Rap1 small GTPases interact with Rap1-GTP-interacting adaptor molecule (RIAM), a member of the MRL (Mig-10/RIAM/Lamellipodin) protein family, to promote talin-dependent integrin activation. Here, we show that MRL proteins function as scaffolds that connect the membrane targeting sequences in Ras GTPases to talin, thereby recruiting talin to the plasma membrane and activating integrins. The MRL proteins bound directly to talin via short, N-terminal sequences predicted to form amphipathic helices. RIAM-induced integrin activation required both its capacity to bind to Rap1 and to talin. Moreover, we constructed a minimized 50-residue Rap-RIAM module containing the talin binding site of RIAM joined to the membrane-targeting sequence of Rap1A. This minimized Rap-RIAM module was sufficient to target talin to the plasma membrane and to mediate integrin activation, even in the absence of Rap1 activity. We identified a short talin binding sequence in Lamellipodin (Lpd), another MRL protein; talin binding Lpd sequence joined to a Rap1 membrane-targeting sequence is sufficient to recruit talin and activate integrins. These data establish the mechanism whereby MRL proteins interact with both talin and Ras GTPases to activate integrins. Rap1 small GTPases interact with Rap1-GTP-interacting adaptor molecule (RIAM), a member of the MRL (Mig-10/RIAM/Lamellipodin) protein family, to promote talin-dependent integrin activation. Here, we show that MRL proteins function as scaffolds that connect the membrane targeting sequences in Ras GTPases to talin, thereby recruiting talin to the plasma membrane and activating integrins. The MRL proteins bound directly to talin via short, N-terminal sequences predicted to form amphipathic helices. RIAM-induced integrin activation required both its capacity to bind to Rap1 and to talin. Moreover, we constructed a minimized 50-residue Rap-RIAM module containing the talin binding site of RIAM joined to the membrane-targeting sequence of Rap1A. This minimized Rap-RIAM module was sufficient to target talin to the plasma membrane and to mediate integrin activation, even in the absence of Rap1 activity. We identified a short talin binding sequence in Lamellipodin (Lpd), another MRL protein; talin binding Lpd sequence joined to a Rap1 membrane-targeting sequence is sufficient to recruit talin and activate integrins. These data establish the mechanism whereby MRL proteins interact with both talin and Ras GTPases to activate integrins. Increased affinity ("activation") of cellular integrins is central to physiological events such as cell migration, assembly of the extracellular matrix, the immune response, and hemostasis (1Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6765) Google Scholar). Each integrin comprises a type I transmembrane α and β subunit, each of which has a large extracellular domain, a single transmembrane domain, and a cytoplasmic domain (tail). Talin binds to most integrin β cytoplasmic domains and the binding of talin to the integrin β tail initiates integrin activation (2Petrich B.G. Fogelstrand P. Partridge A.W. Yousefi N. Ablooglu A.J. Shattil S.J. Ginsberg M.H. J. Clin. Investig. 2007; 117: 2250-2259Crossref PubMed Scopus (105) Google Scholar, 3Petrich B.G. Marchese P. Ruggeri Z.M. Spiess S. Weichert R.A. Ye F. Tiedt R. Skoda R.C. Monkley S.J. Critchley D.R. Ginsberg M.H. J. Exp. Med. 2007; 204: 3103-3111Crossref PubMed Scopus (216) Google Scholar, 4Tadokoro S. Shattil S.J. Eto K. Tai V. Liddington R.C. de Pereda J.M. Ginsberg M.H. Calderwood D.A. Science. 2003; 302: 103-106Crossref PubMed Scopus (961) Google Scholar). A small, PTB-like domain of talin mediates activation via a two-site interaction with integrin β tails (5Wegener K.L. Partridge A.W. Han J. Pickford A.R. Liddington R.C. Ginsberg M.H. Campbell I.D. Cell. 2007; 128: 171-182Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar), and this PTB domain is functionally masked in the intact talin molecule (6Yan B. Calderwood D.A. Yaspan B. Ginsberg M.H. J. Biol. Chem. 2001; 276: 28164-28170Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). A central question in integrin biology is how the talin-integrin interaction is regulated to control integrin activation; recent work has implicated Ras GTPases as critical signaling modules in this process (7Kinbara K. Goldfinger L.E. Hansen M. Chou F.L. Ginsberg M.H. Nat. Rev. Mol. Cell Biol. 2003; 4: 767-776Crossref PubMed Google Scholar).Ras proteins are small monomeric GTPases that cycle between the GTP-bound active form and the GDP-bound inactive form. Guanine nucleotide exchange factors (GEFs) promote Ras activity by exchanging bound GDP for GTP, whereas GTPase-activating proteins (GAPs) 3The abbreviations used are: GAP, GTPase-activating protein; RIAM, Rap1-GTP-interacting adaptor molecule; MRL, Mig-10/RIAM/Lamellipodin; RA, Ras association; PH, pleckstrin homology; HA, hemagglutinin; GFP, green fluorescent protein; GST, glutathione S-transferase; MFI, median fluorescence intensity; Lpd, Lamellipodin.3The abbreviations used are: GAP, GTPase-activating protein; RIAM, Rap1-GTP-interacting adaptor molecule; MRL, Mig-10/RIAM/Lamellipodin; RA, Ras association; PH, pleckstrin homology; HA, hemagglutinin; GFP, green fluorescent protein; GST, glutathione S-transferase; MFI, median fluorescence intensity; Lpd, Lamellipodin. enhance the hydrolysis of Ras-bound GTP to GDP (for review, see Ref. 8Marshall C.J. Curr. Opin. Cell Biol. 1996; 8: 197-204Crossref PubMed Scopus (471) Google Scholar). The Ras subfamily members Rap1A and Rap1B stimulate integrin activation (9Bos J.L. Curr. Opin. Cell Biol. 2005; 17: 123-128Crossref PubMed Scopus (392) Google Scholar, 10Kooistra M.R. Dube N. Bos J.L. J. Cell Sci. 2007; 120: 17-22Crossref PubMed Scopus (253) Google Scholar). For example, expression of constitutively active Rap1 activates integrin αMβ2 in macrophage, and inhibition of Rap1 abrogated integrin activation induced by inflammatory agonists (11Bertoni A. Tadokoro S. Eto K. Pampori N. Parise L.V. White G.C. Shattil S.J. J. Biol. Chem. 2002; 277: 25715-25721Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 12Caron E. Self A.J. Hall A. Curr. Biol. 2000; 10: 974-978Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 13Reedquist K.A. Ross E. Koop E.A. Wolthuis R.M. Zwartkruis F.J. van Kooyk Y. Salmon M. Buckley C.D. Bos J.L. J. Cell Biol. 2000; 148: 1151-1158Crossref PubMed Scopus (363) Google Scholar). Murine T-cells expressing constitutively active Rap1 manifest enhanced integrin dependent cell adhesion (14Sebzda E. Bracke M. Tugal T. Hogg N. Cantrell D.A. Nat. Immunol. 2002; 3: 251-258Crossref PubMed Scopus (244) Google Scholar). In platelets, Rap1 is rapidly activated by platelet agonists (15Franke B. Akkerman J.W. Bos J.L. EMBO J. 1997; 16: 252-259Crossref PubMed Scopus (364) Google Scholar, 16Franke B. van Triest M. de Bruijn K.M. van Willigen G. Nieuwenhuis H.K. Negrier C. Akkerman J.W. Bos J.L. Mol. Cell. Biol. 2000; 20: 779-785Crossref PubMed Scopus (83) Google Scholar). A knock-out of Rap1B (17Chrzanowska-Wodnicka M. Smyth S.S. Schoenwaelder S.M. Fischer T.H. White Jr., G.C. J. Clin. Investig. 2005; 115: 680-687Crossref PubMed Scopus (252) Google Scholar) or of the Rap1GEF, RasGRP2 (18Bergmeier W. Goerge T. Wang H.W. Crittenden J.R. Baldwin A.C. Cifuni S.M. Housman D.E. Graybiel A.M. Wagner D.D. J. Clin. Investig. 2007; 117: 1699-1707Crossref PubMed Scopus (160) Google Scholar), resulted in impairment of αIIbβ3-dependent platelet aggregation, highlighting the importance of Rap1 in platelet aggregation in vivo. Thus, Rap1 GTPases play important roles in the activation of several integrins in multiple biological contexts.Several Rap1 effectors have been implicated in integrin activation (19Katagiri K. Maeda A. Shimonaka M. Kinashi T. Nat. Immunol. 2003; 4: 741-748Crossref PubMed Scopus (397) Google Scholar, 20Lafuente E.M. van Puijenbroek A.A. Krause M. Carman C.V. Freeman G.J. Berezovskaya A. Constantine E. Springer T.A. Gertler F.B. Boussiotis V.A. Dev. Cell. 2004; 7: 585-595Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 21Zhang Z. Rehmann H. Price L.S. Riedl J. Bos J.L. J. Biol. Chem. 2005; 280: 33200-33205Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Rap1-GTP-interacting adaptor molecule (RIAM) is a Rap1 effector that is a member of the MRL (Mig-10/RIAM/Lamellipodin) family of adaptor proteins (20Lafuente E.M. van Puijenbroek A.A. Krause M. Carman C.V. Freeman G.J. Berezovskaya A. Constantine E. Springer T.A. Gertler F.B. Boussiotis V.A. Dev. Cell. 2004; 7: 585-595Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). RIAM contains Ras association (RA) and pleckstrin homology (PH) domains and proline-rich regions, which are defining features of the MRL protein family. In Jurkat cells, RIAM overexpression induces β1 and β2 integrin-mediated cell adhesion, and RIAM knockdown abolishes Rap1-dependent cell adhesion (20Lafuente E.M. van Puijenbroek A.A. Krause M. Carman C.V. Freeman G.J. Berezovskaya A. Constantine E. Springer T.A. Gertler F.B. Boussiotis V.A. Dev. Cell. 2004; 7: 585-595Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar), indicating RIAM is a downstream regulator of Rap1-dependent signaling. RIAM regulates actin dynamics as RIAM expression induces cell spreading; conversely, its depletion reduces cellular F-actin content (20Lafuente E.M. van Puijenbroek A.A. Krause M. Carman C.V. Freeman G.J. Berezovskaya A. Constantine E. Springer T.A. Gertler F.B. Boussiotis V.A. Dev. Cell. 2004; 7: 585-595Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). Whereas RIAM is greatly enriched in hematopoietic cells, Lamellipodin (Lpd) is a paralogue present in fibroblasts and other somatic cells (22Krause M. Leslie J.D. Stewart M. Lafuente E.M. Valderrama F. Jagannathan R. Strasser G.A. Rubinson D.A. Liu H. Way M. Yaffe M.B. Boussiotis V.A. Gertler F.B. Dev. Cell. 2004; 7: 571-583Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar).Recently we used forward, reverse, and synthetic genetics to engineer and order an integrin activation pathway in Chinese hamster ovary cells expressing a prototype activable integrin, platelet αIIbβ3. We found that Rap1 induced formation of an "integrin activation complex" containing RIAM and talin (23Han J. Lim C.J. Watanabe N. Soriani A. Ratnikov B. Calderwood D.A. Puzon-McLaughlin W. Lafuente E.M. Boussiotis V.A. Shattil S.J. Ginsberg M.H. Curr. Biol. 2006; 16: 1796-1806Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). Here, we have established the mechanism whereby Ras GTPases cooperate with MRL family proteins, RIAM and Lpd, to regulate integrin activation. We find that MRL proteins function as scaffolds that connect the membrane targeting sequences in Ras GTPases to talin, thereby recruiting talin to integrins at the plasma membrane.EXPERIMENTAL PROCEDURESAntibodies and Plasmid DNA Constructs—The activation-dependent mouse monoclonal antibody PAC1, activating mouse monoclonal antibody anti-LIBS6 (Ab33) against αIIbβ3, and the αIIbβ3-specificcompetitive inhibitor Ro43-5054 were previously described (23Han J. Lim C.J. Watanabe N. Soriani A. Ratnikov B. Calderwood D.A. Puzon-McLaughlin W. Lafuente E.M. Boussiotis V.A. Shattil S.J. Ginsberg M.H. Curr. Biol. 2006; 16: 1796-1806Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). Anti-HA mouse monoclonal antibody (MMS-101P) was purchased from Covance Research Products Inc. (Denver, PA). Anti-green fluorescent protein (GFP) rabbit polyclonal antibody was obtained from Clontech (Mountain View, CA). Anti-HA mouse monoclonal antibody (12CA5) and anti-integrin αIIb mouse monoclonal antibody (PMI-1) were also described elsewhere (24Shadle P.J. Ginsberg M.H. Plow E.F. Barondes S.H. J. Cell Biol. 1984; 99: 2056-2060Crossref PubMed Scopus (89) Google Scholar). Anti-RhoGDI rabbit polyclonal antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Mammalian expression vector encoding RIAM has been previously described (23Han J. Lim C.J. Watanabe N. Soriani A. Ratnikov B. Calderwood D.A. Puzon-McLaughlin W. Lafuente E.M. Boussiotis V.A. Shattil S.J. Ginsberg M.H. Curr. Biol. 2006; 16: 1796-1806Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). GFP-Lamellipodin was a gift from Dr. F. Gertler (Massachusetts Institute of Technology, Boston, MA). cDNAs encoding full-length and truncated human RIAM or Lamellipodin were amplified by PCR and subcloned into mammalian expression vector pEGFP-C1 and bacterial expression vector pGEX4T-1, respectively. DNA constructs containing membrane-targeting sequences of Rap1A (residues 165–184) were produced by PCR-based cloning. Plasmid constructs containing designated mutations were generated by using QuikChange® II XL site-directed mutagenesis kit from Stratagene (La Jolla, CA) with mutagenic primers. Plasmids encoding for HA-Rap1A (G12V) and HA-Rap1GAP have been described previously (23Han J. Lim C.J. Watanabe N. Soriani A. Ratnikov B. Calderwood D.A. Puzon-McLaughlin W. Lafuente E.M. Boussiotis V.A. Shattil S.J. Ginsberg M.H. Curr. Biol. 2006; 16: 1796-1806Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). Mammalian expression constructs for HA-mouse full-length talin and its mutant (W359A) constructs were previously reported (4Tadokoro S. Shattil S.J. Eto K. Tai V. Liddington R.C. de Pereda J.M. Ginsberg M.H. Calderwood D.A. Science. 2003; 302: 103-106Crossref PubMed Scopus (961) Google Scholar). Bacterial expression vector encoding His-tagged human full-length talin in pET30a was a generous gift from Dr. Liddington (Burnham Institute for Medical Research, La Jolla, CA). The authenticity of constructs was confirmed by DNA sequencing.In Vitro Protein Interaction Assay—Bacterial expression plasmids encoding glutathione S-transferase (GST)-RIAM, GST-Lpd, their mutants, or GST vector were expressed in BL21(DE3) (Novagen, Madison, WI), and recombinant proteins were purified on glutathione-Sepharose beads according to manufacturer's instructions (GE Healthcare). His-tagged full-length talin was expressed in BL21(DE3) with 0.2 mm isopropyl 1-thio-d-galactopyranoside overnight at room temperature and purified using Ni-NTA His-bind® resin (Novagen) affinity matrix. Purified talin was dialyzed overnight in a buffer (50 mm Tris-HCl (pH 7.4), 200 mm NaCl, 0.1% Triton X-100, and 1 mm dithiothreitol). Interaction of GST-RIAM proteins or GST with His-talin was conducted in a reaction buffer (50 mm Tris-HCl (pH 7.4), 200 mm NaCl, 0.1% Triton X-100, 5–10 mg/ml bovine serum albumin, 1 mm phenylmethylsulfonyl fluoride, 10 μm E-64, protease inhibitor mixture (complete mini, Roche Applied Science). 10 μg of purified GST fusion RIAM proteins on affinity matrix was mixed with 20 μg of His-tagged talin and incubated at 4 °C for 1 h. After washing the beads with reaction buffer, samples were fractionated on 4–20% SDS-PAGE gel (Invitrogen). Bound proteins were analyzed by Western blotting or Coomassie Blue staining. Peptide inhibition assay was performed in conditions described above with purified RIAM peptides (GenScript Corp.), wild type (6–30, EDIDQMFSTLLGEMDLLTQSLGVDT), or mutant (6-30-4E, EDIDQEESTEEGEMDLLTQSLGVDT).Transient Transfection and Immunoprecipitation—A5 cells (Chinese hamster ovary cells stably expressing αIIbβ3) were cultured in Dulbecco's modified Eagle's medium supplemented with nonessential amino acids, l-glutamine, 10% fetal calf serum, and antibiotics. Lipofectamine and Plus reagents (Invitrogen) were used for transient transfection according to manufacturer's instruction. For immunoprecipitation, cell lysates were prepared in a lysis buffer (50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 0.5% IGEPAL CA-630, 1 mm phenylmethylsulfonyl fluoride, protease inhibitor mixture (complete mini) (Roche Applied Science). Cell lysates were immunoprecipitated with 5 μg of anti-HA monoclonal antibody (clone 12CA5) for 4 h at 4 °C. Protein G-Sepharose (Invitrogen) was added to the reaction mixture and further incubated for 1 h at 4 °C. After three washes with lysis buffer, beads were mixed with sample buffer and subjected to SDS-PAGE. Bound proteins were detected by Western blotting.Integrin αIIbβ3 Activation—A5 cells were transiently cotransfected with HA-tagged full-length talin and GFP-RIAM, GFP-Lpd, their mutants, or GFP control for 24 h. Three-color cytometry was employed to measure activation-specific antibody PAC1 binding to integrin αIIbβ3 as described (23Han J. Lim C.J. Watanabe N. Soriani A. Ratnikov B. Calderwood D.A. Puzon-McLaughlin W. Lafuente E.M. Boussiotis V.A. Shattil S.J. Ginsberg M.H. Curr. Biol. 2006; 16: 1796-1806Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). In brief, transfected cells were suspended and incubated with 0.1% PAC1 ascites, washed, and stained with R-phycoerythrin-conjugated goat anti-mouse IgM antibody (Biomeda, Foster City, CA) to detect bound PAC1. Cells were further incubated with propidium iodide (1 μg/ml), washed, and analyzed by FACScan flow cytometer (BD Biosciences). Collected data were analyzed using CellQuest software (BD Biosciences). The integrin activation index is defined as 100 × (F - F0)/(Fmax - F0), where F is the median fluorescence intensity (MFI) of PAC1 binding, F0 is MFI of PAC1 binding in the presence of 1 μm αIIbβ3-specific inhibitor Ro43-5054, and Fmax is MFI of PAC1 binding in the presence of 2 μm αIIbβ3-activating antibody anti-LIBS6. Activation index is represented as the mean ± S.E.) for n ≥ 3.Subcellular Fractionation—A5 cells transfected for 24 h were subjected to subcellular fractionation as described (23Han J. Lim C.J. Watanabe N. Soriani A. Ratnikov B. Calderwood D.A. Puzon-McLaughlin W. Lafuente E.M. Boussiotis V.A. Shattil S.J. Ginsberg M.H. Curr. Biol. 2006; 16: 1796-1806Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). Briefly, cells were harvested in a lysis buffer (20 mm Tris-HCl (pH 7.4), 50 mm NaCl, 2 mm MgCl2, 5 mm KCl, 1 mm phenylmethylsulfonyl fluoride, 10 μm E-64, protease inhibitor mixture (Complete mini, Roche Applied Science), and incubated on ice for 10 min. Swollen cells were disrupted by Dounce homogenization, and a fraction (total cell lysate) was saved for analysis. The remainder lysate was centrifuged at 2000 rpm for 10 min to pellet nuclei and unbroken cells. The supernatant was further centrifuged at 14,000 rpm for 30 min to pellet the membrane fraction, which was further washed and extracted in lysis buffer including 1% IGEPAL CA-630 at 4 °C. Total lysate and cytosolic and membrane fractions were fractionated by SDS-PAGE and analyzed for protein expression by Western blotting.Co-clustering of Integrin αIIbβ3 and Talin with RIAM or Lamellipodin—To label αIIbβ3 integrins, A5 cells transfected with mCherry-talin and various GFP-RIAM or GFP-Lpd constructs were incubated with 30 μg/ml D57-Alexa 647 (D57 monoclonal antibody directly conjugated to Alexa-Fluor 647 (Invitrogen)) for 20 min before plating on 10 μg/ml fibrinogen-coated coverslips. The cells were allowed to adhere for 30 min in complete Dulbecco's modified Eagle's medium, rinsed once in phosphate-buffered saline (PBS), and fixed with 3.7% formaldehyde in PBS. In some experiments, A5 cells were co-transfected with HA-Rap1GAP. After fixation and permeabilization in 0.1% Triton X-100 for 5 min, HA-Rap1GAP was immunostained using anti-HA monoclonal antibody (Covance Research Products) that was directly conjugated to Alexa-Fluor 350 (Invitrogen). Coverslips were subsequently mounted in Prolong Gold antifade reagent (Invitrogen) on slides. Epifluorescent images of cells were acquired with a 60× oil immersion objective on a Nikon Eclipse TE2000-U microscope equipped with the appropriate excitation and emission filter sets (Semrock Inc, Rochester, NY). Images as shown are maximal projections of deconvolved images that were acquired at 0.1-μm z-section intervals. Images were deconvolved using the 3D Blind Deconvolution algorithm of Auto-QuantX (Media Cybernetics Inc, Bethesda, MD). Additional post-acquisition processing of images were performed using ImageJ (rsb.info.nih.gov/ij) and Adobe Photoshop.RESULTS AND DISCUSSIONAn N-terminal 103-Residue Fragment of RIAM Binds Talin—RIAM mediates Rap1-dependent integrin activation by forming a complex containing activated Rap1, talin, and the integrin (23Han J. Lim C.J. Watanabe N. Soriani A. Ratnikov B. Calderwood D.A. Puzon-McLaughlin W. Lafuente E.M. Boussiotis V.A. Shattil S.J. Ginsberg M.H. Curr. Biol. 2006; 16: 1796-1806Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). To investigate the mechanism by which RIAM forms this complex, we examined the interaction of various RIAM fragments with talin. cDNAs encoding influenza hemagglutinin epitope-tagged full-length talin (HA-talin) and GFP-tagged full-length RIAM or RIAM fragments were constructed and used to transfect Chinese hamster ovary cells that express recombinant integrin αIIbβ3 (A5 cells) (25Loftus J.C. O'Toole T.E. Plow E.F. Glass A. Frelinger III, A.L. Ginsberg M.H. Science. 1990; 249: 915-918Crossref PubMed Scopus (325) Google Scholar). HA-talin was immunoprecipitated with anti-HA antibody, and associated GFP-RIAM fragments were detected by Western blotting with an anti-GFP antibody. As expected, full-length RIAM interacted with talin (Fig. 1A). RIAM contains several modular domains; thus, we analyzed fragments that specifically deleted one or more of these domains. The C-terminal half of RIAM, containing the principal ENA/VASP binding sites and the PH domain (20Lafuente E.M. van Puijenbroek A.A. Krause M. Carman C.V. Freeman G.J. Berezovskaya A. Constantine E. Springer T.A. Gertler F.B. Boussiotis V.A. Dev. Cell. 2004; 7: 585-595Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar), were dispensable for talin association because fragments lacking these domains (RIAM-(1–421) and RIAM-(1–301)) associated with talin, whereas a construct containing the RIAM C terminus (406–666) associated to a much lesser extent (Fig. 1A). A talin interacting site (1–301) was, thus, localized within a RIAM fragment that contains two coiled-coil regions (63–90 and 150–182) and a RA domain (Fig. 1A). RIAM-(1–301) was well expressed as a GST fusion protein in a prokaryotic expression system, affording us the opportunity to assess the direct binding of this region of RIAM to purified recombinant talin. Purified hexahistidine-tagged full-length recombinant talin (His6-talin) was incubated with GST-tagged fragments of RIAM (GST-RIAM) immobilized on glutathione-Sepharose beads, and bound talin was detected by protein staining after SDS-PAGE fractionation. Three GST-RIAM proteins containing the N terminus residues RIAM 1–301, 1–176, and 1–103 interacted with talin, whereas GST-RIAM-(104–666) or GST alone did not (Fig. 1B). Thus, talin interacts directly with RIAM, and the interaction is mediated by an N-terminal fragment that lacks the Rap1 binding RA domain, the PH domain, and most of the ENA/VASP binding motifs.Both Talin and Rap1 Binding Regions of RIAM Are Required for Integrin Activation—As noted above, RIAM-(1–176), which lacks the Rap1 binding domain, binds directly to talin. Because the activation state of Rap1 controls RIAM-dependent integrin αIIbβ3 activation (23Han J. Lim C.J. Watanabe N. Soriani A. Ratnikov B. Calderwood D.A. Puzon-McLaughlin W. Lafuente E.M. Boussiotis V.A. Shattil S.J. Ginsberg M.H. Curr. Biol. 2006; 16: 1796-1806Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar), we examined the capacity of this fragment to activate integrins in conjunction with talin. We first verified that there was low level spontaneous Rap1 activation in the Chinese hamster ovary cells (supplemental Fig. S1). We co-expressed GFP-RIAM fragments with HA-talin in A5 cells and used flow cytometry to assay the binding of an integrin αIIbβ3 activation-specific antibody, PAC1 (26Shattil S.J. Cunningham M. Hoxie J.A. Blood. 1987; 70: 307-315Crossref PubMed Google Scholar). Cells co-transfected with HA-talin and GFP-RIAM-(1–301), which contains both the talin and Rap1 binding sites, exhibited increased PAC1 binding (Fig. 2, A and B). In contrast, no increase in PAC1 binding was observed when talin was co-transfected with either GFP-RIAM-(1–176), which lacks the RA domain, or GFP-RIAM-(104–666), which contains the RA domain but not the talin binding site (Fig. 2, A and B). In these experiments we confirmed by immunoprecipitation that GFP-RIAM-(1–176) and GFP-RIAM-(1–301) associated with HA-talin in these cells, whereas GFP-RIAM-(104–666) failed to do so (Fig. 2C). Furthermore, as with full-length RIAM (23Han J. Lim C.J. Watanabe N. Soriani A. Ratnikov B. Calderwood D.A. Puzon-McLaughlin W. Lafuente E.M. Boussiotis V.A. Shattil S.J. Ginsberg M.H. Curr. Biol. 2006; 16: 1796-1806Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar), activation induced by RIAM-(1–301) depends on Rap1 activity, as it was blocked by co-transfection with Rap1GAP (Fig. 2D). Thus, RIAM-mediated integrin activation requires RIAM talin and RA binding domains and Rap activity.FIGURE 2The RA and talin binding domains of RIAM are required for integrin αIIbβ3 activation. A, A5 cells were transiently co-transfected with plasmids encoding HA-talin and GFP-RIAM-(1–301), -(1–176), -(104–666) or GFP as indicated. Flow cytometry was used to measure the binding of PAC1, an αIIbβ3 activation-specific antibody, to GFP-expressing cells as described under "Experimental Procedures." B, integrin activation index defined as 100 × (F - F0)/(Fmax - F0), where F is the MFI of PAC1 binding, F0 is MFI of PAC1 binding in the presence of 1 μm αIIbβ3-specific inhibitor Ro43-5054, and Fmax is the MFI of PAC1 binding in the presence of 2 μm αIIbβ3-activating antibody anti-LIBS6 calculated for the cells shown in A. Data are the means ± S.E. for n ≥ 3. Protein expression was assessed by Western blotting using anti-HA or anti-GFP antibodies. C, A5 cells were co-transfected with HA-talin and GFP-RIAM-(1–301), -(1–176), -(104–666), or GFP alone, and HA-talin was immunoprecipitated from the cell lysates with anti-HA antibody (12CA5). Co-immunoprecipitated proteins (IP talin) and protein expression in whole cell lysates (WCL) was assessed by Western blotting with antibodies against GFP for RIAM (upper panel) and against HA for talin (lower panel), as indicated. D, GFP-RIAM-(1–301) induced integrin activation was abolished upon co-transfection with Rap1GAP.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Membrane-targeting Sequences of Ras Proteins Bypass the Requirement of Rap1 Activity for RIAM-dependent Integrin Activation—As noted above, the RA domain and Rap1 activity were needed for RIAM-(1–301) to promote integrin activation. Ras family proteins such as Rap1 contain a C-terminal CAAX box that specifies C-terminal prenylation, proteolytic cleavage, and carboxymethylation (27Gutierrez L. Magee A.I. Marshall C.J. Hancock J.F. EMBO J. 1989; 8: 1093-1098Crossref PubMed Scopus (328) Google Scholar). In combination with nearby polybasic sequences and/or acylated Cys residues, this region of Ras family proteins plays a crucial role in the localization of these proteins to cellular membranes and to particular microdomains within these membranes (28Hancock J.F. Nat. Rev. Mol. Cell Biol. 2003; 4: 373-384Crossref PubMed Scopus (684) Google Scholar). To test the role of the membrane localization region of Rap1A in integrin activation, we joined the C-terminal 20 residues of Rap1A, which contains the CAAX box in combination with a Lys-rich region, with RIAM-(1–176), a fragment that contains the talin binding region but not the RA domain. Expression of GFP-RIAM-(1–176)-CAAX in combination with talin dramatically increased PAC1 binding to αIIbβ3, whereas as before, GFP-RIAM-(1–176) failed to do so (Fig. 3A). Because each RIAM construct was GFP-tagged, we were able to directly compare expression level with effect on integrin activation. The addition of the Rap1 membrane-localization sequence resulted in a >10-fold increase in the capacity of RIAM-(1–176) to activate integrin αIIbβ3 (Fig. 3B). Furthermore, integrin activation induced by GFP-RIAM-(1–176)-CAAX did not require Rap1 activity as it was insensitive to inhibition by Rap1GAP (Fig. 3C), indicating that RIAM-(1–176)-CAAX expression bypasses the requirement for Rap1 activity. The effect of RIAM-(1–176)-CAAX on integrin activation is dependent on the interaction between talin and integrin β3 cytoplasmic tail because GFP-RIAM-(1–176)-CAAX was not able to activate integrins when co-expressed with a talin mutant (talin(W359A)) that is deficient in binding to integrin β cytoplasmic domain (29Garcia-Alvarez B. de Pereda J.M. Calderwood D.A. Ulmer T.S. Critchley D. Campbell I.D. Ginsberg M.H. Liddington R.C. Mol. Cell. 2003; 11: 49-58Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar) (Fig. 3D). These results strongly suggest that one important effect of Rap1 interaction with RIAM is to drive recruitment of a RIAM-talin complex to the plasma membrane.FIGURE 3Tethering membrane targeting sequences of Rap1 to RIAM-(1–176) bypasses Rap1 requirement for integrin activation. A5 cells were co-transfected with HA-talin and GFP-tagged RIAM-(1–176) constructs with or without Rap1GAP, as indicated. Integrin activation was assayed by PAC1 binding and quantified using flow cytometry as described in Fig. 2. A, fusion of the membrane targeting sequences of Rap1 with RIAM-(1–176) to form RIAM-(1–176)-CAAX induced integrin activation. B, the extent of integrin
