CD47 Glycoprotein Interacts with p4.1R and p55 in the Erythrocyte.

CD47 is a 47–50kDa membrane glycoprotein with 5 known isoforms. The role of CD47 within the erythrocyte membrane remains the subject of much research and debate though we recently provided evidence that CD47 may function as an inducer of eryptosis (Head et al, 2005). As both a cytoskeletal linked fraction and a smaller membrane diffuse fraction of CD47 exists, it is most likely that there are a number of protein species that are able to bind to CD47 at its cytoplasmic face. Our research has focused on a study of the molecular interactions of erythrocyte CD47 with erythrocyte membrane skeletal proteins protein 4.1R (p4.1R), protein 4.2 (p4.2) and p55. Here we demonstrate the ubiquitous expression of all CD47 isoforms in haemopoietic cells and tissues using basic and real-time PCR. Via immunoprecipitation of CD47 from mature erythrocyte membranes using the anti-CD47 mAb BRIC-126, yeast two-hybrid analysis and in vitro co-immunoprecipitation of 35[S] labelled peptides in a cell-free translation procedure, a novel ternary complex involving CD47, p55 and p4.1R has been indicated. More specifically, the potential interaction between p55/p4.1R and the cytoplasmic face of CD47 has been localised to the PDZ and FERM domain of these proteins respectively. Though research suggests p4.2 provides the major cytoskeletal attachment of CD47 (Bruce et al, 2002; Mouro-Chanteloup et al, 2003), a direct interaction between CD47 and p4.2 was not suggested by our study and remains undemonstrated. We continue to further the evidence for a functional role of CD47 in eryptosis. We propose p4.1R links CD47 to the apoptotic machinery of the cell and suggest a mechanism whereby cytoskeletal rearrangement and PS exposure occurs. 4.1null cells have been obtained and are currently being investigated. Further characterisation of the eryptotic pathway may offer insight into potential therapies for erythroleukaemia characterised by resistance of the erythroid lineage to apoptosis. This work has equal significance in the stabilization of red cell preparations in the blood transfusion setting.