RESEARCH ARTICLE

The dual structural roles of the membrane distal region of thea-integrin cytoplasmic tail during integrin inside-out activation

Jiafu Liu1,*, Zhengli Wang1,2,*, Aye Myat Myat Thinn1,3, Yan-Qing Ma1,3 and Jieqing Zhu1,3,`

ABSTRACT

Studies on the mechanism of integrin inside-out activation have

been focused on the role of b-integrin cytoplasmic tails, which are

relatively conserved and bear binding sites for the intracellular

activators including talin and kindlin. Cytoplasmic tails for a-integrins

share a conserved GFFKR motif at the membrane-proximal region

and this forms a specific interface with the b-integrin membrane-

proximal region to keep the integrin inactive. The a-integrin

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 a-integrin

cytoplasmic membrane-distal region contributes to maintaining

integrin in the resting state and to integrin inside-out activation.

Complete deletion of the a-integrin membrane-distal region

diminished talin- and kindlin-mediated integrin ligand binding and

conformational change. A proper length and suitable amino acids in

a-integrin membrane-distal region was found to be important for

integrin inside-out activation. Our data establish an essential role for

the a-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.

KEY WORDS: Integrin cytoplasmic domain, Inside-out activation,Talin, Kindlin, Platelet, Leukocyte

INTRODUCTIONIntegrins are cell surface molecules composed of a and bsubunits, each containing a large extracellular domain, a short

transmembrane (TM) domain, and usually a short cytoplasmic tail

(CT). The combination of 18 a-integrin and eight b-integrin

subunits results in 24 human integrins, and these play important

roles in diverse biological processes including cell migration,

morphogenesis, hemostasis and the immune response (Hynes,

2002). Integrins transmit signals between their extracellular ligands

and the intracellular cytoskeleton through bidirectional signaling

across cell membrane (Kim et al., 2011b; Springer and Dustin,

2012). Intracellular stimulations impinging on the 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). Integrin

conformational change was also demonstrated on the cell surface by

the exposure of epitopes (known as ligand-induced-binding sites,

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 aIIbb3 at the bleeding sites and neutrophil arrest mediated

by integrin aLb2 at the inflamed tissues (Lefort et al., 2012).

Integrin inside-out activation is regulated through the CT.

Sequence alignment shows that b-integrin CTs are relatively

conserved at both the membrane-proximal (MP) and membrane-

distal (MD) regions, which have common binding sites for

regulatory proteins like talin and kindlin (supplementary material

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

et al., 2011; Morse et al., 2014). In contrast, a-integrin CTs are only

highly conserved at the MP regions, which have a Gly-Phe-

Phe-Lys-Arg (GFFKR) motif, whereas the MD regions vary

substantially both in sequence and length (supplementary material

Fig. S1). It is known that the a-integrin CT MP region is required

for keeping the 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 a-integrin CT MD

region in integrin activation is not well characterized.

Mutational studies have suggested that the specific associations

of a-integrin and b-integrin TM and 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 structural studies of the aIIbb3 TM and

CT domains performed by the disulfide crosslinking assay 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

1Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI 53226, USA.2College of Marine Science and Engineering, Qingdao Agricultural University,Qingdao 266109, China. 3Department of Biochemistry, Medical College ofWisconsin, Milwaukee, WI 53226, USA.*These authors contributed equally to this work

`Author for correspondence (Jieqing.Zhu@bcw.edu)

Received 29 July 2014; Accepted 2 March 2015

� 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 1718–1731 doi:10.1242/jcs.160663

1718

different interpretations of how the conserved residues, such asb3-K716, contribute to integrin activation (Zhu et al., 2009; Kim

et al., 2011a; Kurtz et al., 2012). The NMR structure in organicsolvent has a very different conformation at the MP region ascompared with the other structures (Fig. 1C). It is not known

whether such structural diversity is due to the differentenvironments used for structure determination or whether itrepresents a dynamic conformational change. The structure of aIIb

CT MD region is not well-defined and varies among all thestructures (Fig. 1A–C). It is not known whether this regioncontributes to maintaining the resting integrin conformation.

In the inside-out integrin activation, the cytosolic protein talin

(which has two isoforms, talin-1 and -2) binds to both the MD andMP regions of the b-integrin CT through its head domain, whichis composed of F0, F1, F2 and F3 subdomains, resulting in

conformational changes in the integrin TM and CT domains,which are propagated to the extracellular domains across cellmembrane (Kim et al., 2011b). Kindlin (which has three isoforms,

kindlin-1, -2 and -3) has been found to cooperate with talin during

integrin activation through direct binding to the b-integrin CTMD 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 integrinconformation have been focused on the b-integrin TM and CT

domains, and have been described in many elegant reviews(Moser et al., 2009a; Shattil et al., 2010; Anthis and Campbell,2011; Kim et al., 2011b; Calderwood et al., 2013; Das et al.,

2014). Recent structural and functional studies suggest that talin-1 binding to the b3 CT changes the tilt angle of b3 TM domain inthe cell membrane, thus disturbing the a-integrin–b-integrininterfaces at the TM and MP regions (Kalli et al., 2011; Kim

et al., 2011a; Kim et al., 2012). A crystal structure of the talin-2F2-F3 domain bound to b1D-CT suggests that there is an ionicinteraction between a lysine residue in the talin-2 F3 domain and

the conserved aspartic acid residue of the b1D integrin CT MPregion (Anthis et al., 2009). This interaction was proposed to beimportant in integrin activation by disrupting the putative salt-

bridge (between aIIb-R995 and b3-D723) at the a-integrin–b-

αβ

PKGPDILVVLLSVMGAILLIGLAALLIWKLLITIHD 723RKEFAKFEEERARA

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50

100

150

200

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W715T

r

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r

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r

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β3 WT

05

10

50

100

150

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indi

ng (n

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Fig. 1. Effect of cytoplasmictruncation on aIIbb3 integrin activation.(A–C) The transmembrane (TM) andcytoplasmic structures of aIIbb3

determined by (A) disulfide crosslinkingand Membrane Rosetta (Zhu et al.,2009), (B) by NMR in lipid bicelles (PDBcode: 2K9J; Lau et al., 2009), (C) byNMR in organic solvent (PDB code:2KNC; Yang et al., 2009). TM regions arered. The cytoplasmic membrane-proximal(MP) and membrane-distal (MD) regionsare cyan and gray, respectively.Interfacial residues are shown as stickswith red oxygens and blue nitrogens or asCa spheres (for glycine), and marked withstars in the aIIbb3 sequences shown. Thethree structures were superimposedbased on the TM region and shown inhorizontal separation on the page.(D) PAC-1 binding of the aIIb CTtruncations (Tr, shown schematicallyabove the bars); (E) PAC-1 binding of theb3 CT truncations. 293FT cells were co-transfected with aIIb truncations and wild-type b3 (WT), or with b3 truncations andaIIb WT. PAC-1 binding was measured byflow cytometry and presented as themean fluorescence intensity (MFI)normalized to integrin expression. Dataare presented as mean6s.e.m. (n53).

RESEARCH ARTICLE Journal of Cell Science (2015) 128, 1718–1731 doi:10.1242/jcs.160663

1719

integrin MP interface that has been suggested to maintain theresting integrin state (Hughes et al., 1996). However, it is not

known if or how the a-integrin CT, and especially the MD region,participates in talin- and kindlin-induced integrin activation.

In this study, we examined the role of aIIb, aV and aL integrinCT MD regions in the regulation of integrin activation. We report

that the a-integrin CT MD region helps maintain integrin in theresting state and is indispensable in talin- and kindlin-inducedintegrin conformational change and ligand binding. The proper

length and proper amino acids are important for the a-integrin CTMD region to exert its effect on integrin activation. During thepreparation of our manuscript, Li et al. (Li et al., 2014) reported

that the aIIb CT MD region is required for aIIbb3 inside-outactivation and proposed a model of ‘steric clashes’ between thetalin-1 head and the aIIb CT MD region that is involved in

integrin activation. Our data of aIIbb3 are consistent with theirresults, but our comprehensive analysis on multiple integrinssuggests a more complicated mechanism by which the a-integrinCT MD region regulates integrin activation.

RESULTSThe CT MP region is the minimum structural requirement formaintaining aIIbb3 integrin in the resting stateTo determine the minimum structure requirement of aIIbb3 CT formaintaining the resting state, we performed a serial truncation

mutagenesis. aIIb CT truncations before F993 greatly reducedintegrin cell surface expression, whereas other truncations had aminor effect (supplementary material Fig. S2A). Similarly, b3 CT

truncations before but not after I719 greatly reduced integrinexpression (supplementary material Fig. S2B). The ligand-mimetic monoclonal antibody (mAb) PAC-1 was used to assessaIIbb3 activation. PAC-1 binding to all the aIIb CT truncations

after R995 was comparable to that for wild-type (WT) integrin(Fig. 1D). Truncation after aIIb-K994 only slightly increasedPAC-1 binding, whereas truncations after aIIb V990, G991, F992

and F993 greatly enhanced PAC-1 binding (Fig. 1D, truncationsare denoted by the suffix Tr). For b3 integrin, there was nodetectable activation for all the b3 CT truncations after I721

(Fig. 1E). b3-W715Tr, b3-K716Tr and b3-L717Tr stronglyinduced PAC-1 binding, and truncation after b3-L719 weaklyinduced PAC-1 binding (Fig. 1E). The combination of the aIIb-K994Tr and b3-I721Tr had comparable PAC-1 binding to WT

aIIbb3 (data not shown). These results demonstrate that the MPsequence, KVGFFK or KVGFFKR in the aIIb CT, and the MPsequence of KLLITI or KLLIT in the b3 CT are the minimum

requirement for maintaining aIIbb3 integrin in the resting state.

Truncations of the aIIb CT MD region amplify the activatingeffect of b3 mutationsTruncations after the aIIb CT MP region might decrease thethreshold for integrin activation because of structure

incompleteness. To test this possibility, we performed a PAC-1binding assay for the aIIb CT truncations co-expressed with theconstitutively active b3 mutants, b3-G708L or b3-K716A. Theseb3 mutations have been known to activate aIIbb3 by disturbing the

TM interaction (Luo et al., 2005; Zhu et al., 2009; Kim et al.,2011a). Compared with WT aIIb, truncation after aIIb-F993,K994, R995, N996, R997 or P998 strongly enhanced PAC-1

binding to both b3-G708L and b3-K716A mutants (Fig. 2A,B).This is in contrast with the very subtle or undetectable activatingeffect of these aIIb truncations when paired with WT b3 (Fig. 1D).

The truncation-amplified activating effect was decreased with the

increase of the length of aIIb CT MD region (Fig. 2A,B). Morestrikingly, the b3-G708A mutation very weakly induced PAC-1

binding when paired with WT aIIb, but its activating effect wasgreatly enhanced upon deletion of the aIIb CT MD region(Fig. 2C). In addition, the enhanced activating effect of aIIb CTMD deletion on the b3-G708L mutation was close to the maximal

level, given that Mn2+ did not further increase PAC-1 binding(Fig. 2C). Similar results were obtained with the active b3-G135A mutant, which facilitates formation of the active

conformation of b3-I domain (Zhang et al., 2013). Completedeletion of the aIIb CT MD region enhanced the activation of b3-G135A (data not shown). These data demonstrate that deletion of

the aIIb CT MD region renders the integrin hyper-reactive to b3-activating mutations. Therefore, the aIIb CT MD regioncontributes to maintaining integrin in the resting state.

Deletion of the aIIb CT MD region abolishes the activatingeffect of aIIb-R995 and b3-D723 mutationsIntegrin activation induced by the aIIb-R995 and b3-D723

mutations has been shown to depend on talin-1 binding to theb3 CT, which is distinct from the activation mechanism promotedby the activating mutations at the TM domain (Wegener et al.,

2007). We asked whether the aIIb CT MD truncations exertdifferent effects on aIIb-R995 and b3-D723 mutations. The b3-D723R mutant constitutively bound PAC-1 when paired with aIIb

WT (Fig. 2D). However, PAC-1 binding was reverted to a WTlevel when b3-D723R was paired with the aIIb truncationsF993Tr, K994Tr, R995Tr or N996Tr, in which all or most of the

MD region is deleted (Fig. 2D). Strikingly, although both aIIb-F993Tr and b3-D723R were constitutively active when pairedwith their WT partners, their combination abolished theactivating effect (Fig. 2D). This is in sharp contrast with the

amplified activation mediated by the aIIb truncations for the b3-G708L and b3-K716A mutants (Fig. 2A,B). Retaining two ormore residues of the aIIb MD region restored or increased integrin

activation induced by the b3-D723R mutation (Fig. 2D).We further verified our results with the aIIb-R995A and aIIb-

R995D mutation. Both of these mutations render aIIbb3 active to

bind PAC-1 in the context of full-length aIIb (Fig. 2E). Theiractivating effects were abolished when the aIIb CT MD regionwas completely deleted (Fig. 2E). By contrast, the activation byaIIb-F992A or aIIb-F993A mutation was enhanced upon the aIIb

CT MD deletion (Fig. 2E). These data suggest that the aIIb CTMD region plays a positive role in integrin inside-out activation.

The a-integrin CT MD region is required for talin-1-inducedintegrin activationThe deactivating effect of aIIb CT MD truncation on aIIb-R995

and b3-D723 mutations raises the intriguing possibility that theaIIb CT MD region might participate in talin-mediated integrinactivation. We performed a well-established integrin activation

assay by overexpressing the EGFP-tagged talin-1 headdomain (EGFP–TH) (Bouaouina et al., 2012). As expected,overexpression of EGFP–TH significantly activated WT aIIbb3 asdetermined by the amount of PAC-1 binding (Fig. 3A). The

EGFP–TH-induced activation was much more substantial forintegrin bearing aIIb-R995A or b3-D723A mutation, indicating asynergetic effect (Fig. 3A). However, EGFP-TH failed to induce

PAC-1 binding to the aIIb-R995Tr mutant, i.e. the aIIb CT MDregion was deleted (Fig. 3A). Remarkably, the strong activatingeffect of EGFP–TH on aIIb-R995A or b3-D723A was also

completely lost in the absence of the aIIb CT MD region

RESEARCH ARTICLE Journal of Cell Science (2015) 128, 1718–1731 doi:10.1242/jcs.160663

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(Fig. 3A). The expression levels of EGFP–TH or integrins werecomparable among all the transfectants (supplementary materialFig. S2C). These results clearly demonstrate the requirement of

aIIb CT MD region in talin-1-head-induced aIIbb3 activation.We next examined whether the CT MD region is also important

for talin-induced activation of other a-integrins. We found that

overexpression of EGFP–TH significantly enhanced the binding offibronectin type III domain 9–10 (Fn9-10) and intercellular adhesionmolecule 1 (ICAM-1) to aVb3 and aLb2, respectively (Fig. 3B,C). In

contrast, when the CT MD region was completely deleted for aV oraL integrin (aV-R993Tr or aL-R1094Tr), the talin-1 head failedto augment ligand binding for these integrins (Fig. 3B,C). Theexpression levels of EGFP–TH or integrin are comparable among

the transfectants (supplementary material Fig. S2D,E). Thus, therequirement for an a-integrin CT MD region for talin-mediatedintegrin activation could be a generalized property of integrins.

The a-integrin CT MD region is required for kindlin-inducedintegrin activationHaving demonstrated the indispensable role of the a-integrin CTMD region in talin-induced integrin activation, we next asked

whether the a-integrin CT MD region is also required for kindlin-mediated integrin activation. Given that integrin activation bykindlin requires the presence of talin (Ma et al., 2008), we

examined the effect of deletion of the a-integrin CT MD region onkindlin-mediated integrin activation with co-expression ofthe talin-1 head. Overexpression of mCherry-tagged kindin-2

(mC–K2) significantly enhanced PAC-1 binding to WT aIIbb3

(Fig. 3D). We also detected a slight increase of PAC-1 bindinginduced by mCherry–kindlin-3 (mC–K3), but it was not statistically

significant compared with the mCherry control (Fig. 3D). In theabsence of aIIb CT MD region (aIIb-R995Tr), mC–K2-mediatedPAC-1 binding was greatly and significantly (P,0.01) reducedcompared with the WT (Fig. 3D). As a control, the talin-1 head and

kindlins failed to induce PAC-1 binding to aIIb–b3-R724Tr owing tothe deletion of the 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 (supplementary materialFig. S3A,B). These data demonstrate that the aIIb CT MD region isrequired for kindlin-mediated aIIbb3 activation.

Similar results were obtained with aVb3 and aLb2 integrins. Inthe presence of the talin-1 head, kindlin-2 but not kindlin-3

αIIb/ 3 W

T

IIb-F99

3Tr/

3

F993T

r

K994T

r

R995T

r

N996T

r

R997T

r

P998T

r

P999T

r

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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

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250

300

+ β3-G708L

PAC

-1 B

indi

ng (n

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MFI

)

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996

994

991

997998

PLEEDDEEGE

10001001

999

10021003

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1004

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-R99

5Tr

05

1015

50

100

150

+ β3 WT

PAC

-1 B

indi

ng (n

omal

ized

MFI

)

KVGFFKA

KVGFFKD995

KVGFFKANRPPLEEDDEEGE

995

KVGFFKRNRPPLEEDDEEGE

995

KVGFFKDNRPPLEEDDEEGGGEE

995 995

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KVGAFKRNRPPLEEDDEEEGE

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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

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)

Fig. 2. aIIb CT truncationssynergize or compensate theactivating effect of thetransmembrane and cytoplasmicmutations. PAC-1 binding of 293FTcells co-transfected with the indicatedaIIb constructs (shown schematicallyabove the bars in A and E) and (A) b3-G708L, (B) b3-K716A, (C) b3-G708Aand b3-G708L, (D) b3-D723R or(E) b3 WT. 293FT transfectants ofaIIbb3 WT and aIIb-F993Tr plus b3 WTare shown as controls in A, B and D.PAC-1 binding was measured by flowcytometry in the buffer containing1 mM Ca2+ and 1 mM Mg2+ (Ca/Mg)(for A–E) or 0.2 mM Ca2+ plus 2 mMMn2+ (Ca/Mn) (for C) and presentedas the MFI normalized to integrinexpression. Data are presented asmean6s.e.m. (n53).

RESEARCH ARTICLE Journal of Cell Science (2015) 128, 1718–1731 doi:10.1242/jcs.160663

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markedly enhanced fibronectin (Fn) binding to WT aVb3 (Fig. 3E).

Both kindlin-2 (P,0.01) and kindlin-3 (P,0.05) significantlyincreased ICAM-1 binding to WT aLb2, but the level of increase bykindlin-3 was significantly (P,0.05) lower than by kindlin-2(Fig. 3F). Deletion of the CT MD region of aV (aV-R993Tr) or aL

(aL-R1094Tr) significantly (P,0.01) and greatly reduced thekindlin-2-mediated ligand binding (Fig. 3E,F). The kindlin-3-induced ICAM-1 binding was also greatly reduced for the aL-

R1094Tr–b2 mutant (Fig. 3F). The expression levels of EGFP–TH,kindlin-2 or kindlin-3 were comparable among the transfectants ofaVb3 (supplementary material Fig. S3C,D) and among the

transfectants of aLb2 (supplementary material Fig. S3E,F). Thesedata strongly demonstrate that the a-integrin CT MD region is alsorequired for kindlin-mediated integrin activation.

The kindlin-2-mediated ligand binding was not completelyabolished in the absence of the a-integrin CT MD region. The lowlevel increase of ligand binding upon the overexpression ofkindlin-2 was statistically significant for aIIb-R995Tr–b3

(P,0.05) and aL-R1094Tr–b2 (P,0.01) (Fig. 3D,F), but notfor aV-R993Tr–b3 (Fig. 3E). In addition, we could detect amoderate increase (P50.05) of kindlin-3-mediated ICAM-1

binding to the aL-R1094Tr–b2 mutant (Fig. 3F). Given thatPAC-1, Fn, and ICAM-1 are all multivalent ligands, the low levelof ligand binding was probably due to the integrin clustering

effect of kindlin as suggested by a recent study (Ye et al., 2013).

The a-integrin CT MD region is required for PMA-stimulatedintegrin activation in K562 cellsWe further validated our data by expressing the aIIbb3 integrin inhuman K562 cells. PMA was used to stimulate integrin activation,which mimics the physiological integrin inside-out activation

cascade (Banno and Ginsberg, 2008). We did not detect PMA-induced PAC-1 binding to WT aIIbb3 (data not shown). However,PMA induced PAC-1 binding to aIIb–b3-D723A in a concentration-

dependent manner (Fig. 4A), indicating that the b3-D723Amutation enhanced integrin sensitivity to PMA stimulation.Consistent with the results obtained upon overexpression of talin-

1 head and kindlins, complete deletion of the aIIb CT MD regionabolished PMA-induced PAC-1 binding (Fig. 4A,B).

Retaining two residues of a-integrin CT MD region partiallyrestores talin-1-induced activation of aIIbb3, but not aVb3 andaLb2 integrinsWe next asked how the a-integrin CT MD region participates in

talin-mediated integrin activation. Structural superposition oftalin bound to the b-CT and the aIIbb3 TM-CT heterodimersuggests that there can be steric clashes between the talin head

and aIIb MD residues that are close to b3 and immediately followthe GFFKR motif (i.e. residues N996–R997) (supplementarymaterial Fig. S4A,B). To test whether the potential steric clash

plays a role in talin-mediated integrin activation, we made

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

KVGFFKANRPPLEEDDEEEGGE

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

KVGFFKR

LKEKMEAGRGVP

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. The a-integrin CT MD region isrequired for talin- and kindlin-inducedintegrin activation. (A–C) Complete deletion ofthe a-integrin CT MD region abolished talin-1-head-induced activation of (A) aIIbb3, (B) aVb3

and (C) aLb2 integrins. Ligand binding wasmeasured by flow cytometry with 293FT cellsco-transfected with the indicated integrinconstructs (also shown schematically above thebars) and EGFP or EGFP–TH. The EGFP andintegrin double-positive cells were analyzed.(D–F) The a-integrin CT MD region is requiredfor kindlin-induced activation of (D) aIIbb3,(E) aVb3, and (F) aLb2 integrins. Ligand bindingwas measured by flow cytometry with 293FTcells co-transfected with the indicated integrinconstructs and EGFP–TH plus mCherry (mC),mC-tagged kindlin-2 (mC–K2), or mC-taggedkindlin-3 (mC–K3). The integrin, EGFP, andmCherry triple-positive cells were analyzed.Data are presented as the MFI of the ligandnormalized to integrin expression. Data arepresented as mean6s.e.m. (n§3). *P,0.05;**P,0.01; ns, not significant (unpaired two-tailed Student’s t-tests).

RESEARCH ARTICLE Journal of Cell Science (2015) 128, 1718–1731 doi:10.1242/jcs.160663

1722

truncated aIIb constructs that had the two native residues,NR997Tr, 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 would expect tosee that the mutations with bulky residues would have a greater

activating effect on talin-mediated integrin activation than themutations with small residues. All the mutants had comparablelevels of PAC-1 binding to that shown by WT aIIb when pairedwith b3 WT, except that the WW997Tr mutant was constitutively

active (Fig. 5A). The talin-1 head augmented PAC-1 binding toall the truncated aIIb mutants co-expressed with WT b3 (Fig. 5B).The WW997Tr mutant had a much higher level of PAC-1 binding

than other constructs owing to its constitutive activation(Fig. 5B). We consistently observed that a lower level of PAC-1 binding was induced by the talin-1 head for the YY997Tr

mutant than for the other constructs (Fig. 5B). We then examinedthe same aIIb mutants in the context of b3-D723A mutation,which is expected to enhance the sensitivity of the assay. The

talin-1 head induced similar levels of PAC-1 binding to aIIb-NR997Tr, aIIb-GG997Tr, and aIIb-AA997Tr mutants co-expressed with b3-D723A, but the binding was significantly(P,0.001) lower than for aIIb-WT co-expressed with b3-D723A

(Fig. 5C). Strikingly, talin-1-head-induced PAC-1 binding to aIIb-YY997Tr mutant was significantly (P,0.01) lower than withaIIb-NR997Tr and the other constructs (Fig. 5C). When the

NR997 was replaced by YY997 in the context of full-length aIIb,the aIIb-YY997 mutation also significantly reduced talin-1-head-mediated activation of b3-D723A (Fig. 5C). We next extended

the C-termini of aIIb CT with a 29-residue tag of V5 epitope plushexahistidine, which would be expected to provide more clasheswith talin-1 head. We found that the response to talin-1-head-

induced activation for V5–His-tagged aIIb was indistinguishablefrom WT aIIb (data not shown). This result, in accordance withthe YY997 mutation data, suggests that it is not plausible toassign a simple ‘steric clash’ model to the mechanism by which

aIIb CT MD region participates in integrin activation.We extended our study to aVb3 and aLb2 integrins. Consistent

with aIIbb3, the presence of b3-D723A or the equivalent b2-

D709A mutation significantly enhanced talin-1-head-inducedbinding of Fn9-10 and ICAM-1 to aVb3 and aLb2, respectively(Fig. 5D,E). Complete deletion of the aV and aL CT MD region

(aV-R993Tr and aL-R1094Tr) totally abolished the activating

effect of the talin-1 head (Fig. 5D,E). However, retaining tworesidues in the aV and aL CT MD region (aV-R995Tr and aL-

R1096Tr) did not restore talin-1-head-induced integrin activation(Fig. 5D,E). This is in contrast to the result showing that retainingtwo residues at the aIIb CT MD region partially restores talin-1-

induced aIIbb3 activation. In addition, retaining eight residuesfor the aL CT MD region (aL-A1102Tr) was still not sufficientto restore talin-1-head-induced integrin activation to the WT aL

level (Fig. 5E). Thus, different a integrins might have different

requirements for the CT MD region in talin-1-mediatedactivation. The suitable amino acids and proper length of a-integrin CT MD region are required for sufficient integrin

activation.

The a-integrin CT MD region is required for talin- and kindin-induced integrin conformational changeIt is generally accepted that talin activates integrin throughinducing conformational change (Anthis and Campbell, 2011;

Kim et al., 2011b). By using conformation-dependent LIBSmAbs, we examined the talin-1-head-induced conformationalchange of integrin ectodomains. Consistent with our previousresults (Zhang et al., 2013), overexpression of the talin-1 head

significantly increased the binding of the b3 LIBS mAb 319.4,but not the aIIb LIBS mAb 370.3 to WT aIIbb3 (Fig. 6A,B).The b3-D723A or aIIb-R995A mutation markedly enhanced the

talin-1-head-induced binding of both mAbs to full-length aIIbb3

(Fig. 6A,B). However, deletion of aIIb CT MD region significantlyreduced talin-1-head-induced mAb binding (Fig. 6A,B). Similarly,

the talin-1 head significantly increased the binding of the b2 LIBSmAbs KIM127 and M24 to WT aLb2, but not to aL-R1094Tr–b2

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

among the transfectants (data not shown). Thus, the a-integrin CTMD region is required for talin-induced integrin conformationalchange.

We next asked whether the a-integrin CT MD region is also

important for kindlin-mediated integrin conformational change.When co-expressed with the talin-1 head, kindlin-2, but notkindlin-3 significantly increased the binding of the mAbs 319.4

and 370.3 to WT aIIbb3 (Fig. 7A,B). This is consistent with thedata that kindlin-2 but not kindlin-3 enhanced ligand binding toWT aIIbb3 (Fig. 3D). Kindlin-2 also significantly enhanced the

binding of KIM127 and M24 to WT aLb2 (Fig. 7C,D). We also

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. The aIIb CT MD region isrequired for PMA-stimulatedintegrin activation in K562 cells.(A) Dose–response curve for PMA-induced PAC-1 binding to aIIbb3

integrin expressed in K562 cells.Transfected K562 cells wereincubated with PMA and then withPAC-1. Flow cytometry was used tomeasure PAC-1 binding. Data arepresented as the MFI of the ligandnormalized to integrin expression.Data are presented as mean6s.e.m.(n54). (B) Representative flowcytometry plots of PMA-induced (at1 mM) PAC-1 binding to K562 cellsexpressing the indicatedaIIbb3 integrins.

RESEARCH ARTICLE Journal of Cell Science (2015) 128, 1718–1731 doi:10.1242/jcs.160663

1723

detected kindlin-3-induced binding of M24 to WT aLb2 (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 tothe truncated aIIb-R995Tr–b3 and aL-R1094Tr–b2 integrins(Fig. 7). The expression levels of the talin-1 head and kindlin-2

or kindlin-3 were comparable among the integrin transfectants(data not shown). These results demonstrate that the a-integrinCT MD region is also required for kindlin-mediated integrinconformational change.

Deletion of the aIIb integrin CT MD region reduced aIIbb3

transmembrane heterodimerization but did not affect thebinding of the talin-1 headTo have direct evidence that the aIIb CT MD region contributes toaIIbb3 TM association, we performed a disulfide crosslinking

assay with integrins on the cell surface. Consistent with ourprevious data (Zhu et al., 2009), disulfide bonds were formed athigh efficiency between the cysteine mutations of interfacialresidues aIIb-G972C and b3-V700C at the TM domains (Fig. 8A),

indicating the close TM association of aIIbb3 (Fig. 1A). However,the disulfide bond formation was significantly reduced upon thedeletion of aIIb CT MD region (Fig. 8A), suggesting a

disassociation of TM domains. As a control, deletion of both theMP and the MD regions of aIIb CT (aIIb-G972C-V990Tr) furtherreduced disulfide bond formation (Fig. 8A). This is consistent with

the ligand-binding assay. Deletion of the aIIb CT MD regiongreatly enhanced PAC-1 binding to the aIIb-G972C mutant but notthe b3-V700C mutant, whereas deletion of the aIIb CT MP region

further enhanced PAC-1 binding due to the increased dissociationof aIIbb3 transmembrane domains (Fig. 8A,B). This data clearlydemonstrate that the aIIb CT MD region contributes to aIIbb3

transmembrane association, which helps to keep integrin inactive.

We next asked whether deletion of the aIIb CT MD affects thebinding of talin-1 head to aIIbb3 integrin. As shown in Fig. 8C, theEGFP–TH was co-immunoprecipitated with both WT aIIbb3 and

aIIb-R995Tr–b3 mutant, suggesting that the loss of talin-mediatedintegrin activation in the absence of the aIIb CT MD region is notdue to the effect on talin-1 head binding.

DISCUSSIONWe present evidence to support the previously unappreciatedstructural roles of the a-integrin CT MD region in integrin

activation. In addition to the well-conserved MP region, the a-integrin CT MD region also contributes to maintaining integrin inthe resting state. Deletion of the aIIb CT MD region lowers the

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

BKVGFFKRWWWWWWWW

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

**

***

*

*

ICA

M-1

Bin

ding

(nom

aliz

ed M

FI)

993

RMGFFKRVR

RMGFFKR

995

αVαL

1094

KVGFFKR

KVGFFKRNL

KVGFFKRNNLKEKMEA

1096

1102

997 997

EGFPEGFP-Talin1-head

P=0

.10

Fig. 5. Retaining two residues atthe a CT MD region partiallyrestores talin-1 head inducedactivation of aIIbb3, but not aVb3 andaLb2 integrins. (A) PAC-1 binding of293FT cells transfected with b3 WTand the indicated aIIb constructsconstructs (also shown schematicallyabove the bars). (B) EGFP–TH-induced PAC-1 binding of the 293FTcells transfected with b3 WT and theindicated aIIb constructs. (C) EGFP–TH-induced PAC-1 binding of 293FTcells transfected with b3-D723A andthe indicated aIIb constructs.(D) EGFP–TH induced Fn9-10binding of 293FT cells transfectedwith aVb3 constructs. (E) EGFP–THinduced ICAM-1 binding of 293FTcells transfected with aLb2 constructs.EGFP and integrin double-positivecells were analyzed by flowcytometry. Ligand binding waspresented as the MFI of the ligandnormalized to integrin expression.Data are presented as mean6s.e.m.(n§3). *P,0.05; **P,0.01;***P,0.001; ns, not significant(unpaired two-tailed Student’s t-tests).

RESEARCH ARTICLE Journal of Cell Science (2015) 128, 1718–1731 doi:10.1242/jcs.160663

1724

threshold of mutation-induced integrin activation. This is due tothe reduced association of aIIbb3 TM domains in the absenceof the aIIb CT MD region as demonstrated by a disulfide

crosslinking assay. Furthermore, we demonstrate that the a-integrin CT MD region is required for talin- and kindlin-inducedinside-out activation of aIIb, aV, and aL integrins, which is

indicative of an essential and conserved mechanism of integrinactivation. The a-integrin CT MD region is dispensable for talinbinding but is required for talin- and kindlin-induced integrinconformational change. Our data explain to a certain extent the

previous observations that deletion of the a-integrin CT MDregion diminished cell adhesion mediated by a1, a2, a4, aV and a6

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 the aL CT MD deletion reduced the

sensitivity of aLb2 integrin to PMA stimulation (Lu and Springer,1997).

It is not readily known how the a-integrin CT MD regioncontributes to maintaining the resting state because current

structural studies show diverse structures for the integrincytoplasmic tails (supplementary material Fig. S4). Severalmodels could be proposed according to the data available

(supplementary material Fig. S4C). First, our data demonstratethat in addition to the MP regions, the aIIb MD region contributesto integrin TM association on the cell surface. A recent report

has shown that complete deletion of the aIIb CT MD regionreduced the interaction of isolated aIIb and b3 TM-CT peptidesco-expressed on the cell surface (Li et al., 2014). Our disulfide

crosslinking experiments (Zhu et al., 2009) and a direct interactionassay (Ginsberg et al., 2001) indicate a close association of aIIb MD

region with b3 cytoplasmic tail. Thus, the aIIb MD region mightcontribute to the structural stability of an aIIbb3 TM-CT heterodimerthrough a direct interaction with the b3 cytoplasmic tail. This

interaction might be sequence specific given that replacing the aIIb

CT with an a5 CT rendered aIIbb3 constitutively active despite aIIb

and a5 having a very similar CT MP region (O’Toole et al., 1991).

Furthermore, replacing the aIIb CT MD region with a penta-alaninesequence reduces the interaction of isolated aIIb and b3 TM-CTpeptides (Li et al., 2014). Second, the C-terminal aIIb CT MDregion is unique with seven acidic amino acids (supplementary

material Fig. S1) that have been shown to interact with the N-terminal aIIb CT MP region in a NMR structure of membrane-anchored aIIb CT peptide (Vinogradova et al., 2000). This

interaction might be important in keeping the resting integrinstate, but the same interaction was not found in the NMR structureof the aIIbb3 TM-CT heterodimer determined in organic solvent

(Yang et al., 2009) (Fig. 1C). Another possible mechanism of a-integrin CT MD region contributing to the inactive state is throughits interaction with the negative integrin regulators. Most of thenegative regulators identified so far bind to the conserved a-

integrin CT MP region (Pouwels et al., 2012; Bouvard et al., 2013;Morse et al., 2014), but the MD region might also contribute to theinteraction to some extent, which helps maintain the resting

integrin state.It has been shown that membrane-permeant peptides

containing the aIIb CT MD sequences specifically blocked

aIIbb3 activation in platelets (Ginsberg et al., 2001; Kolokaet al., 2008). A recent study has suggested that this is because theexogenous aIIb CT MD peptides inhibit the association of talin

with aIIbb3 in thrombin-stimulated platelets (Gkourogianni et al.,2013). However, this is unlikely owing to competition with or

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

25

M24

Bin

ding

(nom

aliz

ed M

FI)

*

ns

EGFPEGFP-TH

B D

P=0.11P=0.09

P=0.07

Fig. 6. Effect of a-integrin CT truncation on talin-1 headinduced integrin conformational change. (A,B) Talin-1-head-induced aIIbb3 epitope exposure for LIBS mAbs 319.4 and370.3. (C,D) Talin-1-head-induced aLb2 epitope exposure formAbs KIM127 and M24. 293FT cells were transfected with theindicated integrin constructs (shown under B and D) and EGFPor EGFP–TH. Cells were first incubated with the biotinylatedLIBS mAbs and then stained with PE-labeled streptavidin andAlexa-Fluor-647-labeled mAb AP3 (for aIIbb3), or stained withAlexa-Fluor-647-labeled streptavidin and PE-labeled TS2/4 (foraLb2). The EGFP and integrin double-positive cells wereanalyzed by flow cytometry. The binding of LIBS mAb ispresented as the MFI normalized to integrin expression. Dataare presented as mean6s.e.m. (n§3). *P,0.05; **P,0.01;***P,0.001; ns, not significant (unpaired two-tailed Student’s t-tests).

RESEARCH ARTICLE Journal of Cell Science (2015) 128, 1718–1731 doi:10.1242/jcs.160663

1725

steric hindrance of the talin-binding sites of b3 CT given that

deletion of the aIIb CT MD region did not increase the binding oftalin-1 head to b3 CT (Fig. 8C and Li et al., 2014). Talin activatesintegrin by disrupting the TM and CT domain association,

whereas the aIIb CT MD region negatively regulates this processby enhancing the structural stability of the aIIbb3 TM-CTheterodimer.

In addition to the negative regulation of a CT MD region in

integrin activation, our data demonstrate a positive role for the a-integrin CT MD region in talin-induced integrin activation.Displacement of a-integrin CT MD region by the b CT-bound

talin-1 head domain might destabilize the integrin TM–CTdimerization, leading to integrin activation. The potential stericclashes between the talin-1 head and aIIb CT MD residues based

on structure superposition have been proposed to play a role inthis process (Yang et al., 2009; Zhu et al., 2009; Ye et al., 2011).This hypothesis was tested recently by replacing the aIIb CT MDregion with a penta-alanine peptide or the CT MD sequence of a5

or aL integrin (Li et al., 2014). It has been shown that the aIIb

chimeras remain responsive to talin-1-head-induced activation.

Based on this result, it has been concluded that the aIIb CT MDregion participates in integrin activation through steric clashingwith the talin-1 head without the requirement of a specificsequence. We examined the potential role of steric clashes using

the truncated aIIb constructs that retain the first two residues ofaIIb CT MD region (i.e. NR997Tr) given that the structuralsuperposition showed major steric clashes between these residues

and the talin-1 head (supplementary material Fig. S4B). Thesteric clash model is not consistent with the results showing thatthe bulky YY997 mutations significantly reduced, rather than

enhancing, the talin-mediated aIIbb3 activation. It should be notedthat both the length and sequence of the aIIb CT MD region areimportant to maintain the resting integrin state. Indeed, truncating

the aIIb CT or swapping the aIIb CT MD region with other aminoacids renders aIIbb3 more active than WT integrin as shown in ourstudy and by Li et al. (Li et al., 2014). In addition, theconformational flexibility of both the aIIb and b3 CT MD region

(Zhu et al., 2009; Metcalf et al., 2010) would eliminate clashes ina talin–aIIbb3 complex. Thus, the steric clash model is not the solemechanism through which the aIIb CT MD region participates in

talin-induced integrin activation.An interaction between aIIb CT and talin has been reported

(Knezevic et al., 1996; Yuan et al., 2006; Gingras et al., 2009;

Raab et al., 2010). Although it might not have a majorcontribution to aIIbb3 and talin interaction, a transient and weakinteraction between aIIb CT and talin might stabilize the active

conformation of aIIb CT during integrin activation. Recentbiochemical and molecular dynamic simulation studies indicatethat the aIIb CT increases its membrane embedding upon integrininside-out activation (Kurtz et al., 2012; Provasi et al., 2014). The

aIIb-YY997 mutation might affect the membrane embedding ofaIIb TM-CT and reduces integrin activation since tyrosineresidues tend to locate at the membrane boundary (Killian and

von Heijne, 2000). A cluster of negatively charged residues at theaIIb CT MD region may disfavor the negatively charged cellmembrane and inhibit the membrane embedding of aIIb TM-CT,

while an interaction between the talin-head and the aIIb CT MDacidic residues as indicated by a molecular dynamic simulationstudy (Provasi et al., 2014) might facilitate the membraneembedding of aIIb TM-CT to activate integrin. In addition, the

capability of talin to activate integrin depends on its binding toboth b-integrin 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 a CT and talin might helptalin maintain a proper orientation for the favorable interactionwith b-integrin CT and cell membrane in order to stabilize the

active conformation of TM-CT (supplementary material Fig.S4C). Considering the significant diversity of the a-integrin CTMD region, it is tempting to propose that this diversity might

regulate integrin inside-out activation in an a-integrin-specificmanner as it has been suggested that the a-integrin CT MDregions determine the specificity of integrin 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 a-integrin CT is through its interacting proteins. Interestingly,

most a-integrin-CT-binding proteins identified so far interactwith the conserved membrane-proximal GFFKR motif andfunction as inhibitors of integrin activation (Pouwels et al.,

2012; Bouvard et al., 2013; Morse et al., 2014), but the

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. Effect of a-integrin CT truncation on kindlin-induced integrinconformational change. (A,B) Binding of LIBS mAb 319.4 and 370.3 toaIIbb3 induced by the overexpression of EGFP–TH plus mCherry (mC)-tagged kindlin-2 (mC–K2) or kindlin-3 (mC–K3). (C,D) Binding of LIBS mAbKIM127 and M24 to aLb2 induced by the overexpression of EGFP–TH plusmC–K2 or mC–K3. 293FT cells were co-transfected with the integrinconstructs (shown under B and D) and EGFP–TH plus mC, mC–K2, or mC–K3. The integrin, EGFP, and mCherry triple-positive cells were analyzed byflow cytometry. The binding of LIBS mAb was presented as the MFInormalized to integrin expression. Data are presented as mean6s.e.m.(n§3). *P,0.05; **P,0.01; ***P,0.001; ns, not significant (unpaired two-tailed t-tests were used for the comparison with mC control group).

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mechanism of their negative regulation is not well-defined. A

Rap1 effector, RapL, has been shown to activate aLb2 integrinthrough binding to the aL CT MD sequence proximal to theGFFKR motif (Katagiri et al., 2003). It is not known how RapL

cooperates with talin and/or kindlin to activate aLb2 integrin. Therequirement of a-integrin CT MD region for integrin activationsuggests that as-yet undiscovered regulatory proteins that interact

with a-integrin CT MD region are involved in integrin activation,probably in an integrin-specific manner.

It has been shown that deletion of the aIIb CT MD region didnot affect aIIbb3-mediated cell adhesion and spreading, but led to

aberrant recruitment of aIIbb3 into focal adhesions formed byother integrins (Ylanne et al., 1993). The underlined mechanismis not known. The same phenomenon was observed with a2

integrin (Kawaguchi et al., 1994). However, deletion of the a CTMD region of a1, a2, a4, aV or a6 integrin diminished celladhesion (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 that there are different regulatory

roles of a CT MD regions among different integrins.Our data on the b3-K716A mutation have important implications

for the current model of integrin inside-out activation. It has beensuggested that the b3-K716 residue helps to maintain a proper tilt

angle for the b3 TM domain in the resting state by placing its basicside chain near negatively charged phospholipid head groups (Kimet al., 2011a). It has been proposed that the b3-K716A mutation or

talin binding to b3 CT induces integrin activation by changing thetilt angle of b3 TM domain to disturb the aIIbb3 TM-CT interaction.

It has also been shown that the tilt angle change of b3 TM domain

can occur upon adding the talin-1 head to the isolated b3 TM-CTpeptide embedded in lipid nanodiscs in the absence of aIIb TM-CTpeptide (Kim et al., 2012). We found that complete deletion of aIIb

CT MD region greatly enhanced the activation of b3-K716A. If thetalin-1 head could induce integrin activation by changing the tiltangle of the b3 TM domain as b3-K716A does, which seems to be

independent of aIIb CT according to the data by Kim et al. (Kimet al., 2012) we would expect to see an enhanced activation mediatedby the talin-1 head in the absence of the aIIb CT MD region.However, an opposite result was obtained. The talin-1 head failed to

activate integrin when the a-integrin CT MD region was completelydeleted. By using a cysteine-scanning accessibility method withintact integrin on the cell surface, another group has suggested that

b3-K716 is embedded in the lipid bilayer both in the inactive andactive conformation. They detected conformational change of theaIIb TM-CT but not the b3 TM-CT domain in activating integrin

mutants (Kurtz et al., 2012). Our structural study revealed thatinteractions between the b3-K716 side chain and the aIIb F992 andK994 backbone carbonyl oxygens are important in maintaining the

resting integrin state (Zhu et al., 2009). These interactions wereobserved in a molecular dynamic simulation study starting with theNMR structure of aIIbb3 TM-CT determined in lipid bicelles (Provasiet al., 2014). The direct interaction between b3-K716 and aIIb CT was

also observed in a NMR study (Metcalf et al., 2010). Clearly, furtherinvestigations are required to reconcile these discrepancies.

It has been proposed that the conserved aIIb-R995 and b3-D723

form a salt bridge that restrains aIIbb3 in the resting state (Hugheset al., 1996). One of the current models suggests that talin-1 head

A B

α

β

α-β

IIb/ 3 W

T

IIb-G

972C

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IIb-G

972C

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700C

IIb-R

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700C

IIb-G

972C

/ 3-V70

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972C

-R99

5Tr/

3-V70

0C

IIb-G

972C

-V99

0Tr/

3-V70

0C0

50

100

150

200

250

Ca/MnCa/Mg

PAC

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indi

ng (n

omal

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MFI

)

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T

IIb-G

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IIb-G

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3-V70

0C

IIb-G

972C

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0Tr/

3-V70

0C0

10

20

30

40 P<0.01C

ross

linki

ng e

ffici

ency

(%)

P<0.01

C

EG

FPE

GFP

-TH

EG

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αIIb

/β3 W

TαI

Ib-R

995T

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IP: αIIbβ3WB: anti-EGFP

WB: anti-β3

Total cell lysateWB: anti-EGFP

Fig. 8. Deletion of aIIb CT MD region reduced aIIb and b3 transmembrane domain association but did not affect the binding of the talin-1 head.(A) Disulfide crosslinking of indicated aIIbb3 constructs expressed in 293FT cells. Immunoprecipitates of [35S]-labeled integrins were subjected to non-reducingSDS-PAGE and autoradiography (upper panel). The quantification of crosslinking efficiency is shown in the lower panel. Data are presented asmean6s.e.m. (n§3). (B) PAC-1 binding of 293FTcells transfected with indicated aIIbb3 constructs in the buffer containing 1 mM Ca2+ and 1 mM Mg2+ (Ca/Mg) or0.2 mM Ca2+ plus 2 mM Mn2+ (Ca/Mn). (C) Co-immunoprecipitation of the talin-1 head with aIIbb3 integrin. 293FT cells were co-transfected with aIIbb3

constructs and EGFP or EGFP–TH. Integrins were immunoprecipitated (IP) with mAb 10E5 and subjected to western blotting (WB) with anti-EGFP and anti-b3 antibodies.

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binding to the b3 CT breaks the putative salt-bridge to induceintegrin activation (Anthis et al., 2009; Anthis and Campbell,

2011). Despite the very disruptive effect of aIIb-R995 and b3-D723 mutations on aIIbb3 TM dimerization observed usingisolated TM-CT peptides (Kim et al., 2009; Lau et al., 2009),their mutations only moderately activated full-length integrins

(Peyruchaud et al., 1998; Ma et al., 2006; Ghevaert et al., 2008;Zhu et al., 2009). Remarkably, we found that the aIIb-K993Tr andaIIb-K994Tr mutation, in which the aIIb-R995 was deleted,

abolished rather than synergized integrin activation induced bythe b3-D723 mutations and/or by the talin-1 head. In sharpcontrast, it was only in the presence of aIIb CT MD region that the

aIIb-R995 or b3-D723 mutations greatly synergized aIIbb3

activation and the conformational change induced by the talin-1head. Thus, although aIIb-R995 and b3-D723 are involved in integrin

activation, they might not be central in structurally maintainingintegrin in the resting state. Mutagenesis data has suggested that theextracellular domains are also involved in stabilizing the aIIbb3 TMdimer as well as their membrane embedding (Lau et al., 2009; Kurtz

et al., 2012). Caution should be taken when interpreting dataobtained using isolated TM-CT peptides.

Natural mutations of aIIb-R995 and b3-D723 (aIIb-R995Q or

aIIb-R995W and b3-D723H) have been identified in patients withthrombocytopenia (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 heresuggest that aIIbb3 integrin with aIIb-R995 or b3-D723 mutationsmight be more responsive to agonist stimulation leading to more

platelet aggregation than in wild type, which mightsimultaneously cause a reduction in the amount of platelets inblood. An equivalent Arg to Ala mutation of a4 integrin CT hasbeen found to aberrantly activate a4 integrin in genetic knock-in

mice (Imai et al., 2008). We also found that the equivalent b2-D709A mutation renders aLb2 integrin hyper-responsive to talin-1-head-induced activation. Interestingly, an equivalent Asp to Ala

mutation in b1 integrin CT did not result in obvious defect in vivo

(Czuchra et al., 2006), despite the fact that the b1 subunit formsheterodimers with 12 a-integrin subunits. Thus, the conserved a-

Arg and b-Asp of the integrin CT might play different roles inregulating integrin activation among different integrins.

Among the caveats of our study is that the effect of aIIb CT MDmutations on talin-1-head-mediated integrin activation was much

more dramatic with the b3-D723A mutant compared with WT b3. Itshould be noted that overexpression of talin-1 head onlymoderately activates WT integrin. Increasing the DNA amount

for transfection did not further increase integrin activation (data notshown). It is possible that overexpression of talin-1 head alonemight not be sufficient to induce maximal integrin activation given

that the overexpressed talin-1 head is not specifically recruited tothe integrin CT and only a small population of integrins encounterthe talin-1 head probably by chance. In the physiological situation,

the active full-length talin might apply traction force to its bound b-integrin CT through the coupled actin cytoskeleton (Zhu et al.,2008; Schurpf and Springer, 2011), and thus exert a moredisruptive effect on the integrin TM-CT interaction than that

mediated by the talin head alone. The b3-D723A mutation or otheractivating mutations like aIIb-R995A and b3-G135A mightfacilitate talin-head-induced integrin activation by decreasing the

energy barrier. The b3-D723A mutation significantly increasestalin-1-head-induced integrin activation by more than 20-foldcompared with WT. Moreover, PMA stimulates a significant

increase in soluble ligand binding to the aIIb–b3-D723A mutant but

not to the WT in K562 cells in suspension. Consistent with thesoluble ligand-binding assay, the b3-D723A or aIIb-R995A

mutation significantly enhanced talin-1-head-induced integrinconformational change. Thus, the combination of b3-D723A andthe aIIb CT MD mutations greatly improves the sensitivity of ourassay. Similarly, the combination of the b3 activating mutations

and the aIIb CT MD truncations enabled us to reveal thecontribution of aIIb CT MD region to maintaining the resting state.

Talin-1-head-induced integrin extension, but not headpiece

opening, has been directly visualized by EM using the purifiedintact aIIbb3 embedded in the lipid nanodiscs (Ye et al., 2010). Wehave recently shown that the talin-1-head-induced integrin

conformational change needs to be propagated to the ligand-binding site, probably through headpiece opening, in order toactivate integrin (Zhang et al., 2013). In this study, by using the

conformation-dependent mAbs that report integrin extension(319.4 for b3 and 370.3 for aIIb; KIM127 for b2) and headpieceopening (M24 for b2), we further demonstrated that the talin-1 headinduced extension and headpiece opening of integrin. In particular,

we detected enhanced integrin conformation change (extension andheadpiece opening) when the talin-1 head was co-expressed withkindlins. Kindlin-2 or -3 exerted more effect on M24 binding than

did KIM127 binding to aLb2. This is consistent with a recent studyshowing that both talin-1 and kindlin-3 were required for inducingthe extended open headpiece conformation of aLb2 (Lefort et al.,

2012). Remarkably, the talin-1-head- and kindlin-induced integrinextension and headpiece opening require the presence of an a-integrin CT MD region. However, because how kindlins induce

integrin activation remains unknown, the requirement of an a-integrin CT MD region for kindlin-mediated integrin activation canonly be interpreted as a secondary effect due to the loss ofeffectiveness of talin according to the current data.

We found that the talin-1-head- and kindlin-induced binding ofb3 LIBS mAb 319.4 is not completely abolished in the absence ofthe aIIb CT MD region. This is in contrast with the binding of aIIb

LIBS mAb 370.3. This indicates that the talin-1 head might exertsome extent of conformational change on the b3 subunit even inthe absence aIIb CT MD region, but that a fully active integrin

conformation induced by talin and kindlin requires theinvolvement of the aIIb CT MD region. In summary, our studyprovides new insights into integrin inside-out activation andsuggests that further structural studies are required to understand

the precise mechanism by which the a-integrin CT MD region isinvolved in talin- and kindlin-mediated integrin activation.

MATERIALS AND METHODSDNA constructsDNA constructs of human aIIbb3, aVb3, aLb2, and the EGFP-tagged

mouse talin-1 head (EGFP–TH) were as described previously (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 ligandsPAC-1 (BD Biosciences) is a ligand-mimetic mAb (IgM) specific for

activated aIIbb3 integrin (Shattil et al., 1985). AP3 is a conformation-

independent anti-b3 mAb (Kouns et al., 1991). 319.4 is a LIBS mAb that

binds to the b3 I-EGF domains (Zhang et al., 2013). 370.3 is a LIBS mAb

that binds to the aIIb calf-1 domain (Zhang et al., 2013). PE-labeled TS2/

4 (BioLegend) is a non-functional anti-aL mAb (Sanchez-Madrid et al.,

1982). KIM127 (which binds to I-EGF-2 domain) and mAb 24 (M24,

which binds to bI domain) are anti-b2 LIBS mAbs (Dransfield and Hogg,

1989; Robinson et al., 1992; Nishida et al., 2006; Chen et al., 2010).

RESEARCH ARTICLE Journal of Cell Science (2015) 128, 1718–1731 doi:10.1242/jcs.160663

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Human Fn9-10 was as described previously (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 with293FT transfectantsPAC-1 binding of 293FT (Life Technologies) cells transfected with

aIIbb3 only were as described previously (Zhang et al., 2013). For the

talin-1-head-induced ligand binding assay, 293FT cells were 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 5 mM EDTA or 1 mM Ca2+ and 1 mM Mg2+ (Ca/Mg)

at 25 C for 30 min with 5 mg/ml PAC-1 for aIIbb3, 20 mg/ml each of

ICAM-1-Fc and biotin-labeled mouse anti-human IgG1 Fc for aLb2, or

50 mg/ml Alexa-Fluor-647-labeled Fn9-10 or Fn for aVb3. Cells were

then washed and incubated in Ca/Mg on ice with the detecting reagents:

10 mg/ml each of biotinylated AP3, PE-labeled streptavidin and Alexa-

Fluor-647-labeled goat anti-mouse IgM for aIIbb3, biotinylated AP3 and

PE-labeled streptavidin for aVb3, PE-labeled TS2/4, and Alexa Fluor

647-labeled streptavidin for aLb2. 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 the normalized MFI, that is ligand MFI (after

subtracting the ligand the MFI in EDTA) as a percentage of integrin MFI.

PMA-induced integrin ligand binding in K562 cellsHuman K562 cells were transfected with aIIbb3 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 the MFI normalized by integrin expression.

LIBS epitope exposureTalin-1-head- and kindlin-induced LIBS epitope exposure was as

described previously (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 aIIbb3, PE-labeled TS2/4 and Alexa-Fluor-647-

labeled streptavidin for aLb2. Integrin and EGFP double-, or integrin,

EGFP and mCherry triple-positive cells were analyzed for calculating the

MFI. LIBS mAb binding is presented as MFI normalized to the integrin

expression, that is the LIBS mAb MFI (after subtracting the background

MFI) as a percentage of the integrin MFI.

Disulfide crosslinking and immunoprecipitationMetabolic labeling with [35S]cysteine/methionine and disulfide

crosslinking using intact cells of 293FT transfectants were as described

previously (Zhu et al., 2009). Formation of disulfide bonds was induced

by treating the cells with CuSO4 and o-phenanthroline on ice. Integrins

were immunoprecipitated with anti-aIIb mAb 10E5 from the cell lysate

and subjected to nonreducing SDS-PAGE and autoradiography. For the

talin-1-head-binding assay, aIIbb3 integrins were immunoprecipitated

with mAb 10E5 from cells co-transfected with EGFP or EGFP–TH.

Binding of the talin-1 head was detected by western blotting with rabbit

anti-EGFP polyclonal antibody (Origene). b3 integrin was detected with

rabbit anti-b3 H-96 (Santa Cruz Biotechnology).

AcknowledgementsWe thank Drs Daniel Bougie, Richard Aster, Barry Coller, Chafen Lu and TimothySpringer for providing antibodies; David Calderwood for providing the DNA

construct of EGFP-tagged mouse talin-1-head domain; Peter Newman for criticalreading of the manuscript.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsJ.L., Z.W., A.M.M.T. and J.Z. performed the experiments and analyzed data.Y-Q.M. made the kindlin constructs. J.Z. designed the study, prepared the figuresand wrote the manuscript.

FundingThis work was supported by a Scientist Development Grant from the AmericanHeart Association [grant number 12SDG12070059 to J.Z.]; and an ASH ScholarAward for junior faculty from the American Society of Hematology [to J.Z.].

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.160663/-/DC1

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