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Integrin α 1 β 1 is an adhesion receptor that binds to collagen and laminin. It regulates cell adhesion, cytoskeletal organization, and migration. The cytoplasmic tail of the α 1 subunit consists of 15 amino acids and contains six positively charged lysine residues. In this study, we present evidence that the α 1 integrin cytoplasmic tail (α 1 CT) directly associates with phosphoinositides, preferentially with phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3 ). Since the association was disrupted by calcium, magnesium and phosphate ions, this interaction appears to be in ionic nature. Here, the peptide-lipid interaction was driven by the conserved KIGFFKR motif. The exchange of both two potential phospholipid-binding lysines for glycines in the KIGFFKR motif increased α 1 β 1 integrin-specific adhesion and F-actin cytoskeleton formation compared to cells expressing the unmodified α 1 subunit, whereas only mutation of the second lysine at position 1171 increased levels of constitutively active α 1 β 1 integrins on the cell surface. In addition, enhanced focal adhesion formation and increased phosphorylation of focal adhesion kinase, but decreased phosphorylation of AKT was observed in these cells. We conclude that the KIGFFKR motif, and in particular lysine1171 is involved in the dynamic regulation of α 1 β 1 integrin activity and that the interaction of α 1 CT with phosphoinositides may contribute to this process., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

T-Cell Protein Tyrosine Phosphatase (TCPTP, PTPN2) is a non-receptor type protein tyrosine phosphatase that is ubiquitously expressed in human cells. TCPTP is a critical component of a variety of key signaling pathways that are directly associated with the formation of cancer and inflammation. Thus, understanding the molecular mechanism of TCPTP activation and regulation is essential for the development of TCPTP therapeutics. Under basal conditions, TCPTP is largely inactive, although how this is achieved is poorly understood. By combining biomolecular nuclear magnetic resonance spectroscopy, small-angle X-ray scattering, and chemical cross-linking coupled with mass spectrometry, we show that the C-terminal intrinsically disordered tail of TCPTP functions as an intramolecular autoinhibitory element that controls the TCPTP catalytic activity. Activation of TCPTP is achieved by cellular competition, i.e., the intrinsically disordered cytosolic tail of Integrin-α1 displaces the TCPTP autoinhibitory tail, allowing for the full activation of TCPTP. This work not only defines the mechanism by which TCPTP is regulated but also reveals that the intrinsically disordered tails of two of the most closely related PTPs (PTP1B and TCPTP) autoregulate the activity of their cognate PTPs via completely different mechanisms., (© 2022. The Author(s).)

Collagens are the most abundant extracellular matrix proteins in vertebrates and have a characteristic triple-helix structure. Hydroxylation of proline residues is critical for helix stability, and diminished prolyl hydroxylase activity causes wide-spread defects in connective tissues. Still, the role of proline hydroxylation in the binding of collagen receptors such as integrins is unclear. Here, we isolated skin collagen from genetically modified mice having reduced prolyl 4-hydroxylase activity. At room temperature, the reduced proline hydroxylation did not affect interactions with the recombinant integrin α2I domain, but at 37 °C, collagen hydroxylation correlated with the avidity of α2I domain binding. Of note, LC-MS/MS analysis of isolated skin collagens revealed no major changes in the hydroxyproline content of the main integrin-binding sites. Thus, the disrupted α2I domain binding at physiological temperatures was most likely due to structural destabilization of the collagenous helix. Integrin α2I binding to the triple-helical GFPGER motif was slightly weaker than to GFOGER (O = hydroxyproline). This phenomenon was more prominent when α1 integrin was tested. Integrin α1β1 expressed on CHO cells and recombinant α1I domain showed remarkably slower binding velocity and weaker avidity to GFPGER when compared with GFOGER. Structural modeling revealed the critical interaction between Arg-218 in α1I and the hydroxyproline residue in the integrin-binding motif. The role of Arg-218 was further validated by testing a variant R218D α1I domain in solid-phase binding assays. Thus, our results show that the lack of proline hydroxylation in collagen can affect integrin binding by a direct mechanism and via structural destabilization of the triple helix., (© 2018 Sipilä et al.)

Integrins are transmembrane cell-extracellular matrix adhesion receptors that impact many cellular functions. A subgroup of integrins contain an inserted (I) domain within the α-subunits (αI) that mediate ligand recognition where function is contingent on binding a divalent cation at the metal ion dependent adhesion site (MIDAS). Ca 2+ is reported to promote α1I but inhibit α2I ligand binding. We co-crystallized individual I-domains with MIDAS-bound Ca 2+ and report structures at 1.4 and 2.15 Å resolution, respectively. Both structures are in the "closed" ligand binding conformation where Ca 2+ induces minimal global structural changes. Comparisons with Mg 2+ -bound structures reveal Mg 2+ and Ca 2+ bind α1I in a manner sufficient to promote ligand binding. In contrast, Ca 2+ is displaced in the α2I domain MIDAS by 1.4 Å relative to Mg 2+ and unable to directly coordinate all MIDAS residues. We identified an E152-R192 salt bridge hypothesized to limit the flexibility of the α2I MIDAS, thus, reducing Ca 2+ binding. A α2I E152A construct resulted in a 10,000-fold increase in Mg 2+ and Ca 2+ binding affinity while increasing binding to collagen ligands 20%. These data indicate the E152-R192 salt bridge is a key distinction in the molecular mechanism of differential ion binding of these two I domains.

The ligand-binding βI and αI domains of integrin are the best-studied von Willebrand factor A domains undergoing significant conformational changes for affinity regulation. In both βI and αI domains, the α1- and α7-helixes work in concert to shift the metal-ion-dependent adhesion site between the resting and active states. An absolutely conserved Gly in the middle of the α1-helix of βI helps maintain the resting βI conformation, whereas the homologous position in the αI α1-helix contains a conserved Phe. A functional role of this Phe is structurally unpredictable. Using α L β 2 integrin as a model, we found that the residue volume at the Phe position in the α1-helix is critical for α L β 2 activation because trimming the Phe by small amino acid substitutions abolished α L β 2 binding with soluble and immobilized intercellular cell adhesion molecule 1. Similar results were obtained for α M β 2 integrin. Our experimental and molecular dynamics simulation data suggested that the bulky Phe acts as a pawl that stabilizes the downward ratchet-like movement of β6-α7 loop and α7-helix, required for high-affinity ligand binding. This mechanism may apply to other von Willebrand factor A domains undergoing large conformational changes. We further demonstrated that the conformational cross-talk between α L αI and β 2 βI could be uncoupled because the β 2 extension and headpiece opening could occur independently of the αI activation. Reciprocally, the αI activation does not inevitably lead to the conformational changes of the β 2 subunit. Such loose linkage between the αI and βI is attributed to the αI flexibility and could accommodate the α L β 2 -mediated rolling adhesion of leukocytes., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Integrins are transmembrane receptors composed of α and β subunits. Although most integrins contain β1, canonical activation mechanisms are based on studies of the platelet integrin, αIIbβ3. Its inactive conformation is characterized by the association of the αIIb transmembrane and cytosolic domain (TM/CT) with a tilted β3 TM/CT that leads to activation when disrupted. We show significant structural differences between β1 and β3 TM/CT in bicelles. Moreover, the 'snorkeling' lysine at the TM/CT interface of β subunits, previously proposed to regulate αIIbβ3 activation by ion pairing with nearby lipids, plays opposite roles in β1 and β3 integrin function and in neither case is responsible for TM tilt. A range of affinities from almost no interaction to the relatively high avidity that characterizes αIIbβ3 is seen between various α subunits and β1 TM/CTs. The αIIbβ3-based canonical model for the roles of the TM/CT in integrin activation and function clearly does not extend to all mammalian integrins., Competing Interests: AP: Reviewing editor, eLife. RF: Reviewing editor, eLife. The other authors declare that no competing interests exist.

Integrin-collagen interactions play a critical role in a myriad of cellular functions that include immune response, and cell development and differentiation, yet their mechanism of binding is poorly understood. There is increasing evidence that conformational flexibility assumes a central role in the molecular mechanisms of protein-protein interactions and here we employ NMR hydrogen-deuterium exchange (HDX) experiments to explore the impact of slower timescale dynamic events. To gain insight into the mechanisms underlying collagen-induced conformational switches, we have undertaken a comparative study between the wild type integrin α1 I and a gain-of-function E317A mutant. NMR HDX results suggest a relationship between regions exhibiting a reduced local stability in the unbound I domain and those that undergo significant conformational changes upon binding. Specifically, the αC and α7 helices within the C-terminus are at the center of such major perturbations and present reduced local stabilities in the unbound state relative to other structural elements. Complementary isothermal titration calorimetry experiments have been performed to derive complete thermodynamic binding profiles for association of the collagen-like triple-helical peptide with wild type α1 I and E317A mutant. The differential energetics observed for E317A are consistent with the HDX experiments and support a model in which intrinsically destabilized regions predispose conformational rearrangement in the integrin I domain. This study highlights the importance of exploring different timescales to delineate allosteric and binding events., (© 2016 The Protein Society.)

Half of the 18 human integrins α subunits have an inserted αI domain yet none have been observed in species that have diverged prior to the appearance of the urochordates (ascidians). The urochordate integrin αI domains are not human orthologues but paralogues, but orthologues of human αI domains extend throughout later-diverging vertebrates and are observed in the bony fish with duplicate isoforms. Here, we report evidence for orthologues of human integrins with αI domains in the agnathostomes (jawless vertebrates) and later diverging species. Sequence comparisons, phylogenetic analyses and molecular modeling show that one nearly full-length sequence from lamprey and two additional fragments include the entire integrin αI domain region, have the hallmarks of collagen-binding integrin αI domains, and we show that the corresponding recombinant proteins recognize the collagen GFOGER motifs in a metal dependent manner, unlike the α1I domain of the ascidian C. intestinalis. The presence of a functional collagen receptor integrin αI domain supports the origin of orthologues of the human integrins with αI domains prior to the earliest diverging extant vertebrates, a domain that has been conserved and diversified throughout the vertebrate lineage.

The α1β1 integrin receptor binds to its main extracellular ligand, collagen, through an inserted domain in its α-subunit called the αI domain (αI). αI contains a metal binding site that allows collagen to coordinate to the domain through a divalent metal ion. Here we report the backbone assignments of the apo and Mg(2+) bound state of the isolated human α1I and the chemical shift changes resulting from metal coordination.

We have determined the structure of the human integrin α1I domain bound to a triple-helical collagen peptide. The structure of the α1I-peptide complex was investigated using data from NMR, small angle x-ray scattering, and size exclusion chromatography that were used to generate and validate a model of the complex using the data-driven docking program, HADDOCK (High Ambiguity Driven Biomolecular Docking). The structure revealed that the α1I domain undergoes a major conformational change upon binding of the collagen peptide. This involves a large movement in the C-terminal helix of the αI domain that has been suggested to be the mechanism by which signals are propagated in the intact integrin receptor. The structure suggests a basis for the different binding selectivity observed for the α1I and α2I domains. Mutational data identify residues that contribute to the conformational change observed. Furthermore, small angle x-ray scattering data suggest that at low collagen peptide concentrations the complex exists in equilibrium between a 1:1 and 2:1 α1I-peptide complex.

Integrin proteins are very important adhesion receptors that mediate cell-cell and cell-extracellular matrix interactions. They play essential roles in cell signaling and the regulation of cellular shape, motility, and the cell cycle. Here, the transmembrane and cytoplasmic (TMC) domains of integrin α1 and β1 were over-expressed and purified in detergent micelles. The structure and backbone relaxations of α1-TMC in LDAO micelles were determined and analyzed using solution NMR. A long helix, extending from the transmembrane region to the cytoplasmic tail, was observed in α1-TMC. Structural comparisons of α1-TMC with reported αIIb-TMC domains indicated different conformations in the transmembrane regions and cytoplasmic tails. An NMR titration experiment indicated weak interactions between α1-TMC and β1-TMC through several α1-TMC residues located at its N-terminal juxta-transmembrane region and C-terminal extended helix region.

We have applied hydrogen-deuterium exchange mass spectrometry, in conjunction with differential scanning calorimetry and protein stability analysis, to examine solution dynamics of the integrin α1 I domain induced by the binding of divalent cations, full-length type IV collagen, or a function-blocking monoclonal antibody. These studies revealed features of integrin activation and α1I-ligand complexes that were not detected by static crystallographic data. Mg(2+) and Mn(2+) stabilized α1I but differed in their effects on exchange rates in the αC helix. Ca(2+) impacted α1I conformational dynamics without altering its gross thermal stability. Interaction with collagen affected the exchange rates in just one of three metal ion-dependent adhesion site (MIDAS) loops, suggesting that MIDAS loop 2 plays a primary role in mediating ligand binding. Collagen also induced changes consistent with increased unfolding in both the αC and allosteric C-terminal helices of α1I. The antibody AQC2, which binds to α1I in a ligand-mimetic manner, also reduced exchange in MIDAS loop 2 and increased exchange in αC, but it did not impact the C-terminal region. This is the first study to directly demonstrate the conformational changes induced upon binding of an integrin I domain to a full-length collagen ligand, and it demonstrates the utility of the deuterium exchange mass spectrometry method to study the solution dynamics of integrin/ligand and integrin/metal ion interactions. Based on the ligand and metal ion binding data, we propose a model for collagen-binding integrin activation that explains the differing abilities of Mg(2+), Mn(2+), and Ca(2+) to activate I domain-containing integrins.

We have analyzed the structure and function of the integrin α(1)I domain harboring a gain-of-function mutation E317A. To promote protein crystallization, a double variant with an additional C139S mutation was used. In cell adhesion assays, the E317A mutation promoted binding to collagen. Similarly, the double mutation C139S/E317A increased adhesion compared with C139S alone. Furthermore, soluble α(1)I C139S/E317A was a higher avidity collagen binder than α(1)I C139S, indicating that the double variant represents an activated form. The crystal structure of the activated variant of α(1)I was solved at 1.9 Å resolution. The E317A mutation results in the unwinding of the αC helix, but the metal ion has moved toward loop 1, instead of loop 2 in the open α(2)I. Furthermore, unlike in the closed αI domains, the metal ion is pentacoordinated and, thus, prepared for ligand binding. Helix 7, which has moved downward in the open α(2)I structure, has not changed its position in the activated α(1)I variant. During the integrin activation, Glu(335) on helix 7 binds to the metal ion at the metal ion-dependent adhesion site (MIDAS) of the β(1) subunit. Interestingly, in our cell adhesion assays E317A could activate collagen binding even after mutating Glu(335). This indicates that the stabilization of helix 7 into its downward position is not required if the α(1) MIDAS is already open. To conclude, the activated α(1)I domain represents a novel conformation of the αI domain, mimicking the structural state where the Arg(287)-Glu(317) ion pair has just broken during the integrin activation.

The activation of α/β heterodimeric integrins is the result of highly coordinated rearrangements within both subunits. The molecular interactions between the two subunits, however, remain to be characterized. In this study, we use the integrin α(L)β(2) to investigate the functional role of the C-linker polypeptide that connects the C-terminal end of the inserted (I) domain with the β-propeller domain on the α subunit and is located at the interface with the βI domain of the β chain. We demonstrate that shortening of the C-linker by eight or more amino acids results in constitutively active α(L)β(2) in which the αI domain is no longer responsive to the regulation by the βI domain. Despite this intersubunit uncoupling, both I domains remain individually sensitive to intrasubunit conformational changes induced by allosteric modulators. Interestingly, the length and not the sequence of the C-linker appears to be critical for its functionality in α/β intersubunit communication. Using two monoclonal antibodies (R7.1 and CBR LFA-1/1) we further demonstrate that shortening of the C-linker results in the gradual loss of combinational epitopes that require both the αI and β-propeller domains for full reactivity. Taken together, our findings highlight the role of the C-linker as a spring-like element that allows relaxation of the αI domain in the resting state and controlled tension of the αI domain during activation, exerted by the β chain.

Collagen is an extracellular matrix structural component that can regulate cellular processes through its interaction with the integrins, α1β1, α2β1, α10β1, and α11β1. Collagen-like proteins have been identified in a number of bacterial species. Here, we used Scl2 from Streptococcus pyogenes serotype M28 strain MGAS6274 as a backbone for the introduction of discrete integrin-binding sequences. The introduced sequences GLPGER, GFPGER, or GFPGEN did not affect triple helix stability of the Scl (Streptococcal collagen-like) protein. Using ELISA and surface plasmon resonance, we determined that Scl2(GLPGER) and Scl2(GFPGER) bound to recombinant human α1 and α2 I-domains in a metal ion-dependent manner and without a requirement for hydroxyproline. We predicted a novel and selective integrin-binding sequence, GFPGEN, through the use of computer modeling and demonstrated that Scl2(GFPGEN) shows specificity toward the α1 I-domain and does not bind the α2 I-domain. Using C2C12 cells, we determined that intact integrins interact with the modified Scl2 proteins with the same selectivity as recombinant I-domains. These modified Scl2 proteins also acted as cell attachment substrates for fibroblast, endothelial, and smooth muscle cells. However, the modified Scl2 proteins were unable to aggregate platelets. These results indicate that Scl2 is a suitable backbone for the introduction of mammalian integrin-binding sequences, and these sequences may be manipulated to individually target α1β1 and α2β1.

Integrin alpha1beta1, the major collagen type IV receptor, is expressed by endothelial cells and plays a role in both physiologic and pathologic angiogenesis. Because the molecular mechanisms whereby this collagen IV receptor mediates endothelial cell functions are poorly understood, truncation and point mutants of the integrin alpha1 subunit cytoplasmic tail (amino acids 1137-1151) were generated and expressed into alpha1-null endothelial cells. We show that alpha1-null endothelial cells expressing the alpha1 subunit, which lacks the entire cytoplasmic tail (mutant alpha1-1136) or expresses all the amino acids up to the highly conserved GFFKR motif (mutant alpha1-1143), have a similar phenotype to parental alpha1-null cells. Pro(1144) and Leu(1145) were shown to be necessary for alpha1beta1-mediated endothelial cell proliferation; Lys(1146) for adhesion, migration, and tubulogenesis and Lys(1147) for tubulogenesis. Integrin alpha1beta1-dependent endothelial cell proliferation is primarily mediated by ERK activation, whereas migration and tubulogenesis require both p38 MAPK and PI3K/Akt activation. Thus, distinct amino acids distal to the GFFKR motif of the alpha1 integrin cytoplasmic tail mediate activation of selective downstream signaling pathways and specific endothelial cell functions.

Collagen receptor integrins alpha 1 beta 1 and alpha 2 beta 1 can selectively recognize different collagen subtypes. Here we show that their alpha I domains can discriminate between laminin isoforms as well: alpha 1I and alpha 2I recognized laminin-111, -211 and -511, whereas their binding to laminin-411 was negligible. Residue Arg-218 in alpha1 was found to be instrumental in high-avidity binding. The gain-of-function mutation E318W makes the alpha 2I domain to adopt the "open" high-affinity conformation, while the wild-type alpha 2I domain favors the "closed" low-affinity conformation. The E318W mutation markedly increased alpha 2I domain binding to the laminins (-111, -211 and -511), leading us to propose that the activation state of the alpha 2 beta 1 integrin defines its role as a laminin receptor. However, neither wild-type nor alpha 2IE318W domain could bind to laminin-411. alpha 2IE318W also bound tighter to all collagens than alpha 2I wild-type, but it showed reduced ability to discriminate between collagens I, IV and IX. The corresponding mutation, E317A, in the alpha 1I domain transformed the domain into a high-avidity binder of collagens I and IV. Thus, our results indicate that conformational activation of integrin alpha 1I and alpha 2I domains leads to high-avidity binding to otherwise disfavored collagen subtypes.

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.

Integrins play a key role in cellular motility; an essential process for embryonic development and tissue morphogenesis, and also for pathological processes such as tumor cell invasion and metastasis. Recently, we showed that the cytoplasmic tail of integrin alpha(1) regulates the formation of focal complexes, F-actin cytoskeleton reorganization, and migration. We now report that the alpha(1) tail directly engages in collagen IV-mediated migration by regulation of the small GTPase Rac1. Deletion variants of the alpha(1) integrin differ in their ability to activate Rac1. Constitutively active Rac1 rescues motility in otherwise immotile cells expressing a truncated alpha(1) integrin without any cytoplasmic tail. In these cells, levels of GTP-Rac1 are constitutively elevated, but kept non-functional in the cytoplasm. The conserved GFFKR motif is sufficient to convey Rac1 activation, but downregulates the amount of GTP-Rac1 in the absence of the alpha(1)-specific sequence PLKKKMEK. This sequence is also required for the recruitment of PI3K to focal adhesions following Rac1 activation. Our results demonstrate that the short alpha(1) cytoplasmic tail is crucial for Rac1 activation and PI3K localization, which in turn results in cytoskeletal rearrangement and subsequent migration.

The alpha subunit-inserted (I) domain of integrin alphaLbeta2 [lymphocyte function-associated antigen-1 (LFA-1)] binds to intercellular adhesion molecule-1 (ICAM-1). The C- and N-termini of the alpha I domain are near one another on the "lower" face, opposite the metal ion-dependent adhesion site (MIDAS) on the "upper face". In conversion to the open alpha I domain conformation, a 7 A downward, axial displacement of C-terminal helix alpha7 is allosterically linked to rearrangement of the MIDAS into its high-affinity conformation. Here, we test the hypothesis that when an applied force is appropriately linked to conformational change, the conformational change can stabilize adhesive interactions that resist the applied force. Integrin alpha I domains were anchored to the cell surface through their C- or N-termini using type I or II transmembrane domains, respectively. C-terminal but not N-terminal anchorage robustly supported cell rolling on ICAM-1 substrates in shear flow. In contrast, when the alphaL I domain was mutationally stabilized in the open conformation with a disulfide bond, it mediated comparable levels of firm adhesion with type I and type II membrane anchors. To exclude other effects as the source of differential adhesion, these results were replicated using alpha I domains conjugated through the N- or C-terminus to polystyrene microspheres. Our results demonstrate a mechanical feedback system for regulating the strength of an adhesive bond. A review of crystal structures of integrin alpha and beta subunit I domains and selectins in high- and low-affinity conformations demonstrates a common mechanochemical design in which biologically applied tensile force stabilizes the more extended, high-affinity conformation.

PRL-3 is a newly identified protein tyrosine phosphatase associated with tumor metastasis. It is over-expressed in various cancers, such as colorectal cancer, gastric cancer, and ovarian cancer, and is correlated with the progression and survival of cancers. Although PRL-3 plays a causative role in promoting cancer cell invasion and metastasis, the molecular mechanism is unknown. To investigate PRL-3's roles in tumorigenesis and signal transduction pathway, we screened the human placenta brain cDNA library with the bait of PRL-3 in yeast two-hybrid system. Then we identified integrin alpha1 as a PRL-3-interacting protein for the first time, and verified this physical association with pull-down and co-immunoprecipitation assays. Furthermore, we found that PRL-3 could down-regulate the tyrosine-phosphorylation level of integrin beta1 and increased the phosphorylation level of Erk1/2. Our present discovery will provide new clues for elucidating the molecular mechanism of PRL-3 in promoting cancer invasion and metastasis.

In vivo, beta(2) integrins and particularly alpha(L)beta(2) (LFA-1) robustly support firm adhesion of leukocytes, but can also cooperate with other molecules in supporting rolling adhesion. Strikingly, a small molecule alpha/beta I-like allosteric antagonist, XVA143, inhibits LFA-1-dependent firm adhesion, while at the same time it enhances adhesion in shear flow and rolling both in vitro and in vivo. XVA143 appears to induce the extended conformation of integrins as shown by increased activation epitope exposure. Fab to the beta(2) I-like domain converts firm adhesion to rolling adhesion, but does not enhance adhesion. Residue alpha(L)-Glu-310 in the linker following the I domain is critical for communication to the beta(2) I-like domain, rolling, integrin extension, and activation by Mn(2+) of firm adhesion. The results demonstrate the importance of integrin extension in rolling, and suggest that rolling and firm adhesion are mediated by extended conformations of alpha(L)beta(2) that differ in the affinity of the alpha(L) I domain for ICAM-1.

Collagen type IV forms a network in the basement membrane into which other constituents of the tissue are incorporated. It also provides cell-adhesion sites that are specifically recognized by cell-surface receptors, i.e., the integrins. Different from the ubiquitous sequential RGD adhesion motif found in most of the matrix proteins, in collagen type IV, the responsible binding sites for alpha1beta1 integrin have been identified as Asp461 of the two alpha1 chains and Arg461 of the alpha2 chain. Because of the heterotrimeric character of this collagen, the spatial geometry of the binding epitope depends not only on the triple-helical fold, but decisively even on the stagger of the chains. To investigate the effects of chain registration on the conformational properties and binding affinities of this adhesion epitope, two synthetic heterotrimeric collagen peptides consisting of the identical three chains were assembled by an artificial cystine knot in two different registers, i.e., in the most plausible alpha2alpha1alpha1' and less probable alpha1alpha2alpha1' chain alignment. A detailed conformational characterization of both trimers allowed to correlate their different binding affinities for alpha1beta1 integrin with the degree of local plasticity of the two different triple helices. Optimal local breathing of the rod-shaped collagens is apparently crucial for selective recognition by proteins interacting with these main components of the extracellular matrix., (Copyright 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2004)

The alpha1 beta1 integrin, an inserted (1) domain containing collagen receptor, is expressed in the cell surface membrane of normal and malignant cells, and may play a role in their migration through tissues or in metastatic spread. Here we report that a functional anti-human alpha1beta1 integrin monoclonal antibody (mAb) (1B3.1) directly and specifically binds plastic bound recombinant human alpha1 I-domain protein containing the collagen binding site. Detection was diminished by acidification of the I-domain protein but was enhanced by increasing concentrations of Mg2+ cation. Furthermore, we detected binding of the mAb to proteins from the ocular fluids of 6 patients, with the highest concentration, corresponding to 22.1 ng/ml of I-domain, found in a sample from the eye of a patient with metastatic lung adenocarcinoma. Interestingly, we found that both SKNSH neuroblastoma cells and virally transformed human T cells adhered specifically to plastic wells coated with either immobilized collagen IV or alpha1 I-domain. MAb I B3.1 inhibited adhesion to collagen IV but not to immobilized I-domain. These results suggest a novel function for cell free alpha1 I-domain as a substrate for cellular adhesion, which may have relevance in tumor spread in vivo.