The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation.

Kindlin-2 (K2), a 4.1R-ezrin-radixin-moesin (FERM) domain adaptor protein, mediates numerous cellular responses, including integrin activation. The C-terminal 15-amino acid sequence of K2 is remarkably conserved across species but is absent in canonical FERM proteins, including talin. In CHO cells expressing integrin αIIbß3, co-expression of K2 with talin head domain resulted in robust integrin activation, but this co-activation was lost after deletion of as few as seven amino acids from the K2 C terminus. This dependence on the C terminus was also observed in activation of endogenous αIIbß3 in human erythroleukemia (HEL) cells and ß1 integrin activation in macrophage-like RAW264.1 cells. Kindlin-1 (K1) exhibited a similar dependence on its C terminus for integrin activation. Expression of the K2 C terminus as an extension of membrane-anchored P-selectin glycoprotein ligand-1 (PSGL-1) inhibited integrin-dependent cell spreading. Deletion of the K2 C terminus did not affect its binding to the integrin ß3 cytoplasmic tail, but combined biochemical and NMR analyses indicated that it can insert into the F2 subdomain. We suggest that this insertion determines the topology of the K2 FERM domain, and its deletion may affect the positioning of the membrane-binding functions of the F2 subdomain and the integrin-binding properties of its F3 subdomain. Free C-terminal peptide can still bind to K2 and displace the endogenous K2 C terminus but may not restore the conformation needed for integrin co-activation. Our findings indicate that the extreme C terminus of K2 is essential for integrin co-activation and highlight the importance of an atypical architecture of the K2 FERM domain in regulating integrin activation.

Epigallocatechin gallate has pleiotropic effects on transmembrane signaling by altering the embedding of transmembrane domains.

Epigallocatechin gallate (EGCG) is the principal bioactive ingredient in green tea and has been reported to have many health benefits. EGCG influences multiple signal transduction pathways related to human diseases, including redox, inflammation, cell cycle, and cell adhesion pathways. However, the molecular mechanisms of these varying effects are unclear, limiting further development and utilization of EGCG as a pharmaceutical compound. Here, we examined the effect of EGCG on two representative transmembrane signaling receptors, integrinαIIbß3 and epidermal growth factor receptor (EGFR). We report that EGCG inhibits talin-induced integrin αIIbß3 activation, but it activates αIIbß3 in the absence of talin both in a purified system and in cells. This apparent paradox was explained by the fact that the activation state of αIIbß3 is tightly regulated by the topology of ß3 transmembrane domain (TMD); increases or decreases in TMD embedding can activate integrins. Talin increases the embedding of integrin ß3 TMD, resulting in integrin activation, whereas we observed here that EGCG decreases the embedding, thus opposing talin-induced integrin activation. In the absence of talin, EGCG decreases the TMD embedding, which can also disrupt the integrin α-ß TMD interaction, leading to integrin activation. EGCG exhibited similar paradoxical behavior in EGFR signaling. EGCG alters the topology of EGFR TMD and activates the receptor in the absence of EGF, but inhibits EGF-induced EGFR activation. Thus, this widely ingested polyphenol exhibits pleiotropic effects on transmembrane signaling by modifying the topology of TMDs.

Bufalin enhances antitumor effect of paclitaxel on cervical tumorigenesis via inhibiting the integrin α2/ß5/FAK signaling pathway.

While Bufalin restrains primary tumorigenesis, the role of Bufalin in cervical cancer remains unclear. Here, we show that Bufalin can inhibit cervical cancer cell proliferation, block cell cycle in G2/M phase, induce cellular apoptosis and reduce cell metastasis through stimulation of p21(waf/cip1), p27(cip/kip), Bax and E-cadherin, and suppression of cyclin A, cyclin B1, CDK2, Bcl-2, Bcl-xl, MMP9 and SNAIL1. Further study suggests that Bufalin has no apparent damage to human normal cervical cells at the low concentration (<20nM), but increases the chemotherapeutic efficacy of paclitaxel. Mechanistic study reveals that Bufalin suppresses the integrin α2/FAK/AKT1/ GSK3ß signaling. Finally, in vivo studies show that Bufalin blocks the Siha-induced xenograft tumor growth without detectable toxicity in the animals at the therapeutic doses, and the combination treatment of Bufalin and paclitaxel more efficiently inhibits xenograft tumor growth. Thus, Bufalin may be developed as a potential therapeutic agent to treat cervical cancer.

Cantharidin and norcantharidin inhibit the ability of MCF-7 cells to adhere to platelets via protein kinase C pathway-dependent downregulation of α2 integrin.

Cancer metastasis is a highly coordinated and dynamic multistep process in which cancer cells interact with a variety of host cells. Morphological studies have documented the association of circulating tumor cells with host platelets, where a surface coating of platelets protects tumor cells from mechanical trauma and the immune system. Cantharidin is an active constituent of mylabris, a traditional Chinese medicine. Cantharidin and norcantharidin are potent protein phosphatase 2A (PP2A) inhibitors that exhibit in vitro and in vivo antitumor activity against several types of cancer, including breast cancer. We investigated whether cantharidin and norcantharidin could repress the ability of MCF-7 breast cancer cells to adhere to platelets. Using MTT, clone formation, apoptosis, adhesion and wound-healing assays, we found that cantharidin and norcantharidin induced apoptosis and repressed MCF-7 cell growth, adhesion and migration. Moreover, we developed a flow cytometry-based analysis of tumor cell adhesion to platelets. We proved that cantharidin and norcantharidin repressed MCF-7 cell adhesion to platelets through downregulation of α2 integrin, an adhesion molecule present on the surface of cancer cells. The repression of α2 integrin expression was found to be executed through the protein kinase C pathway, the activation of which could have been due to PP2A inhibition.

Mapping of potent and specific binding motifs, GLOGEN and GVOGEA, for integrin α1ß1 using collagen toolkits II and III.

Integrins are well characterized cell surface receptors for extracellular matrix proteins. Mapping integrin-binding sites within the fibrillar collagens identified GFOGER as a high affinity site recognized by α2ß1, but with lower affinity for α1ß1. Here, to identify specific ligands for α1ß1, we examined binding of the recombinant human α1 I domain, the rat pheochromocytoma cell line (PC12), and the rat glioma Rugli cell line to our collagen Toolkit II and III peptides using solid-phase and real-time label-free adhesion assays. We observed Mg(2+)-dependent binding of the α1 I domain to the peptides in the following rank order: III-7 (GLOGEN), II-28 (GFOGER), II-7 and II-8 (GLOGER), II-18 (GAOGER), III-4 (GROGER). PC12 cells showed a similar profile. Using antibody blockade, we confirmed that binding of PC12 cells to peptide III-7 was mediated by integrin α1ß1. We also identified a new α1ß1-binding activity within peptide II-27. The sequence GVOGEA bound weakly to PC12 cells and strongly to activated Rugli cells or to an activated α1 I domain, but not to the α2 I domain or to C2C12 cells expressing α2ß1 or α11ß1. Thus, GVOGEA is specific for α1ß1. Although recognized by both α2ß1 and α11ß1, GLOGEN is a better ligand for α1ß1 compared with GFOGER. Finally, using biosensor assays, we show that although GLOGEN is able to compete for the α1 I domain from collagen IV (IC(50) ∼3 µm), GFOGER is much less potent (IC(50) ∼90 µm), as shown previously. These data confirm the selectivity of GFOGER for α2ß1 and establish GLOGEN as a high affinity site for α1ß1.

Multiple alpha subunits of integrin are involved in cell-mediated responses of the Manduca immune system.

The cell-mediated responses of the insect innate immune system-phagocytosis, nodulation, encapsulation-involve multiple cell adhesion molecules of hemocyte surfaces. A hemocyte-specific (HS) integrin and a member of the immunoglobulin (Ig) superfamily (neuroglian) are involved in the encapsulation response of hemocytes in Manduca sexta. In addition, two new integrin alpha (alpha) subunits have been found on these hemocytes. The alpha2 subunit is mainly expressed in epidermis and Malphigian tubules, whereas the alpha3 subunit is primarily expressed on hemocytes and fat body cells. Of the three known alpha subunits, the alpha1 subunit found in HS integrin is the predominant subunit of hemocytes. Cell adhesion assays indicate that alpha2 belongs to the integrin family with RGD-binding motifs, confirming the phylogenetic analysis of alpha subunits based on the amino-acid sequence alignment of different alpha subunits. Double-stranded RNAs (dsRNAs) targeting each of these three integrin alpha subunits not only specifically decreased transcript expression of each alpha subunit in hemocytes, but also abolished the cell-mediated encapsulation response of hemocytes to foreign surfaces. The individual alpha subunits of M. sexta integrins, like their integrin counterparts in mammalian immune systems, have critical, individual roles in cell-substrate and cell-cell interactions during immune responses.

Disulfide bond disruption by a beta 3-Cys549Arg mutation in six Jordanian families with Glanzmann thrombasthenia causes diminished production of constitutively active alpha IIb beta 3.

Alpha IIb beta 3 integrin mediates platelet aggregation following its activation. Its absence or dysfunction causes Glanzmann thrombasthenia (GT), an inherited bleeding disorder that is rare worldwide but relatively frequent in several populations with high rates of consanguinity, including Arabs in Israel and Jordan. Cysteine residues in the beta 3 epidermal growth factor (EGF) domains are involved in alpha IIb beta 3 formation and activation. In this study we present a novel Cys549Arg mutation in beta 3 identified in six Jordanian families, which in the homozygous state is manifested by severe GT. The mutation is located in EGF-3 of beta 3 predicting disruption of a conserved disulfide bond between Cys549 and Cys558. Haplotype analysis disclosed a common founder whose age estimate was 120-150 years. Flow cytometry revealed 1-14% of normal alpha IIb beta 3 expression at the patients' platelet surface. The Cys549Arg or artificial Cys549Ser mutations were introduced into a beta 3 expression vector. Co-transfection of baby hamster kidney cells with normal or mutant beta 3 along with normal alpha IIb demonstrated reduced surface expression of alpha IIb beta 3 by both mutants. The mutants were constitutively active as demonstrated by 20-fold increased binding of the ligand-mimetic antibody PAC-1. Immunoblotting and immunoprecipitation experiments showed reduced beta 3 and alpha IIb beta 3 expression and a higher than normal ratio of pro-alpha IIb to mature alpha IIb. Immunofluorescence experiments showed that beta 3 and alpha IIb beta 3 were mostly retained in the endoplasmic reticulum. In conclusion, the novel ancestral mutation found in a cluster of Jordanian GT patients disrupts a conserved Cys549-Cys558 bond which results in reduced production of constitutively active alpha IIb beta 3.

Functional display of an alpha2 integrin-specific motif (RKK) on the surface of baculovirus particles.

The use of baculovirus vectors shows promise as a tool for gene delivery into mammalian cells. These insect viruses have been shown to transduce a variety of mammalian cell lines, and gene transfer has also been demonstrated in vivo. In this study, we generated two recombinant baculovirus vectors displaying an integrin-specific motif, RKK, as a part of two different loops of the green fluorescent protein (GFP) fused with the major envelope protein gp64 of Autographa californica M nucleopolyhedrovirus. By enzyme linked immunosorbent assays, these viruses were shown to bind a peptide representing the receptor binding site of an alpha2 integrin, the alpha2I-domain. However, the interaction was not strong enough to overcome binding of wild type gp64 to the unknown cellular receptor(s) on the surface of alpha2 integrin-expressing cells (CHO-alpha2beta1) or enhance the viral uptake. After treatment of these cells with phospholipase C, internalization of all viruses was blocked or decreased significantly. However, one of the RKK displaying viruses, AcGFP(K)gp64, was still able to internalize into CHO-alpha2beta1 cells, although at a lower level as compared to non-treated cells. This may indicate the possible utilization of a PLC independent alternative route via, in this case, the alpha2beta1 integrin.