Membrane mobility and clustering of Integrin Associated Protein (IAP, CD47)—Major differences between mouse and man and implications for signaling

Integrin Associated Protein (IAP, CD47) is a ubiquitous integral membrane protein implicated in processes (in mice) that range from inhibiting clearance by phagocytes [Oldenborg et al., Science 2000; Gardai et al., Cell 2005] to neutrophil motility [Lindberg et al., Science 1996]. SIRPα is CD47ʹs main receptor on phagocytes plus a number of other cell types, and SIRPα-CD47 interactions in clusters are believed to mediate signaling. However, considerable species differences in CD47 sequence as well as differences in CD47 extractability from mouse cells versus man motivate a characterization of mobility, clusterability, and kinetics under force of CD47-SIRPα. Despite similar levels of CD47 on red cells from mouse and man, we find an effective avidity of SIRPα-CD47 for mouse appears higher than for human. Both mouse and human CD47 show clustering by multivalent SIRPα complexes, but only mouse cells aggregate with CD47 concentrating at cell–cell contacts. This proves consistent with fluorescence imaged micro-deformation, which indicates near-complete mobility of CD47 on mouse cells compared to only about 30–40% mobility on normal human cells. To qualify the method, we also show that disrupting cellular F-actin dramatically increases the mobility of integral membrane proteins. Furthermore, atomic force microscopy probing of cell membranes with human SIRPα confirms the species-specific interactions and provides evidence of clustering and adhesion on short time scales, but it also shows surprisingly strong forces in detachment for a signaling complex. The results thus highlight major species differences in CD47-SIRPα interactions and CD47 integration, suggesting that signaling by CD47 in man may be qualitatively different from mouse.