CD47 surface stability is sensitive to actin disruption prior to inclusion within the band 3 macrocomplex

CD47 is an important 'marker of self' protein with multiple isoforms produced though alternative splicing that exhibit tissue-specific expression. Mature erythrocytes express CD47 isoform 2 only, with membrane stability of this version dependent on inclusion within the band 3 macrocomplex, via protein 4.2. At present a paucity of information exists regarding the associations and trafficking of the CD47 isoforms during erythropoiesis. We show that CD47 isoform 2 is the predominant version maintained at the surface of expanding and terminally differentiating erythroblasts. CD47 isoforms 3 and 4 are expressed in all cell types tested except mature erythrocytes, but do not reach the plasma membrane in erythroblasts and are degraded by the orthochromatic stage of differentiation. To identify putative CD47 interactants, immunoprecipitation combined with Nano LC-MS/MS mass spectrometry was conducted on the erythroleukaemic K562 cell line, expanding and terminally differentiating primary erythroblasts and mature erythrocytes. Results indicate that prior to incorporation into the band 3 macrocomplex, CD47 associates with actin-binding proteins and we confirm that CD47 membrane stability is sensitive to actin disrupting drugs. Maintenance of CD47 at the cell surface was also influenced by dynamin, with sensitivity to dynamin disruption prolonged relative to that of actin during erythropoiesis.