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]. SIRPalpha is CD47's main receptor on phagocytes plus a number of other cell types, and SIRPalpha-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-SIRPalpha. Despite similar levels of CD47 on red cells from mouse and man, we find an effective avidity of SIRPalpha-CD47 for mouse appears higher than for human. Both mouse and human CD47 show clustering by multivalent SIRPalpha 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 SIRPalpha 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-SIRPalpha interactions and CD47 integration, suggesting that signaling by CD47 in man may be qualitatively different from mouse.
Subramanian
Shyamsundar
S
Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA 19104, USA.
Tsai
Richard
R
Sen
Shamik
S
Dahl
Kris Noel
KN
Discher
Dennis E
DE
D000818:Animals; D051928:CD47 Antigen / Q000378:metabolism*; D019556:COS Cells; D002522:Chlorocebus aethiops; D006801:Humans; D051379:Mice; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D011940:Receptor Aggregation / Q000502:physiology*; D011971:Receptors, Immunologic / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D013045:Species Specificity
D051928:CD47 Antigen; C497894:CD47 protein, human; C497895:Cd47 protein, mouse; D011971:Receptors, Immunologic