Essential diurnal Rac1 activation during retinal phagocytosis requires alpha v beta 5 integrin but not tyrosine kinases focal adhesion kinase or Mer tyrosine kinase

• Published in: Molecular biology of the cell 2012-03, Vol.23 (6), p.1104-1114
• Main Authors: Mao, Yingyu, Finnemann, Silvia C
• Format: Article
• Language: English
• Subjects: Actin; Cdc42 protein; Cell culture; Epidermal growth factor; Focal adhesion kinase; Guanosinetriphosphatase; Integrins; Milk; Phagocytes; Phagocytosis; Photoreceptors; Pigments; Protein-tyrosine kinase; Protein-tyrosine kinase receptors; Rac1 protein; Retina; RhoA protein; Signal transduction; Toxin B
• ISSN: 1059-1524
• DOI: 10.1091/mbc.E11-10-0840

Diurnal phagocytosis of shed photoreceptor outer-segment particles by retinal pigment epithelial (RPE) cells belongs to a group of conserved clearance mechanisms employing alpha v integrins upstream of tyrosine kinases and Rho GTPases. In this study, we tested the interdependence of the tyrosine kinases focal adhesion kinase (FAK) and Mer tyrosine kinase (MerTK) and Rho GTPases during engulfment. RPE cells activated and redistributed Rac1, but not RhoA or Cdc42, during phagocytosis. Toxin B, overexpression of dominant-negative Rac1, or decreasing Rac1 expression prevented particle engulfment. Fluorescence microscopy showed that Rac1 inhibition had no obvious effect on F-actin arrangement in resting RPE but prevented recruitment of F-actin to surface-bound phagocytic particles. Quantification of active GTP-Rac1 in wild-type and mutant RPE in culture and in vivo revealed that Rac1 activation during phagocytosis requires alpha v beta 5 integrin and its ligand milk fat globule EGF factor-8 (MFG-E8) but not the receptor tyrosine kinase MerTK. Abolishing tyrosine kinase signaling downstream of alpha v beta 5 toward MerTK by inhibiting FAK specifically or tyrosine kinases generally neither prevented Rac1 activation nor F-actin recruitment during phagocytosis. Likewise, inhibiting Rac1 had no effect on FAK or MerTK activation. We conclude that MerTK activation via FAK and F-actin recruitment via Rac1 both require MFG-E8-ligated alpha v beta 5 integrin. Both pathways are independently activated and required for clearance phagocytosis.