Abstract
Phagocytosis mediated by the complement receptor CR3 (also known as integrin αMß2 or Mac-1) is regulated by the recruitment of talin to the cytoplasmic tail of the ß2 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 myeloid cell lines HL-60 and THP-1, we determined that knockdown of RIAM impaired αMß2 integrin affinity changes induced by stimuli fMLP and LPS. Phagocytosis of complement-opsonized RBC particles, but not of 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 ß2 integrin 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 the phagocytic complement receptors.
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Abbreviations
- BSA:
-
Bovine serum albumin
- 8CPT:
-
8-pCPT-2-O-Me-cAMP
- FCS:
-
Fetal calf serum
- HRP:
-
Horseradish peroxidase
- PBMC:
-
Peripheral blood mononuclear cells
- PBS:
-
Phosphate buffered saline
- PMA:
-
Phorbol myristate acetate
- RA:
-
Retinoic acid
- FNG:
-
Fibrinogen
- mAb:
-
Monoclonal antibody
- TBS:
-
Tris buffered saline
- SDS-PAGE:
-
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
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Acknowledgments
We are thankful to Joaquin Teixidó and Georgina Coló (CIB; CSIC, Spain) for providing information about RIAM siRNA oligonucleotides, and to José Luis Rodríguez and Jesus Torres (CIB; CSIC, Spain) for their help with siRNA delivery into MDM cells. This work was supported by grants SAF2007-60578 and SAF2012-34561 from MICINN and grants CCG10-UCM/BIO-4795 and CCG08-UCM/SAL-4259 from Comunidad Autonoma de Madrid (to E.M.L.), grants BFU2007-66443/BMC and BFU2010-19144/BMC and SAF2012-34561 from MICINN (to CC), grant SAF2009-08103 from MICINN (to P.A.R.), NIH grants HL107997-01 and R56AI43552 and the Leukemia and Lymphoma Society Translational Research Program TRP 6222-11 (to V.A.B.).
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Medraño-Fernandez, I., Reyes, R., Olazabal, I. et al. RIAM (Rap1-interacting adaptor molecule) regulates complement-dependent phagocytosis. Cell. Mol. Life Sci. 70, 2395–2410 (2013). https://doi.org/10.1007/s00018-013-1268-6
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DOI: https://doi.org/10.1007/s00018-013-1268-6