Allosteric Disulfide Bridges in Integrins: The Molecular Switches of Redox Regulation of Integrin-Mediated Cell Functions
MDPI
Antioxidants
August 202514(8):1005
DOI:10.3390/antiox14081005
LicenseCC BY 4.0
Structure and mechanics of integrin-based cell adhesion
In this communication, we review the structure of integrins in their unliganded and ligand-occupied states, evaluate the conformational rearrangements associated with integrin activation, and describe the structures, conformations, and adhesion dynamics of key cytoskeleton anchoring proteins that transduce outside-in integrin signaling.
The 18 α-subunits and 8 β-subunits of integrins assemble into
24 distinct receptors in mammals and segregate into two groups, one containing and the other lacking an extra von Willebrand factor type A domain (vWFA, known as αA or αI in integrins) in their α-subunits. αA mediates divalent cation binding to extracellular ligands in αA-containing integrins [5]. αA is a GTPase-like domain in which the catalytic site at the apex is replaced with a conserved metal-ion-dependent adhesion site (MIDAS), which is occupied by a divalent cation. αA exists naturally in two conformations closed (low affinity) and open (high affinity) [6, 7]. The open form is distinguished from the closed form by inward movement of the n-terminal α1 helix, restructuring of the F-strand/α7 loop (F/α7 loop) and a two-turn downward movement of the c-terminal α7 helix (reviewed in reference [8]). These tertiary changes produce rearrangements in the three surface loops that form MIDAS, which allow occupancy of MIDAS by an acidic residue from an exogenous ligand that provides the sixth coordination site for the bound metal ion, replacing a water molecule. The closed and open states exist in an equilibrium that favors the former by a ratio of ∼10:1 [9]. Mutations that deform the c-terminal α7 helix [10], its hydrophobic contacts with the central strand [11] or that favor its downward displacement, generate high or intermediate affinity states [12].
10.1016/j.ceb.2007.08.002 ---
The PTB domain from the actin-binding protein tensin binds the β-tail in a talin-like manner, but with lower affinity; the positively charged tyrosine pocket explains why the interaction is indifferent to tyrosine phosphorylation [43]. The recruitment of tensin is crucial in formation of fibrillar adhesions [2]. Crystal structure of the Ig-like 21 domain (IgFLN21) from the actin cross-linking homodimer filamin 1, in complex with integrin β-tail, reveals that the ser/thr-rich membrane distal segment of the β-tail forms an extended β-strand that interacts with strands C and D of IgFLN21. The binding interface extends to the NPxY-binding site of the talin head [49••], precluding the simultaneous binding of both talin and filamin to the β-tail from the same integrin molecule. This competition for binding by filamin may negatively regulate talin-induced integrin activation and may explain the known inhibitory role of filamin on cell migration [61].
While F3 binding to the integrin proximal β3-tail is sufficient to switch the ectodomain into the ligand-competent state, it does not mediate integrin linkage to the cytoskeleton at focal complexes [62]; a second interaction between the rod domain of talin, formed of multiple amphipathic helical bundles, and ligand-occupied integrin β-tails appears necessary [63•].
The structural basis of this integrin–talin rod interaction is unknown; multivalent ligand and force applied on the early matrix–integrin–talin complexes by Rho-activated actomyosin motors may expose a binding site in the β3-tail for the talin rod [52, 64].
The adapter protein paxillin, which is incorporated together with talin in early focal complexes, may link talin to the integrin α-cytoplasmic tail (see below), enhancing resistance of matrix–integrin–talin complexes to mechanical stress [65].
This may explain why paxillin is often found in slow moving membrane protrusions.
The talin- and actin-binding protein vinculin and the focal adhesion tyrosine kinase FAK are incorporated next into focal adhesion complexes, strengthening the ECM–cytoskeleton contacts across the integrin. Cryptic vinculin binding sites (VBS) exposed in the talin rod [46], in cooperation with talin-bound actin [66], activate vinculin by destabilizing its autoinhibitory head–tail intramolecular interaction [67].
Co-crystal and NMR structures of talin-derived VBS peptides in complex with the D1 (Vh1) helical bundle subdomain of the vinculin head show the α-helical VBS inserting and replacing helix 1 of D1 [50, 68]. Increasing force, exerted at the adhesion site by actomyosin contractility probably exposes more talin VBSs (up to 11 sites), leading to more vinculin recruitment.
The vinculin tail domain also interacts with paxillin leucine rich LD motifs [69]. Both interactions probably contribute to the conversion of focal complexes into focal adhesions. FAK plays an important role in signaling networks at focal contacts [70]. It is recruited to ECM-bound integrins through interaction of its c-terminal four-helical bundle FAT domain with paxillin LD motifs [71, 72]. FAK also binds, through the F1 lobe in its n-terminal FERM domain, with the integrin β-tail, an interaction that destabilizes the autoinhibitory state, thus accessing FAK to activation by Src [73]. This interaction has not been structurally characterized.
FAK is also involved in recruiting the adaptor protein p130Cas, a primary mechanical force sensor [74••] (see below).
Recruitment of the actin-bundling homodimer α-actinin is a later event in formation of focal adhesions [3, 75]. A cryoEM study suggests that the region between the spectrin-like α-actinin R1 and R2 repeats in the central α-actinin domain (consisting of four tandem triple-helical bundle repeats) interacts with the proximal helical integrin β-tail already complexed with the talin head, presumably by engaging the opposite side of the β-tail helix [76]. Mechanical stress, exerted by integrin ligation transmitted to the R4 repeat of α-actinin, probably swings out helix 3 from the triple-helical bundle.
The now accessible helix inserts into the vinculin D1 subdomain in a similar manner to talin, but with an inverted orientation relative to talin, eliciting distinct activating structural changes in vinculin [47,
68].
Recruitment of zyxin, through a biochemically but not structurally defined n-terminal interaction with R2/3 repeats of α-actinin [77] enhances Arp2/3-independent actin assembly in focal adhesions through interactions with Ena/VASP family of proteins [78].
The integrin α-cytoplasmic tail is not a passive player in integrin activation or signaling. An interaction between talin and the αIIb cytoplasmic tail has been described [79] but remains structurally uncharacterized.
Further, at least one cytosolic protein, the EF-hand containing calcium and integrin binding protein 1 (CIB1) interacts with hydrophobic residues in the membrane-proximal region of αIIb cytoplasmic tail [80] and interferes with talin binding to an αIIb-tail peptide [81], thus potentially acting as a negative regulator of talin-induced integrin activation.
The α4 (and α9) subunits of ligand-occupied integrins also enhance integrin–cytoskeleton links formed under shear force [65], by indirectly binding to talin through the cytoskeleton adaptor paxillin. The structures of paxillin in complex with talin or the integrin α4-tail have not been determined.
The α4-paxillin interaction is inhibited by phosphorylation of the α4-tail at the leading edge by type I PKA, which is anchored to the α4-tail [82]. This releases paxillin-mediated inhibition of Rac, thus allowing vectorial formation of lamellepodia [83].
Association of the a second Rap1 effector, RAPL, with the αL (CD11a) cytoplasmic tail may act cooperatively with, or perhaps independently of, talin bound to the β2-tail to activate and cluster integrins [84].
Integrin clustering also induces activation of the T cell protein-tyrosine phosphatase (TCPTP) through its association with the integrin α1 cytoplasmic tail; TCPTP-mediated dephosphorylation of the EGF receptor asEGF receptor associated with integrin clusters inhibits anchorage-independent cell proliferation [85].
