The adaptor molecule RIAM integrates signaling events ...the RIAM RA domain was predicted to...

SC I ENCE S I GNAL ING | R EV I EW

CELL B IOLOGY

1Division of Hematology-Oncology, Beth Israel Deaconess Medical Center, HarvardMedical School, Boston, MA 02215, USA. 2Department of Medicine, Beth Israel DeaconessMedical Center, Harvard Medical School, Boston, MA 02215, USA. 3School of Medicine,Unit of Immunology, Complutense University of Madrid, 28040 Madrid, Spain.*Present address: Division of Thoracic Surgery at Tongji Hospital, Tongji MedicalSchool, Huazhong University of Science and Technology, Wuhan, China.†Corresponding author. Email: [email protected]

Patsoukis et al., Sci. Signal. 10, eaam8298 (2017) 22 August 2017

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The adaptor molecule RIAM integrates signaling eventscritical for integrin-mediated control of immunefunction and cancer progressionNikolaos Patsoukis,1,2 Kankana Bardhan,1,2 Jessica D. Weaver,1,2 Duygu Sari,1,2

Alvaro Torres-Gomez,3 Lequn Li,1,2* Laura Strauss,1,2

Esther M. Lafuente,3 Vassiliki A. Boussiotis1,2†

Lymphocyte activation requires adhesion to antigen-presenting cells. This is a critical event linking innate andadaptiveimmunity. Lymphocyte adhesion is accomplished through LFA-1, which must be activated by a process referred toas inside-out integrin signaling. Among the few signalingmolecules that have been implicated in inside-out integrinactivation in hematopoietic cells are the small guanosine triphosphatase (GTPase) Rap1 and its downstreameffectorRap1-interacting molecule (RIAM), a multidomain protein that defined the Mig10-RIAM-lamellipodin (MRL) class ofadaptor molecules. Through its various domains, RIAM is a critical node of signal integration for activation of T cells,recruitsmonomeric and polymerized actin to drive actin remodeling and cytoskeletal reorganization, and promotesinside-out integrin signaling in T cells. As a regulator of inside-out integrin activation, RIAMaffectsmultiple functionsof innate and adaptive immunity. The effects of RIAM on cytoskeletal reorganization and integrin activation haveimplications in cellmigration and trafficking of cancer cells. Weprovide an overview of the structure and interactionsof RIAM, and we discuss the implications of RIAM functions in innate and adaptive immunity and cancer.

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Identification of RIAM
The human Rap1-interacting molecule (RIAM) was identified in ayeast two-hybrid screen for candidate effectors of the small guanosinetriphosphatase (GTPase) Rap1 (1). Before its identification as a Rap1-interacting molecule, RIAM was identified as a binding partner of theamyloidb (A4)precursorprotein–binding, familyB,member1 (APBB1;also known as Fe65) andwas named amyloid b (A4) precursor protein–binding, family B,member 1 interacting protein (APBB1IP) accordingly(2). This interaction is mediated by the WW (tryptophan-tryptophan)domain of Fe65 interacting with the proline-rich regions of RIAM (2).In an independent study, the gene encodingRIAMwas also identified astranscriptionally induced in response to all-trans retinoic acid (ATRA)in the promyeloleukemic HL-60 cell line, and the protein was accord-ingly named retinoic acid–responsive proline-rich protein 1 (RARP-1)(3). In that system, it was found that forced expression in various celltypes suppressed transactivation of activator protein 1 (AP-1) and serumresponse element (SRE), leading to the conclusion that this protein wasfunctionally involved in cell growth arrest. An independent group alsoidentified RIAM as an interactor of Enabled/vasodilator-stimulatedproteins (Ena/VASP) family, which are involved in cell motility and actinpolymerization, and named it proline-rich EVH1 ligand 1 (PREL1) (4).This study reported that RIAM colocalized with Ena/VASP proteins atthe tips of lamellipodia and at focal adhesions in response to epidermalgrowth factor (EGF) treatment of fibroblasts. Because this event coin-cided temporally with Ras activation, the authors suggested that RIAMmight link Ras signaling to cytoskeleton remodeling during cell migra-tion and spreading (4). However, direct evidence for such interactionwas not identified.

Structure and Homologs of RIAMThe open reading frame of RIAM is 1998 base pairs and encodes a pro-tein of 665 amino acids. Structurally, RIAM contains an RA (RalGDS/AF-6 or Ras-association) domain, a PH (pleckstrin homology) domain,and twoproline-rich regions. Twoputative coiled-coil regions are presentat the N terminus (amino acids 62 to 89 and amino acids 149 to 181)(Fig. 1A) (1).

Upon identification as a Rap1-interacting molecule and structuralcharacterization of RIAM, database searches for homologous genes re-vealed that the proteins with highest homology to RIAM are humanlamellipodin (Lpd) (also known as KIAA1681 and AY494951) andLpd-S (a short isoformof human lamellipodin, also known asALS2CR9andBAB69020) (1,5). Furthermore,RIAMis related toproteinsCG11940(AAF49029) in Drosophila melanogaster and Mig-10 (P34400) inCaenorhabditis elegans (Fig. 1A. Comparison of the domain structuresof these proteins indicated that RIAM, Lpd, CG11940, and Mig-10have a proline-rich region at theC terminus and a highly conserved pat-tern of 27 amino acids predicted to be a coiled-coil region immediatelyN-terminal to the RA domain. In addition, comparison of the RA andPH domains in RIAM-related proteins showed regions of these do-mains to be conserved among the proteins (1). Collectively, these pro-teins define the MRL (Mig-10/RIAM/Lpd) family (6). Phylogeneticanalysis showed that the MRL proteins are conserved during evolutionbut Drosophila and C. elegans each only have one gene encoding anMRL family member (1).Mig-10 is the first member of theMRL familyand was identified in a screen for mutations associated with neuronalcell migration defects during C. elegans embryogenesis. Specifically, themig-10 gene is required for the long-range anteroposterior migration ofthe two canal-associated neurons, anterior lateral microtubule cells, andhermaphrodite-specific neurons and for proper development of theexcretory canals (7).

Subsequently, mutations in the Drosophila MRL ortholog CG11940were identified, and the genewas named picodue to the retarded growthphenotype resulting from pico knockdownor loss-of-functionmutation(8). Reduction in pico expression inDrosophila resulted in animals with

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an overall reduction in body size due to decreased cell growth, whereasectopic expression of pico promoted coordinated cell growth and pro-liferation, leading to tissue overgrowth (8). These studies also indicatedthat the cell proliferative effect of pico in Drosophila is conserved withmammalian MRL proteins, because overexpression of Lpd in humancervical carcinoma (HeLa) cells stimulated EGF-mediated growth. Thiseffect appeared to depend on changes in actin dynamics and on theactivation of serum response factor (SRF), a transcription factor thatresponds to reduced globular (G)/filamentous (F) actin ratios throughits cofactor Mal (8).

RIAM is proline-rich (12.9%) and contains six putative profilin-binding motifs (XPPPPP) and six putative Ena/VASP homology 1(EVH1) protein-binding motifs (D/E)(F/L/W/Y)PPPPX(D/E)(D/E)(9). In addition, RIAM contains binding motifs for Src homology 3(SH3) domain– and WW domain–containing proteins (Fig. 1B) (10).The N-terminal structure of RIAM differs from other MRL familymembers and contains a short, proline-rich N-terminal region withtwo putative EVH1-binding sites and one additional coiled-coil do-main. RIAM and Lpd share conserved RA and PH domains, whereas


the N- and C-terminal regions are moredivergent (29.1% amino acid identity inthe N-terminal region and 23.2% aminoacid identity in the C-terminal region).In addition, the C-terminal region of Lpdis 500 amino acids longer than that ofRIAM (1, 5).

Expression and SubcellularLocalization of RIAMNorthern blot analysis has shown thatRIAM is expressedbroadly.Two transcriptsof 5.4 and 2.8 kb were detected in hema-topoietic tissues in approximately equalamounts, whereas in nonhematopoietictissues, the larger transcript predominated.The functional importance of the differen-tial expression of the twoRIAM transcriptsin these tissues has not yet been deter-mined. Nonhematopoietic tissues, inwhichthe RIAM transcript has been identified,include heart, brain, lung, liver, skeletalmuscle, kidney, and pancreas. The abun-dance of the RIAM protein varies amongtissues and cell types, but the greatestamount is detected in cells of hematopoieticorigin (1, 3, 4).

RIAM is constitutively localized to thecytosol and is recruited to sites of actin dy-namics upon cell activation. In humanJurkat T cells, human embryonic kidney(HEK) 293 cells, and mouse fibroblasts[both Swiss Albino 3T3 (SW3T3) andNIH 3T3] overexpressing RIAM, the pro-tein is detected in the cytoplasm but alsolocalizes to the plasma membrane and isconcentrated at the tips of lamellipodia(1, 4). Studies in B16F1 mouse melanomacells transfected with green fluorescent

protein (GFP)–tagged RIAM showed that, in fixed cells seeded on fib-ronectin, RIAM was present at the tips of the lamellipodia and in focaladhesions, whereas in live cells moving on laminin, RIAMwas targetedmainly to the tips of lamellipodia and, to a lesser extent, to focal adhe-sions (4). Treatment of liveNIH3T3mouse fibroblasts expressingGFP-taggedRIAMwith phorbolmyristate acetate orwithmicroinjection of aconstitutively active human Ras mutant (RasV12) also induced recruit-ment of RIAM to focal adhesions and to the tips of lamellipodia, sug-gesting that recruitment of RIAM to these locations may depend uponthe activation state of the cell.

UponT cell activation, RIAM translocates to the actin cytoskeleton,as demonstrated by subcellular fractionation experiments and by con-focal imaging of fixed cells (11). In human platelets plated on fibrino-gen to induce spreading, RIAM localizes to vinculin-rich filopodia andlamellipodial edges (12). In fully spread platelets, RIAM is localizedwith vinculin and actin in structures that resemble focal adhesioncomplexes. In mouse fibroblasts, RIAM localizes to the focal adhesionsin a manner that depends on interaction with the nonphosphorylatedform of VASP (13).

A

B

1 665

1 268

Human RIAM

1 669Mouse RIAM

1 1250Human lamellipodin

1 1162Drosophila CG11940

1 680C. elegans mig-10

1 649Human lamellipodin-S

Human RIAM ΔC(LOC54518)

PR PRPHRA

** **

** **

**

**

**

** **

Fig. 1. Structure of RIAM and its homologs. (A) Schematic representation of the domain structure of humanRIAM with other MRL family proteins. The proline-rich (PR), RA, and PH domains are noted. Asterisks indicatecoiled-coil regions. (B) Human RIAM is proline-rich and contains six putative profilin-binding motifs (gray shading)and six EVH1-binding motifs (underlined).

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Molecular Interactions of RIAMInteractions of the RA and PH domainsThe small GTPase Rap1 of the Ras oncogene superfamily has beenlinked to secretion and cell proliferation and migration. RIAM func-tions downstream of Rap1 in inside-out integrin signaling. In vitro datahave suggested that RIAM interacts with guanosine triphosphate(GTP)–bound Rap1, but not with guanosine diphosphate–boundRap1, and exhibits weak, rather nonspecific binding to other RasGTPases (1, 14). The RA domain of RIAM has also been shown to in-teract with Ras by pull-down experiments using a recombinant gluta-thione S-transferase (GST)–tagged RIAM RA domain and lysates fromNIH 3T3 cells transfected with Myc-tagged RasV12. Although theRIAM RA domain binds to both GTP-bound Rap1 and constitutivelyactive Ras in vitro with similar affinities, only Rap1 controls RIAMtranslocation in intact cells (1, 4).

Interaction of Rap1-GTP with the RA domain of RIAM after T cellreceptor (TCR)–mediated cell activation is required for translocation ofRap1-RIAM to the plasmamembrane (1, 14). The PHdomain of RIAMis also found to be required for Rap1-GTP to interact with RIAMin vitro (1). Although this in vitro finding was initially attributed to thebiochemical properties and folding of the small recombinant proteinsused to test these interactions, the crystal structure of the RIAMRA-PHrevealed that these two domains form a single structural unit that is crit-ical for RIAM function (14). RIAMcolocalizeswithRap1-GTPonly at theplasma membrane and not in any intracellular membrane compartment,in which Rap1-GTP is present, in contrast to the R-ras–binding domain(RBD) of guanine nucleotide dissociation stimulator for Ral (RalGDS-RBD), which colocalizes with Rap1-GTP in both compartments. Plasmamembrane localization of RIAM requires Rap1-GTP but not Ras-GTP.

The affinity of the RIAM PH domain for various members of thephosphatidylinositol (PtdIns) family of lipids was initially investigatedby probing lipid-coatedmembranes with various RIAM domains fusedto GST. These studies showed that the PH domain of RIAM has spec-ificity for phosphatidylinositol monophosphates PtdIns(3)P, PtdIns(4)P,and PtdIns(5)P (4, 5). Subsequently, by using various fluorescentlylabeled phosphatidylinositols (PIPs), recombinant RIAM RA-PH, andfluorescence polarization, it was discovered that RIAM has the highestbinding affinity for phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2;also called PIP2], which is present in the plasma membrane and a sub-strate for phospholipaseC–g1 (PLC-g1),making this PIP themost likelyphysiological target of the RIAM PH domain (14). Analysis of thebinding affinities of the RIAM RA and PH domains showed that boththe RA and PH domains have relatively low affinity for their specificbinding partners Rap1-GTP and PI(4,5)P2, respectively. Furthermore,the crystal structure of RIAM RA-PH showed that the RA and PH do-mains ofRIAMforma single structural unit through an extensiveRA-PHdomain interface, which is further fortified by interactions from residuesin the intervening linker region. On the basis of the crystal structures ofother GTPase-RA domain complexes (15, 16), Lys213 in the a1 helix ofthe RIAM RA domain was predicted to interact with Asp33 of Rap1.Mutation of this lysine residue prevented colocalization of RIAMRA-PH–GFP to the plasma membrane. The amino acid sequence inthe b1-b2 loop region of the PH domain of RIAM conforms to the con-sensus K-Xn-(K/R)-X-R sequence, which is associated with PIP binding(17). Mutation of Lys331, Arg333, and Lys327 within this sequence abro-gated recruitment of RIAM RA-PH–GFP to the plasma membrane(14). These results provide evidence that binding of both componentsof the integrated RA-PH unit to their natural partners and RA-PHstructural integration are required for recruitment of RIAM to the plasma


membrane. This dual binding is likely required because both the RAand the PH domain bind their partners with relatively low affinity.

The crystal structure of RIAM RA-PH in complex with Rap1-GTPrevealed that several side-chain interactions are critical in determiningspecificity of recognition of RIAM by Rap1-GTP (18). In particular,Lys31 of Rap1, which is oppositely charged compared with the Glu31/Asp31 residue in other Ras GTPases, forms a salt bridge with the RIAMresidueGlu212,making it the key specificity determinant of the interaction.Disruption of these interactions results in the reduction of Rap1-RIAMassociation, leading to a loss of coclustering and cell adhesion, consistentwith the finding that an intact Rap1-RIAM module is required forinside-out integrin activation (19).

Interactions of the proline-rich regionsRIAM has an N- and C-terminal proline-rich region, which containswell-defined proline-rich motifs (Fig. 1B). RIAM has six putativeprofilin-binding motifs (XPPPPP) and six putative EVH1-bindingmotifs (D/E)(F/L/W/Y)PPPPX(D/E)(D/E). In addition, RIAMcontainsproline-rich motifs for binding of SH3 domain– and WW domain–containing proteins (10). The proline-rich regions of RIAM mediateinteractionwith theWWdomain of Fe65 as determined by coimmuno-precipitation of endogenous proteins (2).WWdomain–mediated inter-actions have also been identifiedwith formin-bindingprotein 11 (FBP11)and growth arrest–specific protein 7 (Gas7) (20). The proline-richregions of RIAM also interact with profilin and with the EVH1 domainof Ena/VASP proteins, as determined by yeast two-hybrid assays,in vitro associationof recombinant proteins, and coimmunoprecipitationof endogenousproteins in JurkatT cells (1). The interactionofRIAMwithEna/VASP family proteinswas also identified by coimmunoprecipitationof endogenous mammalian Ena (Mena) with GFP-tagged RIAM usingNIH 3T3 cells. Interaction with Ena/VASP proteins was also detectedbypull-down experiments usingGST-taggedEVH1domains and lysatesof NIH 3T3 cells transfected with GFP-tagged N- or C-terminal RIAMconstructs (4). RIAM-VASP interactionpreferentially occurswith thenon-phosphorylated form of VASP, as determined by immunoprecipitationsof endogenous proteins in lysates of mouse fibroblasts isolated fromwild-type and b3 integrin–deficient animals, in which VASP was notphosphorylated due to impaired activation of protein kinaseA (PKA) (13).

Profilin and Ena/VASP family proteins are important regulators of theactin cytoskeleton. Profilin associates with G-actin and promotes nu-cleotide exchange to create profilin-actin(adenosine 5′-triphosphate)complexes. When bound to profilin, actin monomers are added only tothe barbed ends of F-actin (21). The Ena/VASP family members Mena,VASP, and Evl are recruited to sites of actin cytoskeleton remodeling, suchas lamellipodia, filopodia, focal contacts, and the T cell–APC (antigen-presenting cell) contact site (22) but do not have an active role in the for-mation of T cell–APC conjugates (23). Each contains an EVH1 domainthat interacts with the proline-rich motif (D/E)(F/L/W/Y)PPPPX(D/E)(D/E)(abbreviated as FPPPP) present in proteins such as zyxin and vinculinthat target Ena/VASP proteins to focal adhesions (9) or in FYN bindingprotein (FYB-120/130), also known as FYB, ADAP (adhesion- anddegranulation-promoting adapter protein), and SLAP-130 (SLP-76–associated phosphoprotein of 130 kDa) [Fyb/SLAP(ADAP)], which re-cruits Ena/VASP to the T cell–APC interface (24, 25). They also haveproline-rich regions that bind to SH3 domain–containing proteins andprofilin and an EVH2 domain that mediates their tetramerization andinteracts with both G- and F-actin (22). Thus, by its interactions withprofilin and with Ena/VASP family proteins, RIAM is linked to cyto-skeletal modulation.

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The proline-rich C-terminal region of RIAM interacts with the SH3domain of PLC-g1. This interaction was identified by in vitro associa-tion of recombinant RIAM and PLC-g1 proteins and by pull-downexperiments using various GST-tagged domains of PLC-g1 and lysatesfrom primary human T cells and from Jurkat T cells and, conversely,using GST-tagged domains of RIAM and lysates from the same celltypes (11). These findings are intriguing because the RIAMPH domainhas specificity for the PLC-g1 substrate PI(4,5)P2 (14). Functional im-plications of RIAM–PLC-g1 interactions are discussed in the section onT cell–specific signaling interactions of RIAM.

Interaction with talinAfter Rap1 activation and recruitment of RIAM to the plasma mem-brane, RIAM recruits talin through an N-terminal talin-binding (TB)sequence of 103 amino acids that is predicted to form amphipathichelices. This interaction was identified by coimmunoprecipitationusing lysates of Chinese hamster ovary (CHO) cells stably expressingintegrin aIIbb3 and transiently cotransfected with hemagglutinin(HA)–tagged talin and various GFP-tagged human RIAM constructsof different lengths (26). Subsequently, it was discovered that theN-terminal domain of RIAM has two distinct TB sites (TBS1 and TBS2),but only TBS1 can recruit talin to the plasma membrane (27). RIAMTBS1 and TBS2 can recognize multiple sites in talin-R (rod) and talin-H(head) F2F3 regions, suggesting thatmultiple RIAMmolecules bind to asingle talin molecule. The primary RIAM-interacting sites of talin arelocated in the F3 and R8 regions of talin. RIAM binding to talin-F3,which is adjacent to talin’s integrin-binding site, also located in talin-F3,competes with the autoinhibitory R9 domain of talin for binding to thetalin-F3 domain, thereby promoting the unmasking of the integrin-binding site and allowing it to bind to integrin (28). Intriguingly, theTB region of RIAM may also promote RIAM autoinhibition by block-ing the interaction between RIAM and Rap1 (14). Because the TBregion is highly negatively charged, it may mask the positively chargedPI(4,5)P2-binding surface in the PH domain. Additional aspects of theRIAM-talin interaction are discussed below in the section about theintegrin activation machinery.

Interactions with T cell signaling proteinsRIAM participates in TCR signaling events. The TCR-proximal Srcfamily kinases Fyn andLck associatewith and induce tyrosine phospho-rylation of RIAM (29). RIAM is a critical node of signal integrationdownstream of the signalosome containing linker for activation ofT cells (LAT) and SH2 domain–containing leukocyte protein of 76 kDa(SLP-76) and is critical for activation of PLC-g1 downstream of theTCR (11). Through its proline-rich C-terminal region, RIAM interactsconstitutively with the SH3 domain of PLC-g1 (Fig. 2). Upon TCR ac-tivation, RIAMpromotes PLC-g1 recruitment to the actin cytoskeleton,which is essential for PLC-g1 activity (30, 31). Hence, knockdown ofRIAM in T cells results in impaired PLC-g1 activity as determined bya diminished induction of inositol triphosphate production and re-duced intracellular calcium release. Reduced production of these secondmessengers by knockdown of RIAM impairs activation of Ras guanyl-releasing protein 1 (RasGRP1) and calcium- and diacylglycerol-regulatedguanine nucleotide exchange factors (CalDAG-GEFs), resulting in de-fective GTP loading of Ras and Rap1 and reduced nuclear translocationof the nuclear factor of activated T cells (NFAT) family of transcriptionfactors. The combineddefects inTCR-mediated activationof PLC-g1 andRap1-induced activation of integrins when RIAM is knocked downcause a profound reduction of interleukin-2 (IL-2) production inT cells.


The finding that RIAM mediates its effects on PLC-g1 activationdownstream of LAT–SLP-76 signalosome explains why abrogation ofeither SLP-76 or RIAM results in impaired activation of PLC-g1 andproduction of IL-2.

RIAMwas also found to interact constitutively with the scaffold pro-tein Src kinase-associated phosphoprotein of 55 kDa (SKAP55), asdetermined by usingGST-RIAM fusion proteins and lysates from JurkatT cells (32). RIAM binding to SKAP55 did not interfere with RIAMbinding to Rap1, but abrogation of the SKAP55-RIAM interactionled to impaired cell adhesion after TCR activation. This study identifiedthe sequence encompassing the RA-PHdomains of RIAMas the regionthroughwhich association with SKAP55wasmediated. Because the RAdomain of RIAMalso interacts with Rap1 and the PHdomain of RIAMalso interacts with PI(4,5)P2, it remains unclear whether RIAM can in-teract with SKAP55, Rap1, and PI(4,5)P2 simultaneously or whetherseparate intracellular pools of RIAMmight be involved in distinct inter-actions and functions. A summary of the domain-specific interactionsof RIAM is shown in Fig. 2.

RIAM Is an Integral Part of the Integrin Activation MachineryThe Rap1-RIAM module is involved in inside-outintegrin activationAlmost every cell process involves adhesion to the microenvironmentthrough the extracellular matrix (ECM), a process that is mediated byintegrins. Integrins are heterodimeric transmembrane receptors com-posed of one a and one b subunit. They link the cytoskeleton to theECM by binding to ECM proteins through their large ectodomainsand binding to the actin cytoskeletonmicrofilaments through their cyto-plasmic tails. Integrin signaling promotes cell adhesion, proliferation,andmigration andpromotes cell survival throughcross-talkwith receptortyrosine kinases (33–35). In migrating cells, nascent adhesions form atthe leading edge after ECM engagement of integrins and are associatedwith polymerization of actin (36). Some of the nascent adhesions dis-assemble within minutes, whereas other persist, grow, and mature intofocal complexes and then into focal adhesions. This evolution of adhe-sion complexes is driven bymechanical force (37). The ability of integrinsto bind to ECM proteins is controlled by a distinct “inside-out” sig-naling mechanism, wherein a receptor-mediated intracellular signalinduces a conformational change of the integrin cytoplasmic domain,which is relayed through the transmembrane region to the ectodo-main, converting it from a low-affinity to a high-affinity ligand-bindingstate (38).

The small GTPase Rap1 has a key role in the activation of inside-outsignaling that induces b1 and b2 integrin conformational changes,leading to cell adhesion. This function of Rap1 is induced in responseto T cell stimulation by CD31 [also known as platelet endothelial celladhesionmolecule–1 (PCAM-1)] with an anti-CD31–specific antibodybut also with phorbol ester and, in addition to cell adhesion, mediatesT cell migration in response to chemokines (39–41). These in vitro find-ings regarding the role of Rap1 in promoting integrin activation werevalidatedandconfirmedby findings in lymphocytes fromRap1A-deficientmice, which exhibited impaired LFA-1 polarization in vitro, although nomeasurable defects in lymphocyte function were observed in vivo (42).Conversely, T cells from mice expressing constitutively active, GTP-bound Rap1A have increased integrin activation and integrin-mediatedadhesion (43). In contrast to Rap1A, which is ubiquitously expressed,Rap1B (encoded by a different gene) is expressed predominantly inplatelets and is involved in the activation of aIIbb3 integrin (44).

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When RIAM was identified as a Rap1 effector molecule, it was in-vestigated whether the Rap1-RIAM interaction might be involved ininside-out integrin activation. Using in vitro cell adhesion models, itwas shown that RIAM is a Rap1 effector in inside-out signaling and thatRIAMis required forRap1-induced affinity changes in b1 andb2 integrinsin T cells (1). Subsequent studies demonstrated that RIAM is also in-volved in Rap1-mediated activation of aIIbb3 integrin in platelets (45).Platelet aggregation requires incubation with a compound that inducesaIIbb3 activation, which is mediated by Rap1-GTP and talin. Using apathway reconstruction CHO cell experimental system, it was deter-mined that RIAM is mandatory for this process because knockdownof RIAM abrogated aIIbb3 activation in the presence of Rap1-GTP. Inplatelets, RIAM localizes to filopodia, lamellipodia, and focal adhesion–like structures. In primary megakaryocytes, RIAM knockdown blocksaIIbb3 activation induced by thrombin protease-activated receptors,which is mediated in a manner that depends on Rap1-GTP and talin(45). Themolecular and functional properties of RIAM and its selectiveeffect in promoting conformational changes of the integrin b chain dis-tinguish RIAM from the Rap1 adaptor regulator of adhesion and cellpolarization enriched in lymphoid tissues (RapL), which promotesLFA-1 activation by interacting with the integrin a chain (46).

The RIAM-talin module promotes conformational changesrequired for integrin activationTalin is a large protein that is organized into an N-terminal head (1 to433, talin-H; 50 kDa), which contains an F0 domain and a FERM do-main (including F1, F2, and F3 subdomains), and aC-terminal rod (482


to 2541, talin-R; 220kDa),which ismadeupof 13 consecutive helical bundles followedby an actin bindingmotif (Fig. 3A). RIAMrecruits talin through the specific TBS1corresponding to RIAM residues 7 to 30(26). The Rap1-RIAM complex promoteslocalization of talin to the plasma mem-brane through the membrane-anchoringcapacity of RIAM’s RA (to Rap1) andPH [to PI(4,5)P2] domains (14). Multipleinteracting sites between talin-R domainsand RIAMhave been identified. A secondTB site in RIAM (TBS2, residues 50 to 85)was also identified (47). This region caninteract with the R2 and R3 domains oftalin. However, TBS1, but not TBS2, in-teracts with talin in the cytoplasm, andthe R8 domain of talin, which forms hy-drophobic and electrostatic interactionswith RIAM, is the strongest binding sitefor the TBS1 region of RIAM. The inter-action of RIAM TBS1 with talin R8 is re-sponsible for recruiting talin to the plasmamembrane (27). Furthermore,RIAMTBS1binds to talin-H region at the F3 sub-domain, located in close proximity to theintegrin-binding site, which is also locatedin the F3 subdomain of talin (28). The in-teraction of RIAM TBS1 with talin-F3promotes the conformational opening oflatent talin, leading to the binding and ac-tivation of integrin (Fig. 3B).

The multiple binding sites of RIAM on talin-R may cooperativelystrengthen RIAM binding to talin, thus leading to effective RIAM-talincolocalization to themembrane. However, it is the interaction of RIAMTBS1 with the talin-H region that leads to talin activation by stericallypreventing the binding of talin-R domain with the talin-H domain, anautoinhibitory interaction thatmasks the integrin-binding site of talin-H(Fig. 3B) (28). RIAM also binds talin-R through a site in talin-R thatpartially overlaps the binding site for vinculin, a major focal adhesionadaptor. Structural and functional analysis revealed that vinculin candisplace RIAM on talin-R and can function as a switch to promote thematuration of focal adhesions and their turnover. Thus, after integrinactivation, RIAM may be displaced from talin by vinculin, which trig-gers focal adhesion reassembly. The vinculin-talin-integrin and RIAM-talin-integrin modules differ in abundance at various cellular sites, inthe mechanisms that regulate their formation, and in their majorfunctions (47).

The MRL-talin-integrin complex drivesmigratory protrusionsRIAM is abundant at the cell edge and at the lamellipodium, where itpromotes protrusion (48). Protrusive activity is likely due to the abilityof RIAM to increase actin polymerization, most likely due to its inter-actionwith profilin and Ena/VASP family proteins. Consistentwith thishypothesis, overexpression of RIAM in Jurkat T cells and in HEK293cells induced lamellae formation and cell spreading, whereas knock-down of RIAM resulted in defective actin polymerization and reducedthe amount of polymerized actin (1). RIAMseems to interact preferentially

Coiled-coiled region

Proline rich region: Binds WW domain on APBB1EVH1-binding domain

Coiled-coiled region

Talin-binding region: Binds F2, F3, R3, R8 regions on talinRole in integrin “inside-out” activationSelf-inhibitory: blocks RA-PH domain localization to PM

TB

N

C

CC

PR

CC

Domain Description

Interactions and functions of RIAM domains

Proline rich region: FPPPP motif binds Ena/VASP, XPPPP motif binds profilin-actin polymerization sitesBinds SH3 domain of PLCγ1

PR

Pleckstrin homology domain: Binds P1 (4,5) P2

Ras-association domain: Binds Rap1-GTPPM proximity detector: important module for RIAM translocation to the plasma membraneEncompassing region binds SKAP-55

RA

PH

30

263

437

7

176

310

665

Fig. 2. RIAM domains and their molecular interactions. The domains of RIAMmediate multiple distinct interactions,thus involving RIAM in multiple cellular functions.

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with the nonphosphorylated form of VASP, which is a substrate ofPKA. Impaired activation of PKA in mouse fibroblasts deficient in b3integrin resulted in loss of PKA-dependent phosphorylation of VASP,enhanced association of nonphosphorylated VASP with RIAM, andenriched formation of theVASP-RIAMcomplex at focal adhesions, lead-ing to increased binding of talin to b1 integrin. These events resulted inenhanced focal adhesion turnover andmigration (13). RIAMcolocalizeswith talin in lamellipodia and filopodia, and this might play a role inthe localization of activated integrins at these membrane protrusions(12, 26, 45). In contrast to RIAM, vinculin is enriched in maturing focaladhesions, which are present on and at the border of lamellipodia,where vinculin reinforces the ability of the adhesion to transmit andbear force (47, 49).

Live cell imaging combined with indirect bimolecular fluorescencecomplementation indicated that a RIAM–integrin aIIbb3–talin complexis enriched at the tips of growing actin filaments in lamellipodial andfilopodial processes, thus revealing themolecular basis of the formationof the so-called sticky fingers of these protrusions (50). Although RIAMis most abundant at the cell edge and lamellipodium, it is subsequentlyreduced inmature adhesions due to direct competitionwith vinculin forbinding to talin (48). Vinculin stabilizes adhesions and increases theirability to transmit forces, whereas RIAM promotes lamellipodial pro-


trusion. The transition of integrin-basedadhesions from drivers of lamellipodialprotrusion that contain RIAM, but notvinculin, to stable focal adhesions that con-tain vinculin, but not RIAM, delineates amolecular switch in adhesion maturation.Disruption of this complex by mutationsthat affect the TB site in RIAM results inimpaired cell protrusion (48). The protru-sive activity stimulated by RIAM is likelymediated by reorganization of the actincytoskeleton through its binding to bothprofilin and Ena/VASP proteins (1).

A similar result was also observedwhen a5b1 integrin was used instead ofintegrin aIIbb3 or the formation of Lpd-talin-integrin complexes (50). In this ex-perimental approach, talin-bound Lpd,which is a RIAM paralog present in allspecies except C. elegans andDrosophila,also formed a complexwitha5b1 integrinat the tips of actin-based protrusions.Together, these findings lead to the con-clusion that either mammalianMRL pro-tein (RIAM or Lpd) can form a complexwith activated integrin and talin (calledthe MIT complex) at the tips of stickyfingers. In this complex, the N terminusof the MRL protein binds to and recruitstalin to the plasma membrane to induceintegrin activation,whereas theC terminusof the MRL protein increases processiveactin polymerization, in part, by recruitingEna/VASP, thereby propelling the move-ment of the sticky fingers (Fig. 3C).

Involvement of RIAM on Cancer Cell Adhesion, Invasion,Migration, and GrowthRap1 is among the most closely related proteins to members of the Rasoncogene superfamily (51) but serves functions that are distinct fromthose of H-, K-, and N-Ras. Rap1 has been linked to cellular prolifera-tion and migration (52, 53), and aberrant Rap1 activity can lead tointegrin hyperactivity that is linked to tumor development and metas-tasis in several cancer types (54–59). Sustained activation of Rap1 inmouse hematopoietic stem cells causes expansion of hematopoietic pro-genitors, followed by a myeloproliferative disorder mimicking chronicmyeloid leukemia (58). Notably, both a constitutively active Rap1 mu-tant and Rap1 that has been activated in response to EGF1-mediatedstimulation localizes predominantly in the nucleus in epithelial cells(60). It was determined using a high-throughput proteomic approachthat several gene products encoding cytoskeletal regulator proteins,signalingmolecules, transcription factors, viability regulators, and proteintransporters are induced by Rap1-GTP, the active form of Rap1. Notably,under these conditions, fused in sarcoma–translocated in sarcoma (FUS-TLS), which is involved in the regulation of adhesion by the formation ofspreading initiation centers (61), is highly induced by Rap1-GTP (60).

As adhesion receptors, integrins transduce signals in a bidirectionalmanner (62). Integrin signaling canbe triggeredby an inside-outpathway,

Integrinactivation

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Domain organization of talin

nntttiiinnnn

Fig. 3. RIAM is an important component of the integrin activationmachinery. (A) Domain organization of talin. TheN-terminal talin-H is composed of F0, F1, F2, and F3 subdomains. TheC-terminal talin-R is composedof 13helical bundles(R1 to R13) followed by a dimerization domain (DD). (B) Model for talin membrane recruitment and activation. Uponagonist-mediated stimulation, talin is recruited to the plasma membrane by binding to membrane PI(4,5)P2 and theRap1-RIAMcomplex. These interactions promote a conformational change in talinmediatedby twodistinct and synergisticmechanisms that cooperatively unmask the integrin-binding site at the F3 subdomain of the talin head. (C) The N-terminalportion of RIAM (and possibly other MRL proteins) binds to and recruits talin to the integrin cytoplasmic tail to induceintegrin activation. The C-terminal portion of RIAM and other MRL proteins stimulate processive actin polymerization, inpart, by recruiting Ena/VASP, thereby propelling the movement of the “sticky fingers” ends of lamellipodia and filopodia.

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which requires the recruitment of active Rap1, RIAM, and talin to theplasma membrane (1, 26). Conversely, during outside-in signaling,integrin binds to the ECM, forms highly organized clusters, and initiatesdownstream signaling cascades in the cytoplasm (37). As mentionedabove, in migrating cells, nascent adhesions form at the leading edgeafter ECM engagement of integrins, and these are associated with po-lymerizing actin (36). A proportion of these nascent adhesions growand mature into focal adhesions that have a central role in drivingcancer cell migration and invasion. It has been determined that RIAMdepletion in human melanoma cells leads to impairment in persistentcell migration directionality, thus reducing melanoma cell invasion(63). Furthermore, RIAM-depleted melanomas display fewer lungmetastatic lesions in a mouse xenograft model. The compromisedinvasiveness of RIAM knockdown cells correlated with deficient asso-ciation between b1 integrin and talin and with inhibition of b1 integrin–dependent activation of the extracellular signal–regulated kinase 1(ERK1) and ERK2 mitogen-activated protein (MAP) kinases. In anindependent study, RIAM-depletedmelanoma cells were found to haveincreased numbers and stability of focal adhesions, which was inter-preted as resulting from defective focal adhesion disassembly (64). Al-teration of focal adhesion turnover in these cells was associated withdeficient activation of MAP kinase kinase (MEK), and overexpressionof constitutively active forms of MEK rescued focal adhesion dis-assembly and cell invasion.

Additional studies support the intriguing hypothesis that theRap1-RIAM module might play an important role in cancer growthby mediating the recruitment of cancer-promoting myeloid lineagecells to the tumor microenvironment. Recruitment of myeloid lineagecells into the tumor microenvironment promotes angiogenesis, im-munosuppression, and metastasis (65). A key mechanism involvedin the recruitment of CD11b+Gr1lo monocytic lineage cells andCD11b+Gr1hi granulocytic lineage cells by tumor-derived chemo-attractants to the tumor microenvironment involves the activationof the phosphoinositide 3-kinase g (PI3Kg), leading to activation ofPLC-g1 and subsequent initiation of a signaling cascade throughRasGrp1–CalDAG-GEFI and RasGrp1–CalDAG-GEFII, Rap1, andRIAM, which results in a4b1 integrin activation (66). This pathwayhas a mandatory role in tumor progression because genetic depletionof PLC-g, CalDAG-GEFI, CalDAG-GEFII, Rap1a, or RIAM was suf-ficient to prevent integrin a4 activation by chemoattractants or an ac-tivated formof the PI3Kg catalytic subunit (p110gCAAX). In contrast,an activated form of Rap1 (Rap1V12) promoted constitutive integrinactivation and cell adhesion that could only be blocked by inhibition ofRIAM or integrin a4b1. Blockade of PI3Kg, integrin a4b1, or Rap1asuppressed both the recruitment of monocytes and granulocytes totumors and tumor progression. PI3Kg activation appears to functionas a molecular switch that promotes tumor-mediated immuno-suppression, and its inhibition has an impact on therapeutic responsesto checkpoint inhibitor blockade (67, 68). These results stronglysupport a critical role for a PI3Kg-Rap1a-RIAM–dependent pathwayin regulating tumor inflammation, progression, and response to im-munotherapy.

RIAM Has an Active Role in Innate ImmunityRIAM is also a regulator of leukocyte recruitment (adhesion, transmi-gration) and pathogen clearance through complement-mediatedphagocytosis, revealing an important role of RIAM in activationand modulation of innate immune responses.


Complement-mediated phagocytosisOpsonization is the process bywhich antigens aremolecularly tagged toenhance their phagocytosis by immune cells. The b2 integrin phagocyticcomplement receptors CR3 and CR4 are classically involved in the rec-ognition and internalization of particles bound to iC3b, a proteolyticallyinactive cleavage fragment of complement component 3b (C3b) (Fig. 4A).These complement receptors play an important role in eliminatingcomplement-opsonized pathogens and apoptotic particles and contrib-ute to cell homeostasis during tissue remodeling (69). Their role asimportant modulators of the host inflammatory response is high-lighted by the fact that they are exploited by pathogenic bacteria andviruses to potentiate host cell invasion, hijacking signaling down-stream of the receptors to reduce inflammatory cytokine production(70, 71). In a similar fashion, activation ofCR3 indendritic cells duringapoptotic cell clearance promotes intracellular tolerogenic signals(72). Recent reports describe a role of RIAM in the regulation ofCR3 activity.

The CR3 receptor is present mainly in myeloid cells (monocytes,macrophages, neutrophils, and dendritic cells) and in a certain subset ofnatural killer (NK) cells. It is implicated in phagocytosis, cell-mediatedkilling, and chemotaxis through its interaction with structurally un-related ligands [iC3b, intercellular adhesion molecule–1 (ICAM-1),fibrinogen, coagulation factor X, denatured proteins, and variouspathogen-derived molecules] (73). CR3 activity is tightly regulated,requiring inside-out integrin signaling downstream of various receptors,suchasFcgR,TRL4, fMLPreceptor, cytokine receptors, andCD44(74,75).Activation of these receptors culminates in the binding of talin to the shortcytoplasmic tail of the b chain of integrinaMb2 (also calledmacrophage-antigen or Mac-1). Ligand binding further stabilizes the high-affinityconformation of integrin aMb2, which entails separation of the integrincytoplasmic tails, allowing for the recruitment of intracellular effectorproteins (76). Outside-in signaling is then initiated, promoting actincytoskeleton remodeling to ensure particle internalization, cell prolif-eration and survival, or phagocytosis-induced apoptosis (77, 78).

The increase in complement-dependent phagocytosis after inside-out activation of integrin aMb2 by CR3 depends on Rap1 activity. Inmacrophages, expression of the constitutively active mutant Rap1V12triggered complement-mediated phagocytosis in the absence of agoniststimulation, whereas expression of the dominant-negative mutantRap1N17 blocked lipopolysaccharide (LPS)–induced phagocytosis(79). Rap1 activation correlates with talin recruitment to integrinaMb2 during phagocytosis (80), although there is no evidence of a directinteraction between Rap1-GTP and talin. Different Rap1 effectors wereunder consideration as potential candidates to promote talin recruit-ment to phagocytic cups, among them RapL, afadin adherens junctionformation factor 6 (AF-6), Krev interaction trapped protein 1 [Krit-1;also known as cerebral cavernousmalformation-1 (CCM1)], andRIAM(81). RIAM has been shown to promote the acquisition of the high-affinity state of aMb2, as demonstrated by a reduction in the binding ofan activation reporter monoclonal antibody (CBRM1/5) to the aM sub-unit in human promyelocytic cell lines HL-60 and THP-1, in whichRIAM was knocked down by RNA interference. Whereas in controlcells LPS orN-formyl-Met-Leu-Phe ( fMLP) chemotactic peptide stim-ulation resulted in increased CBRM1/5 binding, in RIAM knockdown(KD) cells CBRM1/5 binding was abrogated, which points to RIAM asessential for full aMb2 activation (82).

In accordancewith the defective acquisition of the aMb2 high-affinitystate, this study showed that RIAM knockdown reduced complement-dependent phagocytosis in response to LPSor fMLP treatment as compared

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A B

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αMβ2

Fig. 4. RIAM in innate immune responses. (A) RIAM enhances pathogen clearance through complement-mediated phagocytosis and ROS production. (B) RIAM is requiredfor neutrophil migration, adhesion, extravasation, and polarity in response to chemokines. (C) RIAM is involved in activation of integrin aMb2 (Mac-1), which is a critical step inthe cooperative actions of neutrophils and platelets in producing neutrophil extracellular traps (NETs). (D) RIAM is involved in talin recruitment to the integrin cytoplasmic tailand Pyk2 activation, which are critical events for NK cell cytotoxic function.

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to control cells (82). Moreover, the reduction of aMb2 activation andimpaired phagocytosis in RIAM KD cells was observed even whenRap1 was activated with 8-(4-chlorophenylthio)adenosine 3’,5′-cyclicmonophosphate (8-CPT-cAMP), a cAMP analog that specificallyactivates the Rap1–guanine nucleotide exchange factor exchange nucle-otide protein directly activated by cAMP (EPAC). Similar results wereobtained when RIAM was knocked down in human peripheral bloodmonocyte–derived macrophages, confirming that aMb2 activation andcomplement-dependent phagocytosis induced by Rap1 were mediatedby RIAM. As pointed out above, talin plays a crucial role in stimulatingaMb2 activity by binding to the cytoplasmic tail of the b2 integrin sub-unit and inducing integrin activation. Coimmunoprecipitation exper-iments done in neutrophil-like differentiated HL-60 cells duringcomplement-dependent phagocytosis demonstrated reduced talin re-cruitment to b2 integrin when RIAM was knocked down. Confocalmicroscopy studies also indicated that talin localization to phagocyticcups containing the complement receptor was impaired in RIAMKDHL-60 cells (82). Together, these findings point toward a role of RIAMin linking Rap1 activation to talin recruitment to aMb2 integrin, therebypromoting its activation by CR3 and complement-dependent phago-cytosis (Fig. 4A).

The role of RIAM in promoting complement-dependent phagocy-tosis was assessed in an independent study using RIAM-null polymor-phonuclear leukocytes (PMNs) and serum-opsonized bacteria.RIAM−/−

PMNs presented reduced bacterial uptake compared to wild-typePMNs. Analysis of the respiratory burst in response to tumor necrosisfactor–a (TNF-a) revealed a reduction in free reactive oxygen species(ROS) production in RIAM−/− PMNs, pointing to a role of RIAM inb2 integrin–mediated outside-in signaling (83). These results are in ac-cordance with data obtained from RIAM KD human promyelocyticcells and indicate an essential role of RIAM in b2 integrin–mediatedphagocytosis (82).

Adhesion, extravasation, and motility of innateimmune cellsIntegrinaMb2 activation and ICAM-mediated adhesion of leukocytes tothe endothelium are required for transendothelialmigration and extrava-sation (Fig. 4B). Adhesion and spreading in response to binding ICAM-1,but not to binding vascular cell adhesion molecule–1 (VCAM-1), arereduced in RIAM-null PMNs in vitro, and reduced leukocyte adhesionand extravasation in response to chemokines were observed in vivo.RIAM-null mice exhibit a marked leukocytosis due to an increasednumber of neutrophils in the peripheral blood (83), a hallmark of leu-kocyte adhesion deficiency (LAD) syndromes associated with defectsin leukocyte extravasation (84). A role of RIAM in neutrophil polarityhas also been identified. Specifically, RIAMknockdown in neutrophil-like PLB-985 cells resulted in the loss of a clearly defined leading edgeon two-dimensional (2D) surfaces and impaired directionality (49)toward fMLP, decreasedmigration and chemotaxis velocity, and reduc-tion of protrusion formation in 3DMatrigel (82). These findings con-trast with those from migration analyses of cells in which Kindlin-3,an integrin coactivator that interacts with talin, was knocked down. InKindlin-3 KD cells, polarization was normal, yet cell motility and di-rectionality were impaired because of a reduction in F-actin content atthe leading edge (85). On the basis of these observations, Yamahashi et al.(85) speculated that Kindlin-3 works in parallel with RIAM, becauseKindlin-3 acts through inside-out signaling to facilitate directionality,whereas RIAM enables outside-in signaling to induce leading edgeformation.


The Role of RIAM in Hematopoietic Cells In VivoThe role of RIAM in promoting agonist-mediated activation of integrinaIIbb3 has been well documented by in vitro reconstructive studies.Moreover, RIAMknockdownblocks agonist-mediatedaIIbb3 activationin primarymousemegakaryocytes (12). Despite the compelling in vitrofindings, indicating an important role of the Rap1-RIAM-talin modulein aIIbb3 integrin activation, genetic deletion of RIAM in mice did notaffect platelet developmental, homeostasis, or platelet-related integrinfunctions (83, 86, 87). RIAM protein abundance is low in platelets,whereas Rap1, talin1, and integrins are highly abundant in these cells.Together, these findings indicate that RIAM-independent mechanismsexist for Rap1 to mediate its effects on platelet integrin function underphysiologic conditions. Moreover, Rap1-independent mechanisms ofintegrin activation have been identified in platelets (88), which mighthave a compensatory role in the absence of a functional Rap1-RIAMpathway.

The selective role of RIAM on integrin function in vivo is alsosupported by other findings inRIAM knockoutmice, which display onlya partial defect in the function of b1 integrin but a more pronouncedimpairment of b2 integrin activation. As a consequence, RIAM-deficientmice exhibit leukocytosis due to impaired leukocyte-endothelium adhe-sion and extravasation (83). Consistent with the indispensable role ofRIAM in trafficking of B and T lymphocytes to secondary lymphoidorgans, RIAM knockout mice display impaired humoral responses toT-dependent antigen (87).

Because the mandatory role of RIAM for b2 integrin activationin vivohas been established, there is ample reason to speculate thatRIAMis involved in functions of innate immune cells for which b2 integrinactivation is indispensable. Neutrophils cooperate with platelets to pro-duce NETs, structures composed of histones and DNA that trap extra-cellular pathogens (Fig. 4C). The formation of these structures ismediated by neutrophil aMb2 integrin and is tightly connected toROS production (89). RIAMKD cells have reduced integrin activation,and RIAM−/− neutrophils have reduced phagocytosis and ROS produc-tion (82, 83). Together, these findings are highly suggestive that NETproduction could depend on RIAM.

RIAM−/− macrophages exhibit defective autophosphorylation ofPyk2 at Y402 in response to adhesion on ICAM-1withoutmajor changesin phosphorylation of focal adhesion kinase (FAK) or Src. Pyk2 activityafter b2 integrin activation has also been correlated with neutrophil de-granulation responses and efficient clearance of Staphylococcus aureusinfection (90). Degranulation and ROS production in eosinophils stimu-lated by granulocyte macrophage colony-stimulating factor (GM-CSF)andplatelet activating factor (PAF) are known tobemediatedby integrinaMb2 (91) and correlatewith greater abundance of active eosinophilaMb2integrins, as observed in lung eosinophils from asthmatic patients (92).

In NK cells, LFA-1 engagement with ICAM-1–coated beads wasfound to be necessary and sufficient to produce granule polarizationwhen cells were treated with IL-2 (93). LFA-1–mediated adhesion ofNK cells to insect cells expressing human ICAM-1 resulted in target cellkilling. Cytotoxicity was diminished when the insect cells coexpressedICAM-1 andHLA-Cw4, a ligand for killing-inhibitory receptors onNKcells, indicating that b2 integrin induces early NK activation signals thatcan bemodulated by these inhibitory receptors (93). LFA-1 activation inNK cells also resulted in Pyk2 autophosphorylation and talin enrich-ment at the site of contact between the NK cell and the target cell(Fig. 4D) (94). Moreover, granule polarization and cytotoxicity requirePyk2 activation (95). Given that Pyk2 activation is abrogated in neutro-phils from RIAM-null mice (83) and that recruitment of talin to the

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integrin cytoplasmic tail requires RIAM (27, 50), RIAM could play arole in the activation of NK cell cytotoxic functions.

Thus, multiple lines of evidence converge onto the idea thatRIAM is essential for effective innate immune responses. First,RIAM orchestrates the migration of PMNs andmacrophages to sitesof infection due to its role in regulating adhesion, polarization, direc-tional migration, and extravasation. Second, RIAM plays a critical rolein pathogen clearance by promoting complement-dependent phagocy-tosis and subsequent ROS production. Finally, degranulation in PMNs,eosinophils, and NK cells requires b2 integrin engagement, which, inturn, is dependent on RIAM function. Therefore, an important roleof RIAM in the regulation of innate immune host defense can beinferred.

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Concluding Remarks, Challenges, and Future DirectionsRIAMwas identified as an adaptor defining theMRL family of proteins,a binding partner ofAPBB1, a protein induced in response to retinoic acidin promyelocytic cells, an Ena/VASP interactor, and a Rap1-interactingmolecule and is now a well-established regulator of multiple functionsin immune cells. RIAM is involved in reorganization andmodulation ofthe actin cytoskeleton, activation of integrins, cell adhesion, and migra-tion. In vivo, RIAM seems to have an indispensable role in b2 integrinactivation and function but only aminor impact on activation of b1 andb3 integrins. RIAM is involved in the regulation of innate and adaptiveimmune responses and in the invasion, migration, and metastasis ofcancer cells. RIAM promotes both inside-out and outside-in integrinsignaling. It is also involved in stabilizing PLC-g1 in close proximityto the cytoskeleton and at the plasma membrane and interacts specifi-cally with the PLC-g1 substrate PI(4,5)P2. These properties of RIAMmake it an attractive therapeutic target to selectively modulate inflam-matory cell processes mediated by b2 integrin and block cancer cellmigration.

The selective effect of RIAM on b2 integrin activation in vivo indi-cates that RIAM-independent Rap1-mediated activation of the talin-integrinmodule promotesb1 and b3 integrin activation in vivo.Althoughdetailed studies have determined how RIAM-talin interaction leadsto talin conformational changes and integrin activation as well as re-localization of the RIAM-talin-integrin complex to the leading edge ofcell protrusions during cell migration, it is clear that potent alternativemechanisms for integrin activation are also present. It will be importantto identify suchmechanisms and dissect their contribution in a cell- andtissue-specificmanner. For example, how is talin recruited to the plasmamembrane in the absence of RIAM?How is conformational opening oflatent talin induced, leading to the integrin binding and activation inRIAM-deficient cells, which retain in vivo activation of b1 integrinsalmost intact? One challenge is to determine and quantify the contribu-tionofRIAM-dependent andRIAM-independent pathways downstreamof Rap1 in the function of innate immune cells in the context of infec-tion, inflammation, and cancer. An additional challenge is to identifyhow the multiple molecules and pathways involved in integrin activa-tion converge to regulate cell adhesion and transendothelial migration.In the context of cancer, RIAMmight have a role in regulating not onlycancer cellmigration andmetastasis but also the properties of the tumormicroenvironment by controlling recruitment of tumor-promotingmyeloid cells, which confer resistance to cancer immunotherapy. Theselective effects of RIAMon b2 integrinmight provide a novel therapeutictarget for selectivemodulationof immune cells in certainmicroenviron-mentswhile leavingplatelet function intact. Such therapeutic approaches


might be highly beneficial in autoimmune diseases, inflammation, andcancer.

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Funding: This work was supported by NIH grants CA183605, CA183605S1, and AI098129-01and U.S. Department of Defense grant PC140571 (V.A.B.) and SAF2012-34561 and SAF2016-77096-R (E.M.L.). Author contributions: N.P., K.B., J.D.W., D.S., L.L., and L.S. wrote differentsections of the manuscript and generated two figures. A.T.-G. and E.M.L. wrote a section of themanuscript and generated one figure. V.A.B. generated one figure and integrated the varioussections of the manuscript and figures. All authors reviewed and approved the manuscript.Competing interests: V.A.B. has patents on the PD-1 pathway licensed by Bristol-MyersSquibb, Roche, Merck, EMD Serono, Boehringer Ingelheim, AstraZeneca, Novartis, and Dako.All other authors declare that they have no competing interests.

Submitted 23 January 2017Accepted 25 July 2017Published 22 August 201710.1126/scisignal.aam8298

Citation: N. Patsoukis, K. Bardhan, J. D. Weaver, D. Sari, A. Torres-Gomez, L. Li, L. Strauss,E. M. Lafuente, V. A. Boussiotis, The adaptor molecule RIAM integrates signaling eventscritical for integrin-mediated control of immune function and cancer progression. Sci.Signal. 10, eaam8298 (2017).

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of immune function and cancer progressionThe adaptor molecule RIAM integrates signaling events critical for integrin-mediated control

Esther M. Lafuente and Vassiliki A. BoussiotisNikolaos Patsoukis, Kankana Bardhan, Jessica D. Weaver, Duygu Sari, Alvaro Torres-Gomez, Lequn Li, Laura Strauss,

DOI: 10.1126/scisignal.aam8298 (493), eaam8298.10Sci. Signal.

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