RIAM (Rap1-interacting adaptor molecule) regulates complement-dependent phagocytosis

Abstract

Phagocytosis mediated by the complement receptor CR3 (also known as integrin αMβ2 or Mac-1) is regulated by the recruitment of talin to the cytoplasmic tail of the β2 integrin subunit. Talin recruitment to this integrin is dependent on Rap1 activation. However the mechanism by which Rap1 regulates this event and CR3-dependent phagocytosis remains largely unknown. In the present work we examined the role of the Rap1 effector RIAM, a talin binding protein, in the regulation of complement-mediated phagocytosis. Using the human promyelocytic cell lines HL-60 and THP-1, we determined that knockdown of RIAM impaired αMβ2 integrin affinity changes induced by fMLP. Phagocytosis of complement-opsonized RBC particles, but not IgG-opsonized RBC particles was impaired in RIAM knockdown cells. Rap1 activation via EPAC induced by 8-pCPT-2′-O-Me-cAMP resulted in an increase of complement-mediated phagocytosis that was abrogated by knockdown of RIAM in HL-60 and THP-1 cell lines and in macrophages derived from primary monocytes. Furthermore, recruitment of talin to the phagocytic cup during complement-mediated phagocytosis was reduced in RIAM knockdown cells. These results indicate that RIAM is a critical component of the phagocytosis machinery downstream of Rap1 and mediates its function by recruiting talin to phagocytic complement receptors.

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

“…Analysis of the respiratory burst in response to tumor necrosis factor-a (TNF-a) revealed a reduction in free reactive oxygen species (ROS) production in RIAM −/− PMNs, pointing to a role of RIAM in b 2 integrin-mediated outside-in signaling (83). These results are in accordance with data obtained from RIAM KD human promyelocytic cells and indicate an essential role of RIAM in b 2 integrin-mediated phagocytosis (82).…”

“…In accordance with the defective acquisition of the a M b 2 high-affinity state, this study showed that RIAM knockdown reduced complementdependent phagocytosis in response to LPS or f MLP treatment as compared to control cells (82). Moreover, the reduction of a M b 2 activation and impaired phagocytosis in RIAM KD cells was observed even when Rap1 was activated with 8-(4-chlorophenylthio)adenosine 3',5′-cyclic monophosphate (8-CPT-cAMP), a cAMP analog that specifically activates the Rap1-guanine nucleotide exchange factor exchange nucleotide protein directly activated by cAMP (EPAC).…”

“…Coimmunoprecipitation experiments done in neutrophil-like differentiated HL-60 cells during complement-dependent phagocytosis demonstrated reduced talin recruitment to b 2 integrin when RIAM was knocked down. Confocal microscopy studies also indicated that talin localization to phagocytic cups containing the complement receptor was impaired in RIAM KD HL-60 cells (82). Together, these findings point toward a role of RIAM in linking Rap1 activation to talin recruitment to a M b 2 integrin, thereby promoting its activation by CR3 and complement-dependent phagocytosis (Fig.…”

“…A role of RIAM in neutrophil polarity has also been identified. Specifically, RIAM knockdown in neutrophillike PLB-985 cells resulted in the loss of a clearly defined leading edge on two-dimensional (2D) surfaces and impaired directionality (49) toward f MLP, decreased migration and chemotaxis velocity, and reduction of protrusion formation in 3D Matrigel (82). These findings contrast with those from migration analyses of cells in which Kindlin-3, an integrin coactivator that interacts with talin, was knocked down.…”

“…RIAM has been shown to promote the acquisition of the highaffinity state of a M b 2 , as demonstrated by a reduction in the binding of an activation reporter monoclonal antibody (CBRM1/5) to the a M subunit in human promyelocytic cell lines HL-60 and THP-1, in which RIAM was knocked down by RNA interference. Whereas in control cells LPS or N-formyl-Met-Leu-Phe ( f MLP) chemotactic peptide stimulation resulted in increased CBRM1/5 binding, in RIAM knockdown (KD) cells CBRM1/5 binding was abrogated, which points to RIAM as essential for full a M b 2 activation (82).…”