb) GFP, the cDNA of alpha(IIb) was constructed from p3.1-2b and fused to pEGFP-N1 in frame. When the sequence of palpha(IIb) GFP was confirmed by sequencing it was transferred to Chinese Hamster Ovary (CHO) cells with or without p3.1-3a expressing integrin beta(3). Then the expression of alpha(IIb) GFP fusion protein was confirmed by Western blot and then its subcellular localization was determined with laser confocal scanning microscopy. The results showed that the target gene was cloned into recombinant vector by restriction analysis and sequencing. Overexpression of the fusion protein in the transfected CHO cells was identified with Western blot. Subcellular localization analysis confirmed that alpha(IIb) GFP was expressed in CHO cells and could be transferred from endoplasmic reticulum to Golgi apparatus. It is concluded that the eukaryotic expression plasmid containing alpha(IIb) GFP fusion gene is successfully constructed. GFP fused to the cytoplasmic tail of integrin alpha(IIb) allows the normal expression of alpha(IIb) beta(3) in CHO cells.

Animals↗

PubMed2005

Highly constrained cyclic (S,S) -CXaaC- peptides as inhibitors of fibrinogen binding to platelets.

The Arg-Gly-Asp RGD motif of adhesive proteins is recognized by the activated platelet integrin alpha(IIb)beta3. Binding of fibrinogen (Fg) to activated alpha(IIb)beta3 causes platelet aggregation and thrombus formation. Highly constraint cyclic (S,S) -CXaaC- containing peptides incorporating the (S,S) -CDC- and (S,S) -CRC- motifs were tested for their ability to inhibit platelet aggregation and Fg binding. Our results suggest that the above cyclic scaffolds stabilize a favorable structure for the antiaggregatory activity (IC50-values ranged from 1.7 to 570 microm). The peptides inhibited Fg binding with IC50-values up to 30-fold lower than those determined for the inhibition of the adenosine diphosphate (ADP)-induced platelet aggregation. Importantly, peptides (S,S) PSRCDCR-NH2 (peptide 11) and (S,S) PRCDCK-NH2 (peptide 10) did not inhibit PAC-1 binding to the activated platelets at a concentration in which they completely inhibited Fg binding. Moreover, (S,S) PSRCDCR-NH(2) (peptide 11), one of the more active peptides, inhibited ADP-induced P-selectin exposure. By contrast, peptide (S,S) Ac-RWDCRC-NH2, incorporating the inverse (S,S) -DCRC- sequence (peptide 16), failed to inhibit P-selectin exposure whereas at the same concentration, it effectively inhibited PAC-1 and Fg binding. It is concluded that peptides containing the (S,S) -CDC- as well the (S,S) -CRC- sequences, exhibit a broad range of activities toward platelets, and could be helpful tools for elucidating the structural interaction of Fg with the integrin receptor alpha(IIb)beta3, in its activated form. Furthermore, the (S,S) -RCDC- sequence can be used as a scaffold for developing potent non-RGD-like Fg-binding inhibitors.

Amino Acid Sequence↗

PubMed2000

Integrin expression on neutrophils in a rabbit model of Group B Streptococcal meningitis.

Products released by polymorphonuclear cells (PMNs) during an acute inflammatory response can result in diffuse tissue injury. Integrins are cell surface adhesion proteins that play a pivotal role in inflammation by allowing PMNs to adhere to the endothelium and migrate through the extracellular matrix. We examined the expression of beta1 and beta2 integrins on neutrophils from blood and cerebrospinal fluid (CSF) in an animal model of Group B Streptococcal meningitis. We further evaluated whether integrin expression correlates with pathophysiologic markers of central nervous system inflammation. Our data demonstrate that beta3 and beta2 integrin expression on circulating neutrophils does not significantly increase as a consequence of meningitis. In extravesated CSF neutrophils, a significant increase in expression of both beta1 and beta2 integrins is noted. Furthermore, a majority of the beta1 integrins on extravesated neutrophils have undergone affinity modulation. Using regression analysis, we demonstrated that increasing beta1 integrin expression correlates with decreasing CSF glucose concentration and serum/CSF glucose ratio. Regression analysis approached significance when CSF protein was compared to PMN beta1 integrin expression. Polymorphonuclear leukocytes beta1 integrin expression also showed a direct correlation to myeloperoxidase activity in brain tissue. Beta2 expression on CSF PMNs did not correlate with these markers of inflammation/sequestration. These data demonstrate integrin expression on extravesated neutrophils markedly increases during meningitis and support a role for beta1 integrins on neutrophils in the pathophysiologic consequences of meningitis.

Animals↗

PubMed2006

Anti-angiogenic cancer therapy based on integrin alphavbeta3 antagonism.

Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is a fundamental process during cancer progression. Anti-angiogenic strategies have been pursued for cancer treatment and prevention of cancer recurrence and metastasis. Integrins are a family of cell adhesion molecules consisting of two non-covalently bound transmembrane subunits (alpha and beta). Much research has demonstrated that integrin signaling plays a key role in tumor angiogenesis and metastasis. Integrin alphavbeta3 is highly expressed on activated endothelial cells and tumor cells but is not present in resting endothelial cells and most normal organ systems, which makes it a suitable target for anti-angiogenic cancer therapy. In this review we will focus on cancer therapy targeting integrin alphavbeta3 while other integrins (such as alpha5beta1, alphaIIbbeta3, alphavbeta5, alpha6beta4) will only be briefly mentioned when relevant. MEDI-522 (a humanized anti-human integrin alphavbeta3 monoclonal antibody) and Cilengitide (cyclic peptidic integrin alphavbeta3/alphavbeta5 antagonist) are currently in clinical trials for anti-angiogenic cancer therapy. Small interfering RNA (siRNA) that specifically silences integrin alphav and/or beta3 was reported to cause tumor shrinkage in preclinical xenograft models. Combination of anti-integrin alphavbeta3 therapy and other therapeutic approaches (such as chemotherapy, radiotherapy and gene therapy) has also been applied for cancer treatment. Mounting evidence suggests that there is potentially synergistic effect of combined therapeutic approaches over single modality alone. Lastly, integrin targeted delivery (drugs, genes, and radioisotopes) and imaging (optical, MRI, ultrasound, SPECT, and PET) is discussed in detail.

Angiogenesis Inhibitors↗

PubMed2003

Expression and function of the integrin alpha9beta1 in bovine aortic valve interstitial cells.

BACKGROUND AND AIM OF THE STUDY: Aortic valve interstitial cells (VIC), the most prevalent valve leaflet cells, have not been well studied. However, recent interest in constructing tissue-engineered living heart valves has provided motivation to further an understanding of the molecular and cellular biology of VIC. Since cell-extracellular matrix interactions are critical for tissue morphogenesis, adhesive interactions and integrin function in VIC were investigated. METHODS: Bovine and baboon VIC were isolated and characterized by morphology, cytoskeletal protein expression and integrin expression. The interaction of VIC integrin alpha9beta1 with osteopontin, a ligand known to be up-regulated in the wounded valve, was examined. The ability of bovine VIC alpha9beta1 to mediate adhesion, focal contact formation, migration and proliferation when plated on the immobilized osteopontin substrates, was investigated. RESULTS: Cultured bovine VIC possessed characteristics of both fibroblasts and smooth muscle cells. Like fibroblasts, bovine VIC had an elongated morphology with long cytoplasmic processes. Like smooth muscle cells, bovine VIC expressed alpha-smooth muscle actin and SM22alpha. Using fluorescent flow cytometry, the surface expression of integrins alpha9beta1 and alpha(v)beta3 in bovine VIC and integrins alpha1beta1, alpha2beta1, and alpha5beta1 in baboon VIC was identified. Expression of alpha9beta1 in cultured VIC and in native valve leaflets was further confirmed by Western blot and RT-PCR analysis. Bovine VIC were found to adhere to the 30N fragment of osteopontin in an alpha9beta1-dependent manner, and this interaction was mediated through the SVVYGLR motif. Bovine VIC also formed focal contacts through the alpha9beta1 integrin, but osteopontin (30N) did not stimulate migration or proliferation of bovine VIC through the integrin alpha9beta1. CONCLUSION: Integrin alpha9beta1 appears to be predominantly involved in controlling anchorage rather than movement or proliferation, in bovine VIC. For tissue engineering, knowledge of the VIC integrin profile can be applied to the rational design of scaffolds with the appropriate ligands for tissue formation. The VIC a9beta1-SVVYGLR interaction could be exploited to promote cell anchorage within a tissue engineering scaffold.

Actins↗

PubMed2005

Non-peptidic alpha(v)beta3 antagonists containing indol-1-yl propionic acids.

We describe the synthesis and structure/activity relationship of RGD mimetics that are potent inhibitors of the integrin alpha(v)beta3. Indol-1-yl propionic acids containing a variety of basic moieties at the 5-position, as well as substitutions alpha and beta to the carboxy terminus were synthesized and evaluated. Novel compounds with improved potency have been identified.

Indoles↗

PubMed2000

Cell growth and matrix invasion of EBV-immortalized human B lymphocytes is regulated by expression of alpha(v) integrins.

alpha(v) integrins have been shown to play an important role in epithelial-derived cell migration, cell growth and tumor invasion/metastasis, however their role on cells of hematopoietic origin is less clear. Epstein-Barr virus (EBV), a human herpesvirus associated with several lymphoproliferative disorders in man, induces expression of alpha(v) integrins on transformed B lymphocytes. In the studies reported here, we show that EBV infection increases alpha(v), beta3 and beta5 integrin subunit mRNAs as well as upregulates the expression of the alphavbeta3 integrin protein on human B cells. Among the nine different EBV proteins expressed in latently infected B cells (nuclear and plasma membrane-associated), only LMP1, LMP2A and EBNA2 were shown to selectively transactivate the alpha(v) integrin promoter. Treatment of EBV-transformed B cells with alpha(v) antisense oligonucleotides specifically reduced cell surface expression of alpha(v) integrins, inhibited cell growth in low serum, reduced cell invasion in matrigels and decreased expression of metalloprotease, MMP9. These studies indicate that alpha(v) integrins play a significant role in EBV-induced B-lymphocyte proliferation and invasion. Strategies to interfere with alphav integrin expression and/or function may therefore be of potential value in the treatment of EBV-associated lymphoproliferative disorders.

Antigens, CD↗

PubMedDate not supplied

Expression pattern of integrin adhesion molecules in endometriosis and human endometrium.

Integrins are cell adhesion molecules that undergo cell-specific dynamic changes during the normal menstrual cycle in the human endometrium. Here, using immunohistochemistry, we have investigated the expression pattern of the integrins alphav, alpha2beta1, alpha3beta1, alpha3, alpha6, beta1, beta2 and beta3 in the human ectopic endometrium of 30 patients and in nine cases in the corresponding eutopic endometrium. The biopsies were obtained during the early or late follicular phase (25 cases), during the corpus luteum phase (four cases) and in one case after 6 months' treatment with a gonadotrophin releasing hormone (GnRH) agonist. The integrin expression was independent of the ovarian steroid situation at the time of biopsy. The integrin alpha6 was expressed in all endometriotic and endometrium samples. The integrin alpha3 was absent in all endometrium tissues of patients with endometriosis. However, the corresponding endometriotic lesions re-expressed this adhesion molecule in 15 cases. No change in integrin beta3 expression pattern could be demonstrated in either endometriotic lesions or endometrium samples, regardless of the menstrual cycle phase. A correlation between serum oestradiol and progesterone concentrations and the expression of the investigated integrins was not observed, thus indicating that these two hormones play a minor role in the regulation of the cell adhesion molecules examined. Our investigation suggests that endometriosis is a dedifferentiated disease as it expressed different integrins in comparison with the eutopic endometrium, and independently of the hormonal situation. The ability of endometriotic tissues to express integrins may explain the high recurrence rates in patients with endometriosis, as these samples retain their adhesion potency after retrograde menstruation and are thus able to establish cell-cell and cell-matrix interactions with the surrounding peritoneum.

Biopsy↗

PubMed2000

Alpha v integrin antagonists induce the disassembly of focal contacts in melanoma cells.

In recent years, several antagonists of alpha(v)beta3 have been used to develop therapeutic approaches to the treatment of melanoma neoplasia. We studied the effects of anti-alpha(v)-integrin-blocking antibodies on attached M21 melanoma cells, the cellular distribution of alpha(v)-integrin and the molecular organization of focal structures. Anti-alpha(v)-integrin-blocking antibodies 17E6 and LM609, and an anti-alpha(v)beta3-integrin antagonist peptide cRGD 85189 induced detachment of M21 melanoma cells cultured for 24 hours on various substrates. cRGD was the most effective antagonist, reducing the number of adherent cells by 80%, while 17E6 reduced adhesion by only 30%. Light- and electron microscopy revealed attached cells with a flat shape and well-formed actin cytoskeleton. After treatment, cells became rounded and detached from the culture dish. alpha(v)-Integrins and focal-contact proteins were observed at adhesion sites in focal structures by immunocytochemistry. After treatment, however, cell rounding was accompanied by disorganization of the actin filaments and redistribution of alpha(v)-integrins and most of the focal proteins studied, except vinculin and tensin. Our results indicate that treatment of M21 melanoma cells with a(v)-integrin antagonists disrupts the actin cytoskeleton, redistributes a(v)-integrin and induces molecular disassembly of focal contacts.

Actins↗

PubMed2005

Conformation and concerted dynamics of the integrin-binding site and the C-terminal region of echistatin revealed by homonuclear NMR.

Echistatin is a potent antagonist of the integrins alpha(v)beta3, alpha5beta1 and alpha(IIb)beta3. Its full inhibitory activity depends on an RGD (Arg-Gly-Asp) motif expressed at the tip of the integrin-binding loop and on its C-terminal tail. Previous NMR structures of echistatin showed a poorly defined integrin-recognition sequence and an incomplete C-terminal tail, which left the molecular basis of the functional synergy between the RGD loop and the C-terminal region unresolved. We report a high-resolution structure of echistatin and an analysis of its internal motions by off-resonance ROESY (rotating-frame Overhauser enhancement spectroscopy). The full-length C-terminal polypeptide is visible as a beta-hairpin running parallel to the RGD loop and exposing at the tip residues Pro43, His44 and Lys45. The side chains of the amino acids of the RGD motif have well-defined conformations. The integrin-binding loop displays an overall movement with maximal amplitude of 30 degrees . Internal angular motions in the 100-300 ps timescale indicate increased flexibility for the backbone atoms at the base of the integrin-recognition loop. In addition, backbone atoms of the amino acids Ala23 (flanking the R24GD26 tripeptide) and Asp26 of the integrin-binding motif showed increased angular mobility, suggesting the existence of major and minor hinge effects at the base and the tip, respectively, of the RGD loop. A strong network of NOEs (nuclear Overhauser effects) between residues of the RGD loop and the C-terminal tail indicate concerted motions between these two functional regions. A full-length echistatin-alpha(v)beta3 docking model suggests that echistatin's C-terminal amino acids may contact alpha(v)-subunit residues and provides new insights to delineate structure-function correlations.

Animals↗

PubMed1997

Bombesin stimulates the motility of human prostate-carcinoma cells through tyrosine phosphorylation of focal adhesion kinase and of integrin-associated proteins.

Bombesin-like peptides, including the mammalian homologue gastrin-releasing peptides, are highly expressed and secreted by neuroendocrine cells in prostate carcinoma (PCa) tissues and are likely to be related to the progression of this disease. In the present study, we show that bombesin enhances the migration of androgen-independent PCa cells (PC-3) in vitro, while not affecting their adhesion to extracellular matrix proteins. The bombesin-increased motility of PC-3 cells occurs through its receptor, and, as shown with inhibitors, it likely requires activation of both protein tyrosine kinases (PTKs) and protein kinases C (PKCs). Because the focal adhesion kinase pp125FAK plays a key role in adhesion/motility and is highly expressed in advanced PCa, we examined whether in PC-3 cells bombesin signal transduction triggers the tyrosine phosphorylation of this PTK and of associated integrins and signaling proteins likely to be present in focal adhesion plaques. pp125FAK tyrosine phosphorylation was stimulated by bombesin and mimicked by PKC activation with the tumor-promotor phorbol 12-myristate-13-acetate (PMA). Moreover, this effect of bombesin on pp125FAK tyrosine phosphorylation requires the presence of both active PKC and cytoskeleton integrity since this signal was abolished by down-regulating PKCs induced by prolonged PMA treatment or by PKC inhibition with GF 109203X, as well as by disruption of the cytoskeleton with cytochalasin D. We also show that bombesin increases the tyrosine phosphorylation of a 95-kDa protein (pp95) which was co-immunoprecipitated with the alpha v and beta (3 and 5) subunits, forming integrin receptors with alpha v in PC-3 cells. The protein pp95 is distinct from the endogenously tyrosine-phosphorylated beta3 subunit. In addition, upon bombesin treatment, the beta1, beta3 and beta5 integrin subunits co-immunoprecipitated with pp125FAK and major phosphotyrosine (pY)-containing proteins of 125 and 68-70 kDa, likely corresponding to pp125FAK and paxillin. Together our data suggest that, in addition to PKC activation, tyrosine phosphorylation of pp125FAK and integrin-associated proteins may play an important role in bombesin signaling, triggering the processes of PCa cell motility and invasion.

Bombesin↗

PubMed2003

A well-defined in vitro three-dimensional culture of human endometrium and its applicability to endometrial cancer invasion.

A three-dimensional (3-D) endometrium culture was established, in which human endometrial stromal cells embedded in a mixture of collagen I, a major component of extracellular matrix, and matrigel, a basement membrane material, supports the epithelial cells seeded on top of the collagen/matrigel matrix. The biological growth and differentiation of the epithelial cells were studied microscopically and immunohistochemically. Transmission electron microscopy showed a polarized columnar epithelium in monolayer with basally positioned nuclei. Scanning electron microscopy revealed a confluent epithelium with an abundance of microvilli and cilia as well as pinopodes on the apical surface. An immunohistochemical staining showed that integrin alpha1, alpha4, and beta3 were co-localized with cytokeratin, confirming the epithelial origin of the cells. In contrast, immunoreactivity against cyclooxygenase-1 or -2 was positive in both epithelial and stromal cells. When epithelial cells were replaced by KLE cells, an endometrial cancer cell of epithelial origin, invasion of KLE cells into the stromal fraction was observed. The invasion was closely correlated to expression of matrix metalloproteinases and their tissue inhibitors of metalloproteinases in a manner consistent with paracrine fashion. The present 3-D culture imitates the normal endometrium physiologically as well as morphologically, thus provides an excellent in vitro tissue suitable for reproducing in vivo physiological processes, including endometrial cancer invasion.

Cell Communication↗

PubMed1996

Molecular identification of T cells that respond in a primary bulk culture to a peptide derived from a platelet glycoprotein implicated in neonatal alloimmune thrombocytopenia.

Neonatal alloimmune thrombocytopenia induced by the human platelet alloantigen 1a (HPA1a) is characterized by generation of alloantibodies by a mother who is homozygous for the HPA1b alloantigen and almost always HLA-DRB3*0101. The disease is viewed as B cell mediated but the linkage with HLA is indicative of a role for T cells. The HPA1a and HPA1b allotypes are defined, respectively, by Leu and Pro at amino acid 33 of the beta-chain of the platelet integrin GPIIbIIIa (alpha(IIb)beta3). Under the assumption that the same polymorphism may control both the B cell epitope and constitute the MHC-bound peptide, we restimulated PBMC from a woman with an affected child with a synthetic peptide from this polymorphic region. Molecular analysis of the responding T cell repertoire identified two T cells which predominated in cultures stimulated with the alloantigen peptide and which were absent in cultures with the autoantigen peptide. In spite of the use of different V families, sequence of the CDR3 region of the T cell receptor (TCR) beta-chain revealed the presence of a shared motif, L-P-S/T. Oligonucleotide probes specific for the CDR3 sequence indicated that these T cells were present in the PBMC at the highest levels immediately after delivery of the affected infant and their frequency dropped at later times.

Antigens, Human Platelet↗

PubMed2003

Activation of integrin alphaIIbbeta3 by modulation of transmembrane helix associations.

Transmembrane helices of integrin alpha and beta subunits have been implicated in the regulation of integrin activity. Two mutations, glycine-708 to asparagine-708 (G708N)and methionine-701 to asparagine-701, in the transmembrane helix of the beta3 subunit enabled integrin alphaIIbbeta3 to constitutively bind soluble fibrinogen. Further characterization of the G708N mutant revealed that it induced alphaIIbbeta3 clustering and constitutive phosphorylation of focal adhesion kinase. This mutation also enhanced the tendency of the transmembrane helix to form homotrimers. These results suggest that homomeric associations involving transmembrane domains provide a driving force for integrin activation. They also suggest a structural basis for the coincidence of integrin activation and clustering.

Amino Acid Sequence↗

PubMed2002

Myocardial ischemic injury after heart transplantation is associated with upregulation of vitronectin receptor (alpha(v)beta3), activation of the matrix metalloproteinase induction system, and subsequent development of coronary vasculopathy.

BACKGROUND: Myocardial ischemic injury after heart transplantation is associated with subsequent development of graft vasculopathy. Both vitronectin receptor (integrin alpha(v)beta3) and tissue factor play key roles in vascular endothelial cell injury. Matrix metalloproteinases (MMPs) are activated in ischemic injury models. METHODS AND RESULTS: Thirteen patients developed myocardial ischemic injury within 2 weeks of cardiac transplantation (ischemia group). These were compared with 10 transplantation patients who had no evidence of ischemia (control group). Endomyocardial biopsies were evaluated within 2 weeks of transplantation for alpha(v)beta3, tissue factor, and extracellular MMP inducer (EMMPRIN). At 1 year, MMPs were evaluated, and interstitial myocardial fibrosis was quantified. All patients underwent intravascular ultrasound at 1 month and 1 year after transplantation. Compared with control, the ischemia group demonstrated evidence of significant increased expression of alpha(v)beta3 (3.2-fold, P<0.001), tissue factor (2.5-fold, P<0.001), and EMMPRIN (1.9-fold, P=0.01). At 1 year, the ischemia group had a significant increase in myocardial fibrosis (24+/-1.8% versus 14+/-1.1%, P<0.001) and zymographic activity of MMP-2 (1.4-fold, P<0.001), MMP-3 (1.2-fold, P<0.001), and MMP-9 (1.3-fold, P=0.01). Coronary vasculopathy progression was also more advanced in the ischemia group (change in coronary maximal intimal thickness over 1 year 0.54+/-0.1 versus 0.26+/-0.06 mm; P=0.031). CONCLUSIONS: Myocardial ischemic injury after cardiac transplantation is associated with upregulation of alpha(v)beta3, tissue factor, and activation of the MMP induction system, which may contribute to the subsequent development of allograft remodeling and vasculopathy.

Adult↗

PubMed2006

Identification and characterization of triosephosphate isomerase that specifically interacts with the integrin alphaIIb cytoplasmic domain.

Integrin alphaIIb/beta3 (IIb/IIIa), a platelet fibrinogen receptor, has been shown to play a critical role in thrombosis and hemostasis. However, the mechanisms by which ligands interact with the alphaIIb/beta3 receptor is not very clear at this time. The interaction between the ligand, the receptor and the transmission of extracellular signals may involve the cytoplasmic domains of these integrins. The objective of this investigation was to identify novel proteins that interact with the cytoplasmic tail of alphaIIb. Using alphaIIb cytoplasmic tail as the bait and a yeast two-hybrid system, we have identified three separate clones containing inserts that encoded the same protein with different truncated N-terminals. Sequence analysis showed that the inserts of the three clones encoded a previously identified enzyme: triose phosphate isomerase (TPI). In addition, we demonstrated that TPI failed to interact with the integrin alpha2 tail, beta3 tail and lamin, but showed a weak binding to the alphaV tail which shares the highest homology with alphaIIb tail among the integrin alpha family. Site-directed mutagenesis studies around the homology region indicated that the critical peptide sequence necessary for the interaction between TPI and alphaIIb tail is GFFKRNRPPLEE. Using RT-PCR, we have demonstrated the presence of TPI mRNA in platelets. In addition, experiments were also performed to demonstrate specific binding of TPI to alphaIIb using an ELISA and fusion protein. Taken together, these data suggest that TPI specifically interacts with alphaIIb and may play a critical role in alphaIIb/beta3-mediated platelet function.

Amino Acid Sequence↗

PubMed1997

Lactadherin (formerly BA46), a membrane-associated glycoprotein expressed in human milk and breast carcinomas, promotes Arg-Gly-Asp (RGD)-dependent cell adhesion.

Lactadherin, a major glycoprotein of the human milk fat globule membrane, is abundant in human breast milk and expressed in human breast carcinomas. Previously, we have shown that the mature protein, formerly known as BA46, has three domains: an epidermal growth factor (EGF)-like domain containing an Arg-Gly-Asp (RGD) cell adhesion sequence and C1 and C2 domains similar to those found in coagulation factors V and VIII. An alignment of lactadherin with its bovine (MGP57/53) and murine (MFG-E8) homologs shows that the RGD sequence has been conserved during evolution, suggesting that the RGD sequence is not fortuitous. We demonstrate that lactadherin purified using Triton X-114 phase partitioning promotes RGD-dependent cell attachment of green monkey kidney cells (MA104), mouse fibroblast cells (3T3-L1), and breast carcinoma cells (ELL-G). A lactadherin-specific monoclonal antibody, Mc3, inhibits attachment to purified lactadherin, suggesting that contaminants in the purification are not responsible for binding. In addition, the anti-integrin alpha(v)beta3 monoclonal antibody LM609 inhibits cell attachment of MA104 cells to lactadherin. These results demonstrate that lactadherin promotes RGD-dependent cell adhesion via integrins. Denaturation of lactadherin with heat and reducing conditions diminished cell attachment, suggesting that optimal cell attachment to RGD is dependent on the structural presentation of the sequence.

3T3 Cells↗