Structural and functional characterization of protein 4.1R-phosphatidylserine interaction: potential role in 4.1R sorting within cells.

Erythrocyte protein 4.1R is a multifunctional protein that binds to various membrane proteins and to phosphatidylserine. In the present study, we report two important observations concerning 4.1R-phosphatidylserine interaction. Biochemically, a major finding of the present study is that 4.1R binding to phosphatidylserine appears to be a two-step process in which 4.1R first interacts with serine head group of phosphatidylserine through the positively charged amino acids YKRS and subsequently forms a tight hydrophobic interaction with fatty acid moieties. 4.1R failed to dissociate from phosphatidylserine liposomes under high ionic strength but could be released specifically by phospholipase A(2) but not by phospholipase C or D. Biochemical analyses showed that acyl chains were associated with 4.1R released by phospholipase A(2). Importantly, the association of acyl chains with 4.1R impaired its ability to interact with calmodulin, band 3, and glycophorin C. Removal of acyl chains restored 4.1R binding. These data indicate that acyl chains of phosphatidylserine play an important role in its interaction with 4.1R and on 4.1R function. In terms of biological significance, we have obtained evidence that 4.1R-phosphatidylserine interaction may play an important role in cellular sorting of 4.1R.

New insights into functions of erythroid proteins in nonerythroid cells.

This article presents new insights into potential roles that three erythrocyte cytoskeletal proteins, protein 4.1, ankyrin, and spectrin, may play in nonerythroid nucleated cells. Each of these proteins is encoded by several closely related genes characterized by complex alternative splicing of their pre-mRNA, thus resulting in the cellular expression of a broad repertoire of isoforms that can adopt tissue- and cell-specific distribution. This could account for the presence of skeletal networks in intracellular organelles such as lysosomes, the Golgi apparatus, or the nucleus. In addition to providing structural support to cell membranes, these cytoskeletal proteins regulate the functions of various transmembrane proteins they interact with, in particular ion channels, as well as the activity of membrane-bound enzymes. Thus, they appear to be key players in major unsuspected cell functions such as protein sorting, dynamics of nuclear architecture during mitosis, or regulation of signal transduction pathways.

Molecular and functional characterization of protein 4.1B, a novel member of the protein 4.1 family with high level, focal expression in brain.

Brain-enriched isoforms of skeletal proteins in the spectrin and ankyrin gene families have been described. Here we characterize protein 4.1B, a novel homolog of erythrocyte protein 4.1R that is encoded by a distinct gene. In situ hybridization revealed high level, focal expression of 4.1B mRNA in select neuronal populations within the mouse brain, including Purkinje cells of the cerebellum, pyramidal cells in hippocampal regions CA1-3, thalamic nuclei, and olfactory bulb. Expression was also detected in adrenal gland, kidney, testis, and heart. 4.1B protein exhibits high homology to the membrane binding, spectrin-actin binding, and C-terminal domains of 4.1R, including motifs for interaction with NuMA and FKBP13. cDNA characterization and Western blot analysis revealed multiple spliceoforms of protein 4.1B, with functionally relevant heterogeneity in the spectrin-actin and NuMA binding domains. Regulated alternative splicing events led to expression of unique 4. 1B isoforms in brain and muscle; only the latter possessed a functional spectrin-actin binding domain. By immunofluorescence, 4. 1B was localized specifically at the plasma membrane in regions of cell-cell contact. Together these results indicate that 4.1B transcription is selectively regulated among neuronal populations and that alternative splicing regulates expression of 4.1B isoforms possessing critical functional domains typical of other protein 4.1 family members.

Deciphering the nuclear import pathway for the cytoskeletal red cell protein 4.1R.

The erythroid membrane cytoskeletal protein 4.1 is the prototypical member of a genetically and topologically complex family that is generated by combinatorial alternative splicing pathways and is localized at diverse intracellular sites including the nucleus. To explore the molecular determinants for nuclear localization, we transfected COS-7 cells with epitope-tagged versions of natural red cell protein 4.1 (4.1R) isoforms as well as mutagenized and truncated derivatives. Two distant topological sorting signals were required for efficient nuclear import of the 4.1R80 isoform: a basic peptide, KKKRER, encoded by alternative exon 16 and acting as a weak core nuclear localization signal (4.1R NLS), and an acidic peptide, EED, encoded by alternative exon 5. 4.1R80 isoforms lacking either of these two exons showed decreased nuclear import. Fusion of various 4.1R80 constructs to the cytoplasmic reporter protein pyruvate kinase confirmed a requirement for both motifs for full NLS function. 4.1R80 was efficiently imported in the nuclei of digitonin-permeabilized COS-7 cells in the presence of recombinant Rch1 (human importin alpha2), importin beta, and GTPase Ran. Quantitative analysis of protein-protein interactions using a resonant mirror detection technique showed that 4.1R80 bound to Rch1 in vitro with high affinity (KD = 30 nM). The affinity decreased at least 7- and 20-fold, respectively, if the EED motif in exon 5 or if 4.1R NLS in exon 16 was lacking or mutated, confirming that both motifs were required for efficient importin-mediated nuclear import of 4.1R80.

What do mouse gene knockouts tell us about the structure and function of the red cell membrane?

Recent development of knockout mice with targeted deletion of specific genes encoding various red cell membrane proteins has added valuable armamentarium to red cell membrane structure-function studies. In this chapter we will summarize the various recent developments regarding the structure and function of the red cell membrane derived from studies using knockout mice. In addition to being expressed in red cells, all major red cell membrane proteins are also expressed in cells of various tissues. The potential use of knockout mice to decipher the biological functions of red cell membrane proteins in non-erythroid cells is also explored.

Characterization of multiple isoforms of protein 4.1R expressed during erythroid terminal differentiation.

In erythrocytes, 80-kD protein 4.1R regulates critical membrane properties of deformability and mechanical strength. However, previously obtained data suggest that multiple isoforms of protein 4. 1, generated by alternative pre-mRNA splicing, are expressed during erythroid differentiation. Erythroid precursors use two splice acceptor sites at the 5' end of exon 2, thereby generating two populations of 4.1 RNA: one that includes an upstream AUG-1 in exon 2' and encodes high molecular weight isoforms, and another that skips AUG-1 in exon 2' and encodes 4.1 by initiation at a downstream AUG-2 in exon 4. To begin an analysis of the complex picture of protein 4.1R expression and function during erythropoiesis, we determined the number and primary structure of 4.1R isoforms expressed in erythroblasts. We used reverse-transcription polymerase chain reaction to amplify and clone full-length coding domains from the population of 4.1R cDNA containing AUG-1 and the population excluding AUG-1. We observed an impressive repertoire of 4.1R isoforms that included 7 major and 11 minor splice variants, thus providing the first definitive characterization of 4.1R primary structures in a single-cell lineage. 4.1R isoforms, transfected into COS-7 cells, distributed to the nucleus, cytoplasm, plasma membrane, and apparent centrosome. We confirmed previous studies showing that inclusion of exon 16 was essential for efficient nuclear localization. Unexpectedly, immunochemical analysis of COS-7 cells transfected with an isoform lacking both AUG-1 and AUG-2 documented that a previously unidentified downstream translation initiation codon located in exon 8 can regulate expression of 4.1R. We speculate that the repertoire of primary structure of 4.1R dictates its distinct binding partners and functions during erythropoiesis.

Identification of an interaction between the m-band protein skelemin and beta-integrin subunits. Colocalization of a skelemin-like protein with beta1- and beta3-integrins in non-muscle cells.

Signaling across integrins is regulated by interaction of these receptors with cytoskeletal proteins and signaling molecules. To identify molecules interacting with the cytoplasmic domain of the beta3-integrin subunit (glycoprotein IIIa), a placental cDNA library was screened in the yeast two-hybrid system. Two identical clones coding for a 96-amino acid sequence were identified. This sequence was 100% identical to a sequence in skelemin, a protein identified previously in skeletal muscle. Skelemin is a member of a superfamily of cytoskeletal proteins that contain fibronectin-type III-like motifs and immunoglobulin C2-like motifs and that regulate the organization of myosin filaments in muscle. The amino acid residues in the isolated clones encompassed C2 motifs 4 and 5 of skelemin. A recombinant skelemin protein consisting of C2 motifs 3-7 interacted with beta1- and beta3-integrin cytoplasmic domains expressed as glutathione S-transferase (GST) fusion proteins, but not with GST-beta2-integrin cytoplasmic tail or GST alone. The skelemin-binding region was in the membrane proximal cytoplasmic domains of the integrins. Full-length skelemin interacted with integrin in intact cells as demonstrated by the colocalization of hemagglutinin-tagged skelemin in Chinese hamster ovary (CHO) cells containing alphaIIbbeta3-integrin and by the finding that microinjection of C2 motif 4 of skelemin into C2C12 mouse myoblast cells caused spread cells to round up. A skelemin-like protein was detected in CHO cells, endothelial cells, and platelets, and this protein colocalized with beta1- and beta3-integrins in CHO cells. This study suggests the presence of a skelemin-like protein in non-muscle cells and provides evidence that it may be involved in linking integrins to the cytoskeleton.

Neurobehavioral deficits in mice lacking the erythrocyte membrane cytoskeletal protein 4.1.

The erythrocyte membrane cytoskeletal protein 4.1 (4.1R) is a structural protein that confers stability and flexibility to erythrocytes via interactions with the cytoskeletal proteins spectrin and F-actin and with the band 3 and glycophorin C membrane proteins. Mutations in 4.1R can cause hereditary elliptocytosis, a disease characterized by a loss of the normal discoid morphology of erythrocytes, resulting in hemolytic anemia [1]. Different isoforms of the 4.1 protein have been identified in a wide variety of nonerythroid tissues by immunological methods [2-5]. The variation in molecular weight of these different 4.1 isoforms, which range from 30 to 210 kDa [6], has been attributed to complex alternative splicing of the 4.1R gene [7]. We recently identified two new 4.1 genes: one is generally expressed throughout the body (4. 1G) [8] and the other is expressed in central and peripheral neurons (4.1N) [9]. Here, we examined 4.1R expression by in situ hybridization analysis and found that 4.1R was selectively expressed in hematopoietic tissues and in specific neuronal populations. In the brain, high levels of 4.1R were discretely localized to granule cells in the cerebellum and dentate gyrus. We generated mice that lacked 4.1R expression; these mice had deficits in movement, coordination, balance and learning, in addition to the predicted hematological abnormalities. The neurobehavioral findings are consistent with the distribution of 4.1R in the brain, suggesting that 4.1R performs specific functions in the central nervous system.

Cloning and characterization of 4.1G (EPB41L2), a new member of the skeletal protein 4.1 (EPB41) gene family.

The prototypical erythrocyte membrane skeletal protein 4.1 (HGMW-approved symbol EPB41), here designated 4.1R, is encoded by a large, complexly spliced gene located on human chromosome 1p32-p33. In this paper we report evidence for a second 4.1 gene, 4.1G (HGMW-approved symbol EPB41L2), which maps to human chromosome 6q23 and is widely expressed among human tissues. The complete nucleotide sequence of 4.1G cDNA predicts a 113-kDa protein that exhibits three regions of high homology to 4.1R, including the membrane binding domain, the spectrinactin binding domain, and the C-terminal domain. Interspersed among the shared domains are unique sequences that may define functional differences between 4.1R and 4.1G. Specific isoforms of 4.1R and 4.1G exhibit differential subcellular localizations. These results expand the 4.1 gene superfamily and demonstrate that the diverse cellular complement of 4.1 isoforms results from both alternative splicing and expression of distinct genes.

The 13-kD FK506 binding protein, FKBP13, interacts with a novel homologue of the erythrocyte membrane cytoskeletal protein 4.1.

We have identified a novel generally expressed homologue of the erythrocyte membrane cytoskeletal protein 4.1, named 4.1G, based on the interaction of its COOH-terminal domain (CTD) with the immunophilin FKBP13. The 129-amino acid peptide, designated 4.1G-CTD, is the first known physiologic binding target of FKBP13. FKBP13 is a 13-kD protein originally identified by its high affinity binding to the immunosuppressant drugs FK506 and rapamycin (Jin, Y., M.W. Albers, W.S. Lane, B.E. Bierer, and S.J. Burakoff. 1991. Proc. Natl. Acad. Sci. USA. 88:6677- 6681); it is a membrane-associated protein thought to function as an ER chaperone (Bush, K.T., B.A. Henrickson, and S.K. Nigam. 1994. Biochem. J. [Tokyo]. 303:705-708). We report the specific association of FKBP13 with 4.1G-CTD based on yeast two-hybrid, in vitro binding and coimmunoprecipitation experiments. The histidyl-proline moiety of 4.1G-CTD is required for FKBP13 binding, as indicated by yeast experiments with truncated and mutated 4.1G-CTD constructs. In situ hybridization studies reveal cellular colocalizations for FKBP13 and 4.1G-CTD throughout the body during development, supporting a physiologic role for the interaction. Interestingly, FKBP13 cofractionates with the red blood cell homologue of 4.1 (4.1R) in ghosts, inside-out vesicles, and Triton shell preparations. The identification of FKBP13 in erythrocytes, which lack ER, suggests that FKBP13 may additionally function as a component of membrane cytoskeletal scaffolds.

Structural protein 4.1 in the nucleus of human cells: dynamic rearrangements during cell division.

Structural protein 4.1, first identified as a crucial 80-kD protein in the mature red cell membrane skeleton, is now known to be a diverse family of protein isoforms generated by complex alternative mRNA splicing, variable usage of translation initiation sites, and posttranslational modification. Protein 4.1 epitopes are detected at multiple intracellular sites in nucleated mammalian cells. We report here investigations of protein 4.1 in the nucleus. Reconstructions of optical sections of human diploid fibroblast nuclei using antibodies specific for 80-kD red cell 4.1 and for 4.1 peptides showed 4.1 immunofluorescent signals were intranuclear and distributed throughout the volume of the nucleus. After sequential extractions of cells in situ, 4.1 epitopes were detected in nuclear matrix both by immunofluorescence light microscopy and resinless section immunoelectron microscopy. Western blot analysis of fibroblast nuclear matrix protein fractions, isolated under identical extraction conditions as those for microscopy, revealed several polypeptide bands reactive to multiple 4.1 antibodies against different domains. Epitope-tagged protein 4.1 was detected in fibroblast nuclei after transient transfections using a construct encoding red cell 80-kD 4.1 fused to an epitope tag. Endogenous protein 4.1 epitopes were detected throughout the cell cycle but underwent dynamic spatial rearrangements during cell division. Protein 4.1 was observed in nucleoplasm and centrosomes at interphase, in the mitotic spindle during mitosis, in perichromatin during telophase, as well as in the midbody during cytokinesis. These results suggest that multiple protein 4.1 isoforms may contribute significantly to nuclear architecture and ultimately to nuclear function.

Specific loss of protein kinase activities in senescent erythrocytes.

Rabbit erythrocytes of progressively increasing age were isolated using an avidin-biotin affinity technique and the activity of protein kinases and other enzymes was analyzed in cytosols and membranes from the isolated cells. The activities of cytosolic protein kinase C (PKC), cAMP-dependent kinase (PKA), and casein kinase type I and II (CKI and II) were all found to undergo an age-dependent decrease of twofold to fourfold over the 8-week lifespan of the cells. Membrane-associated tyrosine kinase showed little or no decrease, but membrane-associated CKI showed a dramatic eightfold decrease over the 8-week period. By contrast, various cytosolic enzymes, including lactate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, and acid phosphatase, showed no change in activity over the same time period. Density-separated human erythrocytes showed qualitatively similar decreases in cytosolic protein kinase activities in the densest fractions, which contain the oldest cells. Our results show that aging erythrocytes undergo progressive loss of protein kinases that may adversely affect various cellular processes. The age-dependent loss of kinase activity reported here is one of the most striking manifestations of erythrocyte senescence yet to be reported.

Absence of high-affinity band 4.1 binding sites from membranes of glycophorin C- and D-deficient (Leach phenotype) erythrocytes.

We investigated the role of glycophorins C and D in the association of band 4.1 with the erythrocyte membrane by measuring the binding of band 4.1 to erythrocyte inside-out vesicles stripped of endogenous band 4.1. Vesicles were prepared from either normal erythrocytes or erythrocytes completely lacking glycophorins C and D (Leach phenotype). Band 4.1 binding to vesicles from normal erythrocytes gave rise to a nonlinear Scatchard plot, indicative of two classes of binding sites: a low-capacity, high-affinity class of sites (about 10% of the total) and a high-capacity, low-affinity class of sites. Vesicles prepared from Leach erythrocytes had a binding capacity for band 4.1 that was, on average, 32% lower than that of vesicles from normal erythrocytes. This difference was caused by the complete absence of the high-affinity binding sites as well as by a decrease in the number of low-affinity binding sites. Reduction of membrane phosphatidylinositol 4,5-biphosphate (PIP2) content by adenosine triphosphate depletion or activation of phosphoinositidase C resulted in a decrease in band 4.1 binding capacity to a similar extent in both control and Leach vesicles. The principal effect of PIP2 depletion was a reduction in the number of low-affinity band 4.1 binding sites in control and Leach vesicles. The fact that PIP2 depletion induced a decrease in band 4.1 binding to Leach vesicles shows that glycophorin C or D is not required for the formation of PIP2-sensitive band 4.1 binding sites, and may not be involved in PIP2-sensitive band 4.1 binding sites even when they are present. Our studies give new insights into the involvement of glycophorins and of PIP2 in modulating cytoskeletal-membrane interactions.

Cellular distribution of polyphosphoinositides in rat hepatocytes.

The distribution of total phospholipids, phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) was studied in isolated rat hepatocytes: (i) by mass assay and isotopic labelling in the fractions of plasma membranes, microsomes, mitochondria and nuclei prepared from isolated hepatocytes and (ii) by immunolocalization of PIP2 with a specific antibody (kt3g) in whole hepatocytes and isolated nuclei. Mass measurement and isotopic labelling showed that PIP was distributed in all four fractions. PIP2 was present in the plasma membrane and the nuclei. In whole cells, PIP2 was also detected in the plasma membrane by immunolocalization with the anti-PIP2 antibody kt3g. In unpolarized single hepatocytes, PIP2 distributed evenly throughout the plasma membrane. However, in polarized cell couplets, PIP2 was the most often undetectable in the lateral domain between the cells, and distributed preferentially in the sinusoidal domain of the plasma membrane. These results suggest that hepatocytes segregate PIP2 in particular domains of their plasma membrane. In purified fractions of nuclei, immunolocalization experiments showed that PIP2 was present uniquely in the nuclear envelope.

Characterization of structural and functional phosphoinositide domains in human erythrocyte membranes.

In the erythrocyte membrane, only a fraction (50-60%) of phosphatidylinositol 4,5-bisphosphate (PIP2) and of phosphatidylinositol 4-phosphate (PIP) is rapidly turned over by specific kinases and phosphatases and accessible to hydrolysis by the polyphosphoinositide (PPI)-specific phospholipase C (PLC). To investigate whether the metabolic segregation of PPI resulted from preferential interactions with proteins, we have measured the accessibility of PPI to bee venom phospholipase A2 (PLA2) in native erythrocyte membranes, or after treatments designed to remove peripheral proteins and cytoplasmic domains of integral proteins. In native membranes, PPI, as well as the other major phospholipids, behaved as two distinct fractions (R1 and R2) differing by their sensitivity to PLA2. Such a behavior was not observed in PIP and PIP2 containing artificial vesicles. Evidence was provided that the highly sensitive fraction of PIP and PIP2 (R1) may be identical to the PLC-sensitive and rapidly metabolized pool. Removal of peripheral proteins, followed by proteolysis of the cytoplasmic domain of integral proteins, mainly glycophorins and band 3, led to a reduction of the R1 fraction of PIP and of PIP2. It is proposed that the rapidly metabolized pool of PIP2 and PIP, involved in the regulation of major cellular functions, would be maintained in its functional state through interactions with integral proteins.

The role of inositol phospholipids in the association of band 4.1 with the human erythrocyte membrane.

Band 4.1 is a major protein of the erythrocyte membrane skeleton. It promotes the binding of spectrin to F-actin and may anchor the skeletal network to the plasma membrane via its association with integral membrane proteins. Here, we have investigated the involvement of inositol phospholipids in the binding of band 4.1 to erythrocyte membranes using membrane vesicles stripped of all peripheral proteins at alkaline pH. Trypsinization of these vesicles allows the discrimination of two classes of band 4.1 binding sites: trypsin-sensitive sites (60-65% of the total), largely or exclusively on band 3, and trypsin-resistant sites (35-40% of the total), composed, at least in part, of the glycophorins. ATP depletion or activation of erythrocyte phosphoinositol phospholipase C led to a reduction in membrane phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] content by 20-70% in different experiments. The resulting decrease of band 4.1 binding to vesicles by was variable, but averaged about 15-20%. The same treatments led to an average decrease in the band 4.1 binding capacity of trypsinized vesicles of 55%. Since this is equivalent to a 20% decrease in the binding capacity of non-trypsinized vesicles (consistent with the above result), it indicates that PtdIns(4,5)P2 regulates the binding of band 4.1 only to trypsin-resistant binding sites (and to only a subset of these) accounting for about 15-20% of total band 4.1 binding sites on membranes. We found that hydrolysis of > 95% of PtdIns(4,5)P2 with exogenous phospholipase C-delta (PLC delta) resulted in no further decrease in band 4.1 binding to vesicles than did hydrolysis of 65-70% of PtdIns(4,5)P2 which is accessible to erythrocyte phosphoinositol phospholipase C. This suggests that only 65-70% of total membrane PtdIns(4,5)P2 is involved in regulating band 4.1 binding. Significantly, the pool of PtdIns(4,5)P2 involved is the same pool which can be hydrolysed by erythrocyte phosphoinositol phospholipase C, and which has been shown to be metabolically labile in erythrocytes. The membrane binding capacity for band 4.1 found in this study (averaging 1000 micrograms/mg vesicle protein) is considerably higher than that found in previous studies. The results are consistent with the existence of a binding site for band 4.1 on each copy of the major transmembrane proteins (band 3 and the glycophorins). These results provide new insights into the involvement of membrane inositol phospholipids in cytoskeletal-membrane interactions.