The regulation of integrin function by divalent cations

Abstract

Integrins are a family of α/β heterodimeric adhesion metalloprotein receptors and their functions are highly dependent on and regulated by different divalent cations. Recently advanced studies have revolutionized our perception of integrin metal ion-binding sites and their specific functions. Ligand binding to integrins is bridged by a divalent cation bound at the MIDAS motif on top of either α I domain in I domain-containing integrins or β I domain in α I domain-less integrins. The MIDAS motif in β I domain is flanked by ADMIDAS and SyMBS, the other two crucial metal ion binding sites playing pivotal roles in the regulation of integrin affinity and bidirectional signaling across the plasma membrane. The β-propeller domain of α subunit contains three or four β-hairpin loop-like Ca²⁺-binding motifs that have essential roles in integrin biogenesis. The function of another Ca²⁺-binding motif located at the genu of α subunit remains elusive. Here, we provide an overview of the integrin metal ion-binding sites and discuss their roles in the regulation of integrin functions.

Introduction

Integrins are a major family of cell surface-adhesion receptors that are expressed in all metazoans. In vertebrates, 18 α subunits and 8 β subunits have been discovered, which combine into 24 different heterodimers that recognize overlapping but distinct sets of extracellular ligands (Fig. 1).^1,2 As adhesion molecules, integrin can mediate cell-cell, cell-matrix and cell-pathogen interactions.^3-7 Most integrins are not constitutively active and often expressed on cell surface in an inactive state, in which they neither bind ligands nor signal. The basal low adhesiveness of integrins is very important for their biological functions, such as integrins on circulating blood cells. There are around 1 mM Ca²⁺ and Mg²⁺ in blood and the existence of Ca²⁺ play an important role in keeping integrins in inactive state. Removal of Ca²⁺ or addition of Mn²⁺ will strikingly increase ligand binding affinity and adhesiveness of almost all integrins. In addition to extracellular divalent cations, integrin can also be activated by intracellular proteins or stimuli, such as talin, through a process termed inside-out signaling.^8-13 Moreover, ligand occupancy can induce integrin conformational change and transduce extracellular signals into cytoplasm through outside-in signaling.^9,14 Integrin-mediated adhesion and signaling events are important in normal physiological responses including immune response, tissue morphogenesis, wound healing, hemostasis, cell survival and cell differentiation.^15-18 Conversely, dysregulation of integrins are involved in the pathogenesis of many diseases, including cancer metastasis, auto-immune disease and thrombotic vascular diseases.^15,19-21

Figure 1. Integrin family. Integrins are loosely grouped into three classes that bind basal extracellular matrix (ECM), provisional ECM and cell surface adhesion molecules (CAMs), respectively. Basal ECM mainly includes collagen and laminin. Provisional ECM mainly includes fibrinogen, fibronectin, vitronectin, cryptic collagen and von Willebrand factor. The α I-containing integrins are asterisked.

Integrin Overall Structure and Conformational Changes Associated with Affinity Regulation

Integrins are type I trans-membrane proteins with large ectodomains, short trans-membrane and cytoplasmic domains (except for β4 subunit with exceptional long cytoplasmic domain of ~1,000 amino acids) (Fig. 2).²² There are four extracellular domains, β-propeller, thigh, calf-1 and calf-2, present in all α subunits (Fig. 2). In addition, half of the α subunits incorporate an additional autonomous folding domain of ~200 amino acids termed the inserted domain (I domain) (Figs. 1 and 2).^23,24 Thus, integrins can be classified into two subfamilies, the α I-containing integrins and α I-less integrins. The β subunit ectodomain contains eight domains, β I domain, hybrid, PSI, β-tail and four integrin-EGF domains (Fig. 2). Electron microscopy and X-ray crystal structure studies of several integrins (e.g., αIIbβ3, αVβ3, αXβ2) have demonstrated independently that the overall shape of integrin ectodomain is that of a large “head” on two long “legs” with flexible “knees” (Fig. 2B and C).^25-28 The α head is composed of β-propeller domain and α I domain in α I-containing integrins, while a single β-propeller forms α head in α I-less integrins (Fig. 2B and C). The β I domain, sharing the same overall fold as the α I domain, is present in all β subunits and forms β head (Fig. 2B and C). α I domain is the ligand binding domain in α I-containing integrins whereas the β I domain forms a major ligand binding pocket in α I-less integrins (Fig. 2B and C).^29-31 Integrin affinity regulation is highly related to its global and local conformational rearrangements. It has been demonstrated that integrins exist in at least three conformational states: the bent conformation with closed headpiece, the extended conformation with closed headpiece and the extended conformation with open headpiece, which are corresponding to the low-, intermediate- and high-affinity states, respectively (Fig. 2B and C).¹² Correspondingly, it is proposed that integrin activation is accompanied with a switchblade-like opening of the headpiece-tailpiece interface, which extends the ligand-binding headpiece of the integrin heterodimer away from the plasma membrane (Fig. 2B and C).¹² Cell surface integrins are in the equilibrium among these conformational states which can be shifted by certain stimuli, such as extracellular metal ions.

Figure 2. Schematic of integrin structure and conformational rearrangements. (A) Organization of domains within the primary structure. α I domain inserted in β-propeller is denoted by dash lines. Yellow and red asterisks denote Ca²⁺- and Mg²⁺-binding sites, respectively. Open asterisk denotes the Ca²⁺-binding site in the forth repeat of β-propeller domain in some α subunits. (B and C) Conformational rearrangements of α I-containing (B) and α I-less integrins (C) during activation.

Divalent cations are essential for integrin functions, from stabilizing integrin structure to mediating its interaction with the ligand and modulating integrin-ligand binding in either an enhancing or a suppressing way.^32-40 The removal of divalent cations by EDTA completely inhibits integrin-ligand binding. And the formation of integrin heterodimer complex is also dependent on the presence of at least micromole levels of divalent cations.^33-40 Physiologically, most integrins are in the resting state with the presence of 1 mM Ca²⁺ and 1 mM Mg²⁺ in body fluid. The millimole level Ca²⁺ often has an inhibitory effect on integrin-ligand binding.^38,41-47 By removal of Ca²⁺, the remaining Mg²⁺ alone can promote ligand binding in most integrins. Studies also indicate that micromolar Ca²⁺ can synergize with suboptimal concentration of Mg²⁺ to further activate integrins.^38,41-47 As a non-physiological stimulus, Mn²⁺ can shift integrins into high-affinity conformations (detected by special activation-dependent mAbs) and strikingly activate integrins, even with the presence of millimolar Ca²⁺.^38,41-47

The preliminary information on integrin metal ion-binding sites was obtained from equilibrium dialysis and Tb³⁺ luminescence assay using purified integrin fragments, demonstrating that there are at least five divalent cation binding sites in αIIbβ3.^48,49 Further studies using recombinant integrin fragments identified two functionally distinct classes of metal ion-binding sites in β3.⁵⁰ One site is capable of binding Ca²⁺, Mg²⁺ and Mn²⁺, and is required for integrin-ligand binding, whereas the other site is specific for Ca²⁺ and has an inhibitory effect for ligand binding.⁵⁰ Three kinds of binding sites for Ca²⁺ have been identified in α4β1, which differ in Ca²⁺-binding affinity.⁵¹ The first kind is a moderate-affinity site (ED₅₀ 50–100 μM) that only can be rapidly occupied after integrin binds ligand; the second kind is a ligand binding-independent high-affinity site (ED₅₀ 20 μM); the third kind has the low-affinity for Ca²⁺ and its occupancy can inhibit integrin-ligand binding.⁵¹ Integrin crystal structures have provided new insights into the metal ion-binding sites, and their functions are further revealed by mutagenesis studies. To date, eight divalent cation binding sites have been discovered in integrin ectodomains with five in α subunit and three in β subunit (Fig. 2). The following part will give a general review on integrin divalent cation binding sites.

α Subunit β-Propeller Ca²⁺-Binding Sites

The N-terminal region of integrin α subunit comprises seven homologous repeats of ~60 residues which fold into a 7-bladed β-propeller domain.²⁸ Two concentric rings of predominantly aromatic residues, which line the upper, inner rim of the propeller core, build up a “cage” motif (Fig. 3C).⁵² Each blade is composed of four anti-parallel β-strands, adopting a “W” topology (Fig. 3D).²⁸ Loops connecting the strands extend either above or below the plane of the propeller (Fig. 3B and D).⁵² There are three to four Ca²⁺-binding sites located in the first loops extending between propeller blades 4 to 7 (Fig. 3A and B).²⁸ The Ca²⁺-binding domains were originally described as resembled similar to EF hand structures, but recent computer modeling data and the crystal structures of αVβ3 and αIIbβ3 indicate that these domains are β-hairpin loops, consisting of a charged loop with a glycine hinge, flanked by groups of hydrophobic residues.^28,52-55 The Ca²⁺-binding sites span a nine-residue segment with the consensus sequence Asp/Glu-h-Asp/Asn-x-Asp/Asn-Gly-h-x-Asp/Glu where “h” is hydrophobic and “x” is any residue.²⁸ Ca²⁺ is usually coordinated by oxygen atoms from side chains of residues 1, 3, 5 and 9 and the carbonyl oxygen of residue 7 (Fig. 3A).²⁸ These Ca²⁺-binding sites are located at the bottom β-propeller, opposite to the integrin α/β subunits interface (Fig. 3B), and are unlikely to be accessed by large ligands in intact integrins on cell surface although the synthetic or recombinant fragments containing these calcium binding sites have been reported to bind integrin ligands.^56,57 But together with the N-terminal one-third region of β-propeller, those Ca²⁺-binding sites have been shown to involve in the determination of ligand-recognition specificity in αIIbβ3.⁵⁸ Substitution of the N-terminal portion of αV with the homologous fragment from αIIb endows αVβ3 with the reactivity to αIIbβ3 small ligands and its specific ligand mimetic antibodies.⁵⁸ Moreover, the β-propeller Ca²⁺-binding sites also involve in the regulation of integrin-ligand binding affinity since conservative mutations in those sites of α4 β-propeller significantly decreased the affinity of ligand binding to α4β1.⁵⁹

Figure 3. Ca²⁺-binding sites in α subunit β-propeller domain. (A) Sequence alignment of the Ca²⁺-binding sites in β-propeller domain of 18 human α subunits. The α subunits containing α I domain are asterisked. Residues with metal-coordinating side-chain oxygen atoms are highlighted in red, and residues with metal-coordinating backbone carbonyl oxygen atoms are highlighted in deep purple. The residue numbers are shown in light blue on the left of the first residue of each Ca²⁺ binding sequence. (B) Side-view of the unliganded crystal structure of αIIbβ3 headpiece (pdb3FCS), with αIIb subunit shown in green and β3 in blue. The αIIb β-propeller bound Ca²⁺ ions are denoted in yellow spheres. (C) Schematic of the αIIb β-propeller domain showing the central cage motif (top view).⁵² (D) Schematic of the “W” topology of blade 4 (side view) in αIIb β-propeller domain. The relative locations of the seven mutations found in Glanzmann thrombasthenia patients are shown. The mutant residues are shown in small red spheres and the bound Ca²⁺ are shown in a large yellow sphere.

Another important function of the β-propeller Ca²⁺-binding sites is that they are essential to integrin biogenesis by facilitating the early folding process of αIIb and conferring stability to the cage motif during αIIbβ3-herterodimer formation.⁵² Seven mutations either in or near the αIIb β-propeller Ca²⁺-binding sites have been found in patients who suffer from Glanzman thrombsthenia, an inherited platelet disorder, including Gly242Asp from W4, Val298Phe, Glu324Lys, and Arg327His from W5, Ile374Thr from W6, and Gly418Asp and deletion Val425/Asp426 from W7 (Fig. 3D).^52,60-64 All of these mutations resulted in defective Ca²⁺ binding by either directly interfering with Ca²⁺ coordination or disrupting the electrostatic potential conductive to Ca²⁺ binding. Consequently, β-propeller can’t be folded properly and the stability of the integrin α/β interface will be jeopardized. Furthermore, the malfolded heterodimer will be largely retained within the endoplasmic reticulum and cannot be efficiently transported to Golgi.^52,60-64

α Subunit Genu Ca²⁺-Binding Site

There is one Ca²⁺-binding site located at α subunit genu, which linking thigh domain and calf-1 domain and where the α subunit sharp bending occurs in bent conformation. The epitopes of two Ca²⁺-dependent antibodies that can detect the extention of αL subunit have been mapped to this region and the occupancy of genu Ca²⁺-binding site is required for the binding of the antibodies.⁶⁵ Further mutational studies indicate that genu Ca²⁺-binding site is occupied in integrins in both bent and extended conformations.⁶⁵ Coordinations to genu Ca²⁺ in αIIb are formed by the backbone carbonyl of Cys602 and Val607, the side chain of Asp605 in genu, and the side chain of Glu642 in Calf-1.²⁷ As the acidic patches of the thigh base and that of the calf-1 top will face each other in the extended integrin, it was speculated that a well-coordinated genu Ca²⁺ may help stabilize the extended conformation of high-affinity integrin by neutralizing the negative charge at this interface.²⁸ However, the clear-cut biological function of genu Ca²⁺-binding site remains elusive.

α I Domain MIDAS

The α I domain, which is inserted between blade 2 and 3 of β-propeller, is the ligand binding domain in α I-containing integrins.^55,66,67 The α I domain adopts the dinucleotide-binding Rossmann fold, with the gradually decreased affinity for metal ions in the order of Mn²⁺ > Mg²⁺ > Ca²⁺.^9,23 Structural studies revealed a metal ion-binding site located on the top face of α I domain (Fig. 4A).^9,32 As the metal ion at this site directly coordinates the side chain of an acidic residue that is characteristic of all integrin ligands, it has been designated as the metal ion-dependent adhesion site (MIDAS).^9,68,69 The MIDAS metal ion is coordinated by five side chains from three different loops. The β1-α1 loop contains three coordinating residues in a sequence of the characteristic cation-binding site, Asp-X-Ser-X-Ser (Fig. 4B and C).⁹ The α3-α4 and β4-α5 loops donate the other two coordinating residues of Thr and Asp, respectively (Fig. 4B and C).⁹ The αM I domain has been crystallized in three distinct conformations, termed closed, intermediate, and open, which are corresponding to the low, intermediate and high ligand-binding affinities.^68,70-72 These conformations demonstrate distinct patterns of MIDAS metal ion coordination. In both closed and open αM I domains, the MIDAS metal ion shares the primary coordinating residues of Ser142, Ser144 and secondary coordinating residue of Asp140 in β1-α1 loop (Fig. 4B and C). In the closed conformation, Thr209 in α3-α4 loop and Asp242 in β4-α5 loop form indirect and direct bonds to MIDAS metal ion, respectively, and three water molecules completes the coordination sphere (Fig. 4B).^9,32 In the open structure, the metal ion moves by ~2.3 Å, resulting in a subtle change in its coordination: Thr209 now coordinates the MIDAS metal ion directly, whereas Asp242 is in the secondary coordination sphere (Fig. 4A–C). Moreover, a Glu, contributed by the ligand, replaces one water molecule at the sixth metal ion coordination site (Fig. 4C).⁹ Further studies using surface plasmon resonance suggested an inverse relationship between the affinity of α I domain for metal ions and the affinity for ligand, that is the closed conformation exhibits the highest affinity for metal ions.²³ Compared with the closed conformation, the local changes at the MIDAS face induce downward displacement of C-terminal α7-helix by two helical turns in open conformation (Fig. 4A).^73,74 The axial displacement of the α7-helix represents a critical linkage for transmission of conformational signals from the MIDAS of α I domain to other integrin domains, and vice versa, especially for the allosteric regulation from β I domain, which will be discussed later.^9,32,73,74

Figure 4. Structural rearrangements of integrin α/β I domains and their metal ion-binding sites. (A–C) αM I domain and MIDAS. (A) Superposition of integrin αM I domains in low-affinity (pdb1JLM, cyan) and high-affinity (pdb1IDO, green) conformations. Blue and red spheres denote the MIDAS metal ions in low- and high-affinity conformations, respectively. (B and C) MIDAS from the closed (pdb1JLM) (B) and open (pdb1IDO) (C) αM I domains. The metal ion coordinations are shown by red dashed lines. Glu314 from a neighboring αM I domain in crystal lattice coordinates with the MIDAS Mg²⁺ and is shown in yellow. Large blue and red spheres are Mn²⁺ and Mg²⁺, respectively, and small red spheres are coordinating water-molecule oxygens. (D–F) β3 I domain and its metal ion cluster. (D) Superposition of β subunit I domains from integrin αIIbβ3 in low-affinity (pdb3FCS, cyan) and high-affinity (pdb3FCU, green) conformations. Blue and red spheres denote Mg²⁺ in low- and high-affinity MIDAS, respectively. Orange and yellow spheres denote Ca²⁺ in low- and high-affinity β I domains, respectively. (E) Structure of low-affinity β3 metal ion-binding sites (pdb3FCS). (F) Structure of high-affinity β3 metal ion-binding sites (pdb3FCU). (G) Structure of metal ion-binding sites in low-affinity β2 (pdb3K6S). The linear cluster of β I domain metal ion-binding sites are shown as SyMBS, MIDAS and ADMIDAS from left to right. The metal ion-binding sites are colored as follows: yellow, SyMBS; pink, MIDAS; purple-blue, ADMIDAS. Mg²⁺ ions in low- and high-affinity MIDAS are shown as large blue and red spheres, respectively. The SyMBS and ADMIDAS bound Ca²⁺ ions are shown as large yellow spheres. Coordinating water-molecule oxygens are shown as small red spheres. N and O atoms involved in metal ion-coordinating are colored in blue and red, respectively. Coordinations between O atoms and metal ions are shown by red dashed lines. The cation-π interaction between the aromatic side chain of Y164 (in β3) and SyMBS metal ion is shown by blue dashed line.

For many integrins, millimolar Ca²⁺ often has an inhibitory effect on ligand binding.^38,41,42,75 Hypothesis has been proposed that high concentration of Ca²⁺ may compete with Mg²⁺/Mn²⁺ for α I domain MIDAS occupancy in α I-containing integrins.⁴⁶ However, the hypothesis disagrees with some studies. For example, high concentration Ca²⁺ does not inhibit ligand binding to the isolated α2 I domain.^44,46 Since the MIDAS motif is in a coordination geometry that the larger calcium ion is predicted not to bind, the high concentration Ca²⁺ may exert the inhibitory function in other Ca²⁺-binding sites, probably in β I domain.^44,70,71

β I Domain Metal Ion Cluster

β I domain directly binds ligand in α I-less integrins and allosterically regulates α I domain mediated ligand binding in α I-containing integrins.^68,76 β I domain assumes a similar Rossmann fold as α I domain, with a central six-stranded β sheet surrounded by eight helices.²⁸ Different from α I domain with only one MIDAS site, β I domain contains an interlinked linear array of three metal ion-binding sites, with MIDAS at the center and flanked by two other sites: the synergistic metal ion-binding site (SyMBS) and adjacent to MIDAS (ADMIDAS) (Fig. 4D–G).^{27,28,68,70,73,77-83} Only ADMIDAS is occupied by metal ion in the crystal structure of unliganded αVβ3, whereas both MIDAS and SyMBS are also found occupied by Mn²⁺ in the liganded-αVβ3 crystal structure.^28,73 Thus, SyMBS is previously termed as ligand-associated metal binding site (LIMBS).⁷³ Differently, in the unliganded αIIbβ3 crystal structure, all of the three metal ion-binding sites are occupied when physiologic divalent cations, Ca²⁺ and Mg²⁺, are present (Fig. 4D–F).²⁷ Electron density in crystal structures suggests that MIDAS is occupied by Mg²⁺ and the two flanking sites are loaded with Ca²⁺ (Fig. 4D–F).²⁷ Comparable to α I domain, β I domain C-terminal α7-helix proceeds a similar downward displacement in the transition from low- to high-affinity and allosterically alters the geometry of the metal ion clusters in a way to increase the affinity for ligand binding (Fig. 4).^12,84 And this process is coupled to the outward swing of the hybrid domain relative to β I domain (Fig. 2B and C).^12,84

β I domain MIDAS

Similar to α I domain MIDAS, the β MIDAS also contains a conservative Asp-X-Ser-X-Ser motif, which is essential to coordinating the metal ion and subsequent ligand binding.²⁸ The overall geometry of metal-ion coordination is similar to that of α I MIDAS, e.g., the β3 MIDAS is formed by the side chains of Asp119, Ser121, Ser123, Glu220 and Asp251 (Fig. 4E and F). Removal of MIDAS metal ion by mutations completely abolished ligand binding, demonstrating its essential role in integrin-ligand binding in both α I-containing and α I-less integrins.^30,73,79 Crystal structures of β3 integrins have revealed that the carboxyl group of Asp in the RGD sequence of ligand coordinates with the MIDAS Mg²⁺ of β subunit during ligand binding.⁷³ Although there is no lateral movement of the MIDAS metal ion across the ligand-binding pocket in the transition from low- to high-affinity state of β I domain, the movement of β1-α1 loop and its bound ADMIDAS Ca²⁺ toward the MIDAS Mg²⁺ will make MIDAS more electrophilic for the acidic ligand residue (Fig. 4E and F).^27,28

For α I-containing integrins, the β MIDAS does not directly mediate ligand binding. It is proposed that an invariant Glu (Glu310 in αL), in the linker between the C-terminal α7-helix and β3-sheet of the β-propeller, can bind to the activated α I domain MIDAS as an intrinsic ligand (Fig. 2B).^9,85-87 Therefore, the activation signal can be transferred from β I to α I domain through this intrinsic interaction and induce the activation of α I domain by exerting a downward pull on the α7 helix in α I domain (Fig. 2B). Broken of this intrinsic interaction by mutation of Glu310 in αL inhibits integrin αLβ2 activation.^85-87 By contrast, constitutive linkage of αL I domain α7-helix with β2 MIDAS-loop by an inter-subunit disulfide bond can induce αLβ2 activation.⁸⁷

β I domain ADMIDAS

Based on structural and mutagenesis studies, it is proposed that ADMIDAS is the negative regulatory site responsible for inhibition of integrin activation by high concentration of Ca²⁺ and Mn²⁺ competes with Ca²⁺ at this site to activate integrin.^28,73,79 In the unliganded low-affinity crystal structure of β3 integrin, the ADMIDAS Ca²⁺ coordinates with the backbone carbonyl oxygen of Met335 in β6-α7 loop (Fig. 4E).²⁷ This interaction restrains the downward movement of β I domain α7-helix and stabilizes integrin in the closed conformation. By contrast, in the liganded high-affinity β3 structure, the Met335 coordination is broken with the downward displacement of α7-helix and the side-chain carboxyl of the Asp251 forms a new direct coordination with the ADMIDAS ion (Fig. 4F).⁷³ Loss of the Met335 carbonyl coordination is favored by substitution of Ca²⁺ by Mn²⁺, because Ca²⁺ but not Mn²⁺ has a strong propensity to coordinate carbonyl oxygen atom.⁸⁸ Furthermore, the movement of Asp251 toward ADMIDAS makes the MIDAS more positive, resulting in higher affinity for integrin ligands.²⁷

However, ADMIDAS mutagenesis studies on different integrins produced conflicting effects on ligand binding. For integrins α4β7 and αLβ2 that can mediate both rolling and firm cell adhesions prior- and post-activation, removal of metal ion occupancy at ADMIDAS by mutating its coordination residues induced integrin activation.^79,80,89 Whereas for integrins that can only mediate firm cell adhesion upon activation, e.g, α5β1, α2β1 and αIIbβ3, ADMIDAS mutations decreased ligand binding.^31,78,81,90 Sequence alignment reveals a potential residue that may contribute to the differences. Following the Asp242 (in β2), there is a second Asp in β2 and β7 instead of an Ala in β1 and β3 (Fig. 4E–G). Recently solved αXβ2 crystal structure shows that the second Asp (Asp243) in β2 is close to MIDAS, and has the potential to coordinate MIDAS metal ion in low-affinity β I domain (Fig. 4G).²⁵ Thus, the MIDAS of β2 and β7 is more negative than that of β3 and β1, resulting in a lower affinity for ligand binding. During integrin activation, Asp243 in β2 will move toward the ADMIDAS along with the anterior Asp242, which will reduce the negativity of MIDAS and lead to higher affinity for ligands. Thus, it is speculated that the previously reported β2 and β7 ADMIDAS mutations do not abolish the ADMIDAS metal ion binding, but instead, induce the shift of the two adjacent Asp residues toward ADMIDAS to fulfill the ADMIDAS metal ion coordination, and resulted in a more positive MIDAS which is able to bind ligands with higher affinity than WT.⁷⁸ Despite of the distinct roles in affinity regulation in different integrins, ADMIDAS mutations in all integrins abolished integrin allosteric conformational changes and outside-in signaling.^78,91 All ADMIDAS mutants are in bent conformation with clasped α/β tails, even for the high-affinity β2 and β7 ADMIDAS mutants.⁹¹ Therefore, ADMIDAS plays an important role in linking β I domain and hybrid domain, most likely through the interaction with Met335 (in β3) in the β6-α7 loop, for the propagation of integrin conformational changes and signaling.

The in vivo function of ADMIDAS was studied using integrin β7 knock-in mice with β7 ADMIDAS mutation D146A, which disrupted the divalent cation binding at ADMIDAS.⁹² Similar to the results obtained from the in vitro study,⁷⁹ the ADMIDAS mutation enhanced the lymphocytes adhesion to α4β7 ligand MAdCAM-1 and perturbed lymphocytes migration on MAdCAM-1 substrates in vitro.⁹² Although the ADMIDAS mutation enhanced lymphocytes adhesion to Peyer’s patch venules in vivo, it suppressed the subsequent homing to gut.⁹² Additionally, the disabled β7 ADMIDAS diminished the capacity of T cells to induce colitis.⁹² Thus, ADMIDAS is indispensable for integrin α4β7 mediated lymphocyte homing to gut and trafficking to the inflammation sites.

β I domain SyMBS

SyMBS is the positive regulatory site for integrin-ligand binding. Mutations at SyMBS abolished ligand binding in α2β1, α5β1, αIIbβ3 and αLβ2 integrins, and converted α4β7-mediated high-affinity firm adhesion into low-affinity rolling adhesion in shear flow,^{79-81,83,91,93-96} which demonstrate that integrin activation is dependent on the occupancy of metal ion at SyMBS. In addition, SyMBS metal ion can form a cation-π interaction with a conserved aromatic residue (Tyr or Phe) in the specificity-determining loop (SDL) (Fig. 4E–G), which may help to stabilize SyMBS metal ion coordination and maintain the proper conformation of SDL.⁹⁷ The disruption of this interaction abolished α4β7-MAdCAM-1 high-affinity binding.⁹⁷ SyMBS is important but not absolutely required for integrin activation since the negative effect of SyMBS mutations on integrin-ligand binding can be counteracted by mutations that stabilize integrins in high-affinity conformations.^{31,78,80,91,98,99}

Synergy between MIDAS and SyMBS

In contrast to the inhibitory effect of high concentration (millimole level) of Ca²⁺, low concentration (micromole level) of Ca²⁺ can synergize with suboptimal concentrations of Mg²⁺ to facilitate ligand binding has been discovered in various integrin families.^{31,41,44,50,75,79,80,100-104} For example, low concentration of 50 μM Ca²⁺ greatly augmented α4β7-mediated adhesion in 50 μM Mg²⁺.⁷⁹ Since MIDAS has the propensity to bind Mg²⁺, Ca²⁺ is more likely to exert the positive synergistic effect though either SyMBS or ADMIDAS which prefers Ca²⁺ to Mg²⁺. Mutagenesis studies suggested that the synergy occurred between SyMBS and MIDAS, as SyMBS mutations, but not ADMIDAS mutations, abolished the synergistic adhesion.⁷⁹ αIIbβ3 crystal structures provided a possible structural basis for the synergy between MIDAS and SyMBS.²⁷ In both the unliganded low-affinity state and liganded high-affinity state, the MIDAS Mg²⁺ and SyMBS Ca²⁺ share the side-chain carboxyl of β3 subunit Glu220 (Fig. 4E and F). Therefore, the presence of SyMBS Ca²⁺ may favor the specific orientation of this side-chain and stabilize MIDAS Mg²⁺ occupancy.²⁷

Concluding Remarks

The metal ion dependence of integrin function has been investigated over several decades and progresses have been made to define and locate the metal ion-binding sites and their functions. For now, it seems clear that the α subunit β-propeller have three to four metal ion-binding sites that are deemed to bind Ca²⁺ under physiological conditions, which are important for integrin heterodimer biogenesis and stability. There is one calcium-binding site located at genu in α subunit, but its function is still ill defined.²⁸ Another pivotal function of divalent cations is to regulate integrin ligand-binding, which is mainly through α I domain MIDAS and β I domain metal ion cluster.⁹ MIDAS motif of both α I domain and β I domain prefers to bind Mg²⁺ under physiological conditions, and is essential for ligand binding. Ca²⁺ exerts the biphasic allosteric regulation function through SyMBS and ADMIDAS in β I domain. Mn²⁺ may activate integrins by competing Ca²⁺ binding at multiple metal ion-binding sites, one could be ADMIDAS, but the exact mechanism is not very clear.^14,31 Furthermore, almost all the information about integrin metal ion-binding sites is obtained using purified integrins and cell lines by in vitro study, it is still largely unknown whether divalent cations can regulate integrin ligand-binding affinity in vivo or not. It was reported that extracellular Ca²⁺ and Mg²⁺ might regulate cell migration during cutaneous injury and bone resorption.^42,105 During the early stages of cutaneous injury, to which the inflammatory responses are mainly mediated by integrins, the concentrations of extracellular Ca²⁺ and Mg²⁺ in wound fluids are significantly different from those observed in normal extracellular fluid. Particularly, Mg²⁺ concentration is elevated and Ca²⁺ is reduced, and consequently promotes the migration of keratinocytes, macrophages, fibroblasts and endothelial cells on collagen.¹⁰⁵ But, the mechanism underlying the regulation of cell migration by cations in vivo is still unknown. Thus, to explore the potential regulatory effects of divalent cations on integrins in vivo and their working mechanisms will be of more interest.

Glossary

Abbreviations:

MIDAS

metal ion-dependent adhesion site

ADMIDAS

adjacent to MIDAS

SyMBS

synergistic metal ion-binding site

LIMBS

ligand-associated metal binding site

SDL

specificity-determining loop