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Integrins are transmembrane proteins that transmit bi-directional signals across the cell membrane through global structural rearrangement among three different conformational states: bent, extended- closed, and extended-open conformations. However, the β 8 integrin is distinctive and may adopt only one conformation, that is, extended-closed conformation, with high affinity for ligands under physiological conditions, and may not transmit bi-directional signals like other integrin members. It is unclear how different β 8 domains affect its unique conformation and signaling. We swapped different domains of integrin β 3 with those of β 8 and investigated how they affected integrin ligand binding, global conformation, and outside-in signaling. We found that the β 8 epidermal growth factor (EGF) domains increased integrin ligand binding ability and contributed to its extended conformation. By comparison, the β 8 transmembrane and cytoplasmic domains had little effect on ligand binding or global conformation. The β 8 EGF and transmembrane domains did not affect integrin-mediated cell adhesion, cell spreading, focal adhesion formation, or colocalization of integrin with other proteins, but the cytoplasmic domain had a defective effect on outside-in signaling. Our results showed that different domains of β 8 play various roles on its unique conformation, ligand binding, and signaling, which are considered atypical among integrin members., (© 2022 Wiley Periodicals LLC.)

Many integrins transmit signals through global conformational changes. However, it is unclear whether integrin α v β 8 adopts a similar mechanism during integrin activation and signaling on the cell surface. Here, we showed that disulfide-bonded mutants, which prevented integrin α v β 8 lower leg dissociation, bound ligands with similar level as the wild-type protein, suggesting that α v β 8 ligand binding did not require lower leg disassociation. We further showed that the N-glycosylation mutant at the interface between the β I and hybrid domains did not affect ligand binding, suggesting that the α v β 8 open headpiece was not present on the cell surface. We proposed that α v β 8 integrin may adopt only one state, that is, the extended conformation with a closed headpiece. Our results showed that two lower legs retained heterodimeric interfaces, and this association might be important for stabilizing integrin in the extended conformation. Therefore, α v β 8 may not transmit bidirectional signals across the plasma membrane but instead may serve as an anchoring site with high affinity and high accessibility for extracellular ligands., (© 2021 Wiley Periodicals LLC.)

Integrins are heterodimeric transmembrane proteins that play important roles in various biological processes. Most integrins serve as adhesion molecules and transmit bidirectional signaling across the cell membrane through global conformational changes from the bent closed to the extended open conformation. However, integrin β 8 is distinctive in structure and function. Its cytoplasmic domain lacks the conserved protein-binding sequence, which is important in transmitting inside-out signals, suggesting that integrin β 8 may have a different activation mechanism or lack such signaling. In addition, the ligand-binding or activating metal ion Mn 2+ does not induce a global conformational change in integrin β 8 . It may have only one conformation, that is, an extended, closed conformation, but with high affinity for ligands under physiological conditions, and is, therefore, considered an atypical integrin member. The extended structure and high ligand-binding affinity of integrin α v β 8 make it ideal for encountering and binding ligands expressed on an opposing cell or in the extracellular matrix. In this review, we summarize the progress in integrin β 8 research with a focus on its distinctive function and structure among integrin members., (© 2020 Wiley Periodicals LLC.)

Integrins are heterodimeric cell adhesion molecules that are important in many biological functions, such as cell migration, proliferation, differentiation, and survival. They can transmit bi-directional signals across the plasma membrane. Inside-out activating signal from some cell surface receptors bound with soluble agonists triggers integrins conformational change leading to high affinity for extracellular ligands. Then binding of ligands to integrins results in outside-in signaling, leading to formation of focal adhesion complex at the integrin cytoplasmic tail and activation of downstream signal pathways. This bi-directional signaling is essential for rapid response of cell to surrounding environmental changes. During this process, the conformational change of integrin extracellular and transmembrane/cytoplasmic domains is particularly important. In this review, we will summarize recent progress in both inside-out and outside-in signaling with specific focus on the mechanism how integrins transmit bi-directional signals through transmembrane/cytoplasmic domains., (Copyright © 2012 Wiley Periodicals, Inc.)

Integrins are cell adhesion molecules that play important roles in many biological processes including hemostasis, immune responses, development, and cancer. Their adhesiveness is dynamically regulated through a process termed inside-out signaling. In addition, ligand binding transduces outside-in signals from the extracellular domain to the cytoplasm. Advances in the past several years have shed light on structural basis for integrin regulation and signaling, especially how the large-scale reorientations of the ectodomain are related to the inter-domain and intra-domain shape shifting that changes ligand-binding affinity. Experiments have also shown how the conformational changes of the ectodomain are linked to changes in the α- and β-subunit transmembrane and cytoplasmic domains.

Integrin transmembrane (TM) and/or cytoplasmic domains play a critical role in integrin bidirectional signaling. Although it has been shown that TM and/or cytoplasmic α and β domains associate in the resting state and separation of these domains is required for both inside-out and outside-in signaling, the role of TM homomeric association remains elusive. Formation of TM homo-oligomers was observed in micelles and bacterial membranes previously, and it has been proposed that homomeric association is important for integrin activation and clustering. This study addresses whether integrin TM domains form homo-oligomers in mammalian cell membranes using cysteine scanning mutagenesis. Our results show that TM homomeric interaction does not occur before or after soluble ligand binding or during inside-out activation. In addition, even though the cysteine mutants and the heterodimeric disulfide-bounded mutant could form clusters after adhering to immobilized ligand, the integrin TM domains do not form homo-oligomers, suggesting that integrin TM homomeric association is not critical for integrin clustering or outside-in signaling. Therefore, integrin TM homo-oligomerization is not required for integrin activation, ligand binding, or signaling.

Integrin conformational changes mediate integrin activation and signaling triggered by intracellular molecules or extracellular ligands. Even though it is known that αβ transmembrane domain separation is required for integrin signaling, it is still not clear how this signal is transmitted from the transmembrane domain through two long extracellular legs to the ligand-binding headpiece. This study addresses whether the separation of the membrane-proximal extracellular αβ legs is critical for integrin activation and outside-in signaling. Using a disulfide bond to restrict dissociation of the α-subunit Calf-2 domain and β-subunit I-EGF4 domain, we were able to abolish integrin inside-out activation and outside-in signaling. In contrast, disrupting the interface by introducing a glycosylation site into either subunit activated integrins for ligand binding through a global conformational change. Our results suggest that the interface of the Calf-2 domain and the I-EGF4 domain is critical for integrin bidirectional signaling.

Integrins are cell adhesion receptors that transmit bidirectional signals across plasma membrane and are crucial for many biological functions. Recent structural studies of integrin transmembrane (TM) and cytoplasmic domains have shed light on their conformational changes during integrin activation. A structure of the resting state was solved based on Rosetta computational modeling and experimental data using intact integrins on mammalian cell surface. In this structure, the alpha(IIb) GXXXG motif and their beta(3) counterparts of the TM domains associate with ridge-in-groove packing, and the alpha(IIb) GFFKR motif and the beta(3) Lys-716 in the cytoplasmic segments play a critical role in the alpha/beta association. Comparing this structure with the NMR structures of the monomeric alpha(IIb) and beta(3) (represented as active conformations), the alpha subunit helix remains similar after dissociation whereas beta subunit helix is tilted by embedding additional 5-6 residues into the lipid bilayer. These conformational changes are critical for integrin activation and signaling across the plasma membrane. We thus propose a new model of integrin TM activation in which the recent NMR structure of the alpha(IIb)beta(3) TM/cytoplasmic complex represents an intermediate or transient state, and the electrostatic interaction in the cytoplasmic region is important for priming the initial alpha/beta association, but not absolutely necessary for the resting state., ((c) 2009 Wiley-Liss, Inc.)

Despite extensive evidence that integrin conformational changes between bent and extended conformations regulate affinity for ligands, an alternative hypothesis has been proposed in which a "deadbolt" can regulate affinity for ligand in the absence of extension. Here, we tested both the deadbolt and the extension models. According to the deadbolt model, a hairpin loop in the beta3 tail domain could act as a deadbolt to restrain the displacement of the beta3 I domain beta6-alpha7 loop and maintain integrin in the low affinity state. We found that mutating or deleting the beta3 tail domain loop has no effect on ligand binding by either alphaIIbbeta 3 or alphaVbeta3 integrins. In contrast, we found that mutations that lock integrins in the bent conformation with disulfide bonds resist inside-out activation induced by cytoplasmic domain mutation. Furthermore, we demonstrated that extension is required for accessibility to fibronectin but not smaller fragments. The data demonstrate that integrin extension is required for ligand binding during integrin inside-out signaling and that the deadbolt does not regulate integrin activation.

Integrins are cell adhesion molecules that mediate cell-cell, cell-extracellular matrix, and cell-pathogen interactions. They play critical roles for the immune system in leukocyte trafficking and migration, immunological synapse formation, costimulation, and phagocytosis. Integrin adhesiveness can be dynamically regulated through a process termed inside-out signaling. In addition, ligand binding transduces signals from the extracellular domain to the cytoplasm in the classical outside-in direction. Recent structural, biochemical, and biophysical studies have greatly advanced our understanding of the mechanisms of integrin bidirectional signaling across the plasma membrane. Large-scale reorientations of the ectodomain of up to 200 A couple to conformational change in ligand-binding sites and are linked to changes in alpha and beta subunit transmembrane domain association. In this review, we focus on integrin structure as it relates to affinity modulation, ligand binding, outside-in signaling, and cell surface distribution dynamics.

Integrins are cell adhesion molecules that play critical roles in development, wound healing, hemostasis, immunity and cancer. Advances in the past two years have shed light on the structural basis for integrin regulation and signaling, especially on how global conformational changes between bent and extended conformations relate to the inter-domain and intra-domain shape shifting that regulates affinity for ligand. The downward movements of the C-terminal helices of the alpha I and beta I domains and the swing-out of the hybrid domain play pivotal roles in integrin conformational signaling. Experiments have also shown that integrins transmit bidirectional signals across the plasma membrane by coupling extracellular conformational change with an unclasping and separation of the alpha and beta transmembrane and cytoplasmic domains.

We examined the effect of conformational change at the beta(7) I-like/hybrid domain interface on regulating the transition between rolling and firm adhesion by integrin alpha(4)beta(7). An N-glycosylation site was introduced into the I-like/hybrid domain interface to act as a wedge and to stabilize the open conformation of this interface and hence the open conformation of the alpha(4) beta(7) headpiece. Wild-type alpha(4)beta(7) mediates rolling adhesion in Ca(2+) and Ca(2+)/Mg(2+) but firm adhesion in Mg(2+) and Mn(2+). Stabilizing the open headpiece resulted in firm adhesion in all divalent cations. The interaction between metal binding sites in the I-like domain and the interface with the hybrid domain was examined in double mutants. Changes at these two sites can either counterbalance one another or be additive, emphasizing mutuality and the importance of multiple interfaces in integrin regulation. A double mutant with counterbalancing deactivating ligand-induced metal ion binding site (LIMBS) and activating wedge mutations could still be activated by Mn(2+), confirming the importance of the adjacent to metal ion-dependent adhesion site (ADMIDAS) in integrin activation by Mn(2+). Overall, the results demonstrate the importance of headpiece allostery in the conversion of rolling to firm adhesion.

Conformational communication across the plasma membrane between the extracellular and intracellular domains of integrins is beginning to be defined by structural work on both domains. However, the role of the alpha and beta subunit transmembrane domains and the nature of signal transmission through these domains have been elusive. Disulfide bond scanning of the exofacial portions of the integrin alpha(IIbeta) and beta(3) transmembrane domains reveals a specific heterodimerization interface in the resting receptor. This interface is lost rather than rearranged upon activation of the receptor by cytoplasmic mutations of the alpha subunit that mimic physiologic inside-out activation, demonstrating a link between activation of the extracellular domain and lateral separation of transmembrane helices. Introduction of disulfide bridges to prevent or reverse separation abolishes the activating effect of cytoplasmic mutations, confirming transmembrane domain separation but not hinging or piston-like motions as the mechanism of transmembrane signaling by integrins., Competing Interests: The authors have declared that no conflicts of interest exist.

Conformational change in the integrin extracellular domain is required for high affinity ligand binding and is also involved in post-ligand binding cellular signaling. Although there is evidence to the contrary, electron microscopic studies showing that ligand binding triggers alpha- and beta-subunit dissociation in the integrin headpiece have gained popularity and support the hypothesis that head separation activates integrins. To test directly the head separation hypothesis, we enforced head association by introducing disulfide bonds across the interface between the alpha-subunit beta-propeller domain and the beta-subunit I-like domain. Basal and activation-dependent ligand binding by alpha(IIb)beta(3) and alpha(V)beta(3) was unaffected. The covalent linkage prevented dissociation of alpha(IIb)beta(3) into its subunits on EDTA-treated cells. Whereas EDTA dissociated wild type alpha(IIb)beta(3) on the cell surface, a ligand-mimetic Arg-Gly-Asp peptide did not, as judged by binding of complex-specific antibodies. Finally, a high affinity ligand-mimetic compound stabilized noncovalent association between alpha(IIb) and beta(3) headpiece fragments in the presence of SDS, indicating that ligand binding actually stabilized subunit association at the head, as opposed to the suggested subunit separation. The mechanisms of conformational regulation of integrin function should therefore be considered in the context of the associated alphabeta headpiece.

The affinity of the extracellular domain of integrins for ligand is regulated by conformational changes signaled from the cytoplasm. Alternative types of conformational movement in the ligand-binding headpiece have been proposed. In one study, electron micrograph image averages of the headpiece of integrin aV beta 3 show two different conformations. The open conformation of the headpiece is present when a ligand mimetic peptide is bound and differs from the closed conformation in the presence of an obtuse angle between the beta 3 subunit hybrid and I-like domains. We tested the hypothesis that opening of the hybrid-I-like domain interface increases ligand-binding affinity by mutationally introducing an N-glycosylation site into it. Both beta 3 and beta1 integrin glycan wedge mutants exhibit constitutively high affinity for physiological ligands. The data uniquely support one model of integrin activation and suggest that movement at the interface with the hybrid domain pulls down the C-terminal helix of the I-like domain and activates its metal ion-dependent adhesion site, analogously to activation of the integrin I domain.

Integrins are transmembrane proteins that transmit bi‐directional signals across the cell membrane through global structural rearrangement among three different conformational states: bent, extended‐ closed, and extended‐open conformations. However, the β8 integrin is distinctive and may adopt only one conformation, that is, extended‐closed conformation, with high affinity for ligands under physiological conditions, and may not transmit bi‐directional signals like other integrin members. It is unclear how different β8 domains affect its unique conformation and signaling. We swapped different domains of integrin β3 with those of β8 and investigated how they affected integrin ligand binding, global conformation, and outside‐in signaling. We found that the β8 epidermal growth factor (EGF) domains increased integrin ligand binding ability and contributed to its extended conformation. By comparison, the β8 transmembrane and cytoplasmic domains had little effect on ligand binding or global conformation. The β8 EGF and transmembrane domains did not affect integrin‐mediated cell adhesion, cell spreading, focal adhesion formation, or colocalization of integrin with other proteins, but the cytoplasmic domain had a defective effect on outside‐in signaling. Our results showed that different domains of β8 play various roles on its unique conformation, ligand binding, and signaling, which are considered atypical among integrin members. [ABSTRACT FROM AUTHOR]

Integrins are heterodimeric transmembrane proteins that play important roles in various biological processes. Most integrins serve as adhesion molecules and transmit bidirectional signaling across the cell membrane through global conformational changes from the bent closed to the extended open conformation. However, integrin β8 is distinctive in structure and function. Its cytoplasmic domain lacks the conserved protein‐binding sequence, which is important in transmitting inside‐out signals, suggesting that integrin β8 may have a different activation mechanism or lack such signaling. In addition, the ligand‐binding or activating metal ion Mn2+ does not induce a global conformational change in integrin β8. It may have only one conformation, that is, an extended, closed conformation, but with high affinity for ligands under physiological conditions, and is, therefore, considered an atypical integrin member. The extended structure and high ligand‐binding affinity of integrin αvβ8 make it ideal for encountering and binding ligands expressed on an opposing cell or in the extracellular matrix. In this review, we summarize the progress in integrin β8 research with a focus on its distinctive function and structure among integrin members. [ABSTRACT FROM AUTHOR]

Integrins are heterodimeric transmembrane proteins that play important roles in various biological processes. Most integrins serve as adhesion molecules and transmit bidirectional signaling across the cell membrane through global conformational changes from the bent closed to the extended open conformation. However, integrin β8 is distinctive in structure and function. Its cytoplasmic domain lacks the conserved protein‐binding sequence, which is important in transmitting inside‐out signals, suggesting that integrin β8 may have a different activation mechanism or lack such signaling. In addition, the ligand‐binding or activating metal ion Mn2+ does not induce a global conformational change in integrin β8. It may have only one conformation, that is, an extended, closed conformation, but with high affinity for ligands under physiological conditions, and is, therefore, considered an atypical integrin member. The extended structure and high ligand‐binding affinity of integrin αvβ8 make it ideal for encountering and binding ligands expressed on an opposing cell or in the extracellular matrix. In this review, we summarize the progress in integrin β8 research with a focus on its distinctive function and structure among integrin members. [ABSTRACT FROM AUTHOR]

Integrins play an essential role in hemostasis, thrombosis, and cell migration, and they transmit bidirectional signals. Transmembrane/cytoplasmic domains are hypothesized to associate in the resting integrins; whereas, ligand binding and intracellular activating signals induce transmembrane domain separation. However, how this conformational change affects integrin outside-in signaling and whether the α subunit cytoplasmic domain is important for this signaling remain elusive. Using Chinese Hamster Ovary (CHO) cells that stably expressed different integrin αIIbβ3 constructs, we discovered that an αIIb cytoplasmic domain truncation led to integrin activation but not defective outside-in signaling. In contrast, preventing transmembrane domain separation abolished both inside-out and outside-in signaling regardless of removing the αIIb cytoplasmic tail. Truncation of the αIIb cytoplasmic tail did not obviously affect adhesion-induced outside-in signaling. Our research revealed that transmembrane domain separation is a downstream conformational change after the cytoplasmic domain dissociation in inside-out activation and indispensable for ligand-induced outside-in signaling. The result implicates that the β TM helix rearrangement after dissociation is essential for integrin transmembrane signaling. Furthermore, we discovered that the PI3K/Akt pathway is not essential for cell spreading but spreading-induced Erk1/2 activation is PI3K dependent implicating requirement of the kinase for cell survival in outside-in signaling. [ABSTRACT FROM AUTHOR]

Summary: Structures of intact receptors with single-pass transmembrane domains are essential to understand how extracellular and cytoplasmic domains regulate association and signaling through transmembrane domains. A chemical and computational method to determine structures of the membrane regions of such receptors on the cell surface is developed here and validated with glycophorin A. An integrin heterodimer structure reveals association over most of the lengths of the α and β transmembrane domains and shows that the principles governing association of hetero and homo transmembrane dimers differ. A turn at the Gly of the juxtamembrane GFFKR motif caps the α TM helix and brings the two Phe of GFFKR into the α/β interface. A juxtamembrane Lys residue in β also has an important role in the interface. The structure shows how transmembrane association/dissociation regulates integrin signaling. A joint ectodomain and membrane structure shows that substantial flexibility between the extracellular and TM domains is compatible with TM signaling. [Copyright &y& Elsevier]

Summary: The complete ectodomain of integrin αIIbβ3 reveals a bent, closed, low-affinity conformation, the β knee, and a mechanism for linking cytoskeleton attachment to high affinity for ligand. Ca and Mg ions in the recognition site, including the synergistic metal ion binding site (SyMBS), are loaded prior to ligand binding. Electrophilicity of the ligand-binding Mg ion is increased in the open conformation. The β3 knee passes between the β3-PSI and αIIb-knob to bury the lower β leg in a cleft, from which it is released for extension. Different integrin molecules in crystals and EM reveal breathing that appears on pathway to extension. Tensile force applied to the extended ligand-receptor complex stabilizes the closed, low-affinity conformation. By contrast, an additional lateral force applied to the β subunit to mimic attachment to moving actin filaments stabilizes the open, high-affinity conformation. This mechanism propagates allostery over long distances and couples cytoskeleton attachment of integrins to their high-affinity state. [Copyright &y& Elsevier]

The Asp of the RGD motif of the ligand coordinates with the β I domain metal ion dependent adhesion site (MIDAS) divalent cation, emphasizing the importance of the MIDAS in ligand binding. There appears to be two distinct groups of integrins that differ in their ligand binding affinity and adhesion ability. These differences may be due to a specific residue associated with the MIDAS, particularly the β3 residue Ala252 and corresponding Ala in the β1 integrin compared to the analogous Asp residue in the β2 and β7 integrins. Interestingly, mutations in the adjacent to MIDAS (ADMIDAS) of integrins α4β7 and αLβ2 increased the binding and adhesion abilities compared to the wild-type, while the same mutations in the α2β1, α5β1, αVβ3, and αIIbβ3 integrins demonstrated decreased ligand binding and adhesion. We introduced a mutation in the αIIbβ3 to convert this MIDAS associated Ala252 to Asp. By combination of this mutant with mutations of one or two ADMIDAS residues, we studied the effects of this residue on ligand binding and adhesion. Then, we performed molecular dynamics simulations on the wild-type and mutant αIIbβ3 integrin β I domains, and investigated the dynamics of metal ion binding sites in different integrin-RGD complexes. We found that the tendency of calculated binding free energies was in excellent agreement with the experimental results, suggesting that the variation in this MIDAS associated residue accounts for the differences in ligand binding and adhesion among different integrins, and it accounts for the conflicting results of ADMIDAS mutations within different integrins. This study sheds more light on the role of the MIDAS associated residue pertaining to ligand binding and adhesion and suggests that this residue may play a pivotal role in integrin-mediated cell rolling and firm adhesion. [ABSTRACT FROM AUTHOR]