title: Fractional attachment of CD47 (IAP) to the erythrocyte cytoskeleton and visual colocalization with Rh protein complexes.
abstract: Interactions of CD47 and RhAG and the Rh proteins are visualized between one another and with the cytoskeleton of intact erythrocytes. In a first study, CD47 is labeled with a phycoerythrin (PhE)- tagged antibody, which generates discrete spots that reflect induced clusters of CD47. Rh and RhAG colocalize with each other and to these induced clusters, whereas Band 3 and glycophorin C remain more homogeneously dispersed on the cell periphery. In a second study, red cells are aspirated into a micropipette, and immunofluorescent maps of the surface gradients that develop for CD47 and RhAG determine cytoskeletal connectivity. CD47 and RhAG gradients on normal red cells prove to be nearly identical and also appear intermediate to those found for the fluid bilayer and network-linked glycophorin C. Similar gradients are obtained for CD47 on Rh(null) cells, suggesting that linkage of CD47 to the spectrin-actin skeleton is independent of Rh or RhAG and is not affected by CD47's reduced surface expression on these cells. The results show that CD47 colocalizes with Rh and RhAG but is fractionally attached to the red cell membrane skeleton independent of these and other major integral membrane proteins involved in cytoskeletal attachment. The results imply a homogeneous base distribution of CD47, restrained by cytoskeleton linkages, plus a smaller fraction of CD47, which is able to diffuse in the membrane.
authors.affiliation: School of Engineering and Applied Science, Institute for Medicine and Engineering, and Department of Pathology and Laboratory Medicine, University of Pennsylvania, PA 19104, USA.
authors.forename: Kris Noel
authors.initials: KN
authors.lastname: Dahl
authors.forename: Connie M
authors.initials: CM
authors.lastname: Westhoff
authors.forename: Dennis E
authors.initials: DE
authors.lastname: Discher
mesh_terms: D001457:Anion Exchange Protein 1, Erythrocyte; D015703:Antigens, CD / Q000378:metabolism*; D001798:Blood Proteins; D051928:CD47 Antigen; D002352:Carrier Proteins / Q000378:metabolism*; D004058:Diffusion; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006021:Glycophorins; D006801:Humans; D007519:Isoantigens / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D011485:Protein Binding; D012204:Rh-Hr Blood-Group System / Q000737:chemistry*; D013049:Spectrin / Q000378:metabolism
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D015703:Antigens, CD; D001798:Blood Proteins; D051928:CD47 Antigen; C497894:CD47 protein, human; D002352:Carrier Proteins; D006021:Glycophorins; D007519:Isoantigens; D008562:Membrane Glycoproteins; C498523:RHAG protein, human; D012204:Rh-Hr Blood-Group System; D013049:Spectrin