Structural and mechanistic insights into the recruitment of talin by RIAM in integrin signaling

Abstract

Plasma membrane (PM)-bound GTPase Rap1 recruits the Rap1-interacting-adaptor-molecule (RIAM), which in turn recruits talin to bind and activate integrins. However, it is unclear how RIAM recruits talin and why its close homolog lamellipodin does not. Here, we report that, although RIAM possesses two talin-binding sites (TBS1 and TBS2), only TBS1 is capable of recruiting cytoplasmic talin to the PM, and the R8 domain is the strongest binding site in talin. Crystal structure of an R7R8:TBS1 complex reveals an unexpected kink in the TBS1 helix that is not shared in the homologous region of lamellipodin. This kinked helix conformation is required for the colocalization of RIAM and talin at the PM and proper activation of integrin. Our findings provide the structural and mechanistic insight into talin recruitment by RIAM that underlies integrin activation and explain the differential functions of the otherwise highly homologous RIAM and lamellipodin in integrin signaling.

Figure 1

(A) Schematic diagrams of talin. The head region possesses four N-terminal FERM domains (ovals), and the rod region contains 13 helical bundle domains (R1-R13, each cylinder represents one helix) and a dimerization helix (DH). Vinculin binding helices are colored in red. (B) Schematic diagrams of RIAM. Talin-binding sites (TBS1 and TBS2) are colored in orange; coiled-coil (CC) is in red; poly-proline (PP) is in black; the Ras-association domain (RA) is in yellow; and Pleckstrin-homology domain (PH) is in cyan. Fragments containing residues 1-30 and 27-93 were used in the biochemical characterization and functional assays to represent the TBS1 and TBS2 regions, respectively. (C) GST-tagged TBS1, TBS2, and TBS1-2 (residues 1-93) were used as bait to pull down GFP-talin expressed in HEK293 cells. Bound GFP-talin was analyzed by Western blotting. Input GST-tagged RIAM protein levels were shown by Coomassie staining. (D) GST-tagged TBS1, TBS2, and TBS1-2 were used as a bait to pull down purified recombinant His₆-tagged R2R3 and R7R8 proteins. Bound His-R2R3 and His-R7R8 proteins were analyzed by Western blotting (upper). Input GST-tagged RIAM protein levels were shown by Coomassie staining (lower).

Figure 2

(A) Ribbon diagram representation of the complex structure of the talin R7R8 domains and the RIAM TBS1 peptide. R7 domain is colored in cyan; R8 domain is in green; and the TBS1 peptide is in purple. Left: Top view of the R7R8-TBS1 complex. Right: Only the R8 domain and the TBS1 peptide are shown in the side view. The Ser13 residue located at the kink is shown as a stick representation. The two TBS1-interacting helices in R8 are labeled as α2 and α3. (B) Hydrophobic interactions are represented by a light gray surface. Residues of TBS1 are labeled in black and blue, and residues of the R8 domain are labeled in black and yellow. (C) Hydrogen bonds are denoted by a dotted line. (D) Superposition of the TBS1 peptide (residues 14-25) from the TBS1:R7R8 structure (purple) and the structure of TBS1 in complex with the vinculin Vd1 domain (PDB ID 3ZDL) shows a ~55° kink at Ser13. Vd1 helices (ivory) that interact with TBS1 are labeled (α1, α2, and α4). The hydrogen bond network mediated by the side chain of Ser13 is illustrated in the close-in view (E) GST-tagged TBS1 mutations (D9A, S13G, L15Y and E18A) were used as bait to pull down GFP-talin. Bound GFP-talin was analyzed by Western blotting. Input GST-TBS1 (wild type and mutants) levels were shown by Coomassie staining. (F) Pull-down assay using full-length talin (WT, T1520Y and V1540Y) and the RIAM TBS1 fragment. Input GFP-talin (wild type and mutants) levels were shown in the lower blot. (G) Surface representation of the R8 domain and cartoon diagram of the bound TBS1 peptide. TBS1 interacting residues that are identical in the R3 domain are highlighted in yellow, and similar residues are highlighted in blue. Conserved residues are highlighted in yellow in the structure-based sequence alignment of the R8 domain and the R3 domain. V1540 in the R8 domain and V871 in the R3 domain are indicated by a red dot. (H) Pull-down of His-tagged talin R2R3 domains by GST-TBS1 and its mutants (S13G and L15Y). Input GST-TBS1 (wild type and mutants) levels were shown by Coomassie staining. (I). Pull-down of full-length talin and its mutants (V871Y, V1540 and V871Y/V1540Y) with GST-TBS1. Input GFP-talin (wild type and mutants) levels were shown by Western blotting.

Figure 3. Binding determinants and the helical kink are required for the co-clustering of RIAM and talin at the PM

A5 cells were transiently co-transfected with mCherry-talin and GFP-RIAM-TBS1-CAAX constructs as indicated. 24 hrs post-transfection, the cells were plated on fibrinogen-coated coverslips and allowed to adhere and spread for 1 hr. (A) Fixed cells were imaged using a spinning-disk confocal microscope and representative monochromatic and color-merged images are shown. Note that yellow colors in merged images represent areas of common red and green localizations. Numbers in white correspond to unrefined median PCCs ± SD. (B) Enlarged lamellae sections correspond to the white rectangles marked in A. Median PCC values are shown. Note that small histogram graphs in B are representative of intensity levels of the assorted overexpressed proteins. Bar scales in A and B represent 5 μm. Graphs in C and D respectively correspond to A and B PCC medians (± interquartile range) values. (C) Data calculated from three experimental repetitions counting with 10 images per condition. (D) Data included 9 lamella regions selected from 3 representative cell bodies per condition. *** P < 0.001.

Figure 4. Integrin activity analyses for TBS1 and TBS2

(A) A5 cells were co-transfected with HA-talin and GFP-tagged RIAM-TBS1-CAAX. Non-specific inhibitor of integrin (EDTA) and specific inhibitor of integrin αIIbβ3 (Eptifibatide) were added into the transfected cells before adding PAC-1 antibody. Integrin activation was detected by PAC-1 binding using FACS. The MFI of cells co-transfected with GFP and HA-Talin was defined as 1. *P < 0.001 compared with RIAM-TBS1-CAAX. (B) A5 cells were co-transfected with HA-talin and indicated mutants of GFP-tagged RIAM-TBS1-CAAX. PAC-1 binding was detected as described in A. *P < 0.001 compared with wild type RIAM-TBS1-CAAX. (C) A5 cells were co-transfected with HA-talin and indicated mutants of GFP-tagged RIAM. *P < 0.001 compared with wild type RIAM. (D) PAC-1 binding was detected using GFP-RIAM full length or RIAM with TBS regions deleted constructs. *P < 0.001, ** P < 0.01 compared with full length RIAM. (E) PAC-1 binding was detected in cells expressing TBS1, TBS2 and TBS1-2 constructs fused with a C-terminal CAAX tail. *P < 0.001 compared with co-transfection of GFP and talin. Data shown in A-E are means ± SD, n = 3.

Figure 5

(A) Schematic representation of Lpd domain organization (Lpd-TBS: talin-binding region, orange; CC: coiled-coil region, red; PP: poly-proline region, black; RA: Ras-association domain, yellow; and PH: Pleckstrin homology domain, cyan). Lpd-TBS shares conserved talin-binding residues (highlighted in yellow) with RIAM TBS1. Talin interacting residues are depicted as green dots. A pink cylinder indicates the helical region in TBS1. Blue stars indicate residues that define the binding specificity in RIAM. (B) The L15Y mutant that disrupts TBS1-talin binding and the L15W mutant that mimics Lpd were assessed for talin-binding by pull-down. Bound GFP-talin was analyzed by Western blot. Input GST-TBS1 (wild type and mutants) levels were shown by Coomassie staining. (C) TBS1-S13G of RIAM and Lpd-TBS-G29S of Lpd were assessed for talin-binding by pull-down. Bound GFP-talin was analyzed by Western blot. Input GST-tagged protein levels were shown by Coomassie staining. (D) PAC-1 binding was detected in cells expressing TBS1-CAAX and Lpd-TBS (L60)-CAAX or G29S mutant. *P < 0.01 compared with co-transfection of GFP and talin. ^#P < 0.01 compared with L60-CAAX.