The membrane-distal regions of integrin α cytoplasmic domains contribute differently to integrin inside-out activation

Thinn, Aye Myat Myat; Wang, Zhengli; Zhu, Jieqing

doi:10.1038/s41598-018-23444-w

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Published: 22 March 2018

The membrane-distal regions of integrin α cytoplasmic domains contribute differently to integrin inside-out activation

Aye Myat Myat Thinn^1,2,
Zhengli Wang¹ &
Jieqing Zhu^1,2

Scientific Reports volume 8, Article number: 5067 (2018) Cite this article

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Abstract

Functioning as signal receivers and transmitters, the integrin α/β cytoplasmic tails (CT) are pivotal in integrin activation and signaling. 18 α integrin subunits share a conserved membrane-proximal region but have a highly diverse membrane-distal (MD) region at their CTs. Recent studies demonstrated that the presence of α CTMD region is essential for talin-induced integrin inside-out activation. However, it remains unknown whether the non-conserved α CTMD regions differently regulate the inside-out activation of integrin. Using α_IIbβ₃, α_Lβ₂, and α₅β₁ as model integrins and by replacing their α CTMD regions with those of α subunits that pair with β₃, β₂, and β₁ subunits, we analyzed the function of CTMD regions of 17 α subunits in talin-mediated integrin activation. We found that the α CTMD regions play two roles on integrin, which are activation-supportive and activation-regulatory. The regulatory but not the supportive function depends on the sequence identity of α CTMD region. A membrane-proximal tyrosine residue present in the CTMD regions of a subset of α integrins was identified to negatively regulate integrin inside-out activation. Our study provides a useful resource for investigating the function of α integrin CTMD regions.

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Introduction

Integrins are cell adhesion receptors composed of α and β subunits, each containing a large extracellular domain, a single transmembrane (TM) domain and usually a short cytoplasmic tail (CT). In human, the combinations of 18 α and 8 β subunits form 24 integrin heterodimers that play essential roles in numerous biological activities such as hemostasis, immune responses, and development¹. Aberrant activation of integrin is associated with many pathological conditions including thrombosis, inflammatory diseases, and tumor-driven cell growth, metastasis, and angiogenesis^2,3,4. Therefore, tight regulation of integrin activation is important for normal integrin function. A unique feature of integrins is that they can transmit signals bidirectionally across the cell membrane, so called inside-out and outside-in signaling^5,6. In the inside-out direction, the activating signals impinge on the integrin CT to transform integrin from a resting to an active state by inducing large-scale conformational changes of the extracellular domain⁷. In the outside-in direction, ligand binding to the extracellular domain of active integrin also induces long-range conformational changes that are transmitted to the CT to provoke the association and activation of the kinases and adaptor molecules in the cytosol^8,9. As such, acting as both the receiver and the transmitter of signals, integrin CT is pivotal in integrin activation and signaling.

Largely based on the studies of β₃, β₂, and β₁ integrins, great progress has been made in understanding how the β integrin CT contributes to integrin activation^6,10. Most of the β integrin CTs contain the conserved binding motifs for the common integrin activators, talin and kindlin (Fig. 1A). Structural and functional studies suggested that binding of talin and kindlin to β integrin CT induces integrin activation by disrupting the α-β interactions at the TM and the CT domains¹⁰, which in turn leads to conformational changes of the extracellular domain⁵. A functional role of the α integrin CT in integrin activation had been focused on the highly conserved Gly-Phe-Phe-Lys-Arg (GFFKR) motif at the membrane proximal (MP) region (Fig. 1A), which helps maintain the α-β CT associations^11,12. Notably, the membrane-distal (MD) regions of the α integrin CT differ significantly in both amino acid sequence and length (Fig. 1A) and their roles in integrin activation and signaling remain ill defined. Accordingly, the current available structures of α integrin CTs also show conformational diversity at the MD regions (Fig. 1B). Moreover, even the same α_IIb integrin CTMD region shows different conformations among the reported structures (Fig. 1B). Recent studies from our and other groups demonstrated that the presence of α integrin CTMD region is essential in talin/kindlin induced integrin inside-out activation^13,14. In addition, our study also showed that the length and amino acids of the α integrin CTMD region might be important in regulating integrin inside-out activation^13,14. Given that one β integrin subunit such as β₃, β₂, or β₁ usually heterodimerizes with more than one α subunits (Fig. 1A), an intriguing question is whether the diverse α CTMD regions contribute differently to integrin activation and signaling, which may determine the specific and diverse integrin functions.

In this study, using the platelet-specific α_IIbβ₃, leukocyte-specific α_Lβ₂, and the ubiquitously expressed α₅β₁ as model integrins, we attempted to compare the effect of 17 out of 18 total α integrin CTMD regions on integrin inside-out activation. Our study revealed that the α CTMD regions contribute differently to talin head (TH)-induced integrin activation, evidenced by different levels of ligand binding and conformational changes. This was at least in part determined by the presence of specific residues in the α CTMD regions. Potential mechanisms by which the α CTMD regions participate in integrin activation were discussed.

Results

Design and generation of chimeric α integrins to examine the contributions of the diverse α CTMD regions in integrin inside-out activation

Studies from our group and others have demonstrated the requirement of the presence of α CTMD region in integrin inside-out activation¹³. However, it remains elusive whether and/or how the diverse CTMD regions regulate integrin activation. It has been shown that the α CTMD regions also contribute to maintaining integrin in the resting state possibly by interacting with the β CT^13,14,15. Therefore, a simple mutagenesis or replacement of the α CTMD region with an irrelevant sequence may result in complicated and uninterpretable results. To address this question, we took the advantage that one β subunit usually pairs with more than one α subunits. For example, β₃, β₂, and β₁ subunit can heterodimerize with 2, 4, and 12 different α subunits, respectively (Fig. 1A). Furthermore, in vitro activation assays of α_IIbβ₃, α_Lβ₂, and α₅β₁ have been very well established^13,16,17. Therefore, we used α_IIb, α_L, and α₅ as model α integrins, in which their CTMD regions were replaced by those of α subunits that can pair with β₃, β₂, and β₁ subunits, respectively (Fig. 2A). In such way, we generated the α chimeras that are denoted as α_IIb-α_V, α_L-α_X, α_L-α_D, α_L-α_M, α₅-α_V, α₅-α₁, α₅-α₂, α₅-α₃, α₅-α₄, α₅-α₆, α₅-α₇, α₅-α₈, α₅-α₉, α₅-α₁₀ and α₅-α_11. When these α_IIb, α_L, and α₅ chimeras are co-expressed with β₃, β₂, and β₁ subunits, respectively, the native associations of α and β CT domains are maintained. In addition, comparisons can be made among the α chimeras that share the same β subunit. Any differences seen in the integrin inside-out activation assay would attribute to the diverse CTMD regions. These 15 chimeras together with the wild type (WT) α_IIb, α_L, and α₅ subunits allow our study to cover the CTMD regions of 17 out of 18 human α integrins.

Integrin α_IIb-chimeras bearing various α CTMD regions respond differently to talin head (TH) stimulation

The β₃ integrin’s partners α_IIb and α_V subunits share 6 consensus residues at their CTMD regions, but the α_V CTMD region is about two times longer than that of the α_IIb (Fig. 1A). We have shown that complete deletion of the CTMD regions of α_IIb and α_V subunits abolished TH-induced α_IIbβ₃ and α_Vβ₃ activation¹³. Here, we asked whether the CTMD regions of α_IIb and α_V subunits could be exchangeable and whether they could exert different effect on β₃ integrin inside-out activation. The ligand-mimetic mAb PAC-1 was used to access the α_IIbβ₃ activation induced by the overexpression of GFP-TH in the presence of the β₃ cytoplasmic mutation β₃-D723A, which has been shown to greatly enhance the responsiveness of TH-induced α_IIbβ₃ activation¹³. When the CTMD region of α_IIb was replaced by the α_V CTMD region, the α_IIb-α_V/β₃-D723A chimeric integrin still remained responsive to GFP-TH-induced activation (Fig. 2B). However, the activation of α_IIb-α_V was significantly decreased compared with the α_IIb-WT (Fig. 2B). The reduced activation of α_IIb-α_V chimera was not due to the differences in GFP-TH expression since the lower activity of α_IIb-α_V compared with α_IIb-WT was consistently seen at various levels of GFP-TH expression (Fig. 2C). As a comparison, we replaced the α_IIb CTMD region with those of α₁ and α_L integrins that do not heterodimerize with β₃ integrin. Remarkably, the presence of both the α₁ and α_L CTMD regions significantly enhanced the GFP-TH-induced activation of α_IIbβ₃ integrin (Fig. 2B). The increased activation of α_IIb-α₁ and α_IIb-α_L was also obvious in the absence of TH expression (Fig. 2B), indicating that the mismatch of the α_IIb CTMD mutant with the β₃ CT renders α_IIbβ₃ more active than the wild type. This may be due to the destabilization of α_IIb-β₃ CT interaction, being consistent with the previous observations that the α CTMD regions contribute to maintaining integrin in the resting state^13,14. The expression level of α_IIb-α_L was decreased possibly due to the high integrin activity (Fig. 2B), which is commonly seen among the active integrin mutants¹³. We next asked whether the replacement of α_IIb CTMD region affects the TH-induced conformational change of α_IIbβ₃ integrin. The active conformation-specific mAb 370.3 was used to report the extension of α_IIb integrin. Consistent with the PAC-1 binding assay, the α_IIb-α_V chimera showed decreased while the α_IIb-α₁ and α_IIb-α_L chimeras showed increased binding of mAb 370.3 either in the presence or absence of TH expression (Fig. 2D). This data demonstrates that the CTMD regions of α_IIb and α_V are not completely interchangeable. They can exert different effect on β₃ integrin activation at least in part through regulating the conformational change of integrin.

Replacing the α_L CTMD region with that of α_X, α_D or α_M subunit reduced TH-mediated α_Lβ₂ integrin activation

Integrin β₂ subunit forms heterodimers with α_L, α_M, α_X, and α_D subunits. α_L has the longest while α_M has the shortest CTMD sequence among the four subunits (Fig. 1A). The NMR structures of α_L, α_M and α_X CTs show great structural heterogeneities at their MD regions (Fig. 1B). In addition, we found that deletion of the α_L CTMD region abolished, while truncation of the α_L CTMD region dampened TH-induced α_Lβ₂ activation¹³, arguing a potential regulatory role of the CTMD region. Similar to the α_IIb chimeras, we made the α_L chimeras by replacing the α_L CTMD region with that of α_M, α_X or α_D subunit. Surprisingly, in the TH-induced ICAM-1 binding assay, all the α_L-α_M, α_L-α_X, and α_L-α_D chimeras showed significantly reduced ICAM-1 binding compared with the WT α_L when co-expressed with the β₂-D709A mutation (Fig. 3A,B). The β₂-D709A mutation was used to increase the sensitivity of our assay by greatly enhancing TH-induced α_Lβ₂ activation as shown in our previous study¹³. The expression levels of integrin and GFP-TH were comparable among the α_L transfections (Fig. 3B). Furthermore, as shown for the α_L-α_M chimera in the GFP-TH titration assay, the reduced ICAM-1 binding was obvious when the GFP-TH expression reached a certain level and became independent of the expression level of GFP-TH (Fig. 3C). Similar results were obtained with the α_L-α_D chimera (data not shown). In addition, although all the α_L constructs exhibited increased TH-induced ICAM-1 binding with the increase of ICAM-1 concentration, all the α_L chimeras consistently showed reduced ICAM-1 binding at all the ICAM-1 concentrations tested (Fig. 3D). These data demonstrated that the reduced activation of α_L chimeras was due to the replacement of CTMD region and might attribute to a common feature of α_M, α_X, and α_D CTMD regions.

A conserved tyrosine residue in the CTMD regions of α_M, α_X, and α_D subunits negatively regulates the inside-out activation of β₂ integrin

Sequence alignment revealed a conserved tyrosine residue at the second position of all the CTMD regions of α_M, α_X, and α_D subunits (Fig. 1A). We asked whether this tyrosine contributes to the reduced activation of α_L chimeras. We first tested the tyrosine mutation on α_L-α_X chimera with the co-expression of β₂-D709A mutant. Compared with the α_L-α_X, the tyrosine to phenylalanine mutation at the α_X CTMD region, α_L-α_X-Y1117F, significantly increased the TH-induced ICAM-1 binding and restored it to the WT α_L level (Fig. 4A). Similarly, the α_L-α_D-Y1115F mutation also increased the TH-induced ICAM-1 binding although it was not to the WT level and not statistically significant (Fig. 4B). We further tested the tyrosine mutation on the α_L-α_M chimera. Mutating the tyrosine in the α_M CTMD sequence to phenylalanine (Y1121F), glutamic acid (Y1121E), and alanine (Y1121A) all significantly increased the TH-induced ICAM-1 binding of α_L-α_M chimera (Fig. 4C). Interestingly, the Y1121A mutation exerted a higher level of activation than the Y1121F and Y1121E mutations (Fig. 4C), indicating that both the bulky side chain and the hydroxyl group of tyrosine are important for its negative effect on integrin activation. Sequence alignment also shows a conserved methionine at the fifth position of all the α_M, α_X, and α_D regions (Fig. 1A). We tested whether this methionine residue contributes to the negative effect of CTMD region using the α_L-α_M chimera. We found that the α_L-α_M-M1124A mutation did not significantly increase TH-induced ICAM-1 binding (Fig. 4C). The expression levels of integrin and GFP-TH were comparable among the transfections within the same experimental group (Fig. 4A–C). These data clearly demonstrate that the negative effects of the CTMD regions of α_M, α_X, and α_D subunits on TH-induced β₂ integrin activation at least in part attribute to the presence of a conserved tyrosine.

The position of the tyrosine mutation at the CTMD region is critical for its negative regulation of the α_Lβ₂ integrin inside-out activation

Interestingly, there is only one tyrosine in the CTMD region of α_M, α_X, or α_D subunit and no tyrosine in the α_L CTMD region (Fig. 1A). To further demonstrate the important regulatory role of a tyrosine residue in the CTMD region, we performed a tyrosine scanning mutagenesis for the α_L CTMD region. A tyrosine mutation was placed at the 1^st, 2^nd, 3^rd, 4^th, or 6^th position of the CTMD region after the conserved GFFKR motif (Fig. 5A). As shown in the TH-induced ICAM-1 binding assay, the tyrosine mutation at the 1^st position, α_L-N1095Y, significantly enhanced ICAM-1 binding (Fig. 5B). This is probably due to the disturbance of α-β association at the GFFKR motif, which was known to be important in maintaining integrin in the resting state¹¹. In contrast, the tyrosine mutation at the 2^nd position, α_L-L1096Y, which is equivalent to the native tyrosine of the α_X, α_D and α_M CTMD regions, significantly reduced TH-induced ICAM-1 binding (Fig. 5B). This is consistent with the above data. Remarkably, the tyrosine mutations at the 3^rd and 4^th positions, α_L-K1097Y and α_L-E1098Y, also significantly reduced ICAM-1 binding (Fig. 5B). However, the tyrosine mutation at the 6^th position, α_L-M1100Y, had no significant effect on ICAM-1 binding (Fig. 5B). Among the single tyrosine mutations, the tyrosine mutation at the 3^rd position, α_L-K1097Y, had the most negative effect (Fig. 5B). We next asked whether the presence of multiple tyrosine mutations at the CTMD region has a synergistic effect on its negative regulation of integrin activation. A triple tyrosine repeat was introduced into the 2^nd to 4^th positions of α_L CTMD region (Fig. 5A). The α_L-YYY mutation significantly reduced ICAM-1 binding compared with the wild type, but had no significant difference with the single α_L-K1097Y mutation (Fig. 5B). Furthermore, the negative effect of the tyrosine mutation did not depend on the ICAM-1 concentration (Fig. 5C). These data demonstrate that the presence and the position but not the number of tyrosine mutations at the CTMD region are important in the negative regulation of α_Lβ₂ activation.

The α_L-K1097Y mutation coincidently formed an Yxxɸ motif (x is any amino acid, ɸ is hydrophobic residue), which has been found recently in a subset of α integrins to play a role in the regulation of integrin endocytosis¹⁸. It has been shown that the presence but not the position of the Yxxɸ motif is important for its function in integrin endocytosis¹⁸. To test whether the negative effect of α_L-K1097Y mutation was due to the formation of Yxxɸ motif, we generated another tyrosine mutation, α_L-G1103Y, which formed an Yxxɸ motif (Fig. 5A). In contrast to the α_L-K1097Y mutation, the α_L-G1103Y mutation showed no difference with the α_L-WT in ICAM-1 binding (Fig. 5D), suggesting that the negative effect of α_L-K1097Y mutation is not due to the presence of an Yxxɸ motif.

In our TH-induced ICAM-1 binding assay, all the α_L constructs were co-expressed with the β₂-D709A mutation. To demonstrate that the differences we observed among the α_L mutations in the integrin activation assay are not due to the presence of the β₂-D709A mutation, we did the same assay in the presence of β₂-WT for several representative tyrosine mutations. The results show that all the selected α_L tyrosine mutations reduced TH-induced ICAM-1 binding although not as significant as in the presence of the β₂-D709A mutation (Fig. 5E).

The α CTMD regions contribute to integrin activation by regulating the conformational change of integrin

TH-induced integrin activation is coupled with the large-scale conformational changes of integrin extracellular domain^5,10. We used two mAbs, KIM127 and m24, which report β₂ integrin extension and headpiece opening, respectively¹⁹, to test whether the mutagenesis of α_L CTMD region affect TH-induced α_Lβ₂ conformational change. Consistent with the ICAM-1 binding assay, the α_L-α_M, α_L-α_X, and α_L-α_D chimeras all significantly reduced TH-induced binding of both m24 and KIM127 mAbs when compared with the α_L-WT (Fig. 6A). Similarly, the α_L tyrosine mutations, α_L-L1096Y and α_L-YYY, also decreased the TH-induced m24 or KIM127 binding to α_Lβ₂ (Fig. 6B). These data demonstrate that the α CTMD region contribute to integrin inside-out activation through regulating the large-scale conformational changes.

Having found that introducing a tyrosine residue into the specific position of α_L CTMD region negatively regulates α_Lβ₂ ligand binding and conformational change, the next question is whether the native tyrosine residue present in the α_M, α_X, or α_D CTMD region plays a role in regulating the activation of these integrins. To answer this question, we performed the TH-induced activation assay for α_Mβ₂ integrin by detecting the β₂ integrin headpiece opening using m24. Consistent with the α_Lβ₂, the presence of β₂-D709A mutation greatly enhanced TH-induced binding of m24 to α_Mβ₂ (Fig. 6C). When replacing the conserved tyrosine at the 2^nd position of α_M CTMD region (Y1121) with either leucine or alanine, the TH-induced binding of m24 was further increased significantly in the presence of β₂-D709A (Fig. 6C). The same effect was found when the α_M-Y1121L or α_M-Y1121A was co-expressed with the β₂-WT (Fig. 6D). Interestingly, the enhanced activation by α_M-Y1121A is more obvious than α_M-Y1121L when they were co-expressed with β₂-WT (Fig. 6D), again suggesting that the bulky side chain of tyrosine is important for the negative effect on integrin activation. The expression level of integrin or GFP-TH is comparable among the transfections (Fig. 6C,D). These data, in addition to the α_L tyrosine mutations that exerted the opposite effect on α_Lβ₂ integrin activation, clearly demonstrate that a specific tyrosine residue present in a subset of α integrin CTMD regions negatively regulates β₂ integrin inside-out activation.

Mutagenesis of the α_IIb and α_L CTMD regions does not affect the TH binding to the integrin β₃ and β₂ CTs

Structural and functional studies have demonstrated that TH binds to the integrin β CT to induce integrin activation¹⁰. To test whether the α CTMD region contributes to integrin activation by affecting the TH association with the β CT, we did co-immunoprecipitation assay for the GFP-TH and integrin β subunit co-expressed with the α CTMD mutants. For the α_IIbβ₃ transfectants, the cell lysates were precipitated with anti-GFP antibody and the associated β₃ integrins were detected by the anti-β₃ antibody. The β₃ WT or β₃-D723A was robustly detected in the anti-GFP pull-down only when the GFP-TH but not GFP was present, demonstrating the specific interaction between TH and β₃ subunit (Figs 7A and S1). The expression levels of GFP-TH and β₃ integrin were comparable among the transfectants according to the Western blot of whole cell lysate (Figs 7A and S1). No obvious differences for the TH-bound β₃-D723A were observed among the α_IIb WT and α_IIb chimeras, suggesting that swapping the α_IIb CTMD region with that of α_V, α₁, or α_L does not affect the association of TH with β₃ subunit.

We next performed the same assay for the α_L CTMD mutations. To simultaneously detect both the α_L and β₂ subunits in the anti-GFP pull-down assay, we did the cell surface biotinylation before lysing the cells for co-immunoprecipitation. The presence of α_L and β₂ bands was confirmed by anti-α_L pull-down using the anti-α_L specific mAb TS2/4. As shown in Fig. 7B, two bands that correspond to the α_L and β₂ subunits were readily detected in the anti-GFP pull-down only when both the α_Lβ₂ and GFP-TH were co-expressed. No α_L and β₂ bands were detected in the anti-GFP pull-down in the transfections of GFP-TH alone or α_Lβ₂ plus GFP (Figs 7B and S1). The expression levels of integrin and GFP-TH were comparable among the transfections detected by flow cytometry (data not shown). To compare the association of GFP-TH and the α_Lβ₂ constructs, we quantified the Western blots by normalizing the α_L and β₂ signals to the GFP-TH signals (Fig. 7C). Compared with the α_L WT, only the α_L-α_D chimera shows an obvious increase in the association of integrin and GFP-TH (Fig. 7C). This is in contrast to the decrease in TH-induced α_Lβ₂ activation as shown above. No significant differences were found among the α_L WT, α_L-α_X, α_L-α_M, and the α_L tyrosine mutations, α_L-L1096Y, α_L-K1097Y, and α_L-M1100Y (Fig. 7C). These data indicate that the regulatory function of α CTMD region on integrin activation should not be due to the effect on the TH and β CT association.

Integrin α₅-chimeras bearing various α CTMD regions respond differently to TH stimulation when paired with the same integrin β₁ subunit

Twelve integrin α subunits share the integrin β₁ subunit, making the largest β₁ integrin subfamily (Fig. 1A). The major fibronectin receptor, integrin α₅β₁, has been relatively well studied structurally and functionally. Like α_IIbβ₃ and α_Lβ₂, the α₅β₁ integrin can be activated by the overexpression of TH²⁰. We have demonstrated the important role of the CTMD regions of α_IIb, α_V, and α_L subunits in TH-induced integrin activation¹³. Here, we extended our study to α₅β₁ integrin and asked whether the α₅ CTMD region follows the same rule. Using the α₅β₁-deficient CHO-B2 cell line²¹, we found that complete deletion of the α₅ CTMD region abolished GFP-TH-induced binding of fibronectin fragment Fn9–10 to α₅β₁ integrin (Fig. 8A), demonstrating the requirement of α₅ CTMD region in α₅β₁ inside-out activation. The next question is whether the diverse α CTMD regions of the β₁ integrin family also differently regulate β₁ integrin activation. To answer this question, we compared the function of all the α CTMD regions of the β₁ integrin family in the context of α₅ subunit. Eleven α₅ chimeras were generated by replacing the α₅ CTMD region with that of α subunits as indicated in Figs 2A and 8B. The TH-induced fibronectin-binding assay for α₅β₁ was performed using the α₅β₁-knockout HEK293FT cells. The activating β₁-K732E mutation, located at the transmembrane domain²², was used to enhance the sensitivity of the assay. As shown in Fig. 8B, the β₁-K732E significantly increased TH-induced fibronectin binding compared with the β₁ WT. Among the α₅ chimeras, two groups were identified: one group has no but the other has significant effect on TH-induced fibronectin binding compared with the α₅ WT (Fig. 8B). Remarkably, all the α₅ chimeras, including α₅-α₄, α₅-α₉, α₅-α₃, and α₅-α₆, which have a tyrosine residue adjacent to the GFFKR motif (Fig. 1A), showed comparable fibronectin binding with the α₅ WT that also contains a tyrosine at the CTMD region (Figs 1A and 8B). In contrast, all the α₅ chimeras, including α₅-α₁, α₅-α₇, α₅-α₈, and α₅-α₁₀, α₅-α₁₁, and α₅-α_V, which lack the tyrosine residue adjacent to the GFFKR motif (Fig. 1A), rendered α₅β₁ more active than the WT (Fig. 8B). However, an exception is the α₅-α₂ chimera, which has an equivalent tyrosine at the α₂ CTMD region but significantly increased α₅β₁ activation (Figs 1A and 8B). The similar results were obtained when the selected α₅ chimeras were co-expressed with the β₁ WT in the TH-induced fibronectin binding assay (Fig. 8C). The α₅-α₂ and α₅-α₁₀ significantly increased α₅β₁ activation while the α₅-α₄ had no obvious effect (Fig. 8C). By contrast, the enhanced effect of α₅-α₇ was not detectable when the β₁ WT was used (Fig. 8C), indicating the low sensitivity of the assay. Finally, we correlated the TH-induced α₅β₁ activation with the large-scale conformational change of β₁ ectodomain using the active conformation dependent mAb 9EG7. The presence of β₁-K732E significantly enhanced the TH-induced 9EG7 binding. Consistent with the fibronectin binding assay, the α₅-α₂, α₅-α₇, and α₅-α₁₀ significantly increased 9EG7 binding, but the α₅-α₉ had no such effect compared with the α₅ WT (Fig. 8D). These data suggest that the α CTMD regions of the β₁ family could contribute differently to β₁ integrin inside-out activation. They may also follow the rule of the negative regulation by a tyrosine residue.

Discussion

Compared with the extensive structural and functional studies of the relatively conserved β integrin CT that serve as docking sites for many signaling molecules, little is known about the role of α integrin CT especially the non-conserved MD regions. Since many α integrin subunits share the same β subunit, it is tempting to speculate that on one side the α CTMD regions may be interchangeable; on the other side, they may provide the specificity for integrin function. One of the difficulties in studying the α integrin CTMD regions is the sequence diversity among 18 α subunits. Another challenge is the lack of well-established activation assays for many integrin members, which limits the functional studies of the CTMD regions for many α integrins. Our approach in the current study provides a useful tool to thoroughly examine the potential functions of the CTMD regions of almost all integrin α subunits. By putting the different α CTMD regions in the context of the α_IIb, α_L, or α₅ subunit, this approach made it possible to compare the function of different α CTMD regions.

The potential function of several individual α integrin CTMD regions in integrin activation had been indicated in previous studies more than 20 years ago. They showed that deletion of the α CTMD region diminished cell adhesion or migration mediated by α₁, α₂, α₄, α_V, and α₆ integrins^{23,24,25,26,27,28,29,30} and dampened PMA-induced activation of α_Lβ₂ integrin³¹. Direct evidence for a role of α CTMD region in integrin inside-out activation was provided by the observation that complete deletion of the CTMD region of α_IIb, α_V, or α_L integrin abolished talin and kindlin-mediated integrin ligand binding and conformational changes^13,14. It was suggested that the presence but not the sequence of specific residues was required for the α CTMD region to support talin-induced integrin activation¹⁴. In the current study, we found that replacing the α CTMD region of α_IIb, α_L, or α₅ integrin with those of other α integrins still maintained the capability of integrin inside-out activation mediated by the overexpression of TH, suggesting that the α CTMD region can be interchangeable for this common activation supportive function. However, we observed significant variations of the activation levels among the same α integrins carrying different CTMD regions, indicating that the α CTMD region also plays a regulatory role in integrin inside-out activation.

We found that replacing the α_IIb CTMD region with that of α_V integrin markedly reduced TH-induced activation of α_IIbβ₃. By contrast, the α₁ and α_L CTMD regions rendered α_IIbβ₃ more active than WT, consistent with the previous observation that replacing the α_IIb CT with those of α₂, α₅, α_6A, or α_6B that do not natively pair with β₃ subunit enhanced α_IIbβ₃ activation despite they share the same GFFKR motif ³². Thus, the native pair between the α CTMD region and β CT is important to maintain the resting state of integrin. Although the α_V CTMD region shares six consensus residues with α_IIb CTMD region, which is half of the length of α_IIb CTMD region (Fig. 1A), it exerts different effect on α_IIbβ₃ activation. A predicted β turn structure formed by the PPQEE motif of α_V CTMD region was suggested to regulate the conformation and ligand binding of α_Vβ₃²⁶. A similar motif PPLEE was also found in the α_IIb CTMD region (Fig. 1A). Peptides containing this motif of α_IIb or α_V CTMD region could inhibit the activation of α_IIbβ₃ or α_Vβ₃³³, indicating that it could not be the reason for the different regulation by the α_IIb and α_V CTMD regions. Although the structure of the α_IIb CT has been determined, it shows large conformational variations (Fig. 1B). It is not known if the structural flexibility of α_IIb CT is functionally relevant. The α_IIb CTMD region has a unique tandem acidic residue motif, EEDDEEGE, which is conserved among the α_IIb from different species and not seen in the α_V CTMD region (Fig. 1A and data not shown). These negatively charged residues might regulate the conformation of α_IIb CT through repulsive interactions with the acidic phospholipid head groups at the cytosolic face of cell membrane, or through ionic interactions with the positively charged residues at the membrane-proximal region of α_IIb CT as suggested by a NMR study³⁴. The membrane-permeable peptides containing the α_IIb CTMD region were shown to block α_IIbβ₃ activation in platelets^15,35, and to inhibit the association of talin with α_IIbβ₃ in thrombin-activated platelets³⁶. In addition, the TH domain contains several positively charged surface residues that have been shown to be important for its integrin activating function through interacting with the cell membrane^37,38,39. It is possible that the negatively charged α_IIb CTMD region may also affect the orientation of TH domain when TH encounters with β₃ CT, but a direct interaction between TH and α_IIb remains to be confirmed. Being critical in hemostasis and thrombosis, the activation of α_IIbβ₃ is strictly regulated in platelets⁴⁰. Our data demonstrated that the unique feature of α_IIb CTMD region renders α_IIbβ₃ more susceptible to the signals of inside-out activation, which is in line with the high extent of activation required for α_IIbβ₃ function.

The lymphocyte-specific α_Lβ₂ is another integrin whose activation is highly regulated by the inside-out signals^5,41. The α_L CT has the second longest MD region that folds into α-helical conformation as shown in an NMR structure (Fig. 1A,B). Our recent study showed that deletion of the α_L CTMD region completely abolished TH-induced ICAM-1 binding to α_Lβ₂ integrin¹³. Here we found that replacing the α_L CTMD region with that of α_X, α_D, or α_M all significantly reduced ICAM-1 binding mediated by TH. This was not determined by the differences in the length but by a common tyrosine residue seen in the α_X, α_D, and α_M CTMD regions. Remarkably, we found that the position of the tyrosine at the α CTMD region is important to exert the negative effect on integrin inside-out activation. The bulky side chain and the hydroxyl group of tyrosine are critical for this regulatory function. Interestingly, the same negative effect was observed when introducing the tyrosine mutations into the equivalent position of the α_IIb CTMD region¹³. One caveat of our study is that the negative effect of the tyrosine was found in the context of α_L integrin in which no native tyrosine is present in the CTMD region. However, the physiological relevance of our discovery was built on the same observations on the α_Mβ₂ integrin, in which mutating the native tyrosine residue greatly enhanced the inside-out activation of α_Mβ₂, suggesting that the tyrosine residue negatively regulates the activity of α_Mβ₂. The tyrosine residue is also conserved among the α_M, α_X, or α_D integrins from different species (data not shown). Indeed, in contrast to the α_Lβ₂ integrin that only binds selectively to ICAMs, the α_Mβ₂, α_Xβ₂, and α_Dβ₂ integrins all exhibit multiligand-binding properties^42,43,44. The negative regulation by a tyrosine residue in their α CTMD regions may exert a restraint to avoid hyperactivity (or to balance the activation) of these β₂ integrins, in accordance with their less-selective ligand binding functions.

Several studies have provided evidence demonstrating that the activation of α_Lβ₂ and α_Mβ₂ are differently regulated. Different chemokines and chemoattractants were shown to stimulate inside-out activation of α_Lβ₂ and α_Mβ₂, which was suggested to be mediated by distinct pathways via the α CTs^45,46. Given the β₂ subunit shares the common integrin activation pathway mediated by talin and kindlin, other signaling molecules may be involved in the different regulation of α_Lβ₂ and α_Mβ₂ probably through direct interaction with the α CT. An example is the Rap-1 interacting effector molecule RapL that specifically binds to the α_L CTMD region to support Rap-1-mediated α_Lβ₂ activation⁴⁷. It was known that the α_L-K1097 is involved in RapL binding⁴⁸. Our results show that the α_L-K1097Y mutation rendered α_Lβ₂ the least active among the tyrosine mutations tested. However, RapL is predominantly expressed in immune cells⁴⁹. It should not account for the decreased activation by the α_L-K1097Y mutation since integrin activation was measured in HEK293FT cells lacking RapL expression. A Ser phosphorylation was found in the CTMD regions of both α_L and α_M subunits^50,51. However, mutations of the Ser residue only blocked the conformational changes involved in α_Mβ₂ but not α_Lβ₂ activation, indicating different regulation by the α_M CT. Here, we identified a tyrosine residue at the α_M CTMD region that is also involved in the specific regulation of α_Mβ₂ inside-out activation.

It remains unknown whether the activation of β₁ integrin family members are all subjected to inside-out regulation. Recent structural studies demonstrated that the conformational activation of α₅β₁ integrin could be modulated by many components including the transmembrane and cytoplasmic domains^52,53,54,55. We found that similar to α_IIb, α_V, and α_L integrins the α₅ CTMD region is also required for TH-induced inside-out activation, rationalizing the use of α₅β₁ as a model integrin to study the function of α CTMD regions. Our studies on all the α CTMD regions of the β₁ subfamily suggest a potential regulatory function of these regions in β₁ integrin inside-out activation. Remarkably, a tyrosine residue at the α CTMD region seems to play a similar negative role as seen in α_M integrin in regulating α₅β₁ inside-out activation. All the α CTMD sequences lacking a tyrosine proximal to the GFFKR motif promoted α₅β₁ inside-out activation, while the α CTMD sequences having the tyrosine had no such effect, with the exception of α₂ CTMD region. Notably, the α₇ CTMD region contains a tyrosine at the 11^th position distal from the GFFKR motif (Fig. 1A), but the α₅-α₇ exhibits a high level of integrin activation, consistent with the hypothesis that the membrane-proximal location of the tyrosine is important to exert an inhibitory effect. Interestingly, the α₁₀ CTMD region that is abundant in negatively charged residues as seen in α_IIb CTMD region exerted the most dramatic effect on α₅β₁ activation. Consistently, the α_V CTMD region increased α₅β₁ activation due to the lack of the tyrosine residue when compared with α₅ WT, but it decreased α_IIbβ₃ activation due to the lack of the cluster of acidic residues when compared with α_IIb WT. We found that α₂ CTMD region rendered α₅β₁ more active despite having the consensus tyrosine, suggesting that other residues of the α₂ CTMD region may counteract with the negative effect of tyrosine in regulating integrin activation.

Our study raised the question of the mechanism by which the α CTMD regions contribute to integrin inside-out activation. Previous and our current data demonstrated that the integrin α CTMD region is involved in the associations at the α/β transmembrane and CT domains, required to maintain integrin in the resting state¹³. In line with this function, our current data suggest two functional aspects of α CTMD region in integrin inside-out activation, i.e. the activation-supportive function and the activation-regulatory function. Structure analysis suggested that when binding to the β₃ CT, the talin 1 head domain might encounter steric hindrance with the α_IIb CTMD residues immediately following the GFFKR motif^{13,14,56,57,58}. Such interactions may disrupt the integrin α/β association at the cytoplasmic as well as transmembrane domains, leading to an active ectodomain conformation capable of high-affinity ligand binding. This non-specific interaction is required for the activation-supportive function of α CTMD region, which could be independent of the amino acid sequences. In addition, a minimal length of two amino acids of the α CTMD region could support the integrin inside-out activation although at a reduced level¹³. This model is consistent with our observation that the α CTMD regions are interchangeable for the activation-supportive function that is not sensitive to the diversities of sequence and length.

In contrast, the activation-regulatory function of α CTMD region is dependent on certain amino acids. Several potential mechanisms are involved in this regulation. We found that the differences of the α CTMD regions in regulating integrin activation were not due to the effect of talin binding to the β CT since truncation or swapping the α CTMD region did not reduce the amount of TH bound to the β CT¹³. The specific amino acids of the α CTMD region may directly affect the conformational change of α cytoplasmic as well as transmembrane domains induced by the binding of integrin activators such as talin and kindlin. Recent studies provided evidence suggesting the conformational change of α_IIb transmembrane and cytoplasmic domains in the context of full-length integrin on the cell surface⁵⁹. In addition, different levels of integrin activation had been observed when introducing mutations into the transmembrane or cytoplasmic domains, which correlate with the different levels of ectodomain conformational changes (unpublished data). Certain amino acids such as the acidic residue clusters present in α_IIb and α₁₀ CTMD regions and the tyrosine present in a subset of α CTMD regions may either facilitate or restrain the conformational change of α subunit as demonstrated by the active conformation-specific mAbs. The regulation of receptor activity by a tyrosine residue at the cytoplasmic domains has been seen in many cell surface receptors⁶⁰. It was proposed that the tyrosine residue could be buried in the cell membrane to restrain the movement of the cytoplasmic domain in the resting state. Such restraints could be released upon the tyrosine phosphorylation. This mechanism may also be applied to the specific tyrosine of α integrin CTMD regions. Structures of α₄, α_M, and α_X CT indicate that the tyrosine could be buried in the cell membrane (Fig. 1B). The tyrosine may restrain the piston like movement of α CT or the conformational plasticity of the GFFKR region as suggested by structural studies^59,61,62,63. This negative effect depends on the position of tyrosine as shown by our current data. Whether the tyrosine can be phosphorylated to release its negative effect on integrin activation clearly requires further investigation on individual integrins. Another potential mechanism by which the α integrin CTMD regions regulate the levels of integrin activation is through their interacting proteins. Several α integrin CT binding proteins have been identified to function as either negative or positive regulators for integrin activation^8,64. Some of the regulators such as SHARPIN, MDGI, and filamin interact with a subset of α integrins while the others bind to specific α integrins, such as Nischarin for α₅ and CIB1 for α_IIb^{54,62,65,66,67}. Interestingly, most of the current α CT binding proteins interact with the membrane-proximal region containing the conserved GFFKR motif ⁸. More novel integrin activation regulators interacting with the α CTMD region are yet to be identified.

There are accumulating data showing that the diverse α integrin CTMD regions can specify the cellular function of integrins. Novel functions of the α CTMD regions have been identified in recent years. For example, a subset of α integrin CTMD regions was found to regulate integrin internalization through interacting with the endocytic clathrin adaptor AP2¹⁸. Specific interaction between integrin α₅ CTMD region with phosphodiesterase-4D5 (PDE4D5) was found to regulate endothelial inflammatory signaling⁶⁸. It is tempting to speculate that more novel functions of α integrin CTMD regions are yet to be identified. Our large-scale analysis of the function of α integrin CTMD regions provokes new hypotheses that need to be tested on individual integrins. In addition, our approach provides a valuable tool and resource to study the integrin signaling events that are specified by the α CTMD regions.

Materials and Methods

DNA constructs

Plasmid DNA constructs for human α_IIbβ₃, α_Lβ₂, α₅β₁, and EGFP-tagged mouse talin-1-head (GFP-TH) were as described^20,69,70. Mutations were introduced by PCR following the protocol of the QuikChange XL site-directed mutagenesis kit (Agilent Technologies). α₅-CRISPR/Cas9 and β₁-CRISPR/Cas9 plasmids were purchased from Santa Cruz Biotechnology. Human ICAM-1 cDNA was obtained from Addgene. The cDNA of ICAM-1 extracellular domain was amplified by PCR and subcloned into a modified pIRES2-EGFP vector with a tag of human IgG1 Fc region at the C-terminus (denoted as ICAM-1-Fc).

The integrin α_IIb-chimeras were generated by the overlap PCR to replace the cDNA of α_IIb CTMD region with the cDNA of the CTMD region of α_V, α₁, or α_L integrin. The chimeric full-length α_IIb cDNA was cloned into the pEF1/V5-HisA vector using the 5′ EcoRV and 3′ XbaI restriction sites. A stop codon was added right before the 3′ XbaI site. The integrin α_L-chimeras were constructed using the 5′ Bsp1407I site right before the cDNA of KVGFFKR motif and the 3′ XbaI site preceded by a stop codon. The extra Bsp1407I and XbaI sites in the WT α_L vector were silenced by site-directed mutagenesis. A set of sense and antisense overlapping primers were designed to encode the sequence of KVGFFKR followed by the CTMD region of α_D, α_M, or α_X integrin. The 5′ Bsp1407I and 3′ XbaI sites as well as a stop codon were included in the primers. The chimeric cDNA fragments were obtained by mixing the primers for PCR amplification and subcloned into the α_L construct using the Bsp1407I and XbaI sites. The full-length cDNA of human α_M integrin was cloned into the pcDNA3.1 vector using the 5′ KpnI and 3′ XbaI sites. Mutations at the α_M CTMD region were introduced by PCR. The integrin α₅-chimeras was constructed using a 5′ HindIII site preceding the GFFKR-coding sequence and a 3′ MluI site preceded by a stop codon. A set of sense and antisense overlapping primers were designed to encode the sequence of GFFKR followed by the CTMD region of α_V, α₁, α₂, α₃, α₄, α₆, α₇, α₈, α₉, α₁₀, or α₁₁ integrin. The chimeric cDNA fragments were generated by PCR and subcloned into the WT α₅ vector. All the DNA constructs were validated by DNA sequencing.

Antibodies, inhibitors and ligands

PAC-1 (BD Biosciences) is a ligand-mimetic IgM mAb that is specific to activated α_IIbβ₃⁷¹. AP3 is non-blocking anti-β₃ mAb⁷². mAb 370.3 is specific for the extended conformation of α_IIb^13,70. PE-labeled or unlabeled TS2/4 (BioLegend) is non-blocking anti-α_L mAb. m24 (Biolegend) and KIM127 are anti-β₂ conformation-specific mAbs that report β₂ integrin headpiece opening and extension, respectively^19,73,74,75. 2LPM19c is an anti-α_M mAb (Santa Cruz Biotechnology). PE-labeled MAR-4 (BD Biosciences) is a non-blocking anti-β₁ mAb. 9EG7 (BD Biosciences) is a rat anti-β₁ conformation-specific mAb that reports β₁ integrin extension⁷⁶. Rabbit anti-GFP antibody was from Immunology Consultants Laboratory. Rabbit anti-β₃ antibody (H-96) was from Santa Cruz Biotechnolgy. Anti-β-actin mAb was purchased from Sigma. 9EG7 were conjugated with Alexa Fluor-647 (Life Technologies). AP3 was conjugated with R-PE using the R-PE antibody conjugation kit (Solulink). 370.3, m24, and KIM127 were biotinylated using the EZ-Link Sulfo-NHS-Biotin (Thermo Scientific). A286982 (Santa Cruz Biotechnology) is a α_Lβ₂-specific inhibitor. Eptifibatide acetate (Santa Cruz Biotechnology) is a α_IIbβ₃-specific inhibitor.

Human ICAM-1-Fc was expressed as the secreted form in HEK293FT cells via transient transfection using polyethylenimine (PEI). The transfected cells were cultured for 10 days before the culture supernatant was collected. The concentration of ICAM-1-Fc in the supernatant was determined by ELISA using the anti-human ICAM-1 mAb (SinoBiological, Inc.) and the peroxidase-conjugated anti-human IgG1 (Fc specific) (Jackson ImmunoResearch Laboratories, Inc.). The culture supernatant was used for the ICAM-1 binding assay. Human fibronectin type III domains 9^th−10^th fragment (Fn9–10) was expressed in E. coli and purified as described before⁷⁷. Human fibronectin (Fn) was purchased from Sigma. Fn9–10 and Fn were conjugated with Alexa Fluor-647.

Cell lines

HEK293FT cells (ThermoFisher Scientific) were cultured in DMEM plus 10% FBS at 37 °C supplied with 5% CO₂. The α₅ and β₁ integrin double-knockout HEK293FT (HEK293FT-α₅β₁-KO) cells were generated by the CRISPR/Cas9 gene editing technology as described in our previous study⁷⁸. α₅β₁ integrin deficient CHO-B2 cells were as described before²¹.

Soluble ligand binding assay by flow cytometry

GFP-TH induced ligand binding assay of HEK293FT cells transfected with α_IIbβ_{3 or} α_Lβ₂ integrin was as described previously¹³. In brief, HEK293FT cells were co-transfected with integrin constructs and GFP or GFP-TH for at least 24 hours. Ligand binding was performed in HBSGB buffer (25 mM HEPES pH 7.4, 150 mM NaCl, 2.75 mM glucose, and 0.5% BSA) plus 10 μM eptifibatide acetate (for α_IIbβ₃), 50 μM A286982 (for α_Lβ₂), or 1 mM Ca²⁺ and 1 mM Mg²⁺ (Ca/Mg). The cells were first incubated at 25 _°C for 30 min with 5 μg/ml PAC-1 and 10 μg/ml biotinylated AP3 for α_IIbβ₃, 42 μg/ml of ICAM-1-Fc and 45 μg/ml biotin-conjugated goat anti-human IgG Fc (Novex, Life Technologies) for α_Lβ_2, and then washed and incubated in Ca/Mg on ice with the detecting reagents: PE-labeled streptavidin and Alexa Fluor-647-labeled goat anti-mouse IgM for α_IIbβ₃, PE-labeled TS2/4 and Alexa Fluor 647-labeled streptavidin for α_Lβ₂. For α₅β₁ integrin, either CHO-B2 or HEK293FT-α₅β₁-KO cells were transfected with α₅β₁ integrin plus GFP or GFP-TH. The cells were first incubated with 50 μg/ml Alexa-Fluor-647-labeled Fn9–10 or 15 μg/ml Alexa-Fluor-647-labeled Fn in HBSGB buffer plus 5 mM EDTA or 1 mM Ca/Mg, and then washed and incubated in Ca/Mg on ice with PE-labeled MAR-4. Integrin and GFP double-positive cells were acquired for calculating the mean fluorescence intensity (MFI) by flow cytometry. Ligand binding was presented as the normalized MFI, that is ligand MFI (after subtracting the ligand MFI in the inhibitor or EDTA condition) as a percentage of integrin MFI.

Conformation-specific antibody binding

Integrin conformational changes detected by the conformation-specific mAbs on the cell surface was as described previously^13,70. In brief, HEK293FT transfectants were first incubated with the biotinylated conformation-specific mAb in Ca/Mg at 25 _°C for 30 mins, washed and then incubated with the detecting reagents: Alexa-Fluor-647-labeled streptavidin plus R-PE-labeled AP3 for α_IIbβ₃ or PE-labeled TS2/4 for α_Lβ₂. For α₅β₁ integrins, HEK293FT-α₅β₁-KO transfectants were incubated with rat 9EG7 mAb in Ca/Mg at 25 _°C for 30 mins, washed and then incubated with PE-labeled MAR-4 and Alexa-Fluor-647-labeled goat anti-rat IgG (cross-absorbed) (Abcam). Integrin and GFP double-positive cells were analyzed for calculating the MFI of mAb binding. The binding of conformation-specific mAb is presented as their MFI normalized to the MFI of integrin expression.

For m24 binding with α_Mβ₂ integrin, HEK293FT transfectants were incubated with m24 or mouse anti-α_M mAb (2LPM19c) separately, washed and incubated with Alexa-Fluor-647-labeled anti-mouse IgG. GFP-positive cells were analyzed for calculating the MFI of bound m24 or 2LPM19c. The m24 binding was presented as the m24 MFI normalized to the 2LPM19c MFI reporting α_Mβ₂ expression.

Co-immunoprecipitation

To detect the TH-binding to integrin β₃ CT, the α_IIbβ₃ constructs were co-transfected with GFP-TH or GFP into HEK293FT cells. The cells were lysed to perform co-immunoprecipitation with rabbit anti-GFP antibody. The GFP-TH associated β₃ subunit was detected by immunoblot using rabbit anti-β₃ antibody H-96 and HRP-labeled anti-rabbit IgG antibody. The amount of GFP or GFP-TH in pull-down samples was detected by rabbit anti-GFP antibody and HRP-labeled anti-rabbit IgG antibody. Total expression levels of integrin β₃ and GFP-TH in the whole cell lysates were detected by immunoblot. β-actin was blotted with anti-β-actin mAb as a loading control.

To detect the TH binding to integrin β₂ CT, the α_L constructs were co-transfected with β₂-D709A and GFP-TH into HEK293FT cells. GFP-TH only or α_Lβ₂ plus GFP transfectants were used as negative controls. The cells were washed with PBS at pH 8.0 and the cell surface molecules were biotinylated using 2 mM biotin reagent (EZ-Link™ Sulfo-NHS-Biotin, Thermo Scientific) in PBS at 25 °C for 30 min. The cells were washed and lysed for co- immunoprecipitation with anti-GFP antibody. As a positive control for integrin α_L and β₂ bands, α_Lβ₂ was immunoprecipitated with anti-α_L mAb TS2/4. Integrin α_L and β₂ subunits that associated with GFP-TH were quantitatively detected by blotting with IRDye® 800CW Streptavidin (LI-COR). The immunoprecipitated GFP or GFP-TH were detected by Western blot using anti-GFP antibody. Integrin α_L and β₂ signals were normalized to the precipitated GFP or GFP-TH, and shown as a percentage of the normalized α_L-WT signal. The cell surface expression of α_Lβ₂ integrins and GFP-TH were detected by flow cytometry.

Statistical Analysis

Data are expressed as mean ± s.e.m from at least three independent experiments (n ≥ 3 in each group) unless specified. Statistical analyses were performed with GraphPad Prism using parametric Student’s t-test (two-tailed).

Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank Drs. Chafen Lu, Timothy Springer, Daniel Bougie, and Richard Aster for providing antibodies. This work was supported by grants HL122985 and HL131836 (to J. Zhu) from the Heart, Lung, and Blood Institute of the National Institute of Health.

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Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI, 53226, USA
Aye Myat Myat Thinn, Zhengli Wang & Jieqing Zhu
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, 53226, USA
Aye Myat Myat Thinn & Jieqing Zhu

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Aye Myat Myat Thinn
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Zhengli Wang
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A. Thinn performed the experiments, analyzed data, and wrote the manuscript. Z. Wang performed the experiments and analyzed data. J. Zhu designed the study, analyzed data and wrote the manuscript.

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Correspondence to Jieqing Zhu.

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Thinn, A.M.M., Wang, Z. & Zhu, J. The membrane-distal regions of integrin α cytoplasmic domains contribute differently to integrin inside-out activation. Sci Rep 8, 5067 (2018). https://doi.org/10.1038/s41598-018-23444-w

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Received: 21 November 2017
Accepted: 13 March 2018
Published: 22 March 2018
DOI: https://doi.org/10.1038/s41598-018-23444-w

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