Conformational resemblance between the structures of integrin-activating pentapetides derived from betaig-h3 and RGD peptide analogues in a membrane environment

The peptides NKDIL and EPDIM, respectively derived from the 2nd and 4th domains of betaig-h3, were fully active in mediating cell adhesion through interactions with alpha3beta1 integrin [Biochem. Biophys. Res. Commun. 294 (2002) 940; J. Biol. Chem. 275 (2000) 30907]. Here, the conformational differences between NKDIL and EPDIM in water and in membrane environments were studied using CD spectroscopy, and their structures in sodium dodecylsulfate micelles were determined by NMR. The two peptides adopt beta-turn structures like RGD peptides, and have more regular structures in micelles than in aqueous buffers. EPDIM shows a distorted type I beta-turn for the PDIM segment in a membrane environment. The structure of NKDIL is similar with the standard type I' beta-turn, but shows large backbone flexibility even in a membrane environment. The conformational change of the 4th repeated domain of betaig-h3 in micelle solutions suggests that the Asp-Ile motif of the 4th fas-1 domain (EPDIM) would be solvent-exposed and could interact with integrin alpha3beta1 in a membrane environment. The present study provides a structural basis of betaig-h3 function and information for the development of integrin-regulating drugs involving the wound healing protein.     

Identification of motifs in the fasciclin domains of the transforming growth factor-beta-induced matrix protein betaig-h3 that interact with the alphavbeta5 integrin

betaig-h3 is a TGF-beta-induced matrix protein known to mediate the adhesion of several cell types. In this study, we found that all four of the fas-1 domains in betaig-h3 mediate MRC-5 fibroblast adhesion and that this was specifically inhibited by a function-blocking monoclonal antibody specific for the alphavbeta5 integrin. Using deletion mutants of the fourth fas-1 domain revealed the MRC-5 cell adhesion motif (denoted the YH motif) is located in amino acids 548-614. Experiments with substitution mutants showed that tyrosine 571, histidine 572, and their flanking leucine and isoleucine amino acids, which are all highly conserved in many fas-1 domains, are essential for mediating MRC-5 cell adhesion. A synthetic 18-amino acid peptide encompassing these conserved amino acids could effectively block MRC-5 cell adhesion to betaig-h3. Using HEK293 cells stably transfected with the beta5 integrin cDNA, we confirmed that the alphavbeta5 integrin is a functional receptor for the YH motif. In conclusion, we have identified a new alphavbeta5 integrin-interacting motif that is highly conserved in the fas-1 domains of many proteins. This suggests that fas-1 domain-containing proteins may perform their biological functions by interacting with integrins.     

Identification of the alphavbeta3 integrin-interacting motif of betaig-h3 and its anti-angiogenic effect

betaig-h3 is an extracellular matrix protein that mediates adhesion and migration of several cell types through interaction with integrins. In the present study, we tested whether betaig-h3 mediates endothelial cell adhesion and migration, thereby regulating angiogenesis. In this study, we demonstrate that not only betaig-h3 itself but also all four fas-1 domains of betaig-h3 mediate endothelial cell adhesion and migration through interaction with the alphavbeta3 integrin. We found that the alphavbeta3 integrin-interacting motif of the four fas-1 domains of betaig-h3 is the same YH motif that we reported previously to interact with alphavbeta5 integrin. The YH peptide inhibited endothelial cell adhesion and migration in a dose-dependent manner. We demonstrate that the YH peptide has anti-angiogenic activity in vitro and in vivo using an endothelial cell tube formation assay and a Matrigel plug assay, respectively. Our results reveal that betaig-h3 bears alphavbeta3 integrin-interacting motifs that mediate endothelial cell adhesion and migration and, therefore, may regulate angiogenesis.     

TGF-beta1 enhances betaig-h3-mediated keratinocyte cell migration through the alpha3beta1 integrin and PI3K

betaig-h3 is an extracellular matrix (ECM) protein whose expression is highly induced by transforming growth factor beta1 (TGF-beta1). We previously demonstrated that betaig-h3 has two alpha3beta1 integrin-interacting motifs, which promote adhesion, migration, and proliferation of human keratinocytes. Both betaig-h3 and TGF-beta1 have been suggested to play important roles in the healing of skin wounds. In this study, we demonstrate that TGF-beta1 enhances keratinocyte adhesion and migration toward betaig-h3 through the alpha3beta1 integrin. TGF-beta1 did not increase the amount of the alpha3beta1 integrin on the cell surface, but rather increased its affinity for betaig-h3. LY294002, an inhibitor of PI3K, blocked the basal and TGF-beta1-enhanced cell migration but not adhesion to betaig-h3. A constitutively active mutant of PI3K stimulated cell migration but not adhesion to betaig-h3. The PI3K pathway is also not associated with the affinity of the alpha3beta1 integrin to betaig-h3. TGF-beta1 induced phosphorylation of AKT and FAK. Taken together, these data suggest that TGF-beta1 increases affinity of the alpha3beta1 integrin to betaig-h3, resulting in enhanced adhesion and migration of keratinocytes toward betaig-h3. TGF-beta1 also enhances migration through PI3K, but PI3K is not associated with either the binding affinity of the alpha3beta1 integrin or its adhesion to betaig-h3.     

Transforming growth factor-beta-induced gene product, as a novel ligand of integrin alphaMbeta2, promotes monocytes adhesion, migration and chemotaxis

Monocyte recruitment from the blood in response to chemoattractant gradients is a key phenomenon in inflammation. Various extracellular matrix proteins, at the site of inflammation, have chemoattractant activity and mediate monocyte adhesion and migration as ligands of integrins. In this report, we demonstrate that transforming growth factor-beta-induced gene product (betaig-h3/TGFBIp), as an extracellular matrix protein, mediates monocytes adhesion under both static and flow conditions mainly through integrin alphaMbeta2. Fasciclin 1 domains of betaig-h3/TGFBIp are responsible for the interaction with integrin alphaMbeta2, not only enhances monocyte migration in both chemotactic and haptotactic manners but also mediates their transendothelial migration and subendothelial matrix invasion. These activities are also mediated through integrin alphaMbeta2. Intraperitoneal injection of betaig-h3/TGFBIp promotes the recruitment of monocytes but not neutrophils. Our results demonstrate that betaig-h3/TGFBIp produced at inflammatory sites is a novel chemoattractant for monocytes and interacts with integrin alphaMbeta2 to serve as a substrate for their migration, suggesting that betaig-h3/TGFBIp plays an important role in inflammation.     

Solution structure of the Mycobacterium tuberculosis complex protein MPB70: from tuberculosis pathogenesis to inherited human corneal desease

The closely related mycobacteria responsible for tuberculosis produce an unusually high number of secreted proteins, many of which are clearly implicated in pathogenesis and protective immunity. Falling within this category are the closely related proteins MPB70 and MPB83. The structure of MPB70 reveals a complex and novel bacterial fold, which has clear structural homology to the two C-terminal FAS1 domains of the cell adhesion protein fasciclin I, whose structures were reported very recently. Assessment of the surface features of MPB70, the sequence divergence between MPB70 and MPB83, the conservation of residues across a group of FAS1 domains, and the locations of disease-inducing mutations in betaig-h3 strongly suggests that MPB70 and MPB83 contain two functional surfaces on opposite faces, which are probably involved in binding to host cell proteins. This analysis also suggests that these functional surfaces are retained in the FAS1 proteins associated with mediating interactions between cells and the extracellular matrix (fasciclin I, periostin, and betaig-h3) and furthermore that some of the human corneal disease-inducing substitutions identified in betaig-h3 will perturb interactions at these sites.     

Contrasting roles for domain 4 of VCAM-1 in the regulation of cell adhesion and soluble VCAM-1 binding to integrin alpha4beta1

Cell adhesion mediated by the interaction between integrin alpha4beta1 and VCAM-1 is important in normal physiologic processes and in inflammatory and autoimmune disease. Numerous studies have mapped the alpha4beta1 binding sites in VCAM-1 that mediate cell adhesion; however, little is known about the regions in VCAM-1 important for regulating soluble binding. In the present study, we demonstrate that 6D VCAM-1 (an alternatively spliced isoform of VCAM-1 lacking Ig-like domain 4) binds alpha4beta1 with a higher relative affinity than does the full-length form of VCAM-1 containing 7 Ig-like extracellular domains (7D VCAM-1). In indirect binding assays, the EC50 of soluble 6D VCAM-1 binding to alpha4beta1 on Jurkat cells (in 1 mM MnCl2) was 2 x 10(-9) M, compared with 7D VCAM-1 at 11 x 10(-9) M. When used in solution to inhibit alpha4beta1 mediated cell adhesion, the IC50 of 6D VCAM-1 was 13 x 10(-9) M, compared with 7D VCAM-1 measured at 150 x 10(-9) M. Removal of Ig-like domains 4, 5, or 6, or simply substituting Asp328 in domain 4 of 7D VCAM-1 with alanine, caused increased binding of soluble 7D VCAM-1 to alpha4beta1. In contrast, cells adhered more avidly to 7D VCAM-1 under shear force, as it induced cell spreading at lower concentrations than did 6D VCAM-1. Finally, soluble 6D VCAM-1 acts as an agonist through alpha4beta1 by augmenting cell migration and inducing cell aggregation. These results indicate that the domain 4 of VCAM-1 plays a contrasting role when VCAM-1 is presented in solution or as a cell surface-expressed adhesive substrate.     

Novel fold revealed by the structure of a FAS1 domain pair from the insect cell adhesion molecule fasciclin I

Fasciclin I is an insect neural cell adhesion molecule consisting of four FAS1 domains, homologs of which are present in many bacterial, plant, and animal proteins. The crystal structure of FAS1 domains 3 and 4 of Drosophila fasciclin I reveals a novel domain fold, consisting of a seven-stranded beta wedge and a number of alpha helices. The two domains are arranged in a linear fashion and interact through a substantial polar interface. Missense mutations in the FAS1 domains of the human protein betaig-h3 cause corneal dystrophies. Many mutations alter highly conserved core residues, but the two most common mutations, affecting Arg-124 and Arg-555, map to exposed alpha-helical regions, suggesting reduced protein solubility as the disease mechanism.     

Analysis of the roles of RGD-binding integrins, alpha(4)/alpha(9) integrins, alpha(6) integrins, and CD9 in the interaction of the fertilin beta (ADAM2) disintegrin domain with the mouse egg membrane

Fertilin beta (also known as ADAM2), a mammalian sperm protein that mediates gamete cell adhesion during fertilization, is a member of the ADAM protein family whose members have disintegrin domains with homology to integrin ligands found in snake venoms. Fertilin beta utilizes an ECD sequence within its disintegrin domain to interact with the egg plasma membrane; the Asp is especially critical. Based on what is known about different integrin subfamilies and their ligands, we sought to characterize fertilin beta binding sites on mouse eggs, focusing on integrin subfamilies that recognize short peptide sequences that include an Asp residue: the alpha(5)/alpha(8)/alpha(v)/alpha(IIb) or RGD-binding subfamily (alpha(5)beta(1), alpha(8)beta(1), alpha(V)beta(1), alpha(V)beta(3), alpha(V)beta(5), alpha(V)beta(6), alpha(V)beta(8), and alpha(IIb)beta(3)) and the alpha(4)/alpha(9) subfamily (alpha(4)beta(1), alpha(9)beta(1), and alpha(4)beta(7)). We tested peptide sequences known to perturb interactions mediated by these integrins in two different assays for fertilin beta binding. Peptides with the sequence MLDG, which perturb alpha(4)/alpha(9) integrin-mediated interactions, significantly inhibit fertilin beta binding to eggs, which suggests a role for a member of this integrin subfamily as a fertilin beta receptor. RGD peptides, which perturb alpha(5)/alpha(8)/alpha(v)/alpha(IIb) integrin-mediated interactions, have partial inhibitory activity. The anti-alpha(6) antibody GoH3 has little or no inhibitory activity. An antibody to the integrin-associated tetraspanin protein CD9 inhibits the binding of a multivalent presentation of fertilin beta (immobilized on beads) but not soluble fertilin beta, which we speculate has implications for the role of CD9 in the strengthening of fertilin beta-mediated cell adhesion but not in initial ligand binding.     

Synthetic peptides from the carboxy-terminal globular domain of the A chain of laminin: their ability to promote cell adhesion and neurite outgrowth, and interact with heparin and the beta 1 integrin subunit

The large carboxy-terminal globular domain (G domain; residues 2,110-3,060) of the A chain of murine-derived laminin has been shown to promote heparin binding, cell adhesion, and neurite outgrowth. This study was conducted to define the potential sequence(s) originating from the G domain of laminin with any of these functional activities. A series of peptides were synthesized from the G domain, termed GD peptides, each approximately 20 amino acids long and containing multiple positively charged amino acids. In direct 3H-heparin binding assays, peptides GD-1 and GD-2 bound high levels of 3H-heparin, while peptides GD-3 and GD-4 bound lower levels of 3H-heparin, and GD-5 bound essentially no 3H-heparin. The binding of 3H-heparin to peptides GD-1 and GD-2 appeared to be of high affinity, since significant binding of 3H-heparin to these two peptides was still observed even when the NaCl concentration was raised to 1.0 M. Four of the peptides, GD-1, GD-2, GD-3, and GD-4, directly promoted the adhesion and spreading of HT-1080 human fibrosarcoma cells as well as the outgrowth of neurites from chick spinal cord and dorsal root ganglia neurons. In addition, solutions of these peptides or antibodies generated against these peptides inhibited laminin-mediated HT-1080 cell adhesion. Antibodies against the beta 1 integrin subunit inhibited HT-1080 cell adhesion and neurite outgrowth on surfaces adsorbed with peptides GD-3 and GD-4. Therefore, laminin appears to have multiple, independent sequences in the G domain that serve a similar cell adhesion promoting function for different cell types. Furthermore, these results suggest that the sequences comprising peptides GD-3 and GD-4 use an integrin as a receptor, of which the beta 1 integrin subunit is a component for these various cell types.     

Activity-independent cell adhesion to tissue-type transglutaminase is mediated by alpha4beta1 integrin

Transglutaminases (TGases) are enzymes which catalyze cross-link formation between glutamine residues and lysine residues in substrate proteins. We have previously reported that one of the TGases, blood coagulation factor XIIIa (FXIIIa), is capable of mediating adhesion of various cells. In this paper, we report for the first time that tissue-type transglutaminase (TGc) also has cell adhesion activity. TGc-coated plastic surface promoted adhesion and spreading of cells in a TGc concentration-dependent manner. However, there are some obvious differences between cell adhesion mediated by TGc and FXIIIa. As was reported previously, the adhesion to FXIIIa is dependent on its TGase activity. In contrast, the TGc-mediated cell adhesion is independent of its TGase activity: 1) The modification of the active center cysteine with iodoacetamide blocked the enzyme activity without any effect on cell adhesion; 2) the addition of Mg2+ did not induce the enzyme activity, but it was as effective as Ca2+ for cell adhesion; 3) the addition of NH4+ inhibited the enzyme activity but did not affect the cell adhesion significantly. The integrins involved in these cell adhesions are quite different. In the case of FXIIIa, alpha vbeta3 and alpha5beta1 integrins are involved and consequently the RGD peptide substantially inhibited the adhesion. On the other hand, the cell adhesion to TGc is mediated by alpha4beta1 integrin but not alpha5beta1; a CS-1 peptide, which represents the binding site of fibronectin to alpha4beta1 integrin, completely inhibited the cell adhesion to TGc. It is possible that TGc and FXIIIa may mediate cell adhesion under different physiological and pathological situations.     

The cell adhesion and proliferation activities of a peptide derived from human tenascin-C are dependent on two Ile residues

A tenascin-C derived peptide (TNIIIA2 peptide, 1) stimulated β1 integrin-mediated cell adhesion via binding to syndecan-4. Ala-substituted peptides were synthesized to understand the structure-activity relationship. Peptides in which basic amino acids were substituted showed reduced cell adhesion activity, but their proliferation activities were similar to or higher than those mediated by peptide 1. In contrast, peptides in which the Ile residues of peptide 1 were replaced were inactive, indicating that the Ile residues are critical for the peptide's activity. CD analysis suggested that the Ile residues are necessary for the formation of a specific conformation required for binding to syndecan-4.     

DOK1 mediates SHP-2 binding to the alphaVbeta3 integrin and thereby regulates insulin-like growth factor I signaling in cultured vascular smooth muscle cells

Recruitment of the Src homology 2 domain tyrosine phosphatase (SHP-2) to the phosphorylated beta3 subunit of the alphaVbeta3 integrin is required for insulin-like growth factor I (IGF-I)-stimulated cell migration and proliferation in vascular smooth muscle cells. Because SHP-2 does not bind directly to beta3, we attempted to identify a linker protein that could mediate SHP-2/beta3 association. DOK1 is a member of insulin receptor substrate protein family that binds beta3 and contains YXXL/I motifs that are potential binding sites for SHP-2. Our results show that IGF-I induces DOK1 binding to beta3 and to SHP-2. Preincubation of cells with synthetic peptides that blocked either DOK1/beta3 or DOK1/SHP-2 association inhibited SHP-2 recruitment to beta3. Expression of a DOK1 mutant that does not bind to beta3 also disrupts SHP-2/beta3 association. As a result of SHP-2/beta3 disruption, IGF-I dependent phosphorylation of Akt and p44/p42 mitogen-activated protein kinase and its ability to stimulate cell migration and proliferation were significantly impaired. These results demonstrate that DOK1 mediates SHP-2/beta3 association in response to IGF-I thereby mediating the effect of integrin ligand occupancy on IGF-IR-linked signaling in smooth muscle cells.     

Betaig-h3 supports keratinocyte adhesion,migration, and proliferation through alpha3beta1 integrin

betaig-h3 is an extracellular matrix protein and its expression is highly induced by TGF-beta and it has also been suggested to play important roles in skin wound healing. In this paper, we demonstrate that betaig-h3 is present in the papillary layer of dermis and synthesized in the basal keratinocytes in vivo and its expression is induced by TGF-beta in normal human keratinocytes (NHEK) and HaCaT cells. betaig-h3 mediates not only adhesion and spreading of keratinocytes but also supports migration and proliferation. These activities are mediated through interacting with alpha3beta1 integrin. Previously identified two alpha3beta1 integrin-interacting motifs of betaig-h3, EPDIM, and NKDIL, are responsible for these activities. The results suggest that betaig-h3 may regulate keratinocyte functions in normal skin and potentially during wound-healing process.     

The minimal essential sequence for a major cell type-specific adhesion site (CS1) within the alternatively spliced type III connecting segment domain of fibronectin is leucine-aspartic acid-valine

Fibronectin contains at least two major domains that support cell adhesion. One is the central cell-binding domain that is recognized by a variety of cell types via the integrin alpha 5 beta 1. The second, originally identified by its ability to support melanoma cell adhesion, is located in the alternatively spliced type III connecting segment (IIICS). A dominant cell type-specific adhesion site within the IIICS has been localized to a peptide designated as CS1 comprising its amino-terminal 25 residues. The receptor for CS1 is the integrin alpha 4 beta 1. We have synthesized a variety of peptides with overlapping sequences in order to identify the minimum active amino acid sequence of this major cell adhesion site. A peptide comprising the carboxyl-terminal 8 amino acids of CS1, EILDVPST, was found to support melanoma cell spreading, while all peptides without this sequence had little or no activity. Two smaller overlapping pentapeptides, EILDV and LDVPS, were also active, whereas EILEV, containing a conservative substitution of Glu for Asp, was inactive. These data suggested that the minimum sequence for cell adhesion activity is Leu-Asp-Val, the tripeptide sequence common to both active peptides. This prediction was confirmed by the observed ability of the Leu-Asp-Val peptide itself to block spreading on fibronectin, whereas Leu-Glu-Val was inactive. Interspecies amino acid sequence comparison also supports the importance of the LDV sequence, since it is completely conserved in the IIICS regions of human, rat, bovine, and avian fibronectins.     

The PPFLMLLKGSTR motif in globular domain 3 of the human laminin-5 alpha3 chain is crucial for integrin alpha3beta1 binding and cell adhesion

Laminin-5 regulates various cellular functions, including cell adhesion, spreading, and motility. Here, we expressed the five human laminin alpha3 chain globular (LG) domains as monomeric, soluble fusion proteins, and examined their biological functions and signaling. Recombinant LG3 (rLG3) protein, unlike rLG1, rLG2, rLG4, and rLG5, played roles in cell adhesion, spreading, and integrin alpha3beta1 binding. More significantly, we identified a novel motif (PPFLMLLKGSTR) in the LG3 domain that is crucial for these responses. Studies with the synthetic peptides delineated the PPFLMLLKGSTR peptide within LG3 domain as a major site for both integrin alpha3beta1 binding and cell adhesion. Substitution mutation experiments suggest that the Arg residue is important for these activities. rLG3 protein- and PPFLMLLKGSTR peptide-induced keratinocyte adhesion triggered cell signaling through FAK phosphorylation at tyrosine-397 and -577. To our knowledge, this is the first report demonstrating that the PPFLMLLKGSTR peptide within the LG3 domain is a novel motif that is capable of supporting integrin alpha3beta1-dependent cell adhesion and spreading.     

Coordinate role for cell surface chondroitin sulfate proteoglycan and alpha 4 beta 1 integrin in mediating melanoma cell adhesion to fibronectin

Cellular recognition and adhesion to the extracellular matrix (ECM) has a complex molecular basis, involving both integrins and cell surface proteoglycans (PG). The current studies have used specific inhibitors of chondroitin sulfate proteoglycan (CSPG) synthesis along with anti-alpha 4 integrin subunit monoclonal antibodies to demonstrate that human melanoma cell adhesion to an A-chain derived, 33-kD carboxyl-terminal heparin binding fragment of human plasma fibronectin (FN) involves both cell surface CSPG and alpha 4 beta 1 integrin. A direct role for cell surface CSPG in mediating melanoma cell adhesion to this FN fragment was demonstrated by the identification of a cationic synthetic peptide, termed FN-C/H-III, within the fragment. FN-C/H-III is located close to the amino terminal end of the fragment, representing residues #1721-1736 of intact FN. FN-C/H-III binds CSPG directly, can inhibit CSPG binding to the fragment, and promotes melanoma cell adhesion by a CSPG-dependent, alpha 4 beta 1 integrin-independent mechanism. A scrambled version of FN-C/H-III does not inhibit CSPG binding or cell adhesion to the fragment or to FN-C/H-III, indicating that the primary sequence of FN-C/H-III is important for its biological properties. Previous studies have identified three other synthetic peptides from within this 33-kD FN fragment that promote cell adhesion by an arginyl-glycyl-aspartic acid (RGD) independent mechanism. Two of these synthetic peptides (FN-C/H-I and FN-C/H-II) bind heparin and promote cell adhesion, implicating cell surface PG in mediating cellular recognition of these two peptides. Additionally, a third synthetic peptide, CS1, is located in close proximity to FN-C/H-I and FN-C/H-II and it promotes cell adhesion by an alpha 4 beta 1 integrin-dependent mechanism. In contrast to FN-C/H-III, cellular recognition of these three peptides involved contributions from both CSPG and alpha 4 integrin subunits. Of particular importance are observations demonstrating that CS1-mediated melanoma cell adhesion could be inhibited by interfering with CSPG synthesis or expression. Since CS1 does not bind CSPG, the results suggest that CSPG may modify the function and/or activity of alpha 4 beta 1 integrin on the surface of human melanoma cells. Together, these results support a model in which the PG and integrin binding sites within the 33-kD fragment may act in concert to focus these two cell adhesion receptors into close proximity on the cell surface, thereby influencing initial cellular recognition events that contribute to melanoma cell adhesion on this fragment.     

Transition from rolling to firm adhesion is regulated by the conformation of the I domain of the integrin lymphocyte function-associated antigen-1

The integrin lymphocyte function-associated antigen-1 (alpha(L)beta(2)), which is known for its ability to mediate firm adhesion and migration, can also contribute to tethering and rolling in shear flow. The alpha(L) I domain can be mutationally locked with disulfide bonds into two distinct conformations, open and closed, which have high and low affinity for the ligand intercellular adhesion molecule 1 (ICAM-1), respectively. The wild type I domain exists primarily in the lower energy closed conformation. We have measured for the first time the effect of conformational change on adhesive behavior in shear flow. We show that wild type and locked open I domains, expressed in alpha(L)beta(2) heterodimers or as isolated domains on the cell surface, mediate rolling adhesion and firm adhesion, respectively. alpha(L)beta(2) is thus poised for the conversion of rolling to firm adhesion upon integrin activation in vivo. Isolated I domains are surprisingly more effective than alpha(L)beta(2) in interactions in shear flow, which may in part be a consequence of the presence of alpha(L)beta(2) in a bent conformation. Furthermore, the force exerted on the C-terminal alpha-helix appears to stabilize the open conformation of the wild type isolated I domain and contribute to its robustness in supporting rolling. An allosteric small molecule antagonist of alpha(L)beta(2) inhibits both rolling adhesion and firm adhesion, which has important implications for its mode of action in vivo.     

Significance of beta116 His (G18) at alpha1beta1 contact sites for alphabeta assembly and autoxidation of hemoglobin

The role of heterotetramer interaction sites in assembly and autoxidation of hemoglobin is not clear. The importance of beta(116His) (G-18) and gamma(116Ile) at one of the alpha1beta1 or alpha1gamma1 interaction sites for homo-dimer formation and assembly in vitro of beta and gamma chains, respectively, with alpha chains to form human Hb A and Hb F was assessed using recombinant beta(116His)(-->)(Asp), beta(116His)(-->)(Ile), and beta(112Cys)(-->)(Thr,116His)(-->)(Ile) chains. Even though beta chains (e.g., 116 His) are in monomer/tetramer equilibrium, beta(116Asp) chains showed only monomer formation. In contrast, beta(116Ile) and beta(112Thr,116Ile) chains showed homodimer and homotetramer formation like gamma-globin chains which contain 116 Ile. Assembly rates in vitro of beta(116Ile) or beta(112Thr,116Ile) chains with alpha chains were 340-fold slower, while beta(116Asp) chains promoted assembly compared to normal beta-globin chains. These results indicate that amino acid hydrophobicity at the G-18 position in non-alpha chains plays a key role in homotetramer, dimer, and monomer formation, which in turn plays a critical role in assembly with alpha chains to form Hb A and Hb F. These results also suggest that stable dimer formation of gamma-globin chains must not occur in vivo, since this would inhibit association with alpha chains to form Hb F. The role of beta(116His) (G-18) in heterotetramer-induced stabilization of the bond with oxygen in hemoglobin was also assessed by evaluating autoxidation rates using recombinant Hb tetramers containing these variant globin chains. Autoxidation rates of alpha(2)beta(2)(116Asp) and alpha(2)beta(2)(116Ile) tetramers showed biphasic kinetics with the faster rate due to alpha chain oxidation and the slower to the beta chain variants whose rates were 1.5-fold faster than that of normal beta-globin chains. In addition, NMR spectra of the heme area of these two hemoglobin variant tetramers showed similar resonance peaks, which are different from those of Hb A. Oxygen-binding properties of alpha(2)beta(2)(116His)(-->)(Asp) and alpha(2)beta(2)(116His)(-->)(Ile), however, showed slight alteration compared to Hb A. These results suggest that the beta116 amino acid (G18) plays a critical role in not only stabilizing alpha1beta1 interactions but also in inhibiting hemoglobin oxidation. However, stabilization of the bonds between oxygen and heme may not be dependent on stabilization of alpha1beta1 interactions. Tertiary structural changes may lead to changes in the heme region in beta chains after assembly with alpha chains, which could influence stability of dioxygen binding of beta chains.     

Human colon carcinoma cells use multiple receptors to adhere to laminin: involvement of alpha 6 beta 4 and alpha 2 beta 1 integrins.

In this study, we used clone A, a human colon carcinoma cell line, to characterize those integrins that mediate colon carcinoma adhesion to laminin. Monoclonal antibodies specific for the human beta 1 subunit inhibited clone A adhesion to laminin. They also precipitated a complex of surface proteins that exhibited an electrophoretic behavior characteristic of alpha 2 beta 1 and alpha 3 beta 1. A monoclonal antibody specific for alpha 2 (PIH5) blocked clone A adhesion to laminin, as well as to collagen I. An alpha 3-specific antibody (P1B5) had no effect on clone A adhesion to laminin, even though it can block the adhesion of other cell types to laminin. Thus, the alpha 2 beta 1 integrin can function as both a laminin and collagen I receptor on clone A cells. Although these cells express alpha 3 beta 1, an established laminin receptor, they do not appear to use it to mediate laminin adhesion. In addition, the monoclonal antibody GoH3, which recognizes the alpha 6 integrin subunit, also inhibited carcinoma adhesion to laminin but not to fibronectin or collagen I. This antibody precipitated the alpha 6 subunit in association with the beta 4 subunit. There was no evidence of alpha 6 beta 1 association on these cells. In summary, the results obtained in this study indicate that multiple integrin alpha subunits, in association with two distinct beta subunits, are involved in colon carcinoma adhesion to laminin. Based on the behavior of alpha 3 beta 1 and alpha 2 beta 1, the results also suggest that cells can regulate the ability of a specific integrin to mediate adhesion.