Abstract
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.
Highlights
CD47 is a ubiquitously expressed ‘marker of self ’ protein, originally identified as integrin-associated protein (IAP) following co-purification with β3 integrins from placenta[1, 2]
The specificity of the different CD47 isoform antibodies available to us was confirmed using a panel of lysates prepared from untransfected HEK293T cells, HEK293T cells expressing the empty vector alone, or cells transfected with CD47 isoforms 2, 3, 4 or a version of CD47 with the whole C-terminus deleted (CD47 Δ Ct; Fig. 1B), which were separated by SDS-PAGE and immunoblotted
Using a panel of human cell lines, primary cells and mature erythrocytes we confirmed that CD47 isoform 2 is restricted to haematopoietic and endothelial cells, being detected in Jurkat T cells, mature erythrocytes, K562 cells and Human Umbilical Vein Endothelial Cell (HUVEC) (Supplementary Fig. S1A)
Summary
CD47 is a ubiquitously expressed ‘marker of self ’ protein, originally identified as integrin-associated protein (IAP) following co-purification with β3 integrins from placenta[1, 2]. This ~52 kDa glycoprotein possesses an immunoglobulin variable (IgV) like N-terminal domain, five transmembrane domains and an alternatively spliced. The ‘marker of self ’ function of CD47 was originally elucidated in murine erythrocytes[6]. We have previously found that during terminal differentiation CD47 is independent of protein 4.2 until the basophilic erythroblast stage (48 hours post-differentiation20, 23), suggesting that CD47 is dependent on alternative membrane stabilising interactions early during erythroblast development
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