The protein 4.1 family comprises a group of skeletal proteins structurally related to the erythroid membrane skeletal protein, 4.1R, that plays a critical role in determining the morphology and mechanical stability of the red cell plasma membrane. These proteins are characterized by the presence of three main conserved structural/functional domains. A 30-kDa

Lutheran blood group glycoproteins (Lu gps) are receptors for the extracellular matrix protein, laminin. Studies suggest that Lu gps may contribute to vasoocclusion in sickle cell disease and it has recently been shown that sickle cells adhere to laminin isoforms containing the ␣5 chain (laminin 10/11). Laminin ␣5 is present in the subendothelium and is also a constituent of bone marrow sinusoids, suggesting a role for the Lu/ laminin interaction in erythropoiesis. The objectives of the current study were to define more precisely the molecular interactions of the extracellular and intracellular regions of human Lu and to clone and characterize a mouse homologue. To this end, complementary DNA and genomic clones for the mouse homologue were sequenced and the mouse Lu gene mapped to a region on chromosome 7 with conserved synteny with human 19q13.2. Mouse and human Lu gps are highly conserved (72% identity) at the amino acid sequence level and both mouse and human Lu gps specifically bind laminin 10/11 with high affinity. Furthermore, the first 3, N-terminal, immunoglobulin superfamily domains of human Lu are critical for this interaction. The results indicated that the cytoplasmic domain of BRIC 221-labeled human Lu gp is linked with the spectrin-based skeleton, affording the speculation that this interaction may be critical for signal transduction. These results further support a role for Lu gps in sickle cell disease and indicate the utility of mouse models to explore the function of Lu gp-laminin 10/11 interaction in normal erythropoiesis and in sickle cell disease. IntroductionThe Lutheran blood group is composed of a complex set of antigens expressed on 2 integral membrane glycoprotein isoforms of 85 and 78 kd. 1,2 The complementary DNA (cDNA) encoding the 85-kd isoform has been cloned, 3 and the predicted structure is that of a type 1 membrane protein. There are 5 disulfide-bonded, extracellular, immunoglobulin superfamily (IgSF) domains, a single hydrophobic membrane span, and a cytoplasmic tail of 59 residues. 3 The composition of the extracellular IgSF domains puts Lutheran blood group glycoproteins (Lu gps) in the subset of adhesion molecules that includes the human tumor marker MUC18/ MCAM4 and the chicken neural adhesion molecule Gicerin. [4][5][6] Chicken gicerin binds neurite outgrowth factor, a variant of the extracellular matrix (ECM) protein laminin 7,8 and, interestingly, recent studies suggest that Lu gp also functions as a laminin receptor. [9][10][11] The Lu gp cytoplasmic tail contains an SH3 binding motif and 5 potential phosphorylation sites, consistent with receptor signaling function. Of note, differences in the structure of the cytoplasmic tail distinguish the 2 isoforms. The 78-kd isoform (also termed B-CAM 12 or Lu[v13] 13 ) is generated by alternative splicing of intron 13 and differs from the larger form by having a truncated cytoplasmic tail lacking the SH3 binding motif as well as the potential phosphorylation sites. A recent study in epithelial cells 14 suggests that the cytop...

During erythroblast enucleation, nuclei surrounded by plasma membrane separate from erythroblast cytoplasm. A key aspect of this process is sorting of erythroblast plasma membrane components to reticulocytes and expelled nuclei. Although it is known that cytoskeletal elements actin and spectrin partition to reticulocytes, little is understood about molecular mechanisms governing plasma membrane protein sorting. We chose glycophorin A (GPA) as a model integral protein to begin investigating proteinsorting mechanisms. Using immunofluorescence microscopy and Western blotting we found that GPA sorted predominantly to reticulocytes. We hypothesized that the degree of skeletal linkage might control the sorting pattern of transmembrane proteins. To explore this hypothesis, we quantified the extent of GPA association to the cytoskeleton in erythroblasts, young reticulocytes, and mature erythrocytes using fluorescence imaged microdeformation (FIMD) and observed that GPA underwent dramatic reorganization during terminal differentiation. We discovered that GPA was more connected to the membrane cytoskeleton, either directly or indirectly, in erythroblasts and young reticulocytes than in mature cells. We conclude that skeletal protein association can regulate protein sorting during enucleation. Further, we suggest that the enhanced rigidity of reticulocyte membranes observed in earlier investigations results, at least in part, from increased connectivity of GPA with the spectrinbased skeleton. IntroductionDuring mammalian erythroid terminal differentiation, the plasma membrane and cytoskeleton are in a state of dynamic reorganization. We and others have determined that changes in protein expression and membrane protein assembly occur that involve both integral and skeletal membrane components. [1][2][3][4][5][6][7] At the conclusion of terminal differentiation, erythroblasts expel their nuclei and become reticulocytes. During enucleation, nuclei surrounded by plasma membrane separate from erythroblast cytoplasm. A key aspect of this process is the sorting of erythroblast plasma membrane components to the plasma membranes of the nascent reticulocyte and the expelled nucleus. Although approximately 2 million reticulocytes are generated each second, amazingly little is known about molecular mechanisms governing protein sorting during enucleation. It is known that actin, spectrin, tubulin, ankyrin, and protein 4.1 partition to young reticulocytes, leaving extruded nuclei devoid of skeletal elements. 3,[8][9][10] Earlier studies also report that nonsialated glycoproteins are enriched in membranes of extruded nuclei, while sialoglycoproteins are enriched in membranes of young reticulocytes. 11 However, the redistribution of a large number of well-characterized integral membrane proteins and the mechanism(s) underlying their redistribution are unexplored.Since glycophorin A (GPA) is a well-defined, major sialoglycoprotein in the mature erythrocyte (as reviewed in Chasis and Mohandas 12 ), we chose it as a model integral membrane ...

Protein 4.1R, a multifunctional structural protein, acts as an adaptor in mature red cell membrane skeletons linking spectrin-actin complexes to plasma membrane-associated proteins. In nucleated cells protein 4.1 is not associated exclusively with plasma membrane but is also detected at several important subcellular locations crucial for cell division. To identify 4.1 domains having critical functions in nuclear assembly, 4.1 domain peptides were added to Xenopus egg extract nuclear reconstitution reactions. Morphologically disorganized, replication deficient nuclei assembled when spectrin-actin-binding domain or NuMA-binding C-terminal domain peptides were present. However, control variant spectrin-actin-binding domain peptides incapable of binding actin or mutant C-terminal domain peptides with reduced NuMA binding had no deleterious effects on nuclear reconstitution. To test whether 4.1 is required for proper nuclear assembly, 4.1 isoforms were depleted with spectrin-actin binding or C-terminal domain-specific antibodies. Nuclei assembled in the depleted extracts were deranged. However, nuclear assembly could be rescued by the addition of recombinant 4.1R. Our data establish that protein 4

One hypothesis to explain the age-dependent clearance of red blood cells (RBCs) from circulation proposes that denatured/oxidized hemoglobin (hemichromes) arising late during an RBC’s life span induces clustering of the integral membrane protein, band 3. In turn, band 3 clustering generates an epitope on the senescent cell surface leading to autologous IgG binding and consequent phagocytosis. Because dog RBCs have survival characteristics that closely resemble those of human RBCs (ie, low random RBC loss, ≈115-day life span), we decided to test several aspects of the above hypothesis in the canine model, where in vivo aged cells of defined age could be evaluated for biochemical changes. For this purpose, dog RBCs were biotinylated in vivo and retrieved for biochemical analysis at various later dates using avidin-coated magnetic beads. Consistent with the above hypothesis, senescent dog RBCs were found to contain measurably elevated membrane-bound (denatured) globin and a sevenfold enhancement of surface-associated autologous IgG. Interestingly, dog RBCs that were allowed to senesce for 115 days in vivo also suffered from compromised intracellular reducing power, containing only 30% of the reduced glutathione found in unfractionated cells. Although the small quantity of cells of age ≥110 days did not allow direct quantitation of band 3 clustering, it was nevertheless possible to exploit single-cell microdeformation methods to evaluate the fraction of band 3 molecules that had lost their normal skeletal linkages and were free to cluster in response to hemichrome binding. Importantly, band 3 in RBCs ≥112 days old was found to be 25% less restrained by skeletal interactions than band 3 in control cells, indicating that the normal linkages between band 3 and the membrane skeleton had been substantially disrupted. Interestingly, the protein 4.1a/protein 4.1b ratio, commonly assumed to reflect RBC age, was found to be maximal in RBCs isolated only 58 days after labeling, implying that while this marker is useful for identifying very young populations of RBCs, it is not a very sensitive marker for canine senescent RBCs. Taken together, these data argue that several of the readily testable elements of the above hypothesis implicating band 3 in human RBC senescence can be validated in an appropriate canine model.

One hypothesis to explain the age-dependent clearance of red blood cells (RBCs) from circulation proposes that denatured/oxidized hemoglobin (hemichromes) arising late during an RBC’s life span induces clustering of the integral membrane protein, band 3. In turn, band 3 clustering generates an epitope on the senescent cell surface leading to autologous IgG binding and consequent phagocytosis. Because dog RBCs have survival characteristics that closely resemble those of human RBCs (ie, low random RBC loss, ≈115-day life span), we decided to test several aspects of the above hypothesis in the canine model, where in vivo aged cells of defined age could be evaluated for biochemical changes. For this purpose, dog RBCs were biotinylated in vivo and retrieved for biochemical analysis at various later dates using avidin-coated magnetic beads. Consistent with the above hypothesis, senescent dog RBCs were found to contain measurably elevated membrane-bound (denatured) globin and a sevenfold enhancement of surface-associated autologous IgG. Interestingly, dog RBCs that were allowed to senesce for 115 days in vivo also suffered from compromised intracellular reducing power, containing only 30% of the reduced glutathione found in unfractionated cells. Although the small quantity of cells of age ≥110 days did not allow direct quantitation of band 3 clustering, it was nevertheless possible to exploit single-cell microdeformation methods to evaluate the fraction of band 3 molecules that had lost their normal skeletal linkages and were free to cluster in response to hemichrome binding. Importantly, band 3 in RBCs ≥112 days old was found to be 25% less restrained by skeletal interactions than band 3 in control cells, indicating that the normal linkages between band 3 and the membrane skeleton had been substantially disrupted. Interestingly, the protein 4.1a/protein 4.1b ratio, commonly assumed to reflect RBC age, was found to be maximal in RBCs isolated only 58 days after labeling, implying that while this marker is useful for identifying very young populations of RBCs, it is not a very sensitive marker for canine senescent RBCs. Taken together, these data argue that several of the readily testable elements of the above hypothesis implicating band 3 in human RBC senescence can be validated in an appropriate canine model.

Mutations affecting the conversion of spectrin dimers to tetramers result in hereditary elliptocytosis (HE), whereas a deficiency of human erythroid ␣-or ␤-spectrin results in hereditary spherocytosis (HS). All spontaneous mutant mice with cytoskeletal deficiencies of spectrin reported to date have HS. Here, the first spontaneous mouse mutant, sph Dem / sph Dem , with severe HE is described. The sph Dem mutation is the insertion of an intracisternal A particle element in intron 10 of the erythroid ␣-spectrin gene. This causes exon skipping, the inframe deletion of 46 amino acids from repeat 5 of ␣-spectrin and alters spectrin dimer/tetramer stability and osmotic fragility. The disease is more severe in sph Dem /sph Dem neonates than in ␣-spectrin-deficient mice with HS. Thrombosis and infarction are not, as in the HS mice, limited to adults but occur soon after birth. Genetic background differences that exist between HE and HS mice are suspect, along with red blood cell morphology differences, as modifiers of thrombosis timing. sph Dem