Changes in erythroid membrane proteins during erythropoietin‐mediated terminal differentiation

Abstract

Membrane and membrane skeleton proteins were examined in erythroid progenitor cells during terminal differentiation. The employed model system of erythroid differentiation was that in which proerythroblasts from mice infected with the anemia-inducing strain of Friend virus differentiate in vitro in response to erythropoietin (EP). With this system, developmentally homogeneous populations of cells can be examined morphologically and biochemically as they progress from proerythroblasts through enucleated reticulocytes. alpha and beta spectrins, the major proteins of the erythrocyte membrane skeleton, are synthesized in the erythroblasts both before and after EP exposure. At all times large portions of the newly synthesized spectrins exist in and are turned over in the cytoplasm. The remaining newly synthesized spectrin is found in a cellular fraction containing total membranes. Pulse-chase experiments show that little of the cytoplasmic spectrins become membrane associated, but that the proportion of newly synthesized spectrin which is membrane associated increases as maturation proceeds. A membrane fraction enriched in plasma membranes has significant differences in the stoichiometry of spectrin accumulation as compared to total cellular membranes. Synthesis of band 3 protein, the anion transporter, is induced only after EP addition to the erythroblasts. All of the newly synthesized band 3 is membrane associated. A two-dimensional gel survey was conducted of newly synthesized proteins in the plasma membrane enriched fraction of the erythroblasts as differentiation proceeded. A majority of the newly synthesized proteins remain in the same proportion to each other during maturation; however, a few newly synthesized proteins greatly increase following EP induction while others decrease markedly. Of the radiolabeled proteins observed in two dimensional gels, only the spectrins, band 3 and actin become major proteins of the mature erythrocyte membrane. Examination of total proteins of the plasma membrane enriched fractions of EP-treated erythroblasts using silver staining and 32P autoradiography show that many proteins and phosphoproteins are selectively eliminated from this fraction late in the course of differentiation during the reticulocyte stage. The selective removal of many proteins at the reticulocyte stage of development combined with previous selective synthesis and accumulation of some specific proteins such as alpha and beta spectrin and band 3 in the differentiating erythroblasts lead to the final mammalian erythrocyte membrane structure.

Ineffective erythropoiesis with reduced red blood cell survival in serotonin-deficient mice

“…They confirm that the underlying cause for the macrocytosis of 5-HT-deficient mice is decreased proliferation of erythroid precursors leading to the production of macrocytic RBCs, because these cell divisions are tightly linked with a reduction in cell size (33).…”

“…+ cells in mutant animals but imply a low production efficiency of RBC precursors. Moreover, during erythroid differentiation, cell division is tightly linked with reduction in cell size (33), and reduced cell division could result in the macrocytosis observed in TPH1 −/− mice. By analogy, in humans, macrocytic anemia may give rise to defective DNA synthesis in the BM, leading to alteration of the normal differentiation-proliferation pathway of the erythroid lineage and release of proper enucleation.…”

Maturation and enucleation of primitive erythroblasts during mouse embryogenesis is accompanied by changes in cell-surface antigen expression

“…63 Studies of definitive erythroid progenitors have shown that cytoskeletal components such as actin and spectrin [64][65][66] are partitioned into reticulocytes after the vimentin-based microfilaments are degraded, allowing the nucleus to move freely about the cell. 64 The expelled nuclei are encapsulated by a thin membrane into which cell-surface molecules such as glycophorin A and phosphatidylserine are selectively partitioned.…”

Caspase-activated ROCK-1 allows erythroblast terminal maturation independently of cytokine-induced Rho signaling

“…Terminal erythroid differentiation is characterized by hemoglobin synthesis and major morphological changes, including chromatin condensation, cell size reduction and finally, nucleus extrusion to generate reticulocytes that enter in the blood circulation. 1 This differentiation process involves the transcription factor GATA-1, 2,3 which positively regulates promoters of erythroid genes such as glycophorin A, the erythropoietin receptor (EPO-R) and hemoglobin chains.…”