Structure of Rap1b bound to talin reveals a pathway for triggering integrin activation

Abstract

Activation of transmembrane receptor integrin by talin is essential for inducing cell adhesion. However, the pathway that recruits talin to the membrane, which critically controls talin's action, remains elusive. Membrane-anchored mammalian small GTPase Rap1 is known to bind talin-F0 domain but the binding was shown to be weak and thus hardly studied. Here we show structurally that talin-F0 binds to human Rap1b like canonical Rap1 effectors despite little sequence homology, and disruption of the binding strongly impairs integrin activation, cell adhesion, and cell spreading. Furthermore, while being weak in conventional binary binding conditions, the Rap1b/talin interaction becomes strong upon attachment of activated Rap1b to vesicular membranes that mimic the agonist-induced microenvironment. These data identify a crucial Rap1-mediated membrane-targeting mechanism for talin to activate integrin. They further broadly caution the analyses of weak protein-protein interactions that may be pivotal for function but neglected in the absence of specific cellular microenvironments.

Fig. 1

Domain organization and a linear structural model of talin. The model was generated from known crystal and NMR structures of talin fragments and shown in cartoon representation. Critical talin-binding partners involved in talin membrane recruitment and activation are indicated in the figure. PIP2 phosphatidylinositol-4,5-bisphosphate, RIAM-N the N terminus of RIAM, PIPKIγ type I phosphatidylinositol phosphate kinase isoform-γ

Fig. 2

Rap1b/talin-F0 interaction is GTP-dependent and specific but is of weak affinity. a The HSQC spectra of 50 μM ¹⁵N-labeled talin-F0 in the absence (black) and presence of 125 μM GMP-PNP loaded Rap1b (red). b The HSQC spectra (four representative residues were shown) of 50 μM ¹⁵N-labeled talin-F0 in the absence (black) and presence of 125 μM GDP loaded Rap1b (blue) or GMP-PNP loaded Rap1b (red). Note that Rap1b-GDP induced the same overall pattern of chemical shift changes of ¹⁵N-labeled talin-F0 as Rap1b-GMP-PNP does but with less peak shifts and broadenings, indicating a weaker affinity. c The affinity of Rap1b (GMP-PNP)/talin-F0 interaction measured by HSQC titration. d The HSQC spectra of 50 μM ¹⁵N-labeled kindlin2-F0 in the absence (black) and presence of 125 μM GMP-PNP loaded Rap1b (red)

Fig. 3

Rap1b recognizes F0 domain of talin in a GTP-dependent manner. a The HSQC spectra (representative regions were shown) of 45 μM GMP-PNP loaded ¹⁵N-labeled Rap1b (1–167) in the absence (black) and presence of 90 μM talin-F0 (red), 225 μM talin-F0 (blue), 90 μM talin-H (red), or 45 μM full-length talin (red). Note that the peak of I27 was broadened in the presence of talin-H or full-length talin. b GST pull-down assays to show that Rap1b interacts with both talin-H and full-length talin in a GTP-dependent manner. Full blot/gel images are shown in Supplementary Figs. 11 and 12

Fig. 4

Solution structure of Rap1b/talin-F0 complex by NMR. a Superposition of 20 calculated Rap1b/talin-F0 complex structures with lowest energies (shown in ribbon representation). b Cartoon representation of the Rap1b/talin-F0 complex structure with the lowest energy. c Current solved complex structures of Rap1 and its effector proteins (shown in cartoon representation). d Structure-based sequence alignment of talin-F0 and RA domain containing Rap1b effector proteins or kindlin2-F0 (only binding interfaces were shown). Residues involved in the binding interface with a cutoff of 4 Å are highlighted in cyan. Conserved residues are colored in red. e Detailed interaction diagram between the α1 helix and switch I of Rap1b and the α2 helix of talin-F0. Hydrogen bond or salt bridge is represented by red dashed line

Fig. 5

The Rap1b binding to talin is crucial for integrin activation. a The HSQC spectra of 50 μM ¹⁵N-labeled talin-F0_DM (K15A, R35A) in the absence (black) and presence of 125 μM GMP-PNP loaded Rap1b (red). b Integrin activation assay in CHO A5 cells, which stably express integrin α_IIbβ₃. Double mutations (K15A, R35A) in talin-H substantially decreases integrin activation. The data are shown as means ± S.E.M. from four independent experiments. *p < 0.05. c Static adhesion of talin^1/2dko fibroblasts, expressing either ypet alone, ypet-tagged talin WT, or ypet-tagged talin DM (K15A, R35A). Number of adherent cells was quantified by measuring absorbance of crystal violet staining. Values measured for talin WT-transduced cells were set to one in each of six independent experiments. Values are given as mean ± S.E.M. *p < 0.05 and **p < 0.01. d Spreading of talin^1/2dko fibroblasts expressing ypet (green), ypet-tagged talin WT (red), or ypet-tagged talin DM (blue) on a fibronectin-coated surface, measured 5, 15, 30 min, 1, 2 and 4 h after plating. N = 5. e Confocal images of ypet-tagged talin WT or ypet-tagged talin DM cells on FN-coated round micropatterns stained for paxillin (red) and F-actin (blue). Ypet signal is shown in green. Scale bar 10 μm. Focal adhesion area per cell area (f), focal adhesion number per cell (g), and focal adhesion size (h) of ypet-tagged talin WT and ypet-tagged talin DM cells on FN-coated micropatterns. i Ypet fluorescence intensity within paxillin-positive focal adhesion area in relation to the cellular ypet fluorescence. N = 5; 10–15 talin WT and talin DM-expressing cells in each measurement were analyzed. All values are given as mean ± S.E.M. *p < 0.05 and **p < 0.01

Fig. 6

Mechanism of talin recruitment by Rap1. a Protein expression levels of talin, RIAM, Rap1, Ras GTPases, and GAPDH in various types of cell lines indicated in the figure. Full blots are shown in Supplementary Fig. 13. b A model of membrane-associated Rap1b recruiting talin for integrin activation. Proteins are shown in cartoon representation. Talin F1 loop is colored in maroon, the positively charged residues of talin-F2F3 domains (green), which interact with PIP2, are colored in deep blue and shown in surface representation, and the negatively charged residues of talin-R9 (cyan) are colored in magenta and shown in surface representation. c GST pull-down assay to show the robustly enhanced interaction between membrane-anchored Rap1b and talin-H. Rap1b was loaded with GMP-PNP in this assay and “LUV-Rap1b” represents that Rap1b was anchored to large unilamellar vesicles (LUVs). LUV-Rap1b could be easily pulled down by GST-talin-H and the band was visible in Coomassie blue staining gel (boxed in red) and intense in WB analysis, while free Rap1b pulled down by GST-talin-H was hardly seen in the same condition. Full blot/gel images are shown in Supplementary Fig. 14. d Left, a representative vesicle co-sedimentation assay showing that the interaction between talin-H_WT and membrane is enhanced by around 1.5-folds when Rap1b (GMP-PNP) is attached to membrane but not for talin-H_DM. “MLV + Rap1b” represents that Rap1b remained as free form in solution without being attached to multilamellar vesicles (MLVs), which were pre-incubated with β-mercaptoethanol. “MLV-Rap1b” represents that Rap1b was anchored to MLVs. Right, the quantification of four independent experiments. The intensity of talin-H_WT or talin-H_DM band was normalized to that of corresponding “MLV only” group and the data are shown in means ± S.E.M. ***p < 0.0001. Full gel image is shown in Supplementary Fig. 15. e Left, a representative vesicle co-sedimentation assay showing that the interaction between full-length talin and membrane is significantly enhanced by around 3-folds when Rap1b (GMP-PNP) is attached to membrane. Right, the quantification of four independent experiments. The intensity of talin band was normalized to that of the “MLV only” group and the data are shown as means ± S.E.M. ***p < 0.0001. Full gel image is shown in Supplementary Fig. 15