RIAM (Rap1-Interactive Adaptor Molecule)
RIAM was identified in a search of new effector molecules of the small guanosine triphosphatase (GTPase) Rap1, using the yeast two-hybrid system screen. Due to its interaction with Rap1, the newly identified Rap1-interacting effector was named RIAM (Rap1-interacting adaptor molecule) (Lafuente et al. 2004). Before its identification as a Rap1-interacting molecule, RIAM was found as a binding partner of the amyloid beta (A4) precursor protein-binding, family B, member 1 (APBB1) – also known as neural Fe65 protein – and was termed amyloid beta (A4) precursor protein-binding, family B, member 1 interacting protein (APBB1IP) (Ermekova et al. 1997). In an independent study, RIAM was identified as a protein whose expression was induced in response to all-trans retinoic acid (ATRA) in the promyeloleukemic HL-60 cell line (Inagaki et al. 2003). The protein was named retinoic acid-responsive proline-rich protein 1 (RARP-1). An independent group also identified the protein as an Ena/VASP interactor and named it proline-rich EVH1 ligand 1 (PREL1) (Jenzora et al. 2005). This study reported that RIAM co-localized with the Ena/VASP proteins at the tips of lamellipodia and at focal adhesions in response to EGF treatment of fibroblasts, an event that coincided temporally with Ras activation.
Structure and Homologues of RIAM
RIAM is proline-rich (12.9%) and contains six putative profilin-binding motifs (XPPPPP) and six putative EVH1-binding motifs (D/E)(F/L/W/Y)PPPPX(D/E)(D/E), which interact with profilin and EVH1 domain-containing proteins, respectively (Niebuhr et al. 1997). In addition, RIAM contains binding motifs for SH3 and WW domain-containing proteins (Holt and Koffer 2001). RIAM and Lpd share conserved RA and PH domains, whereas the N-terminus and C-terminus regions are more divergent (29.1% amino acid identity in the N-terminal and 23.2% amino acid identity in the C-terminal). In addition, the C-terminus region of Lpd is 500 amino acids longer than that of RIAM (Lafuente et al. 2004; Krause et al. 2004).
Expression of RIAM
Northern blot analysis has shown that RIAM is expressed broadly. RIAM is expressed predominantly in hematopoietic tissues, where a larger transcript of 5.4 Kb predominates. It is also expressed to a lesser extend in nonhematopoietic tissues, where a smaller 2.8 Kb predominates (Lafuente et al. 2004; Inagaki et al. 2003; Jenzora et al. 2005). RIAM is constitutively localized in the cytosol and is recruited to sites of actin dynamics. In Jurkat T cells, HEK293 cells, and mouse fibroblasts overexpressing RIAM, the protein localizes in the cytoplasm and at the plasma membrane and concentrates at the tips of lamellipodia (Lafuente et al. 2004; Krause et al. 2004). Studies in B16F1 mouse melanoma cells transfected with GFP-tagged RIAM showed that RIAM was targeted mainly to the tips of lamellipodia and, to a lesser extent, to focal adhesions (Krause et al. 2004). Upon T-cell activation, RIAM translocates to the actin cytoskeleton, as identified by subcellular fractionation experiments and by confocal imaging of fixed cells (Patsoukis et al. 2009). In human platelets, RIAM was localized in vinculin-rich filopodia and lamellipodial edges (Watanabe et al. 2008). In mouse fibroblasts, RIAM was detected to localize at the focal adhesions in a manner dependent on the interaction with the dephosphorylated form of VASP (Worth et al. 2010).
Interactions of RIAM
Interactions of RA and PH Domains
In vitro data have suggested that RIAM interacts with GTP-bound Rap1 but not with GDP-bound Rap1 and retains weak, rather nonspecific binding capability with other Ras GTPases (Lafuente et al. 2004; Wynne et al. 2012). The RA domain of RIAM has also been shown to interact with Ras by pull-down experiments using a recombinant glutathione-S-transferase (GST)-tagged RA domain of RIAM and lysates of NIH 3 T3 cells transfected with a constitutively active, myc-tagged Ras mutant (RasV12). Although the RA domain binds to both GTP-bound Rap1 and Ras in vitro with similar affinities, only Rap1 controls RIAM subcellular distribution in intact cells (Lafuente et al. 2004; Jenzora et al. 2005; Wynne et al. 2012). The Rap1/RIAM module translocates to the plasma membrane, and interaction of Rap1-GTP with the Ras association (RA) domain of RIAM is required for this event. Importantly, for interaction with Rap1-GTP, not only the RA but also the PH domain of RIAM is required (Lafuente et al. 2004). The crystal structure of the RIAM RA-PH revealed that these two domains form a single structural unit (Wynne et al. 2012). RIAM co-localizes with Rap1-GTP only at the plasma membrane and not in any intracellular membrane compartment in which Rap1-GTP is present.
The PH domain of RIAM has significant binding affinity for PI(4,5)P2,which is present in the plasma membrane, making this PIP the most likely physiological target for the RIAM PH domain (Wynne et al. 2012). The crystal structure of RIAM RA-PH showed that the RA and PH domains of RIAM form a single structural unit through an extensive RA-PH domain interface, which is further fortified by interactions from residues in the intervening linker region. Moreover, binding of both components of the integrated RA-PH unit to their natural partners and RA-PH structural integration is co-required for recruitment of RIAM to the plasma membrane. This dual binding is likely required because both the RA and the PH domain bind their partners, Rap1-GTP and PI(4,5)P2, respectively, with relatively low affinity.
The crystal structure of RIAM RA-PH in complex with Rap1-GTP revealed that several side-chain interactions are critical in determining specificity of recognition of RIAM by Rap1-GTP (Zhang et al. 2014). Disruption of these interactions results in reduction of Rap1/RIAM association, leading to a loss of co-clustering and cell adhesion (Lafuente and Boussiotis 2006), consistent with the finding that an intact Rap1/RIAM module is required for integrin activation.
Interactions of the Proline-Rich Regions
RIAM contains six putative profilin-binding motifs (XPPPPP) and six putative EVH1-binding motifs (D/E)(F/L/W/Y)PPPPX(D/E)(D/E), binding motifs for SH3 and WW domain-containing proteins (Holt and Koffer 2001). Via its proline-rich regions, RIAM interacts with the WW domain of Fe65 (Ermenkova et al. 1997). WW domain-mediated interactions have also been identified with formin-binding protein 11 (FBP11) and growth arrest-specific protein 7 (Gas7) (Ingham et al. 2005). The proline-rich regions of RIAM also interact with profilin and with the EVH1 domain of Ena/VASP proteins (Lafuente et al. 2004).
Profilin and Ena/VASP family proteins are important regulators of the actin cytoskeleton. Profilin associates with monomeric G-actin and promotes nucleotide exchange to create profilin-actin (ATP) complexes. When profilin-bound, actin monomer is added only to the barbed ends of F-actin (Pollard and Borisy 2003). The Ena/VASP family members Mena, VASP, and Evl are recruited to sites of actin cytoskeleton remodeling such as lamellipodia, filopodia, focal contacts, and the T cell/APC contact site (Krause et al. 2003). They contain an EVH1 domain that interacts with a proline-rich motif (FPPPP) present in proteins such as zyxin and vinculin that target Ena/VASP proteins to focal adhesions (Niebuhr et al. 1997) or in Fyb/SLAP that recruits Ena/VASP to the T cell/APC interface (Renfranz and Beckerle 2002). They also have proline-rich regions that bind to SH3 domain-containing proteins and profilin, as well as an EVH2 domain that mediates their tetramerization and interacts with G- and F-actin (Krause et al. 2003). The interactions with profilin and with Ena/VASP family proteins link RIAM to cytoskeletal modulation. The proline-rich C-terminal region of RIAM interacts with the SH3 domain of PLCγ1 (Patsoukis et al. 2009). This finding is intriguing because the RIAM PH domain has specificity for phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], the phospholipase C gamma 1 (PLCγ1) substrate (Wynne et al. 2012).
Interaction with Talin
After Rap1 activation and membrane translocation, RIAM recruits talin via an N-terminal talin-binding (TBS1) sequence (103 amino acids, predicted to form amphipathic helices) (Lee et al. 2009). A second talin-binding site is also present in the N-terminal domain of RIAM (TBS2), but only TBS1 can recruit talin to the plasma membrane (Chang et al. 2014). RIAM TBS1 and TBS2 can recognize multiple sites in talin-R (rod) as well as in talin-H (head) F2F3 regions. The primary RIAM-interacting sites of talin are located in talin-F3 and talin-R8. RIAM binding to talin-F3, which is next to the integrin-binding site of talin, competes with the auto-inhibitory talin-R9 for binding to talin-F3 and promotes unmasking of the integrin-binding site of talin, which allows its binding to integrin leading to integrin activation (Yang et al. 2014).
The TCR-proximal Src family kinases Fyn and Lck associate with RIAM and induce its tyrosine phosphorylation (Sari et al. 2014). RIAM is a critical node of signal integration downstream of the LAT/SLP-76 signalosome with a mandatory role for activation of PLCγ1 in T cells (Patsoukis et al. 2009). Specifically, via its proline-rich C-terminal region, RIAM interacts constitutively with the Src homology 3 (SH3) domain of PLCγ1. Upon TCR triggering, RIAM regulates PLCγ1 recruitment to the actin cytoskeleton, a process that is essential for PLCγ1 activity (Yang et al. 1994). Hence, knockdown of RIAM expression in T cells results in impaired PLCγ1 activity as leading to diminished generation of inositol triphosphate (IP3) and intracellular calcium release. Reduced levels of these second messengers by knockdown of RIAM expression result in impaired activation of Ras guanyl-releasing protein 1 (RasGRP1) and calcium and diacylglycerol-regulated guanine nucleotide exchange factors (CalDAG-GEFs), defective Ras and Rap1-GTP loading and reduced transcriptional activation of NFAT, leading to a profound defect on interleukin-2 (IL-2) production.
RIAM was also found to interact with the scaffold protein Src kinase-associated phosphoprotein of 55 kDa (SKAP55) (Menasche et al. 2007). Abrogation of SKAP55/RIAM interaction led to impaired cell adhesion after TCR activation. This study identified the sequence encompassing the RA-PH domains of RIAM as the region via which association with SKAP55 was mediated. Because the RA domain of RIAM interacts with Rap1 and the PH domain of RIAM interacts with PI(4,5)P2, it remains unclear how these distinct interactions might be mediated by the same regions of RIAM.
RIAM Regulates Integrin Activation
RIAM Is Involved in Inside-Out Integrin Activation
It was first determined in T cells that RIAM is a Rap1 effector implicated in “inside-out” signaling and was shown that RIAM regulates Rap1-induced affinity changes in β1 and β2 integrins in T cells (Lafuente et al. 2004). Subsequent studies demonstrated that RIAM is also involved in Rap1-mediated activation of αIIbβ3 integrin in platelets (Han et al. 2006). Platelet aggregation requires agonist-induced αIIbβ3 activation, a process mediated by Rap1-GTP and talin. Using a pathway reconstruction Chinese hamster ovary (CHO) cell experimental system, it was determined that RIAM is mandatory for this process because knockdown of RIAM abrogated αIIbβ3 activation in the presence of Rap1-GTP. In primary megakaryocytes, RIAM knockdown blocks αIIbβ3 activation mediated by thrombin protease-activated receptors (PARs) that is mediated in a Rap1-GTP- and talin-dependent manner (Han et al. 2006).
Interaction of RIAM with Talin Regulates Conformational Changes of Integrins
The MRL-Talin-Integrin Complex Localizes at the Tips of Growing Protrusions
RIAM is abundant at the cell edge and at the lamellipodium, where it supports protrusion (Lee et al. 2013). Protrusive activity is likely due to the ability of RIAM to increase actin polymerization, most likely due to its interaction with profilin and ENA/VASP family proteins. RIAM co-localizes with talin in lamellipodia and filopodia, which might reflect an event involved in the localization of activated integrins at these membrane protrusions (Watanabe et al. 2008; Lee et al. 2009; Han et al. 2006). In contrast to RIAM, vinculin is enriched in maturing focal adhesions, which begin at the lamellum/lamellipodial border where vinculin reinforces the ability of the adhesion to transmit and bear force (Goult et al. 2013; Ziegler et al. 2006). The RIAM-integrin-talin complex is enriched at the tips of growing actin filaments in lamellipodial and filopodial protrusions, corresponding to the tips of “sticky fingers” (Lagarrigue et al. 2015). In this complex, the N-terminus of the MRL protein binds and recruits talin to the plasma membrane to induce integrin activation, whereas the C-terminal of the MRL protein increases processive actin polymerization in part by recruiting ENA/VASP, thereby propelling the movement of the “sticky fingers.”
RIAM Is Involved in Cancer Cell Adhesion and Migration
As adhesion receptors, integrins transduce signals in a bidirectional manner (Kim et al. 2003). Integrin signaling can be triggered via an inside-out pathway, which requires the recruitment of active Rap1, RIAM, and talin to the plasma membrane (Lafuente et al. 2004; Lee et al. 2009). Conversely, during outside-in signaling, integrin binds to the extracellular matrix, forms highly organized clusters, and initiates downstream signaling cascades in the cytoplasm. RIAM depletion in human melanoma cells leads to impairment in persistent cell migration directionality, causing deficient melanoma cell invasion (Hernández-Varas et al. 2011). Furthermore, RIAM-depleted melanoma cells display significant inhibition of lung metastasis in a mouse xenograft model. The defective invasion of RIAM knockdown cells was found to correlate with deficient association between β1 integrin and talin and with inhibition of β1 integrin-dependent activation of the Erk1/Erk2 mitogen-activated protein (MAP) kinases. In an independent study (Colo et al. 2012), RIAM-depleted melanoma cells were found to have increased numbers and stability of focal adhesions, which was interpreted as the result of defective focal adhesion disassembly.
RIAM Has an Active Role in Innate Immunity
The increase in complement-dependent phagocytosis after inside-out activation of CR3/αMβ2 depends on Rap1 activity. Rap1 activation correlates with talin recruitment to αMβ2 integrin during phagocytosis (Lim et al. 2007) although there is no evidence of a direct interaction between Rap1-GTP and talin. RIAM regulates the acquisition of αMβ2 high affinity state, as demonstrated by a significant reduction in the binding of an activation reporter monoclonal antibody (CBRM1/5) to the αM subunit in RIAM shRNA interfered (RIAM KD) human promyelocytic cell lines HL-60 and THP-1 (Medrano-Fernandez et al. 2013). RIAM KD cells have reduced complement-dependent phagocytosis in response to LPS or fMLP treatment. Similar results were obtained when RIAM expression was knocked down in human peripheral blood monocyte-derived macrophages (MDM), confirming that αMβ2 activation and complement-dependent phagocytosis induced by Rap1 were mediated by RIAM. Co-immunoprecipitation experiments done in neutrophil-like differentiated HL-60 cells during complement-dependent phagocytosis demonstrated reduced talin recruitment to β2 integrin when RIAM expression was knocked down. Confocal microscopy studies also indicated that talin localization at the complement receptor phagocytic cups is impaired in RIAM KD neutrophil-like cells. Together these findings point toward a role of RIAM in linking Rap1 activation to talin recruitment to CR3/αMβ2 receptor, promoting its activation and complement-dependent phagocytosis. Consistent with these findings, PMNs from RIAM-deficient mice have significantly reduced bacterial uptake and reduced levels of free reactive oxygen species (ROS) in response to TNF-α indicating a role of RIAM in β2 integrin-mediated outside-in signaling (Klapproth et al. 2015).
Adhesion, Extravasation, and Motility of Innate Immune Cells
Integrin αMβ2 activation and ICAM-mediated adhesion of leukocytes to the endothelium are required for transendothelial migration and extravasation. Adhesion and spreading to ICAM-1 but not VCAM-1 are reduced in RIAM-null PMNs in vitro, and a reduced leukocyte adhesion and extravasation in response to chemokines are observed in vivo (Fig. 3b) (Klapproth et al. 2015). A role of RIAM in neutrophil polarity has also been identified. Specifically, RIAM knockdown in neutrophil-like PLB-985 cells resulted in the loss of a clearly defined leading edge on 2D surfaces and impaired directionality toward fMLP, decreased migration and chemotaxis velocity, and reduction of protrusion formation on 3D Matrigel (Yamahashi et al. 2015).
Despite the compelling in vitro findings indicating an important role of the Rap1/RIAM/talin module in αIIbβ3 integrin activation (Watanabe et al. 2008), genetic deletion of RIAM in mice did not affect development, homeostasis, or platelet integrin functions (Klapproth et al. 2015; Stritt et al. 2014; Su et al. 2015). These findings indicate that RIAM-independent mechanisms exist for Rap1 to mediate its effects on platelet integrin function under physiologic conditions. The selective role of RIAM on integrin function in vivo is also supported by a partial defect in the function of β1 integrin but a significant impairment of β2 integrin activation observed in RIAM-deficient mice. As a consequence, RIAM-deficient mice exhibit significant leukocytosis due to impaired leukocyte-endothelium adhesion extravasation (Klapproth et al. 2015). Consistent with the indispensable role of RIAM in trafficking of B and T lymphocytes to secondary lymphoid organs, RIAM knockout mice display impaired humoral response to T-dependent antigen (Su et al. 2015). Thus, multiple lines of evidence converge into the idea that RIAM is essential for effective innate and adaptive immune responses.
RIAM is a Rap1-interacting molecule and a well-established regulator of multiple functions in immune cells. RIAM is involved in reorganization and modulation of the actin cytoskeleton, activation of integrins, cell adhesion, and migration. In vivo, RIAM seems to have an indispensable role selectively on β2 integrin activation and function but only a minor impact on β1 and β3 integrin activation. RIAM is involved in the regulation of innate and adaptive immune responses and in the invasion and migration of cancer cells. RIAM regulates both inside-out and outside-in signaling linked to integrin activation. These properties of RIAM make it an attractive therapeutic target not only to selectively modulate immune cell processes mediated via β2 integrin interactions but also to block cancer cell migration and metastasis.
This work was supported by NIH grants CA183605, CA183605S1, and AI098129-01 and the DoD grant PC140571 (VAB), SAF2012-34561, and SAF2016-77096- R (EML).
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