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Integrin and Integrin-associated protein

Integrin associated protein (IAP) is a 50-kD membrane protein that copurifies with the integrin alpha v beta 3 from placenta and coimmunoprecipitates with beta 3 from platelets. IAP also is functionally associated with signal transduction from the leukocyte response integrin. Using tryptic peptide sequence, human and murine IAP cDNAs have been isolated. The protein has an extracellular amino-terminal immunoglobulin domain that binds all monoclonal anti-IAP antibodies. The carboxy-terminal region is highly hydrophobic with either three or five membrane-spanning segments and a short hydrophilic tail. Immunofluorescence microscopy suggests that this hydrophilic tail is located on the inside of the cytoplasmic membrane. Monoclonal anti-IAP antibody inhibits the binding of vitronectin-coated beads to alpha v beta 3 on human erythroleukemia cells, and polyclonal anti-IAP recognizes hamster IAP on CHO cells and inhibits vitronectin bead binding. When CHO cells are transfected with human IAP, monoclonal anti-human antibody completely inhibits vitronectin bead binding. These data suggest a model in which ligand binding by alpha v beta 3 is regulated by IAP (Lindberg, 1993).

The CD47 glycoprotein has been isolated from human erythrocytes by immunoprecipitation using monoclonal antibody (mAb) BRIC-125. Enzymic deglycosylation of the protein shows it contains N-linked oligosaccharides, and trypsin proteolysis of the protein in situ in the erythrocyte membrane cleaves it into two portions, one of which is glycosylated. Both the intact protein and the glycosylated fragment have blocked N-termini. The protein sequence reveals it to be very similar to or identical with OA3, a multispanning membrane protein. The protein also appears to be the same as the integrin-associated protein, which has a role in cell adhesion in non-erythroid cells. CD47 has six potential N-glycosylation sites, five of which are in an Ig superfamily domain. Three of these sites carry N-glycans in erythrocytes. Immunocytochemical staining of human tissues shows that CD47 is broadly distributed on mesenchyme and epithelia at multiple sites. Reactivity is particularly prominent in surface and ductular epithelia, and in the brain (Mawby, 1994).

Using a K562 cell transfection model, a novel relationship has been made between the integrins alpha v beta 3 and alpha 5 beta 1. alpha v beta 3 ligation is able to inhibit alpha 5 beta 1-mediated phagocytosis without effect on alpha 5 beta 1-mediated adhesion. The alpha v beta 3-dependent inhibition apparently requires a signal transduction cascade, since it is reversed by inhibitors of serine/threonine kinases. The mechanisms of signal transduction in this system have been studied and the beta 3 cytoplasmic tail has been found to be both necessary and sufficient for initiation of the signal leading to inhibition of alpha 5 beta 1 phagocytosis. Ligation of integrin-associated protein (IAP), which has been implicated in alpha v beta 3 signal transduction, mimics the effects of alpha v beta 3 ligation only when the beta 3 integrin with an intact cytoplasmic tail is present. Although fibronectin-mediated phagocytosis requires the high affinity conformation of alpha 5 beta 1, ligation of alpha v beta 3/IAP does not prevent acquisition of this high affinity state. It is concluded that alpha v beta 3/IAP ligation initates a signal transduction cascade, dependent on the beta 3 cytoplasmic tail, which inhibits the phagocytic function of alpha 5 beta 1 at a step subsequent to modulation of integrin affinity (Blystone, 1995).

Integrin-associated protein (IAP/CD47) is physically associated with the alpha v beta 3 vitronectin (Vn) receptor and a functionally and immunologically related integrin on neutrophils (PMN) and monocytes. Anti-IAP antibodies inhibit multiple phagocyte functions, including Arg-Gly-Asp (RGD)-initiated activation of phagocytosis, chemotaxis, and respiratory burst; PMN adhesion to entactin; and PMN transendothelial and transepithelial migration at a step subsequent to tight intercellular adhesion. Anti-IAP antibodies also inhibit binding of Vn-coated particles to many cells expressing alpha v beta 3. However, prior studies with anti-IAP did not directly address IAP function because they could not distinguish between IAP blockade and antibody-induced signaling effects on cells. To better determine the function of IAP, an IAP-deficient human cell line was characterized and used. Despite expressing alpha v integrins, these cells do not bind Vn-coated particles unless transfected with IAP expression constructs. Increasing the level of alpha v beta 3 expression or increasing Vn density on the particle does not overcome the requirement for IAP. All known splice variants of IAP restore Vn particle binding equivalently. Indeed, the membrane-anchored IAP Ig variable domain suffices to mediate Vn particle binding in this system, while the multiple membrane-spanning and cytoplasmic domains are dispensable. In all cases, adhesion to a Vn-coated surface and fibronectin particle binding through alpha 5 beta 1 fibronectin receptors are independent of IAP expression. These data demonstrate that some alpha v integrin ligand-binding functions are IAP independent, whereas others require IAP, presumably through direct physical interaction between its Ig domain and the integrin (Lindberg, 1996).

The C-terminal "cell-binding domain" (CBD) of thrombospondin-1 (TS1) is a binding site for many cell types. Cell-binding peptides based on the sequence RFYVVM from the CBD of TS1 affinity label a 52-kDa cell surface glycoprotein, which is integrin-associated protein (IAP or CD47). IAP associates with alpha v beta 3 and thereby modulates the activity of several integrins. Cells that express IAP bind strongly to TS1, as well as the CBD and its active cell-binding peptides, while IAP negative cells do not. The 52-kDa protein is affinity labeled on IAP-positive but not IAP-negative cells, and monoclonal antibodies against IAP specifically immunoprecipitate the affinity-labeled 52-kDa protein from lysates of IAP-positive cells. Consistent with the association of IAP with alpha v beta 3 integrin, the labeled 52-kDa protein is immunoprecipitated by an anti-alpha v beta 3 antibody. Endothelial cells exhibit chemotaxis toward TS1 and RFYVVM peptides. Chemotaxis to both agents is specifically inhibited by a function blocking anti-IAP monoclonal antibody. These data establish IAP (CD47) as a receptor for the CBD of TS1 and suggest a mechanism for the well established effects of the CBD on cell motility (Gao, 1996).

Integrin-associated protein (IAP or CD47) is a receptor for the cell/platelet-binding domain (CBD) of thrombospondin-1 (TS1), the most abundant protein of platelet alpha granules. Although it associates with alphaIIbbeta3, IAP has no known function in platelets. TS1, the CBD, and an IAP agonist peptide (4N1K) from the CBD of TS1 activate the platelet integrin alphaIIbbeta3, resulting in platelet spreading on immobilized fibrinogen, stimulation of platelet aggregation, and enhanced tyrosine phosphorylation of focal adhesion kinase. 4N1K peptide selectively stimulates the phosphorylation of LYN and SYK and their association with FAK. The phosphorylation of SYK is blocked by pertussis toxin, implicating a Gi-like heterotrimeric G protein. IAP solublized from membranes of unstimulated platelets binds specifically to an affinity column of 4N1K peptide. Both alphaIIb and beta3 integrin subunits and c-Src bind along with IAP. This complex of proteins is also detected with immunoprecipitation. Activation of platelets with the agonist peptide 4N1K results in the association of FAK with the IAP-alphaIIbbeta3 complex. Thus an important function of TS1 in platelets is that of a secreted costimulator of alphaIIbbeta3 whose unique properties result in its localization to the platelet surface and the fibrin clot (Chung, 1997).

The PCR differential display method was adapted to identify cDNA clones associated with memory formation in rats. The one-way inhibitory avoidance learning task was used as the behavioral paradigm. Total RNA isolated from the hippocampus of rats with poor-memory (<80 sec) and good-memory (600 sec) 3 hr after training was used for comparison. Three cDNA fragments corresponding to different spliced forms of integrin-associated protein (IAP) mRNA were found to be differentially expressed in the hippocampus of good-memory rats. Quantitative reverse transcription-PCR reveals approximately four fold higher levels of IAP mRNA level in good-memory rats. This result was confirmed further by in situ hybridization analysis, and the major difference is in the dentate gyrus. It has been demonstrated that this difference in IAP mRNA expression is not attributable to different sensitivities of individual rats to electric shock. Rapid amplification of cDNA ends obtains the full-length IAP cDNA, which is 1192 bp in length excluding the poly(A+) tail. The IAP mRNA expression is significantly upregulated by NMDA and amphetamine injections to the dentate gyrus of the hippocampus. In contrast, injection of antisense oligonucleotide complementary to the IAP transcript markedly impairs memory retention in rats and decreases the amplitude and slope of EPSP in the in vivo long-term potentiation paradigm. These results together suggest that IAP gene expression plays an important role in memory formation and synaptic plasticity in rat hippocampus (Huang, 1998).

The carboxyl-terminal domain of thrombospondin-1 enhances the migration and proliferation of smooth muscle cells. Integrin-associated protein (IAP or CD47) is a receptor for the thrombospondin-1 carboxyl-terminal cell-binding domain and binds the agonist peptide 4N1K (kRFYVVMWKk) from this domain. 4N1K peptide stimulates chemotaxis of both human and rat aortic smooth muscle cells on gelatin-coated filters. The migration on gelatin is specifically blocked by monoclonal antibodies against IAP and a beta1 integrin, rather than alphav beta3 as found previously for 4N1K-stimulated chemotaxis of endothelial cells on gelatin. Both human and rat smooth muscle cells display a weak migratory response to soluble type I collagen; however, the presence of 4N1K peptide or intact thrombospondin-1 provokes a synergistic chemotactic response that is partially blocked by antibodies to alpha2 and beta1 integrin subunits and to IAP. A combination of antialpha2 and IAP monoclonal antibodies completely blocks chemotaxis. RGD peptide and antialphav beta3 antibody are without effect. 4N1K and thrombospondin-1 do not augment the chemotactic response of smooth muscle cells to fibronectin, vitronectin, or collagenase-digested type I collagen. Complex formation between alpha2 beta1 and IAP is detected by the coimmunoprecipitation of both alpha2 and beta1 integrin subunits with IAP. These data suggest that IAP can associate with alpha2 beta1 integrin and modulate its function (Wang, 1998).

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myospheroid: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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