RIAM-VASP Module Relays Integrin Complement Receptors in Outside-In Signaling Driving Particle Engulfment

Abstract

The phagocytic integrins and complement receptors αMβ2/CR3 and αXβ2/CR4 are classically associated with the phagocytosis of iC3b-opsonized particles. The activation of this receptor is dependent on signals derived from other receptors (inside-out signaling) with the crucial involvement of the Rap1-RIAM-Talin-1 pathway. Here, we analyze the implication of RIAM and its binding partner VASP in the signaling events occurring downstream of β2 integrins (outside-in) during complement-mediated phagocytosis. To this end, we used HL-60 promyelocytic cell lines deficient in RIAM or VASP or overexpressing EGFP-tagged VASP to determine VASP dynamics at phagocytic cups. Our results indicate that RIAM-deficient HL-60 cells presented impaired particle internalization and altered integrin downstream signaling during complement-dependent phagocytosis. Similarly, VASP deficiency completely blocked phagocytosis, while VASP overexpression increased the random movement of phagocytic particles at the cell surface, with reduced internalization. Moreover, the recruitment of VASP to particle contact sites, amount of pSer157-VASP and formation of actin-rich phagocytic cups were dependent on RIAM expression. Our results suggested that RIAM worked as a relay for integrin complement receptors in outside-in signaling, coordinating integrin activation and cytoskeletal rearrangements via its interaction with VASP.

Complement Receptors and Their Role in Leukocyte Recruitment and Phagocytosis

“…These lead to inside-out activation via the Rap1-RIAM-Talin pathway. Genetic ablation of these proteins leads to defects in complement-mediated phagocytosis, as illustrated by impaired adhesion, phagocytosis and ROS production by RIAM-deficient phagocytes in vitro , and signs of LAD in RIAM knockout mice in vivo (Klapproth et al, 2015 ; Torres-Gomez et al, 2020b ). Inside-out signaling drives the phosphorylation of CD18 on serine residues, but not of the alpha chains (CD11b or CD11c), which are constitutively phosphorylated (Chatila et al, 1989 ; Fagerholm et al, 2006 ).…”

Dynamin-2 controls complement receptor 3- mediated phagocytosis completion and closure

“…Integrins activity is highly regulated by conformational changes. During the "inside-out" phase of integrin activation, there is an actomyosin-dependent stretching of talin that allows the release of RIAM and subsequent binding to vinculin (Torres-Gomez et al, 2020b;Vigouroux et al, 2020), which reinforces the association to the β2 tail and its activation. Interestingly, dynamin-2 was not implicated in regulating these actin-dependent steps of integrin activation in human macrophages, as the association of macrophages to the complement-opsonised particles was not modified when dynamin-2 activity was perturbed (Figure 2).…”

The Connection Between Rap1 and Talin1 in the Activation of Integrins in Blood Cells

“…Similar to RIAM, Lpd contains talin1 binding sites and triggers integrin activation ( Watanabe et al, 2008 ; Lee et al, 2009 ). In addition to regulating talin1-dependent integrin activation, RIAM also contributes to β2 integrin-mediated outside-in signaling in neutrophils and macrophages ( Klapproth et al, 2015 ; Torres-Gomez et al, 2020 ). On the other hand, the role of Lpd in the outside-in signaling of integrins is poorly understood and deserves to be explored.…”