1

The dual structural roles of the membrane distal region of α integrin cytoplasmic

tail in integrin inside-out activation

Jiafu [Liu]1, *, Zhengli [Wang]1, 3, *, Aye Myat Myat [Thinn]1, 2, Yan-Qing [Ma]1, 2, and

Jieqing [Zhu]1, 2, §

1Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI 53226

2Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226

3College of Marine Science and Engineering, Qingdao Agricultural University, Qingdao

266109, China

*These authors contributed equally to this work

§Author for correspondence:

Jieqing Zhu, Ph.D.

Associate Investigator

Blood Research Institute, BloodCenter of Wisconsin

8727 Watertown Plank Road

Milwaukee, WI 53226

Jieqing.Zhu@bcw.edu

Phone #: 414-937-3867

Fax #: 414-937-6284

Running tile: α cytoplasmic tail in integrin activation

Keywords: integrin cytoplasmic domain, inside-out activation, talin, kindlin, platelet,

leukocyte

© 2015. Published by The Company of Biologists Ltd.Jo

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JCS Advance Online Article. Posted on 6 March 2015

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Abstract

Studies on the mechanism of integrin inside-out activation have been focused on the role

of β cytoplasmic tails that are relatively conserved and bear binding sites for the

intracellular activators including talin and kindlin. Integrin α cytoplasmic tails share a

conserved GFFKR motif at the membrane-proximal region forming specific interface

with β membrane-proximal region that keeps integrin inactive. The α membrane-distal

regions after the GFFKR motif are diverse both in length and sequence and their roles in

integrin activation have not been well-defined. In this study, we report that the α

cytoplasmic membrane-distal region contributes to maintaining integrin in the resting

state and to integrin inside-out activation. Complete deletion of the α membrane-distal

region diminished talin and kindlin mediated integrin ligand binding and conformational

change. A proper length and amino acids of α membrane-distal region is important for

integrin inside-out activation. Our data establish an essential role of the α integrin

cytoplasmic membrane-distal region in integrin activation and provide new insights into

how talin and kindlin induce the high affinity integrin conformation that is required for

fully functional integrins.

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Introduction

Integrins are cell surface molecules composed of α and β subunits, each containing a

large extracellular domain, a short transmembrane (TM) domain, and usually a short

cytoplasmic tail (CT). The combination of 18 α and 8 β integrin subunits results in 24

human integrins that play important roles in diverse biological processes including cell

migration, morphogenesis, hemostasis, and the immune response (Hynes, 2002).

Integrins communicate between extracellular ligands and intracellular cytoskeleton

through bidirectional signaling across cell membrane (Kim et al., 2011; Springer and

Dustin, 2012). Intracellular stimulations impinging on integrin CT induce conformational

changes that transform resting integrin into a high affinity, ligand binding-competent

state (known as integrin activation or inside-out signaling). Ligand binding to the

extracellular domain induces integrin conformational rearrangement and transmits signals

to the cytoplasmic side (known as integrin outside-in signaling). The integrin TM and CT

domains play pivotal roles in the bidirectional signaling process.

The large scale integrin conformational rearrangements, including the changing from a

bent to an extended, and from a closed to an open headpiece conformation, have been

directly visualized by electron microscopy (EM) (Takagi et al., 2002; Nishida et al.,

2006; Ye et al., 2010; Eng et al., 2011; Zhu et al., 2012), crystallography (Xiao et al.,

2004; Zhu et al., 2013), and small angle x-ray scattering (Mould et al., 2003; Eng et al.,

2011), and indicated by exposing the epitopes (known as Ligand-Induced-Binding-Site,

LIBS) that are masked in the inactive state (Frelinger et al., 1991; Sims et al., 1991;

Humphries, 2004; Byron et al., 2009). It has been demonstrated that both integrin

extension and headpiece opening are required for high-affinity ligand binding (Chen et

al., 2010; Springer and Dustin, 2012), which is crucial for platelet aggregation mediated

by integrin αIIbβ3 at the bleeding sites and neutrophil arrest mediated by integrin αLβ2 at

the inflamed tissues (Lefort et al., 2012).

Integrin inside-out activation is regulated through the CT. Sequence alignment shows that

β integrin CTs are relatively conserved at both the membrane-proximal (MP) and

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membrane-distal (MD) regions bearing common binding sites for regulatory proteins like

talin and kindlin (Fig. S1) (Anthis and Campbell, 2011; Kim et al., 2011; Margadant et

al., 2011; Morse et al., 2014). In contrast, α integrin CTs are only highly conserved at the

MP regions having a Gly-Phe-Phe-Lys-Arg (GFFKR) motif, whereas the MD regions are

diverse significantly both in sequence and length (Fig. S1). It is known that the α CT MP

region is critical for keeping integrin inactive (O'Toole et al., 1991; O'Toole et al., 1994;

Hughes et al., 1995; Hughes et al., 1996; Lu and Springer, 1997; Zhu et al., 2009), but the

role of α CT MD region in integrin activation is not well characterized.

Mutational studies suggested that the specific associations of α and β TM-CT domains

maintain integrin in the resting state (O'Toole et al., 1994; Hughes et al., 1995; Luo et al.,

2005; Partridge et al., 2005; Berger et al., 2010). This was confirmed by the αIIbβ3 TM-CT

structures determined by disulfide crosslinking using intact integrin on the cell surface

(Zhu et al., 2009) (Fig. 1A) and by NMR using the isolated peptides either in lipid

bicelles (Lau et al., 2009) (Fig. 1B) or in organic solvent (Yang et al., 2009) (Fig. 1C).

The three structures share a similar interface at the TM domain, but differ at the CT (Fig.

1A-C). Although the disulfide-based and the NMR structure in lipid bicelles have a

similar conformation at the CT MP regions (Fig. 1A, B), there are different

interpretations of how the conserved residues such as β3-K716 contribute to integrin

activation (Zhu et al., 2009; Kim et al., 2012b; Kurtz et al., 2012). The NMR structure in

organic solvent has very different conformation at the MP region with the other structures

(Fig. 1C). It is not known if such structural diversity is due to the different environments

used for structure determination or represents a dynamic conformational change. The

structure of αIIb CT MD region was not well-defined and varies among all the structures

(Fig. 1A-C). It is not known if this region contributes to maintaining the resting integrin

conformation.

In the inside-out integrin activation, the cytosolic protein, talin (-1 or -2), through its head

domain that is composed of F0, F1, F2 and F3 subdomains, binds to both the MD and MP

regions of β CT resulting in conformational changes that are propagated to the

extracellular domains across cell membrane (Kim et al., 2011). Kindlin (-1, -2 or -3) was

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found to cooperate with talin in integrin activation through direct binding to the β CT

MD region (Ma et al., 2008; Moser et al., 2008; Harburger et al., 2009; Malinin et al.,

2009; Moser et al., 2009b; Svensson et al., 2009; Bledzka et al., 2012). Studies on how

talin changes integrin conformation have been focused on the β TM-CT domain, which

have been described in many elegant reviews (Moser et al., 2009a; Shattil et al., 2010;

Anthis and Campbell, 2011; Kim et al., 2011; Calderwood et al., 2013; Das et al., 2014).

Recent structural and functional studies suggest that talin-1 binding to the β3 CT changes

the tilt angel of β3 TM domain in the cell membrane, thus disturbing the α-β interfaces at

the TM and MP regions (Kalli et al., 2011; Kim et al., 2012b; Kim et al., 2012a). A

crystal structure of talin-2 F2-F3/β1D-CT suggests an ionic interaction between a lysine of

talin-2 F3 domain and the conserved Asp of β1D integrin CT MP region (Anthis et al.,

2009). This interaction was proposed to be important in integrin activation by disrupting

the putative salt-bridge (between αIIb-R995 and β3-D723) at the α-β MP interface that was

suggested to maintain the resting integrin state (Hughes et al., 1996). However, it is not

known if or how α CT, especially the MD region participates in talin- and kindlin-

induced integrin activation.

In this study, we examined the role of αIIb, αV, and αL integrin CT MD regions in the

regulation of integrin activation. We report that the α CT MD region helps maintain

integrin in the resting state and is indispensible in talin- and kindlin-induced integrin

conformational change and ligand binding. The proper length and amino acids are

important for the α CT MD region to exert its effect on integrin activation. During the

preparation of our manuscript, Li et al (Li et al., 2014) reported that αIIb CT MD is

required for αIIbβ3 inside-out activation and proposed a model of “steric clashes” between

talin-1 head and αIIb CT MD region that is involved in integrin activation. Our data of

αIIbβ3 are consistent with their results, but our comprehensive analysis on multiple

integrins suggests a more complicated mechanism by which α CT MD region regulates

integrin activation.

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Results

The CT MP region is the minimum structural requirement for maintaining αIIbβ3

integrin in the resting state

To determine the minimum structure requirement of αIIbβ3 CT for maintaining the resting

state, we did serial truncation mutagenesis. αIIb CT truncations before F993 greatly

reduced integrin cell surface expression, while other truncations had minor effect (Fig.

S2A). Similarly, β3 CT truncations before but not after I719 greatly reduced integrin

expression (Fig. S2B). Ligand-mimetic mAb PAC-1 was used to assess αIIbβ3 activation.

PAC-1 binding to all the αIIb CT truncations after R995 is comparable to wild type (WT)

integrin (Fig. 1D). Truncation after αIIb-K994 only slightly increased PAC-1 binding,

while truncations after αIIb V990, G991, F992, and F993 greatly enhanced PAC-1 binding

(Fig. 1D). For β3 integrin, there was no detectable activation for all the β3 CT truncations

after I721 (Fig. 1E). Truncations after β3-W715, β3-K716, and β3-L717 strongly, and

truncation after β3-L719 weakly induced PAC-1 binding (Fig. 1E). The combination of

αIIb-K994Tr and β3-I721Tr had comparable PAC-1 binding to WT αIIbβ3 (data not

shown). These results demonstrate that the MP sequence, KVGFFK or KVGFFKR of αIIb

CT, and the MP sequence of KLLITI or KLLIT of β3 CT are the minimum requirement

for maintaining αIIbβ3 integrin in the resting state.

αIIb CT MD truncations amplify the activating effect of β3 mutations

Truncations after αIIb CT MP region may decrease the threshold for integrin activation

because of structure incompleteness. To test this possibility, we did PAC-1 binding assay

for the αIIb CT truncations co-expressed with the β3 active mutants, β3-G708L or β3-

K716A. These β3 mutations are known to activate αIIbβ3 by disturbing the TM interaction

(Luo et al., 2005; Zhu et al., 2009; Kim et al., 2012b). Compared with WT αIIb, truncation

after αIIb-F993, K994, R995, N996, R997, or P998 robustly enhanced PAC-1 binding to

both β3-G708L and β3-K716A mutants (Fig. 2A,B). This is in contrast with the very

subtle or undetectable activating effect of these αIIb truncations when paired with β3 WT

(Fig. 1D). The truncation-amplified activating effect was decreased with the increase of

the length of αIIb CT MD region (Fig. 2A,B). More strikingly, the β3-G708A mutation

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very weakly induced PAC-1 binding when paired with WT αIIb, but its activating effect

was greatly enhanced upon deletion of αIIb CT MD region (Fig. 2C). In addition, the

enhanced activating effect by αIIb CT MD deletion on β3-G708L mutation was close to

the maximal level since Mn2+ did not further increase PAC-1 binding (Fig. 2C). Similar

results were obtained with the active β3-G135A mutant, which facilitates the active

conformation of β3-I domain (Zhang et al., 2013). Complete deletion of the αIIb CT MD

region enhanced the activation of β3-G135A (not shown). These data demonstrate that

deletion of the αIIb CT MD region renders integrin hyper-reactive to β3 activating

mutations. Therefore, the αIIb CT MD region contributes to maintaining integrin in the

resting state.

αIIb CT MD truncation abolishes the activating effect of αIIb-R995 and β3-D723

mutations

Integrin activation induced by the αIIb-R995 and β3-D723 mutations was shown to depend

on talin-1 binding to β3 CT, which is distinct from the active mutations at the TM domain

(Wegener et al., 2007). We asked whether the αIIb CT MD truncations exert different

effect on αIIb-R995 and β3-D723 mutations. The β3-D723R mutant constitutively bound

PAC-1 when paired with αIIb WT (Fig. 2D). However, PAC-1 binding was reverted to

WT level when β3-D723R was paired with the αIIb truncation of F993Tr, K994Tr,

R995Tr, or N996Tr, in which all or most of the MD region was deleted (Fig. 2D).

Strikingly, although both αIIb-F993Tr and β3-D723R were constitutively active when

paired with their WT partners, their combination abolished the activating effect (Fig. 2D).

This is in sharp contrast with the amplified activation by the αIIb truncations for the β3-

G708L and β3-K716A mutants (Fig. 2A,B). Retaining two or more residues of the αIIb

MD region restored or increased integrin activation induced by β3-D723R mutation (Fig.

2D).

We further verified our results with the αIIb-R995A and αIIb-R995D mutation. Both of

these mutations render αIIbβ3 active to bind PAC-1 in the context of full-length αIIb (Fig.

2E). Their activating effects were abolished when the αIIb CT MD region was completely

deleted (Fig. 2E). By contrast, the activation by αIIb-F992A or αIIb-F993A mutation was

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enhanced upon the αIIb CT MD deletion (Fig. 2E). These data suggest that the αIIb CT MD

region play a positive role in integrin inside-out activation.

α integrin CT MD region is required for talin-1-induced integrin activation

The deactivating effect of αIIb CT MD truncation on αIIb-R995 and β3-D723 mutations

raises the intriguing possibility that the αIIb CT MD region may participate in talin-

mediated integrin activation. We performed the well-established integrin activation assay

by overexpression of EGFP-talin-1-head domain (EGFP-TH) (Bouaouina et al., 2012).

As expected, overexpression of EGFP-TH significantly activated WT αIIbβ3 for binding

PAC-1 (Fig. 3A). The EGFP-TH-induced activation was much more robust for integrin

bearing αIIb-R995A or β3-D723A mutation, indicating a synergetic effect (Fig. 3A).

However, EGFP-TH failed to induce PAC-1 binding to the αIIb-R995Tr mutant, i.e. the

αIIb CT MD region was deleted (Fig. 3A). Remarkably, the strong activating effect of

EGFP-TH on αIIb-R995A or β3-D723A was also completely lost in the absence of the αIIb

CT MD region (Fig. 3A). The expression levels of EGFP-TH or integrins were

comparable among all the transfectants (Fig. S2C). These results clearly demonstrate the

requirement of αIIb CT MD region in talin-1-head induced αIIbβ3 activation.

We next examined whether the CT MD region is also important for talin-induced

activation of other α integrins. We found that overexpression of EGFP-TH significantly

enhanced the binding of Fn9-10 (fibronectin type III domain 9-10) and ICAM-1

(intercellular adhesion molecule 1) to αVβ3 and αLβ2, respectively (Fig. 3B,C). In contrast,

when the CT MD region was completely deleted for αV or αL integrin, i.e. αV-R993Tr or

αL-R1094Tr, talin-1-head failed to augment ligand binding for these integrins (Fig.

3B,C). The expression levels of EGFP-TH or integrin are comparable among the

transfectants (Fig. S2D,E). Thus, the requirement of α CT MD region for talin-mediated

integrin activation could be a generalized mechanism for integrins.

α integrin CT MD region is required for kindlin-induced integrin activation

Having demonstrated the indispensable role of α CT MD region in talin-induced integrin

activation, we next asked if the α CT MD region is also required for kindlin-mediated

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integrin activation. Since integrin activation by kindlin requires the presence of talin (Ma

et al., 2008), we examined the effect of α CT MD deletion on kindlin-mediated integrin

activation with co-expression of talin-1-head. Overexpression of mCherry-kindin-2 (mC-

K2) significantly enhanced PAC-1 binding to WT αIIbβ3 (Fig. 3D). We also detected a

slight increase of PAC-1 binding induced by mCherry-kindlin-3 (mC-K3), but it was not

statistically significant compared with the mCherry control (Fig. 3D). In the absence of

αIIb CT MD region (αIIb-R995Tr), mC-K2-mediated PAC-1 binding was greatly and

significantly (P < 0.01) reduced compared with the WT (Fig. 3D). As a control, talin-1-

head and kindlins failed to induce PAC-1 binding to αIIb/β3-R724Tr due to the deletion of

binding sites for talin-1 and kindlin (Fig. 3D). The expression levels of EGFP-TH, mC-

K2, or mC-K3 were comparable among all the transfectants (Fig. S3A,B). These data

demonstrate that αIIb CT MD region is required for kindlin-mediated αIIbβ3 activation.

Similar results were obtained with αVβ3 and αLβ2 integrins. In the presence of talin-1-

head, kindlin-2 but not kindlin-3 markedly enhanced fibronectin (Fn) binding to wild

type αVβ3 (Fig. 3E). Both kindlin-2 (P < 0.01) and kindlin-3 (P < 0.05) significantly

increased ICAM-1 binding to WT αLβ2, but the level of increase by kindlin-3 was

significantly (P < 0.05) lower than by kindlin-2 (Fig. 3F). Deletion of the CT MD region

of αV (αV-R993Tr) or αL (αL-R1094Tr) significantly (P < 0.01) and greatly reduced the

kindlin-2 mediated ligand binding (Fig. 3E,F). The kindlin-3 induced ICAM-1 binding

was also greatly reduced for αL-R1094Tr/β2 mutant (Fig. 3F). The expression levels of

EGFP-Talin-1-head, kindlin-2, or kindlin-3 were comparable among the transfectants of

αVβ3 (Fig. S3C,D) or αLβ2 (Fig. S3E,F). These data strongly demonstrated that α CT MD

region is also required for kindlin-mediated integrin activation.

The kindlin-2 mediated ligand binding was not completely abolished in the absence of α

CT MD region. The low level increase of ligand binding by kindlin-2 was statistically

significant for αIIb-R995Tr/β3 (P < 0.05) and αL-R1094Tr/β2 (P < 0.01) (Fig. 3D, F), but

not for αV-R993Tr/β3 (Fig. 3E). In addition, we could detect a moderate increase (P =

0.05) of kindlin-3 mediated ICAM-1 binding to αL-R1094Tr/β2 mutant (Fig. 3F). Since

PAC-1, Fn, and ICAM-1 are all multivalent ligands, the low level of ligand binding was

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probably due to integrin clustering effect of kindlin as suggested by a recent study (Ye et

al., 2013).

α integrin CT MD region is required for PMA-stimulated integrin activation in

K562 cells

We further validated our data with αIIbβ3 integrin expressed in human K562 cells. PMA

was used to stimulate integrin activation, which mimics the integrin inside-out activation

cascade in physiological situation (Banno and Ginsberg, 2008). We didn’t detect PMA-

induced PAC-1 binding to WT αIIbβ3 (data not shown). However, PMA induced PAC-1

binding to αIIb/β3-D723A in a concentration-dependent manner (Fig. 4A), indicating that

the β3-D723A mutation enhanced integrin sensitivity to PMA stimulation. Consistent

with the results obtained by overexpression of talin-1 head and kindlin, complete deletion

of αIIb CT MD region abolished PMA-induced PAC-1 binding (Fig. 4A,B).

Retaining two residues of α CT MD region partially restores talin-1-induced

activation of αIIbβ3, but not αVβ3 and αLβ2 integrins

We next asked how α CT MD region participates in talin-mediated integrin activation.

Structure superposition of talin/β-CT and αIIbβ3 TM-CT complexes suggests steric clashes

between talin-head and αIIb MD residues that are close to β3 and immediately follow the

GFFKR motif, i.e. residues N996-R997 (Fig. S4A,B). To test whether the potential steric

clash plays a role in talin-mediated integrin activation, we made truncated αIIb constructs

by keeping two native residues, NR997Tr, or two small residues, GG997Tr and

AA997Tr, or two bulky residues, YY997Tr and WW997Tr at the MD region (Fig. 5A). If

the steric clashes are important, we expect to see the mutations with bulky residues would

exert more activating effect on talin-mediated integrin activation than the mutations with

small residues. All the mutants had comparable levels of PAC-1 binding as αIIb WT when

paired with β3 WT, except the WW997Tr mutant was constitutively active (Fig. 5A).

Talin-1-head augmented PAC-1 binding to all the truncated αIIb mutants co-expressed

with WT β3 (Fig. 5B). The WW997Tr mutant had much higher level of PAC-1 binding

than other constructs due to its constitutive activation (Fig. 5B). We consistently

observed the lower PAC-1 binding induced by talin-1-head for the YY997Tr mutant than

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others (Fig. 5B). We then examined the same αIIb mutants in the context of β3-D723A

mutation, which is expected to enhance the sensitivity of the assay. Talin-1-head induced

similar levels of PAC-1 binding to αIIb-NR997Tr, αIIb-GG997Tr, and αIIb-AA997Tr

mutants co-expressed with β3-D723A, but the binding was significantly (P < 0.001)

lower than αIIb-WT co-expressed with β3-D723A (Fig. 5C). Strikingly, talin-1-head-

induced PAC-1 binding to αIIb-YY997Tr mutant was significantly (P < 0.01) lower than

αIIb-NR997Tr and other mutants (Fig. 5C). When the NR997 was replaced by YY997 in

the context of full-length αIIb, the αIIb-YY997 mutation also significantly reduced talin-1-

head-mediated activation of β3-D723A (Fig. 5C). We next extended the C-termini of αIIb

CT with a 29-residue tag of V5 epitope plus hexahistidine, which is expected to provide

more clashes with talin-1 head. The V5-His-tagged αIIb is indistinguishable with WT αIIb

in response to talin-1-head-induced activation (data not shown). This result, in

accordance with the YY997 mutation data, suggests that it is not plausible to assign a

simple “steric clash” model to the mechanism by which αIIb CT MD region participates in

integrin activation.

We extended our study to αVβ3 and αLβ2 integrins. Consistent with αIIbβ3, the presence of

β3-D723A or the equivalent β2-D709A mutation significantly enhanced talin-1-head-

induced binding of Fn9-10 and ICAM-1 to αVβ3 and αLβ2, respectively (Fig. 5D, E).

Complete deletion of αV and αL CT MD region (αV-R993Tr and αL-R1094Tr) totally

abolished the activating effect of talin-1-head (Fig. 5D, E). However, retaining two

residues for αV and αL CT MD region (αV-R995Tr and αL-R1096Tr) did not restore

talin-1-head-induced integrin activation (Fig. 5D, E). This is in contrast to the result that

retaining two residues at the αIIb CT MD region partially restores talin-1-induced αIIbβ3

activation. In addition, retaining eight residues for the αL CT MD region (αL-A1102Tr)

was still not sufficient to restore talin-1-head-induced integrin activation to the WT αL

level (Fig. 5E). Thus, different α integrins may have different requirement of the CT MD

region for talin-1-mediated activation. The proper amino acids and length of α CT MD

region are required for sufficient integrin activation.

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α integrin CT MD region is required for talin- and kindin-induced integrin

conformational change

It has been generally accepted that talin activates integrin through inducing

conformational change (Anthis and Campbell, 2011; Kim et al., 2011). By using

conformation-dependent LIBS mAbs, we examined talin-1-head-induced conformational

change of integrin ectodomains. Consistent with our previous results (Zhang et al., 2013),

overexpression of talin-1-head significantly increased the binding of β3 LIBS mAb 319.4,

but not αIIb LIBS mAb 370.3 to WT αIIbβ3 (Fig. 6A,B). The β3-D723A or αIIb-R995A

mutation markedly enhanced the talin-1-head-induced binding of both mAbs to full-

length αIIbβ3 (Fig. 6A,B). However, deletion of αIIb CT MD region significantly reduced

talin-1-head-induced mAb binding (Fig. 6A,B). Similarly, talin-1-head significantly

increased the binding of β2 LIBS mAbs KIM127 and M24 to WT αLβ2, but not to αL-

R1094Tr/β2 mutant (Fig. 6C, D). The expression levels of talin-1 head were comparable

among the transfectants (data not shown). Thus, the α CT MD region is required for talin-

induced integrin conformational change.

We next asked whether the α CT MD region is also important for kindlin-mediated

integrin conformational change. When co-expressed with talin-1-head, kindlin-2, but not

kindlin-3 significantly increased the binding of 319.4 and 370.3 to WT αIIbβ3 (Fig. 7A,

B). This is consistent with the data that kindlin-2 but not kindlin-3 enhanced ligand

binding to WT αIIbβ3 (Fig. 3D). Kindlin-2 also significantly enhanced the binding of

KIM127 and M24 to WT αLβ2 (Fig. 7C,D). We also detected kindlin-3-induced binding

of M24 to WT αLβ2 (P < 0.05) (Fig. 7D). The binding of KIM127 was also increased by

kindlin-3, but it is not statistically significant (Fig. 7C). By contrast, both kindlin-2 and

kindlin-3 failed to induce the binding of mAbs to the truncated αIIb-R995Tr/β3 and αL-

R1094Tr/β2 integrins (Fig. 7). The expression levels of talin-1 head and kindlin-2 or

kindlin-3 were comparable among the integrin transfectants (data not shown). These

results demonstrate that the α CT MD region is also required for kindlin-mediated

integrin conformational change.

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Deletion of αIIb integrin CT MD region reduced αIIbβ3 transmembrane

heterodimerization, but did not affect the binding of talin-1-head. To have direct

evidence that αIIb CT MD region contributes to αIIbβ3 TM association, we did disulfide

crosslinking assay with integrins on the cell surface. Consistent with our previous data

(Zhu et al., 2009), disulfide bond was formed at high efficiency between the cysteine

mutations of interfacial residues αIIb-G972C and β3-V700C at the TM domains (Fig. 8A),

indicating the close TM association of αIIbβ3 (Fig. 1A). However, the disulfide bond

formation was significantly reduced upon the deletion of αIIb CT MD region (Fig. 8A),

suggesting a disassociation of TM domains. As a control, deletion of both the MP and the

MD regions of αIIb CT (αIIb-G972C-V990Tr) further reduced disulfide bond formation

(Fig. 8A). This is consistent with the ligand-binding assay. Deletion of αIIb CT MD region

greatly enhanced PAC-1 binding to αIIb-G972C mutant but not β3-V700C mutant, while

deletion of αIIb CT MP region further enhanced PAC-1 binding due to the increased

dissociation of αIIbβ3 transmembrane domains (Fig. 8A, B). This data clearly

demonstrates that αIIb CT MD region contributes to αIIbβ3 transmembrane association,

which helps maintaining integrin inactive. We next asked if αIIb CT MD deletion affects

the binding of talin-1-head with αIIbβ3 integrin. As shown in Fig. 8C, EGFP-talin-1-head

was co-immunoprecipitated with both WT αIIbβ3 and αIIb-R995Tr/β3 mutant, suggesting

that the loss of talin-mediated integrin activation in the absence of αIIb CT MD region is

not due to the effect on talin-1-head binding.

Discussion

We present evidence to support the previously unappreciated structural roles of α integrin

CT MD region in integrin activation. In addition to the well-conserved MP region, the α

integrin CT MD region also contributes to maintaining integrin in the resting state.

Deletion of the αIIb CT MD region lowers the threshold of mutation-induced integrin

activation. This is due to the reduced association of αIIbβ3 transmembrane domains in the

absence of αIIb CT MD region as demonstrated by disulfide crosslinking assay. On the

other hand, we demonstrate that the α integrin CT MD region is required for talin and

kindlin induced inside-out activation of αIIb, αV, and αL integrins, indicative of an

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essential and conserved mechanism of integrin family. The α integrin CT MD region is

dispensable for talin binding but is required for talin and kindlin induced integrin

conformational change. Our data explain to a certain extent the previous observations that

deletion of the α CT MD region diminished cell adhesion mediated by α1, α2, α4, αV, and

α6 integrins (Kassner and Hemler, 1993; Kawaguchi and Hemler, 1993; Shaw and

Mercurio, 1993; Filardo and Cheresh, 1994; Kassner et al., 1994; Kawaguchi et al., 1994;

Yauch et al., 1997; Abair et al., 2008) and that αL CT MD deletion reduced the sensitivity

of αLβ2 integrin to PMA stimulation (Lu and Springer, 1997).

It is not readily known how the α integrin CT MD region contributes to maintaining the

resting state according to the current structural information due to the diverse structures

observed for integrin cytoplasmic tails (Fig. S4). Several models could be proposed

according to the data available (Fig. S4C). First, our data demonstrate that in addition to

the MP regions, the αIIb MD region contributes to integrin TM association on the cell

surface. A recent report showed that complete deletion of the αIIb CT MD region reduced

the interaction of isolated αIIb and β3 TM-CT peptides co-expressed on the cell surface (Li

et al., 2014). Our disulfide crosslinking experiments (Zhu et al., 2009) and a direct

interaction assay (Ginsberg et al., 2001) indicate a close association of αIIb MD region

with β3 cytoplasmic tail. Thus, the αIIb MD region might contribute to the structural

stability of αIIbβ3 TM-CT heterodimer through direct interaction with β3 cytoplasmic tail.

This interaction might be sequence specific since replacing the αIIb CT with α5 CT

rendered αIIbβ3 constitutively active despite αIIb and α5 have very similar CT MP region

(O'Toole et al., 1991). Furthermore, replacing the αIIb CT MD region with a penta-alanine

sequence reduced the interaction of isolated αIIb and β3 TM-CT peptides (Li et al., 2014).

Second, the C-terminal αIIb CT MD region is unique with seven acidic amino acids (Fig.

S1) that had been shown to interact with the N-terminal αIIb CT MP region in a NMR

structure of membrane-anchored αIIb CT peptide (Vinogradova et al., 2000). This

interaction may be important in keeping the resting integrin state, but the same interaction

was not found in the NMR structure of αIIbβ3 TM-CT heterodimer determined in organic

solvent (Yang et al., 2009) (Fig. 1C). Another possible mechanism of α CT MD region

contributing to the inactive state is through its interaction with the negative integrin

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regulators. Most of the negative regulators identified so far bind to the conserved α CT

MP region (Pouwels et al., 2012; Bouvard et al., 2013; Morse et al., 2014), but the MD

region may also contribute to the interaction to some extent, which helps maintain the

resting integrin state.

It was shown that membrane-permeant peptides containing the αIIb CT MD sequences

specially blocked αIIbβ3 activation in platelets (Ginsberg et al., 2001; Koloka et al., 2008).

A recent study suggested that this is because the exogenous αIIb CT MD peptides inhibit

the association of talin with αIIbβ3 in thrombin-stimulated platelets (Gkourogianni et al.,

2013). However, this is unlikely due to the competition with or the steric hindrance of the

talin binding sites of β3 CT since deletion of αIIb CT MD region did not increase the

binding of talin-1 head to β3 CT (Fig. 8C and (Li et al., 2014). Talin activates integrin by

disrupting the α/β TM-CT association, while the αIIb CT MD region negatively regulate

this process through enhancing the structural stability of αIIbβ3 TM-CT heterodimer.

In addition to the negative regulation of α CT MD region in integrin activation, our data

demonstrate a positive role of α CT MD region in talin-induced integrin activation.

Displacement of α CT MD region by the β CT-bound talin-1 head domain might

destabilize the α/β TM-CT dimerization, leading to integrin activation. The potential

steric clashes between talin-1 head and αIIb CT MD residues based on structure

superposition have been proposed to play a role in this process (Yang et al., 2009; Zhu et

al., 2009; Ye and Ginsberg, 2011). This hypothesis was tested recently by replacing the

αIIb CT MD region with a penta-alanine peptide or the CT MD sequence of α5 or αL

integrin (Li et al., 2014). It was shown that the αIIb chimeras remain responsive to talin-1-

head-induced activation. Based on this result, it was concluded that the αIIb CT MD

region participates in integrin activation through steric clashing with talin-1 head without

the requirement of a specific sequence. We examined the potential role of steric clashes

using the truncated αIIb constructs that retain the first two residues of αIIb CT MD region

(i.e. NR997Tr) since structural superposition showed major steric clashes of these

residues with talin-1 head (Fig. S4B). The steric clash model is not consistent with the

results that the bulky YY997 mutations significantly reduced but not enhanced talin-

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mediated αIIbβ3 activation. It should be noted that both the length and sequence of αIIb CT

MD region are important in maintaining the resting integrin state. Indeed, truncating the

αIIb CT or swapping the αIIb CT MD region with other amino acids renders αIIbβ3 more

active than WT as shown in our study and by Li et al (Li et al., 2014). In addition, the

conformational flexibility of both the αIIb and β3 CT MD region (Zhu et al., 2009; Metcalf

et al., 2010) would eliminate clashes in a talin/αIIbβ3 complex. Thus, the steric clash

model is not the sole mechanism of αIIb CT MD region participating in talin-induced

integrin activation.

An interaction of αIIb CT and talin has been reported (Knezevic et al., 1996; Yuan et al.,

2006; Gingras et al., 2009; Raab et al., 2010). Although it may not have a major

contribution to αIIbβ3 and talin interaction, a transient and weak interaction between αIIb

CT and talin might stabilize the active conformation of αIIb CT during integrin activation.

Recent biochemical and molecular dynamic simulation studies indicated that the αIIb CT

increases its membrane embedding upon integrin inside-out activation (Kurtz et al., 2012;

Provasi et al., 2014). The αIIb-YY997 mutation might affect the membrane embedding of

αIIb TM-CT and reduces integrin activation since tyrosine residues tend to locate at the

membrane boundary (Killian and von Heijne, 2000). A cluster of negatively charged

residues at αIIb CT MD region may disfavor the negatively charged cell membrane and

inhibit the membrane embedding of αIIb TM-CT, while an interaction between talin-head

and αIIb CT MD acidic residues as indicated by a molecular dynamic simulation study

(Provasi et al., 2014) might facilitate the membrane embedding of αIIb TM-CT to activate

integrin. In addition, talin’s capability of activating integrin depends on its binding to

both β CT and cell membrane (Wegener et al., 2007; Anthis et al., 2009; Moore et al.,

2012; Song et al., 2012; Kalli et al., 2013). The interaction between α CT and talin may

help talin maintain a proper orientation for the favorable interaction with β CT and cell

membrane in order to stabilize the active conformation of TM-CT (Fig. S4C).

Considering the significant diversity of α CT MD region, it is tempting to propose that

this diversity may regulate integrin inside-out activation in α integrin-specific manner as

it has been suggested that the α CT MD regions determine the specificity of integrin

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outside-in signaling (Chan et al., 1992; Shaw et al., 1995; Sastry et al., 1999; Na et al.,

2003; Goel et al., 2014).

Another potential mechanism of regulating integrin activation by α CT is through its

interacting proteins. Interestingly, most α CT binding proteins identified so far interact

with the conserved membrane-proximal GFFKR motif and function as inhibitors of

integrin activation (Pouwels et al., 2012; Bouvard et al., 2013; Morse et al., 2014), but the

mechanism of their negative regulation is not well-defined. A Rap1 effector, RapL, was

shown to activate αLβ2 integrin through binding to the αL CT MD sequence proximal to

the GFFKR motif (Katagiri et al., 2003). It is not known how RapL cooperates with talin

and/or kindlin to activate αLβ2 integrin. The requirement of α CT MD region for integrin

activation suggests that as-yet undiscovered regulatory proteins that interact with α CT

MD region are involved in integrin activation probably in an integrin-specific manner.

It has been shown that deletion of the αIIb CT MD region did not affect αIIbβ3-mediated

cell adhesion and spreading, but led to aberrant recruitment of αIIbβ3 into focal adhesions

formed by other integrins (Ylanne et al., 1993). The underlined mechanism is not known.

The same phenomenon was observed with α2 integrin (Kawaguchi et al., 1994).

However, deletion of the α CT MD region of α1, α2, α4, αV, or α6 integrin diminished cell

adhesion (Kassner and Hemler, 1993; Kawaguchi and Hemler, 1993; Shaw and Mercurio,

1993; Filardo and Cheresh, 1994; Kassner et al., 1994; Kawaguchi et al., 1994; Yauch et

al., 1997; Abair et al., 2008), indicating the different regulatory roles of α CT MD regions

among different integrins.

Our data of β3-K716A mutation have important implications on the current model of

integrin inside-out activation. The β3-K716 was suggested to help maintain a proper tilt

angle of β3 TM domain in the resting state by placing its basic side chain near negatively

charged phospholipid head groups (Kim et al., 2012b). It was proposed that the β3-

K716A mutation or talin binding to β3 CT induces integrin activation by changing the tilt

angle of β3 TM domain to disturb the αIIbβ3 TM-CT interaction. It was also shown that

the tilt angle change of β3 TM domain could occur upon adding talin-1-head to the

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isolated β3 TM-CT peptide embedded in lipid nanodiscs in the absence of αIIb TM-CT

peptide (Kim et al., 2012a). We found that complete deletion of αIIb CT MD region

greatly enhanced the activation of β3-K716A. If talin-1 head could induce integrin

activation by changing the tilt angle of β3 TM domain as β3-K716A does, which seems to

be independent of αIIb CT, we expect to see an enhanced activation mediated by talin-1

head in the absence of αIIb CT MD region. However, an opposite result was obtained.

Talin-1 head failed to activate integrin when α CT MD region was completely deleted.

By using the cysteine scanning accessibility method with intact integrin on the cell

surface, another group suggested that β3-K716 was embedded in the lipid bilayer both in

the inactive and active conformation. They detected conformational change of αIIb TM-

CT but not β3 TM-CT domain with active integrin mutants (Kurtz et al., 2012). Our

structural study revealed interactions between the β3-K716 side chain and the αIIb

F992/K994 backbone carbonyl oxygens that are important in maintaining the resting

integrin state (Zhu et al., 2009). These interactions were observed in a molecular dynamic

simulation study starting with the NMR structure of αIIbβ3 TM-CT determined in lipid

bicelles (Provasi et al., 2014). The direct interaction between β3-K716 and αIIb CT was

also observed by a NMR study (Metcalf et al., 2010). Clearly, further investigations are

required to reconcile these discrepancies.

It has been proposed that the conserved αIIb-R995 and β3-D723 form a salt bridge that

restrains αIIbβ3 in the resting state (Hughes et al., 1996). One of the current models

suggests that talin-1 head binding to the β3 CT breaks the putative salt-bridge to induce

integrin activation (Anthis et al., 2009; Anthis and Campbell, 2011). Despite the very

disruptive effect of αIIb-R995 and β3-D723 mutations on αIIbβ3 TM dimerization observed

using isolated TM-CT peptides (Kim et al., 2009; Lau et al., 2009), their mutations only

moderately activated integrin in the context of full-length integrin (Peyruchaud et al.,

1998; Ma et al., 2006; Ghevaert et al., 2008; Zhu et al., 2009). Remarkably, we found that

αIIb-K993Tr and αIIb-K994Tr mutation, in which the αIIb-R995 was deleted, abolished but

not synergized integrin activation induced by β3-D723 mutations and/or by talin-1-head.

In sharp contrast, only in the presence of αIIb CT MD region, the αIIb-R995 or β3-D723

mutations greatly synergize αIIbβ3 activation and conformational change induced by talin-

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1 head. Thus, although αIIb-R995 and β3-D723 involve in integrin activation, they might

not be central in structurally maintaining integrin in the resting state. Mutagenesis data

suggested that the extracellular domains are also involved in stabilizing αIIbβ3 TM dimer

as well as their membrane embedding (Lau et al., 2009; Kurtz et al., 2012). Cautions

should be taken when interpreting data obtained using isolated TM-CT peptides.

The natural mutations of αIIb-R995 and β3-D723 (αIIb-R995Q or αIIb-R995W and β3-

D723H) have been identified in patients with thrombocytopenia (a relative decrease of

platelets in blood) (Peyruchaud et al., 1998; Ghevaert et al., 2008; Kunishima et al.,

2011). Although it was not studied in these patients, our data here suggest that patient

αIIbβ3 integrin with αIIb-R995 or β3-D723 mutations might be more responsive to agonist

stimulation leading to more platelet aggregation than wild type, which may

simultaneously cause a reduction of platelet amount in blood. An equivalent Arg to Ala

mutation of α4 integrin CT was found to aberrantly activate α4 integrin in genetic knock-

in mice (Imai et al., 2008). We also found that the equivalent β2-D709A mutation renders

αLβ2 integrin hyper-responsive to talin-1-head-induced activation. Interestingly, an

equivalent Asp to Ala mutation of β1 integrin CT did not result in obvious defect in vivo

(Czuchra et al., 2006), despite β1 subunit forms heterodimers with 12 α integrin subunits.

Thus, the conserved α-Arg and β-Asp of integrin CT might play different roles in

regulating integrin activation among different integrins.

Among the caveats of our study is the effect of αIIb CT MD mutations on talin-1-head-

mediated integrin activation was much dramatic with β3-D723A mutant compared with β3

WT. It should be noted that overexpression of talin-1 head only moderately activates WT

integrin. Increasing the DNA amount for transfection did not further increase integrin

activation (data not shown). It is possible that overexpression of talin-1 head alone may

not be sufficient to induce maximal integrin activation due to the lack of the recruitment

process of talin-1 head to the integrin tail. In the physiological situation, the active full-

length talin may apply traction force to its bound β CT through coupled actin

cytoskeleton (Zhu et al., 2008; Schurpf and Springer, 2011), and thus exerts more

disruptive effect on integrin TM-CT interaction than talin-head alone. The β3-D723A

mutation or other active mutations like αIIb-R995A and β3-G135A may facilitate talin-

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head-induced integrin activation by decreasing the energy barrier. It significantly

increases talin-1-head-induced integrin activation by more than 20-fold compared with

WT. Moreover, PMA stimulates significant increase in soluble ligand binding to αIIb/β3-

D723A mutant but not WT in suspension K562 cells. Consistent with the soluble ligand-

binding assay, the β3-D723A or αIIb-R995A mutation significantly enhanced talin-1-head-

induced integrin conformational change. Thus, the combination of β3-D723A and αIIb CT

MD mutations greatly improves the sensitivity of our assay. Similarly, the combination of

the β3 activating mutations and αIIb CT MD truncations enabled us to reveal the

contribution of αIIb CT MD region in maintaining the resting state.

Talin-1-head-induced integrin extension, but not headpiece opening has been directly

visualized by EM using the purified intact αIIbβ3 embedded in the lipid nanodiscs (Ye et

al., 2010). We recently showed that talin-1-head-induced integrin conformational change

needs to be propagated to the ligand-binding site probably through headpiece opening in

order to activate integrin (Zhang et al., 2013). In this study, by using the conformation-

dependent mAbs that report integrin extension (319.4 for β3 and 370.3 for αIIb; KIM127

for β2) and headpiece opening (M24 for β2), we further demonstrated talin-1-head-

induced extension and headpiece open of integrin. In particular, we detected enhanced

integrin conformation change (extension and headpiece opening) when talin-1 head was

co-expressed with kindlins. Kindlin-2 or -3 exerts more effect on M24 binding than

KIM127 binding to αLβ2. This is consistent with a recent study showing that both talin-1

and kindlin-3 were required for inducing the extended open headpiece conformation of

αLβ2 (Lefort et al., 2012). Remarkably, the talin-1-head and kindlin-induced integrin

extension and headpiece opening require the presence of α CT MD region. However,

since how kindlins induce integrin activation remains unknown, the requirement of α CT

MD region for kindlin-mediated integrin activation can only be interpreted as a secondary

effect due to the loss of effectiveness of talin according to the current data.

We found that the talin-1-head and kindlin-induced binding of β3 LIBS mAb 319.4 is not

completely abolished in the absence of αIIb CT MD region. This is in contrast with the

binding of αIIb LIBS mAb 370.3. It indicates that talin-1 head might exert some extent of

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conformational change on β3 subunit even in the absence αIIb CT MD region, but a fully

active integrin conformation induced by talin and kindlin requires the involvement of αIIb

CT MD region. In summary, our study provides new insights into integrin inside-out

activation and suggests that further structural studies are required to understand the

precise mechanism by which the α CT MD region is involved in talin and kindlin-

mediated integrin activation.

Materials and methods

DNA constructs

DNA constructs of human αIIbβ3, αVβ3, αLβ2, and EGFP-tagged mouse talin-1-head

(EGFP-TH) were as described (Zhu et al., 2007; Bouaouina et al., 2008; Zhang et al.,

2013). Human kindlin-2 or kindlin-3 was cloned into the pmCherry-C1 vector (Clontech).

Mutations were introduced by site-directed mutagenesis with the QuikChange kit

(Agilent Technologies).

Antibodies and ligands

PAC-1 (BD Bioscience) is a ligand-mimetic mAb (IgM) specific for activated αIIbβ3

integrin (Shattil et al., 1985). AP3 is a conformation-independent anti-β3 mAb (Kouns et

al., 1991). 319.4 is a LIBS mAb that binds to β3 I-EGF domains (Zhang et al., 2013).

370.3 is a LIBS mAb that binds to the αIIb calf-1 domain (Zhang et al., 2013). PE-labeled

TS2/4 (BioLegend) is a non-functional anti-αL mAb (Sanchez-Madrid et al., 1982).

KIM127 (binds to I-EGF-2 domain) and mAb 24 (M24, binds to βI domain) are anti-β2

LIBS mAbs (Dransfield and Hogg, 1989; Robinson et al., 1992; Nishida et al., 2006;

Chen et al., 2010). Human Fn9-10 was as described (Takagi et al., 2001). Human

fibronectin and ICAM-1 (with a C-terminal human IgG1 Fc tag, ICAM-1-Fc) were

purchased from Sigma-Aldrich and Sino-Biological, respectively.

Soluble ligand binding assay by flow cytometry with 293FT transfectants

PAC-1 binding of 293FT (Life Technologies) cells transfected with αIIbβ3 only was as

described (Zhang et al., 2013). For talin-1-head-induced ligand binding, 293FT cells were

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co-transfected with integrin constructs and EGFP or EGFP-TH for at least 24 hours. For

kindlin-induced ligand binding, 293FT cells were co-transfected with integrin constructs

plus EGFP-TH and mCherry or mCherry-kindlin for at least 24 hours. Ligand binding

was performed in HBSGB buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 5.5 mM

glucose, and 1% BSA) plus 5mM EDTA or 1mM Ca2+/Mg2+ (Ca/Mg) at 25°C for 30 min:

5 μg/ml PAC-1 for αIIbβ3; 20 μg/ml each of ICAM-1-Fc and biotin-labeled mouse anti-

human IgG1 Fc for αLβ2; 50 μg/ml Alexa Fluor 647-labeled Fn9-10 or Fn for αVβ3. Cells

were then washed and incubated in Ca/Mg on ice with the detecting reagents: 10 μg/ml

each of biotinylated AP3, PE-labeled streptavidin, and Alexa Fluor 647-labeled goat anti-

mouse IgM for αIIbβ3; biotinylated AP3 and PE-labeled streptavidin for αVβ3; PE-labeled

TS2/4 and Alexa Fluor 647-labeled streptavidin for αLβ2. Integrin and EGFP double-, or

integrin, EGFP, and mCherry triple-positive cells were acquired for calculating mean

fluorescence intensity (MFI) by flow cytometry. Ligand binding was presented as

normalized MFI, i.e. ligand MFI (after subtracting the ligand MFI in EDTA) as a

percentage of integrin MFI.

PMA-induced integrin ligand binding in K562 cells

Human K562 cells were transfected with αIIbβ3 constructs by Lipofectamine 2000 (Life

Technologies). Cells were incubated in RPMI-1640 medium plus 1% BSA with PMA for

10 minutes followed by a 10-minute incubation with PAC-1. Cells were then washed and

incubated with Alexa Fluor 488-labeled AP3 and Alexa Fluor 647-labeled goat anti-

mouse IgM on ice for 30 minutes. Integrin-positive cells were acquired for calculating

MFI by flow cytometry. Ligand binding was presented as MFI normalized by integrin

expression.

LIBS epitope exposure

Talin-1-head and kindlin-induced LIBS epitope exposure was as described (Zhang et al.,

2013). In brief, 293FT transfectants were first incubated with or without (as background)

the biotinylated LIBS mAb in Ca/Mg at 25°C for 30 mins, and then washed and

incubated with the detecting reagents: Alexa fluor 647-labeled AP3 and PE-labeled

streptavidin for αIIbβ3, PE-labeled TS2/4 and Alexa fluor 647-labeled streptavidin for

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αLβ2. Integrin and EGFP double-, or integrin, EGFP, and mCherry triple-positive cells

were analyzed for calculating the MFI. LIBS mAb binding was presented as MFI

normalized by integrin expression, i.e. LIBS mAb MFI (after subtracting the background

MFI) as a percentage of integrin MFI.

Disulfide crosslinking and immunoprecipitation

Metabolic labeling with [35S]cysteine/methionine and disulfide crosslinking using intact

cells of 293FT transfectants were as described (Zhu et al., 2009). Formation of disulfide

bonds was induced by treating the cells with CuSO4/o-phenanthroline on ice. Integrins

were immunoprecipitated with anti-αIIb mAb 10E5 from the cell lysate and subjected to

nonreducing SDS-PAGE and autoradiography. For talin-1-head binding assay, αIIbβ3

integrins were immunoprecipitated with mAb 10E5 from cells co-transfected with EGFP

or EGFP-talin-1-head. Binding of talin-1-head was detected by western blotting with

rabbit anti-EGFP polyclonal antibody (Origene). β3 integrin was detected with rabbit anti-

β3 H-96 (Santa Cruz Biotechnology).

Acknowledgements

We thank Drs. Daniel Bougie, Richard Aster, Barry Coller, Chafen Lu, and Timothy

Springer for providing antibodies; David Calderwood for providing the DNA construct of

EGFP-tagged mouse talin-1-head domain; Peter Newman for critical reading of the

manuscript.

Author contributions

J. Liu, Z. Wang, A. Thinn, and J. Zhu performed the experiments and analyzed data. Y-Q.

Ma made the kindlin constructs. J. Zhu designed the study, prepared the figures, and

wrote the manuscript.

Funding

This work was supported by a Scientist Development Grant from the American Heart

Association [grant number 12SDG12070059 to J.Z.]; and an ASH Scholar Award for

junior faculty from the American Society of Hematology [to J.Z.].

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Disclosure of Conflict-of-interest: The authors declare no competing financial interests.

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Figure Legends

Figure 1. Effect of cytoplasmic truncation on αIIbβ3 integrin activation. (A-C) The

transmembrane (TM) and cytoplasmic structures of αIIbβ3 determined by (A) disulfide

crosslinking and Membrane Rosetta, (B) by NMR in lipid bicelles (PDB code: 2K9J), (C)

by NMR in organic solvent (PDB code: 2KNC). TM regions are red. The cytoplasmic

membrane-proximal (MP) and membrane-distal (MD) regions are cyan and grey,

respectively. Interfacial residues are shown as sticks with red oxygens and blue nitrogens

or as Cα spheres (for glycine), and marked with stars in the αIIbβ3 sequences shown. The

three structures were superimposed based on the TM region and shown in horizontal

separation on the page. (D) PAC-1 binding of the αIIb CT truncations (Tr); (E) PAC-1

binding of the β3 CT truncations. 293FT cells were co-transfected with αIIb truncations

and β3 wild type (WT), or with β3 truncations and αIIb WT. PAC-1 binding was measured

by flow cytometry and presented as the mean fluorescence intensity (MFI) normalized to

integrin expression. Data are presented as mean ± s.e.m (n = 3).

Figure 2. αIIb CT truncations synergize or compensate the activating effect of the

transmembrane and cytoplasmic mutations. PAC-1 binding of 293FT cells co-

transfected with the indicated αIIb constructs and (A) β3-G708L, (B) β3-K716A, (C) β3-

G708A and β3-G708L, (D) β3-D723R, or (E) β3 WT. 293FT transfectants of αIIbβ3 WT

and αIIb-F993Tr plus β3 WT were shown as controls in A, B, and D. PAC-1 binding was

measured by flow cytometry in the buffer containing 1 mM Ca2+/Mg2+ (Ca/Mg) (for A-E)

or 0.2 mM Ca2+ plus 2 mM Mn2+ (Ca/Mn) (for C) and presented as the MFI normalized

to integrin expression. Error bars represent mean ± s.e.m (n = 3).

Figure 3. The α integrin CT MD region is required for talin- and kindlin-induced

integrin activation. (A-C) Complete deletion of the α integrin CT MD region abolished

talin-1-head-induced activation of (A) αIIbβ3, (B) αVβ3, and (C) αLβ2 integrins. Ligand

binding was measured by flow cytometry with 293FT cells co-transfected with the

indicated integrin constructs and EGFP or EGFP-tagged talin-1 head (EGFP-TH). The

EGFP and integrin double-positive cells were analyzed. (D-F) The α integrin CT MD

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region is required for kindlin-induced activation of (D) αIIbβ3, (E) αVβ3, and (F) αLβ2

integrins. Ligand binding was measured by flow cytometry with 293FT cells co-

transfected with the indicated integrin constructs and EGFP-TH plus mCherry (mC), mC-

tagged kindlin-2 (mC-K2), or mC-tagged kindlin-3 (mC-K3). The integrin, EGFP, and

mCherry triple-positive cells were analyzed. Data are presented as the ligand’s MFI

normalized to integrin expression. Error bars are mean ± s.e.m (n � 3). Unpaired two-

tailed t-tests were used for comparison. * P < 0.05; ** P < 0.01; ns P > 0.05.

Figure 4. αIIb CT MD region is required for PMA-stimulated integrin activation in

K562 cells. (A) Dose-response of PMA-induced PAC-1 binding to αIIbβ3 integrin

expressed in K562 cells. Transfected K562 cells were incubated with PMA and then with

PAC-1. Flow cytometry was used to measure PAC-1 binding. Data are presented as the

ligand’s MFI normalized to integrin expression. Error bars are mean ± s.e.m (n = 4). (B)

Representative flow cytometry plots of PMA-induced (at 1μM) PAC-1 binding to K562

cells expressing αIIbβ3 integrins.

Figure 5. Retaining two residues at the α CT MD region partially restores talin-1

head induced activation of αIIbβ3, but not αVβ3 and αLβ2 integrins. (A) PAC-1 binding

of 293FT cells transfected with β3 WT and the indicated αIIb constructs. (B) EGFP-Talin-

1-head (EGFP-TH)-induced PAC-1 binding of the 293FT cells transfected with β3 WT

and the indicated αIIb constructs. (C) EGFP-TH-induced PAC-1 binding of 293FT cells

transfected with β3-D723A and the indicated αIIb constructs. (D) EGFP-TH induced Fn9-

10 binding of 293FT cells transfected with αVβ3 constructs. (E) EGFP-TH induced

ICAM-1 binding of 293FT cells transfected with αLβ2 constructs. EGFP and integrin

double-positive cells were analyzed by flow cytometry. Ligand binding was presented as

the ligand’s MFI normalized to integrin expression. Error bars are mean ± s.e.m (n � 3).

* P < 0.05; ** P < 0.01; *** P < 0.001; ns P > 0.05.

Figure 6. Effect of α integrin CT truncation on talin-1 head induced integrin

conformational change. (A, B) Talin-1 head induced αIIbβ3 epitope exposure for LIBS

mAbs 319.4 and 370.3. (C, D) Talin-1 head induced αLβ2 epitope exposure for mAbs

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KIM127 and M24. 293FT cells were transfected with the indicated integrin constructs

and EGFP or EGFP-Talin-1-head (EGFP-TH). Cells were first incubated with the

biotinylated LIBS mAbs and then stained with PE-labeled streptavidin and Alexa Fluor

647-labeled mAb AP3 (for αIIbβ3), or stained with Alexa Fluor 647-labeled streptavidin

and PE-labeled TS2/4 (for αLβ2). The EGFP and integrin double-positive cells were

analyzed by flow cytometry. The binding of LIBS mAb was presented as the MFI

normalized to integrin expression. Error bars are mean ± s.e.m (n � 3). * P < 0.05; ** P <

0.01; *** P < 0.001; ns P > 0.05.

Figure 7. Effect of α integrin CT truncation on kindlin-induced integrin

conformational change. (A) Binding of LIBS mAb 319.4 and 370.3 to αIIbβ3 induced by

the overexpression of EGFP-Talin1-head (EGFP-TH) plus mCherry (mC)-tagged kindlin-

2 (mC-K2) or kindlin-3 (mC-K3). (B) Binding of LIBS mAb KIM127 and M24 to αLβ2

induced by the overexpression of EGFP-TH plus mC-K2 or mC-K3. 293FT cells were

co-transfected with the integrin constructs and EGFP-TH plus mC, mC-K2, or mC-K3.

The integrin, EGFP, and mCherry triple-positive cells were analyzed by flow cytometry.

The binding of LIBS mAb was presented as the MFI normalized to integrin expression.

Error bars are mean ± s.e.m (n � 3). Unpaired two-tailed t-tests were used for the

comparison with mC control group. * P < 0.05; ** P < 0.01; ns P > 0.05.

Figure 8. Deletion of αIIb CT MD region reduced αIIb and β3 transmembrane domain

association, but did not affect the binding of talin-1-head. (A) Disulfide crosslinking

of indicated αIIbβ3 constructs expressed in 293FT cells. Immunoprecipitates of 35S-labeled

integrins were subjected to non-reducing SDS-PAGE and autoradiography. The

quantification of crosslinking efficiency was shown in the lower panel. Error bars are

mean ± s.e.m (n � 3). (B) PAC-1 binding of 293FT cells transfected with indicated αIIbβ3

constructs in the buffer containing 1 mM Ca2+/Mg2+ (Ca/Mg) or 0.2 mM Ca2+ plus 2 mM

Mn2+ (Ca/Mn). (C) Co-immunoprecipitation of talin-1-head with αIIbβ3 integrin. 293FT

cells were co-transfected with αIIbβ3 constructs and EGFP or EGFP-talin-1-head (EGFP-

TH). Integrins were immunoprecipitated (IP) with mAb 10E5 and subjected to western

blot (WB) with anti-EGFP and anti-β3 antibodies.

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αβ

P KGPDILVVLLSVMGAILLIGLAALLIWKLLITIHD 723RKEFAKFEEERARA

αIIb

693

995

LEERAIPIWWVLVGVLGGLLLLTILVLAMWKVGFFKRNRP

β3

*

*

*

*

**

*

*

**

**

*

*

***

*

*

*

*

992993

996

730

727726

697

701700

704705

983

712

708

984

980

976

968

972

716

*969

994

991719*

*

A

C E

997*998

**

*

β βα α

PLEEDDEEGE

.

.

.

10001001

999

10021003

10051006

1004

10071008

G708

K716

D723 R995

G991G991

K716 K716

D723 R995

G991

G708 G708

D723R995

E1008

P998 P998

G972 G972 G972

737

A737

F727

G976 G976 G976

N743

A B C

762

TM

MP

MD

995

KVGFFKRNRP

992993

996

994

991

997998

PLEEDDEEGE

10001001

999

1002100310051006

1004

10071008

KV

KVGF

KVG

990KVGFF

KVGFFK

KVGFFKR

KVGFFKRN

KVGFFKRN

KVGFFKRNRP

KVGFFKRNRPP

KVGFFKRNRPPL

KVGFFKRNRPPLE

R

αIIbKLLITIHD 723RKEFAKFEEERARA

730

727726

716

719

.

.

.

737

762

K KL

KLLI

KLLITI

KLLITIH

KLLITIHDR

KLLITIHDRKEFA

W 715W 715

722

724

721

728

β3D E

V990T

r

G991T

r

F992T

r

F993T

r

K994T

r

R995T

r

N996T

r

R997T

r

P998T

r

P999T

r

L1000

Tr

E1001

Tr

αIIb W

T05

10

50

100

150

200

250

+ 3 WT

PAC

-1 B

indi

ng (n

omal

ized

MFI

)

W715T

r

K716T

r

L717T

r

I719T

r

I721T

r

H722T

r

R724T

r

A728T

r

αIIb W

T05

10

50

100

150

200

250

+ IIb WT

PAC

-1 B

indi

ng (n

omal

ized

MFI

)

Fig. 1

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αIIb/ 3 W

T

IIb-F99

3Tr/

3

F993T

r

K994T

r

R995T

r

N996T

r

R997T

r

P998T

r

P999T

r

L1000

Tr

E1001

Tr

IIb W

T0

2

4

6

8

10

12

14

+ β3-D723R

PAC

-1 B

indi

ng (n

omal

ized

MFI

)

0

50

100

150

200

250

+ β3-K716APAC

-1 B

indi

ng (n

omal

ized

MFI

)

0

50

100

150

200

250

300

+ β3-G708L

PAC

-1 B

indi

ng (n

omal

ized

MFI

)

995

KVGFFKRNRP

992993

996

994

991

997998

PLEEDDEEGE

10001001

999

10021003

10051006

1004

10071008

990KVGFFKR

KVGFFK

KVGFFKRN

KVGFFKRNR

KVGFFKRNRP

KVGFFKRNRPP

KVGFFKRNRPPL

KVGFFKRNRPPLE

KVGFF

A CαIIb

IIb W

T

R995A

R995A

Tr

R995D

R995D

Tr

F992A

F992A

-R99

5Tr

F993A

F993A

-R99

5Tr

05

1015

50

100

150

+ β3 WT

PAC

-1 B

indi

ng (n

omal

ized

MFI

)

KVGFFKA

KVGFFKD995

KVGFFKANRPPLEEDDEEGE

995

KVGFFKRNRPPLEEDDEEGE

995

KVGFFKDNRPPLEEDDEEGE

995 995

B

D995

KVGAFKRNRPPLEEDDEEGE

995

KVGAFKR 995

KVGFAKRNRPPLEEDDEEGE

995

KVGFAKR

992 992993 993

αIIbE

IIb/ 3 W

T

IIb/ 3-G

708A

IIb-R

995T

r/3-G

708A

IIb/ 3-G

708L

IIb-R

995T

r/3-G

708L

0

50

100

150

200

250

Ca/MnCa/Mg

PAC

-1 B

indi

ng (n

omal

ized

MFI

)

Fig. 2

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A B

EGFP EGFP-TH

V/ 3

V-R99

3Tr/

30

2

4

6 *

ns

Fn9-

10 B

indi

ng (n

omal

ized

MFI

)

IIb W

T

R995T

r

R995A

R995A

Tr

IIb W

T

R995T

r0123455

25

45

65

+ 3-D723A+ 3 WT

PAC

-1 B

indi

ng (n

omal

ized

MFI

) KVGFFKA

KVGFFKR

KVGFFKANRPPLEEDDEEGE

995

KVGFFKRNRPPLEEDDEEGE

KVGFFKRNRPPLEEDDEEGE

995 995

KVGFFKR 995 993

RMGFFKRVRPPQEEQEREQL

RMGFFKR

.

.

.

αVαIIbC

L/ 2

L-R10

94Tr/

20

1

2

3

4

5 **

ns

ICA

M-1

Bin

ding

(nom

aliz

ed M

FI)

1094

KVGFFKRNLKEKMEAGRGVP

KVGFFKR

.

.

.

αL

*ns

**

nsns

**

D EGFP-TH + mCEGFP-TH + mC-K2EGFP-TH + mC-K3

E F

IIb3 W

T

IIb-R

995T

r/3

IIb/ 3-R

724T

r0

10

20

30

40**

*ns

**

ns ns

ns

*

PAC

-1 B

indi

ng (n

omal

ized

MFI

)

V/ 3

V-R99

3Tr/

30

10

20

30

40

**

ns

ns

P =

0.10

**

*

Fn B

indi

ng (n

omal

ized

MFI

)

L/ 2

L-R10

94Tr/

20

10

20

30

40**

**

**

P =

0.05*

*

ICA

M-1

Bin

ding

(nom

aliz

ed M

FI)

Fig. 3

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A

B

0 0.1 0.5 1 2 5 200

20

40

60

80IIb/ 3-D723AIIb-R995Tr/ 3-D723A

PMA (µM)PAC

-1 b

indi

ng (n

orm

aliz

ed M

FI)

PAC

-1

αIIbβ3 (AP3 binding)

- PMA + PMA

αIIb

/β3-

D72

3AαI

Ib-R

995T

r/β3-

D72

3A

Fig. 4

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PAC

-1 B

indi

ng (n

omal

ized

MFI

)

KVGFFKRNR

KVGFFKRGG

KVGFFKRAA

KVGFFKRYY

0

1

2

35

15253545

KVGFFKRNR

KVGFFKRGG

KVGFFKRAA

KVGFFKRYY

KVGFFKRWW

A

C

BKVGFFKRWW

αIIb W

T

NR997T

r

GG997T

r

AA997T

r

YY997T

r

WW997T

r

D + β3 WT

αIIb αIIb

αIIb W

T

NR997T

r

GG997T

r

AA997T

r

YY997T

r

WW997T

r012345

10203040506070

EGFPEGFP-Talin1-head

PAC

-1 B

indi

ng (n

omal

ized

MFI

)

+ β3 WT

V WT

V-R99

3Tr

V-R99

5TrV W

T

V-R99

3Tr

V-R99

5Tr

0

10

20

30

*

+ 3-D723A+ 3 WT

*

ns P=0

.10

ns

Fn9-

10 B

indi

ng (n

omal

ized

MFI

)

**E

IIb W

T

NR997T

r

GG997T

r

AA997T

r

YY997T

r

YY997

R995T

r0

10

20

30

40

50

60

+ 3-D723A

*

**

***

PAC

-1 B

indi

ng (n

omal

ized

MFI

)

nsns

L2 W

TL W

T

L-L1094

Tr

L-L1096

Tr

L-A11

02Tr

0

5

10

15 ***

ns

+ 2-D709A

ns

**

***

*

*IC

AM

-1 B

indi

ng (n

omal

ized

MFI

)

993

RMGFFKRVR

RMGFFKR

995

αVαL

1094

KVGFFKR

KVGFFKRNL

KVGFFKRNLKEKMEA

1096

1102

997 997

EGFPEGFP-Talin1-head

P=0

.10

Fig. 5

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A C** **

0

10

20

30

40

**

319.

4 B

indi

ng (n

omal

ized

MFI

)

* *

*****

IIb/ 3

IIb-R

995T

r/3

IIb/ 3-D

723A

IIb-R

995T

r/3-D

723A

IIb-R

995A

/ 3

IIb-R

995A

Tr/3

0

10

20

30

40

nsns ns ns

370.

3 B

indi

ng (n

omal

ized

MFI

)

**

**0

20

40

60

KIM

127

Bin

ding

(nom

aliz

ed M

FI)

*

ns

L/ 2

L-R10

94Tr/

20

5

10

15

20

25M

24 B

indi

ng (n

omal

ized

MFI

)*

ns

EGFPEGFP-TH

B D

P=0.11P=0.09

P=0.07

Fig. 6

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EGFP-TH + mCEGFP-TH + mC-K2EGFP-TH + mC-K3

A C

0

5

10

15

**

nsns

319.

4 B

indi

ng (n

omal

ized

MFI

)

IIb/ 3

IIb-R

995T

r/3

0

5

10

15

ns

**

ns ns

370.

3 B

indi

ng (n

omal

ized

MFI

)

0

10

20

30

40

50 *

nsns

KIM

127

Bin

ding

(nom

aliz

ed M

FI)

L/ 2

L-R10

94Tr/

20

10

20

30

40

50**

*

nsns

M24

Bin

ding

(nom

aliz

ed M

FI)

P=0

.16 P=0

.89

B D

Fig. 7

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A B

α

β

α-β

IIb/ 3 W

T

IIb-G

972C

/ 3

IIb-G

972C

-R99

5Tr/

3

IIb/ 3-V

700C

IIb-R

995T

r/3-V

700C

IIb-G

972C

/ 3-V70

0C

IIb-G

972C

-R99

5Tr/

3-V70

0C

IIb-G

972C

-V99

0Tr/

3-V70

0C0

50

100

150

200

250

Ca/MnCa/Mg

PAC

-1 B

indi

ng (n

omal

ized

MFI

)

IIb/ 3 W

T

IIb-G

972C

/ 3-V70

0C

IIb-G

972C

-R99

5Tr/

3-V70

0C

IIb-G

972C

-V99

0Tr/

3-V70

0C0

10

20

30

40 P<0.01

Cro

sslin

king

effi

cien

cy (%

)

P<0.01

C

EG

FPE

GFP

-TH

EG

FPE

GFP

-TH

αIIb

/β3 W

TαI

Ib-R

995T

r/β3

IP: αIIbβ3WB: anti-EGFP

WB: anti-β3

Total cell lysateWB: anti-EGFP

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