New PI(4,5)P2- and membrane proximal integrin-binding motifs in the talin head control beta3-integrin clustering

Abstract

Integrin-dependent adhesion sites consist of clustered integrins that transmit mechanical forces and provide signaling required for cell survival and morphogenesis. Despite their importance, the regulation of integrin clustering by the cytoplasmic adapter protein talin (Tal) and phosphatidylinositol (PI)-4,5-biphosphate (PI(4,5)P(2)) lipids nor their dynamic coupling to the actin cytoskeleton is fully understood. By using a Tal-dependent integrin clustering assay in intact cells, we identified a PI(4,5)P(2)-binding basic ridge spanning across the F2 and F3 domains of the Tal head that regulates integrin clustering. Clustering requires a new PI(4,5)P(2)-binding site in F2 and is negatively regulated by autoinhibitory interactions between F3 and the Tal rod (Tal-R). The release of the Tal-R exposes a new beta3-integrin-binding site in F3, enabling interaction with a membrane proximal acidic motif, which involves the formation of salt bridges between K(316) and K(324) with E(726) and D(723), respectively. This interaction shields the beta-integrin tail from reassociation with its alpha subunit, thereby maintaining the integrin in a substrate-binding and clustering-competent form.

Figure 1.

The F2 and F3 domains of Tal-H are required for β3-integrin clustering. (A and B) Scheme of the Tal-dependent β3-integrin clustering protocol (A) and ECFP-tagged Tal fragments (B). (C) Western blot with anti-EGFP antibodies of equivalent amounts of cell lysates (50% for ECFP) of transiently transfected B16F1 cells. (D–I) Representative TIRF images (EGFP channel) of Mn²⁺-stimulated B16F1 cells plated on serum-coated coverslips and double transfected with β3-EGFP-integrin (D–I) and ECFP–FL-Tal (D), ECFP–Tal-H (E), ECFP–Tal-F2/3 (F), ECFP–Tal-F3 (G), ECFP-tagged Tal-R (H), and ECFP only (I). Magnified views of the boxed areas and ECFP epifluorescence are shown in the insets. (J) Averaged (n > 25) fluorescence intensity histograms of β3-EGFP-integrin transfected cells as shown in D–I. The dashed line represents the fluorescence intensity threshold (>200 12-bit gray levels) that was used to calculate the integrin clustering index (K). Histograms are from one representative experiment, whereas the integrin clustering index (K) is the mean of n > 3 (SEM) experiments. Bars: (D and E) 25 µm; (F–I) 20 µm.

Figure 2.

The K318A mutation in FL-Tal increases β3-integrin clustering. (A and B) Western blots of equivalent amounts of cell lysates probed with an anti-Tal mAb from wild-type (wt) or mutant mCherry– or EGFP–FL-Tal–transfected B16F1 cells. (C) Schematic view of Mn²⁺-induced integrin activation and association with wild-type or K318A FL-Tal. (D) Integrin clustering index (percentage of pixels with >200 gray levels) of wild-type and K318A FL-Tal–transfected cells (n = 4; SEM). (E and F) Representative TIRF images of Mn²⁺-stimulated B16F1 cells grown on serum-coated coverslips and double transfected with β3-EGFP-integrin (E and F) and wild-type (E) or K318A mCherry (mCh)–FL-Tal (F). Magnified views of the boxed areas are shown in E′–E‴ and F′–F‴. Bar, 20 µm.

Figure 3.

The F2 and F3 domains of Tal-H contain PI(4,5)P₂-binding sites. (A) Electrostatic surface map of the Tal-F2/3 structure (García-Alvarez et al., 2003) showing basic (blue) and acidic (red) amino acids. Mutations of basic amino acids are indicated in brackets. (B and C) Structure and sequence comparison of the F2 (B) and F3 (phosphotyrosine binding [PTB]) domain (C) of Tal1 (cyan) and radixin (pink). Basic motifs are indicated by asterisks, and conserved residues are shown in the structure. Note the basic patch in Tal-F2, replacing a turn of helix 3 of radixin (B) and the extended loop between β-sheets 1 and 2 in Tal-F3 (C). In the sequence alignments, identical and similar amino acids are indicated by dark and light gray shading, respectively. (D) SPR analysis of 0.5 µM GST–Tal-H binding to reconstituted liposomes composed of 10% PI(4,5)P₂, 30% PC, 40% PE, and 20% PS. Note that mutations of the basic residues in Tal-F2 and/or Tal-F3 reduce PI(4,5)P₂ binding.

Figure 4.

Mutations in the PI(4,5)P₂-binding sites of Tal-H affect integrin clustering. (A–C) Representative TIRF images (EGFP channel) of Mn²⁺-stimulated B16F1 cells grown on serum-coated coverslips and double transfected with β3-EGFP-integrin and ECFP–Tal-H carrying PI(4,5)P₂-binding mutations K320A/K322A (A), K322A/K324A (B), and K272A/K274Q/R277E (C). Magnified views of the boxed areas and ECFP–Tal-H expression are shown in the insets. (D and E) Averaged (n > 25) intensity histograms (D) and mean clustering index (n > 3; SEM; E) of β3-EGFP-integrin fluorescence as a function of Tal-H expression. The vertical dashed line in D represents the fluorescence intensity threshold (>200 12-bit gray levels) that was used to calculate the integrin clustering index. (F) Scheme of basic regions involved in Tal-R interaction, PI(4,5)P₂ binding, and integrin clustering. (G) Overlay of the proposed structure of the Tal-R–Tal-F3 complex (PDB ID 2KGX; Goult et al., 2009) with the Tal-F2/3 structure (PDB ID 1MK7; García-Alvarez et al., 2003), indicating PI(4,5)P₂ (K²⁷², K²⁷⁴, R²⁷⁷, K³²², and K³²⁴) as well as Tal-R–binding residues (K³¹⁸, K³²⁰ [hidden], K³²² [hidden], and K³²⁴; Goult et al., 2009). wt, wild type. Bar, 20 µm.

Figure 5.

Mutations of E⁷²⁶ and E⁷³³ affect integrin activation, Tal-H binding, and Tal-H–dependent integrin clustering. (A) β3-EGFP-integrin cytoplasmic tail sequence with critical amino acids involved in integrin activation. (B) Soluble integrin ligand binding capacity (integrin activation index; n > 3; SEM) of different integrin mutants. (C) GST–Tal-H pull-down of wild-type (wt) and mutant β3-EGFP-integrins from lysates of transiently transfected COS-7 cells, involving DelW739-T762, W739A/Y747A, D723A/E726A/E733A, and D723K/E726K and as controls, α6-EGFP-integrin and the high-affinity NPLY integrin mutation (SPLH) according to Wegener et al. (2007). (D–I) Representative TIRF images (EGFP channel) of Mn²⁺-stimulated B16F1 cells cotransfected with wild-type ECFP–Tal-H (insets) and wild-type (D) or mutant β3-EGFP-integrin Y747A (E), F730A (F), E726K (G), E733K (H), or E726K/E733K (I) and cultured on serum-coated coverslips. (J) Mean cell surface reactivity with anti–β3-integrin mAb (n > 3; SEM), as measured by FACS. Note that endogenous β3-integrin levels correspond to 22% of wild-type β3-EGFP-integrin–transfected cells. (K) Mean clustering index (n > 25 cells; SD) taken from one representative out of three similar experiments. Bar, 20 µm.

Figure 6.

Complementation of integrin clustering by charge-inversion mutants. (A–D) Representative TIRF images (EGFP channel) of Mn²⁺-stimulated B16F1 cells cultured on serum-coated coverslips and coexpressing wild-type (wt; A and B) or E726K mutant (C and D) β3-EGFP-integrin together with wild-type (A and C) or K316E mutant (B and D) ECFP–Tal-H. Magnified views of the boxed areas and ECFP–Tal-H expression by epifluorescence are shown in the insets. Note the extensive integrin clustering in the wild-type/wild-type (A) and E726K/K316E condition (D). (E) Averaged (n > 25) histograms of cells as shown in A–D. The dashed vertical line indicates the threshold used to calculate the integrin clustering index. (F) Mean integrin clustering index (n > 3; SEM) of conditions as in A–D. Bar, 20 µm.

Figure 7.

Complementation of focal adhesion formation by E726K β3-integrin and K316E FL-Tal. (A–L) Representative TIRF images of transiently transfected B16F1 cells cultured on serum-coated coverslips in the absence of Mn²⁺. Wild-type (wt; A and D) and E726K mutant (G and J) β3-EGFP-integrin were coexpressed with either wild-type (B and H) or K316E mutant (E and K) mCherry (mCh)–FL-Tal. The merged images demonstrate perfect colocalization in large focal adhesions in the wild-type/wild-type (C) and E726K/K316E (L) conditions. (E) In contrast, K316E FL-Tal was inefficiently recruited to focal adhesions in the presence of wild-type integrins. (G–I) Similarly, the E726K β3-integrin perturbed efficient cell spreading, causing irregular cell shapes and recruitment only to small focal adhesions. (M) Per cell quantification of the ratio of integrin to Tal fluorescence within focal adhesions. Each point corresponds to the mean of 5–15 contacts per cell. The horizontal bar represents the mean and SD of n > 20 cells. (N) Mean β3-EGFP-integrin fluorescence in focal adhesions, which is reduced for the E726K mutant integrin (n > 20 cells; mean and SD of 300–400 contacts per condition). Bar, 20 µm.

Figure 8.

Docking and MD analysis of the integrin–Tal interface. (A) Side view of the MP helix of β3-integrin docked to Tal-F3, revealing putative charge–charge interactions (dotted lines) of residues required for Tal-dependent integrin clustering (D⁷²³–K³²⁴; E⁷²⁶–K³¹⁶; E⁷³³–K³⁶⁴). (B) MD analysis starting from the model in A, after manual connection (magenta) to the Tal-F3–bound W⁷³⁹/NPLY⁷⁴⁷ motif. Snapshot after 300 ps of MD analysis, showing the position maintained for another 1500 ps. (C) Details of peptide position at 300 ps, indicating amino acids involved in interactions between D⁷²³–K³²⁴; E⁷²⁶–K³¹⁶; F⁷³⁰–L³²⁵ (L³²⁵ not depicted) and E⁷³³ with S³⁶⁵, S³⁷⁹, and Q³⁸¹. A PDB file of this model is available in the supplemental data. (D) Overlay and shifts in localization (arrows) of relevant amino acids between the NMR-derived model (dark green; Wegener et al., 2007) and the structure shown in C (light green).

Figure 9.

Mechanical and biochemical control of Tal–integrin association during focal adhesion formation. Multiparametrical regulation of the integrin–Tal association. The autoinhibited form of Tal (top left) can interact with PI(4,5)P₂-enriched membranes, resulting in Tal-R dissociation from Tal-H (bottom left). In turn, PI(4,5)P₂-bound Tal-H associates with clasped integrin receptors via the NPLY motif of the cytoplasmic domain of β-integrins (middle left). Integrin transmembrane domain unclasping by the basic finger in Tal-F3 interacting with the MP acidic motif in the β-integrin tail, creating a competition between K³²⁴ of Tal-H with R⁹⁹⁵ of the α subunit for D⁷²³ association. This competition is illustrated by the overlay of two exclusive structures (magnified inset; top middle). Stable Tal-H association with the MP acidic motif requires the open integrin conformation stabilized by integrin ligand occupancy (middle right). In the absence of F-actin, maintaining the Tal–integrin complex requires Mn²⁺ or mutational activation of integrins (e.g., salt bridge mutation). In the presence of F-actin, the C terminus of Tal-R (pink tube) can be captured, potentially reducing the autoinhibitory interaction of Tal-R (1,655–1,822) with Tal-H (middle right). Force-dependent stretching of Tal-R creates new binding sites for vinculin, stabilizing the integrin–F-actin linkage, preventing Tal autoinhibition (right).