Differential nm23 gene expression at the fetal-maternal interface

The product of the nm23 gene has been proposed as a candidate tumour metastasis suppressor protein. A strong association has been observed between reduced expression of the nm23 gene and acquisition of metastatic behaviour in some tumour cells, including breast cancer and melanoma, but not in others, such as neuroblastoma and colon, cervical and thyroid cancers. During the early gestation period both human and murine trophoblast cells exhibit in vitro invasive properties similar to those of neoplastic cells. Such invasive properties, however, disappear in the late stage of gestation. In the present study, we examined the abundance of nm23 mRNA from various fetal-maternal interface tissues (uterus, decidua, placenta and embryo) during early (day 8), mid (day 14) and late (day 18) stages of gestation in CD1 mice, in order to determine whether nm23 plays any anti-invasive and/or biological roles during gestation. nm23 was found to be expressed in all the tissues during the early and mid stages of gestation. The expression levels were, however, variable among different tissues and development stages. In the early stage, nm23 mRNA levels were the highest and similar among tissues from the uterus, decidua, placenta and embryo. In the mid stage, the mRNA levels were reduced significantly in the uterus, decidua and placenta, but not in the embryo. In the late stage, nm23 mRNA was further reduced to the extent that it could not be seen in the decidua, was barely seen in the uterus and was weakly present in the placenta. However, the mRNA level of the embryo in the late stage was still high and similar to the early stage. We also examined nm23 expression in trophoblast cells from normal human term placenta and a highly metastatic human choriocarcinoma cell line, JAR. nm23 expression was significantly higher in JAR than in normal placenta, indicating that nm23 does not appear to have an anti-metastatic function in this cell line. Several cytokines--interleukin 2 (IL-2), tumour necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma)--and prostaglandin E2 (PGE2) known to modulate tumour growth and metastasis were examined to determine whether they regulate nm23 expression in JAR in vitro. The B16F10 melanoma cell line was used as control. No effect was found in the JAR cell line, whereas TNF-alpha, IFN-gamma and PGE2 down-regulated nm23 expression in the B16F10 cell line.(ABSTRACT TRUNCATED AT 400 WORDS)     

Overexpression of the HIP gene coding for a heparin/heparan sulfate-binding protein in human thyroid carcinomas

A subtractive library screening was performed to identify changes in gene expression that occur during the process of neoplastic transformation of thyroid cells. A cDNA library was constructed from a human thyroid papillary carcinoma cell line (NPA) subtracted with cDNAs from normal thyroid cells (HTC 2). The differential screening of this library lead to the isolation of 39 cDNA clones; six of them showed homology with a recently isolated gene, named HIP, that codes for a protein belonging to a novel class of heparin/heparan sulfate-binding proteins. Northern blot analysis revealed HIP gene overexpression in all of the human thyroid carcinoma cell lines analyzed, as compared to the HTC 2 cells. HIP expression was particularly abundant in the anaplastic carcinoma-derived cell lines. The analysis of surgically removed thyroid tumors showed overexpression of HIP gene in all of the carcinomas, independent of the histotype, although the largest increase in HIP expression was observed in the undifferentiated forms. In contrast, none of the benign adenomas or normal thyroid tissues showed HIP overexpression. To establish the role of HIP overexpression in cell transformation, the NPA cell line was transfected with an eukaryotic expression vector carrying the HIP gene in the antisense orientation. Stable transfectants expressed reduced HIP mRNA levels and showed morphological changes, such as becoming spindle-shaped and growing scattered. The growth rate of the antisense clones was greatly reduced compared to the NPA cells transfected with the backbone vector. Taken together, these results indicate that HIP gene overexpression is associated with thyroid carcinogenesis and strongly suggest its involvement in thyroid cell growth regulation.     

Importance of specific amino acids in protein binding sites for heparin and heparan sulfate

The present study tests whether lesions small enough to allow the rapid reestablishment of a normally aligned tract glial framework would provide a permissive environment for the regeneration of cut adult CNS axons. We made penetrating microlesions which cut a narrow beam of axons in the adult rat cingulum, but caused minimal damage to the tract glial framework and no cavitation. The proximal tips of cut axons were identified by enhanced immunoreactivity for low affinity neurotrophin receptor, p75. From 1 day they became expanded into large growth-cone-like structures. At later times some axons turned back and extended in the reverse direction. Up to 14 days (after which time p75 could no longer be used as a marker), no axons advanced beyond the line of the lesion. From 1 to 2 days, OX42 immunostaining and electron microscopy showed that the lesion site was densely infiltrated by macrophages, which disappeared by 3 to 4 days. This was followed by a local hypertrophy of the OX42 immunoreactive resident tract microglial cells and an increase in both GFAP and vimentin immunoreactivity of the tract astrocytes. These responses were greatly reduced by 8 days, when the longitudinal alignment of glial processes across the lesion site was similar to that of an undamaged tract. The large growth-cone-like structures formed at the ends of the cut axons resemble those of developing axons exposed to chemorepulsive factors. This suggests that cellular elements in adult tract lesions may also exert chemorepulsive influences blocking regeneration of axons even in an apparently "open" tract framework.     

Reg1ulatory role and molecular interactions of a cell-surface heparan sulfate proteoglycan (N-syndecan) in hippocampal long-term potentiation

The cellular mechanisms responsible for synaptic plasticity involve interactions between neurons and the extracellular matrix. Heparan sulfates (HSs) constitute a group of glycosaminoglycans that accumulate in the beta-amyloid deposits in Alzheimer's disease and influence the development of neuron-target contacts by interacting with other cell surface and matrix molecules. However, the contribution of HSs to brain function is unknown. We found that HSs play a crucial role in long-term potentiation (LTP), a finding that is consistent with the idea that converging molecular mechanisms are used in the development of neuron-target contacts and in activity-induced synaptic plasticity in adults. Enzymatic cleavage of HS by heparitinase as well as addition of soluble heparin-type carbohydrates prevented expression of LTP in response to 100 Hz/1 sec stimulation of Schaffer collaterals in rat hippocampal slices. A prominent carrier protein for the type of glycans implicated in LTP regulation in the adult hippocampus was identified as N-syndecan (syndecan-3), a transmembrane proteoglycan that was expressed at the processes of the CA1 pyramidal neurons in an activity-dependent manner. Addition of soluble N-syndecan into the CA1 dendritic area prevented tetanus-induced LTP. A major substrate of src-type kinases, cortactin (p80/85), and the tyrosine kinase fyn copurified with N-syndecan from hippocampus. Moreover, association of both cortactin and fyn to N-syndecan was rapidly increased after induction of LTP. N-syndecan may thus act as an important regulator in the activity-dependent modulation of neuronal connectivity by transmitting signals between extracellular heparin-binding factors and the fyn signaling pathway.     

The cell-surface heparan sulfate proteoglycan glypican-1 regulates growth factor action in pancreatic carcinoma cells and is overexpressed in human pancreatic cancer

Heparan sulfate proteoglycans (HSPGs) play diverse roles in cell recognition, growth, and adhesion. In vitro studies suggest that cell-surface HSPGs act as coreceptors for heparin-binding mitogenic growth factors. Here we show that the glycosylphosphatidylinositol- (GPI-) anchored HSPG glypican-1 is strongly expressed in human pancreatic cancer, both by the cancer cells and the adjacent fibroblasts, whereas expression of glypican-1 is low in the normal pancreas and in chronic pancreatitis. Treatment of two pancreatic cancer cell lines, which express glypican-1, with the enzyme phosphoinositide-specific phospholipase-C (PI-PLC) abrogated their mitogenic responses to two heparin-binding growth factors that are commonly overexpressed in pancreatic cancer: fibroblast growth factor 2 (FGF2) and heparin-binding EGF-like growth factor (HB-EGF). PI-PLC did not alter the response to the non-heparin-binding growth factors EGF and IGF-1. Stable expression of a form of glypican-1 engineered to possess a transmembrane domain instead of a GPI anchor conferred resistance to the inhibitory effects of PI-PLC on growth factor responsiveness. Furthermore, transfection of a glypican-1 antisense construct attenuated glypican-1 protein levels and the mitogenic response to FGF2 and HB-EGF. We propose that glypican-1 plays an essential role in the responses of pancreatic cancer cells to certain mitogenic stimuli, that it is relatively unique in relation to other HSPGs, and that its expression by pancreatic cancer cells may be of importance in the pathobiology of this disorder.     

Binding, internalization, and degradation of antiproliferative heparan sulfate by human embryonic lung fibroblasts

Pretargeting methodologies can produce high tumor:blood ratios, but their role in cancer radioimmunotherapy (RAIT) is uncertain. A pretargeting method was developed using a streptavidin (StAv) conjugate of MN-14 IgG, an anti-carcinoembryonic antigen (CEA) murine monoclonal antibody (mab) as the primary targeting agent, an anti-idiotype antibody (WI2 IgG) as a clearing agent, and DTPA- or DOTA-conjugated biotin as the radiolabeled targeting agent. A variety of reagents and conditions were examined to optimize this method. At 3 h, 111In-DTPA-peptide-biotin tumor uptake was 3.9 +/- 0.8% per gram and tumor:blood ratios were > 11:1. By 24 h, this ratio was 178:1, but tumor accretion declined in accordance with the gradual loss of StAv-MN-14 from the tumor. Tissue retention was highest in the liver and kidneys, but their tumor:organ ratios were > 2:1. Dosimetry predicted that radiolabeled MN-14 alone would deliver higher tumor doses than this pretargeting method. Increasing the specific activity and using DOTA-biotin in place of DTPA increased tumor uptake nearly 2-fold, but analysis of StAv-MN-14's biotin-binding capacity indicated over 90% of the initial biotin-binding sites were blocked within 24 h. Animals fed a biotin-deficient diet had 2-fold higher 111In-DOTA-biotin uptake in the tumor, but higher uptake also was observed in all normal tissues. Although exceptionally adept at achieving high tumor:blood ratios rapidly, the tumor uptake of radiolabeled biotin with this pretargeting method is significantly (p < 0.0001) lower than that with a radiolabeled antibody. Endogenous biotin and enhanced liver and kidney uptake may limit the application of this method to RAIT, especially when evaluating the method in animals, but with strategies to overcome these limitations, this pretargeting method could be an effective therapeutic alternative.     

Role of the extracellular domain of human herpesvirus 7 glycoprotein B in virus binding to cell surface heparan sulfate proteoglycans

In an attempt to identify the human herpesvirus 7 (HHV-7) envelope protein(s) involved in cell surface binding, the extracellular domain of the HHV-7 glycoprotein B (gB) homolog protein was cloned and expressed as a fusion product with the Fc domain of human immunoglobulin G heavy chain gamma1 (gB-Fc) in an eukaryotic cell system. Indirect immunofluorescence followed by flow cytometric analysis revealed specific binding of gB-Fc to the membrane of SupT1 cells but not to other CD4+ T-lymphoblastoid cell lines, such as Jurkat or PM1, clearly indicating that gB-Fc did not bind to the CD4 molecule. This was also suggested by the ability of gB-Fc to bind to CD4-negative fibroblastoid Chinese hamster ovary (CHO) cells. The binding was abrogated by enzymatic removal of cell surface heparan sulfate proteoglycans by heparinase and heparitinase but not by treatment with condroitinase ABC. In addition, binding of the gB-Fc fusion protein to CHO cells was severely impaired in the presence of soluble heparin, as well as when heparan sulfate-deficient mutant CHO cells were used. Consistent with these findings, soluble heparin was found to block HHV-7 infection and syncytium formation in the SupT1 cell line. Although the CD4 antigen is a critical component of the receptor for the T-lymphotropic HHV-7, these findings suggest that heparin-like molecules also play an important role in HHV-7-cell surface interactions required for infection and that gB represents one of the HHV-7 envelope proteins involved in the adsorption of virus-to-cell surface proteoglycans.     

A single protein catalyzes both N-deacetylation and N-sulfation during the biosynthesis of heparan sulfate

Heparan sulfate is a highly sulfated carbohydrate polymer that binds to and modulates the activities of numerous proteins. The formation of these protein-binding domains in heparan sulfate is dependent on a series of biosynthetic reactions that modify the polysaccharide backbone; the initiating and rate-limiting steps of this process are the N-deacetylation and N-sulfation of N-acetylglucosamine residues in the polymer. We now report that in the rat liver, biosynthesis of heparan sulfate utilizes a single protein that possesses both N-deacetylase and N-sulfotransferase activities. This was accomplished by demonstrating that both activities resided in a purified soluble fusion protein containing the Golgi-lumenal portion of the enzyme. We propose that this protein be renamed the rat liver Golgi heparan sulfate N-deacetylase/N-sulfotransferase.     

Structural modification of fibroblast growth factor-binding heparan sulfate at a determinative stage of neural development

Heparan sulfate (HS) glycosaminoglycans are essential modulators of fibroblast growth factor (FGF) activity and appear to act by coupling particular forms of FGF to appropriate FGF receptors. During neural development, one particular HS proteoglycan is able to rapidly switch its potentiating activity from FGF-2, as neural precursor cell proliferation occurs, to FGF-1, as neuronal differentiation occurs. Using various analytical techniques, including chemical and enzymatic cleavage, low pressure chromatography, and strong anion-exchange high performance liquid chromatography, we have analyzed the different HSs expressed during these crucial developmental stages. There are distinct alterations in patterns of 6-O-sulfation, total chain length, and the number of sulfated domains of the HS from the more mature embryonic brain. These changes correlate with a switch in the ability of the HS to potentiate the actions of FGF-1 in triggering cell differentiation. It thus appears that each HS pool is designed to function in the modulation of an intricate interaction with a specific growth factor and its cognate receptor, and suggests tightly regulated expression of specific, bioactive disaccharide sequences. The data can be used to construct a simple model of controlled variations in HS chain structure which have functional consequences at a crucial stage of neuronal maturation.     

Characterization of the heparan sulfate and chondroitin sulfate assembly sites in CD44

Isoforms of CD44 are differentially modified by the glycosaminoglycans (GAGs) chondroitin sulfate (CS), heparan sulfate (HS), and keratan sulfate. GAG assembly occurs at serines followed by glycines (SG), but not all SG are utilized. Seven SG motifs are distributed in five CD44 exons, and in this paper we identify the HS and CS assembly sites that are utilized in CD44. Not all the CD44 SG sites are modified. The SGSG motif in CD44 exon V3 is the only HS assembly site; this site is also modified with CS. HS and CS attachment at that site was eliminated by mutation of the serines in the V3 motif to alanine (AGAG). Exon E5 is the only other CD44 exon that supports GAG assembly and is modified with CS. Using a number of recombinant CD44 protein fragments we show herein that the eight amino acids located downstream of the SGSG site in V3 are responsible for the specific addition of HS to this site. If the eight amino acids located downstream from the first SG site in CD44 exon E5 are exchanged with those located downstream of the SGSG site in exon V3, the SG site in E5 becomes modified with HS and CS. Likewise if the eight amino acids found downstream from the first SG in E5 are placed downstream from the SGSG in V3, this site is modified with CS but not HS. We also show that these sequences cannot direct the modification of CD44 with HS from a distance. Constructs containing CD44 exon V3 in which the SGSG motif was mutated to AGAG were not modified with HS even though they contained other SG motifs. Thus, a number of sequence and structural requirements that dictate GAG synthesis on CD44 have been identified.     

The heparan sulfate binding sequence of interferon-gamma increased the on rate of the interferon-gamma-interferon-gamma receptor complex formation

Interferon-gamma (IFNgamma), in common with a number of growth factors, binds both to heparan sulfate or heparin-related molecules and to a specific high affinity receptor (IFNgammaR). Using surface plasmon resonance technology, kinetic analysis of the IFNgamma. IFNgammaR complex formation was performed with the extracellular part of IFNgammaR immobilized on a sensor chip. At the sensor chip surface, IFNgamma was bound by two IFNgammaR molecules with an affinity in the nanomolar range (0.68 nM). This binding was characterized by an important on rate, kon = 7.3 x 10(6) M-1.s-1, and an off rate, koff = 5 x 10(-3).s-1. This binding assay was used to investigate a possible role of heparin in the IFNgamma.IFNgammaR complex formation. In contrast to growth factors for which binding to heparin is usually required for high affinity receptor interaction, we found in this study that IFNgamma bound to heparin displayed a strongly reduced affinity for its receptor. This is consistent with the fact that a cluster of basic amino acids (KTGKRKR, called the C1 domain) in the carboxyl-terminal sequence of the cytokine was involved both in heparin and receptor recognition. To understand how a single domain of IFNgamma could be implicated in two discrete functions (i.e. binding to heparin and to IFNgammaR), we also analyzed in a detailed manner the role of the IFNgamma carboxyl-terminal sequence in receptor binding. Using forms of IFNgamma, with carboxyl terminus truncations of defined regions of the heparin binding sequence, we found that the C1 domain functioned by increasing the on rate of the IFNgamma.IFNgammaR binding reaction but was not otherwise required for the stability of the complex. Interactions between the IFNgamma carboxyl-terminal domain and IFNgammaR could increased the association rate of the reaction either by increasing the number of encounters between the two molecules or by favoring productive collisions. The mechanisms by which heparan sulfate regulates IFNgamma activity may thus include both control of selective protease cleavage events, which directly affect the cytokine activity, and also an ability to modulate the interaction of IFNgamma with the IFNgammaR via competitive binding to the C1 domain.     

Differential binding of fibroblast growth factor-2 and -7 to basement membrane heparan sulfate: comparison of normal and abnormal human tissues

Fibroblast growth factors (FGFs) play multiple roles during development and in adult tissues as paracrine regulators of growth and differentiation. FGFs signal through transmembrane receptor tyrosine kinases, but heparan sulfate is also required for signaling by members of the FGF family. In addition, heparan sulfate may be involved in determining tissue distribution of FGFs. Using biotinylated FGF-2 and FGF-7 (KGF) as probes, we have identified specific interactions between FGFs and heparan sulfates in human tissues. Both FGF species bind to tissue mast cells and to epithelial cell membranes. Binding to basement membrane heparan sulfate is tissue source dependent and specific. Although FGF-2 strongly binds to basement membrane heparan sulfate in skin and most other tissue sites examined, FGF-7 fails to bind to basement membrane heparan sulfate in most locations. However, in subendothelial matrix in blood vessels and in the basement membrane of a papillary renal cell carcinoma, strong FGF-7 binding is seen. In summary, distinct and specific affinities of heparan sulfates for different FGFs were identified that may affect growth factor activation and local distribution. Heparan sulfate may have a gatekeeper function to either restrict or permit diffusion of heparin-binding growth factors across the basement membrane.     

Characterization of heparin and heparan sulfate domains binding to the long splice variant of platelet-derived growth factor A chain

Platelet-derived growth factors (PDGFs) are homo- or heterodimers of two related polypeptides, known as A and B chains. The A chain exists as two splice variants due to the alternative usage of exons 6 (PDGF-AL, longer) and 7 (PDGF-AS, shorter). Exon 6 encodes an 18-amino acid sequence rich in basic amino acid residues, which has been implicated as a cell retention signal. Several lines of evidence indicate that the retention is due to binding of PDGF-AL to glycosaminoglycans, especially to heparan sulfate. We have analyzed the saccharide domains of smooth muscle cell-derived heparan sulfate involved in this interaction. Furthermore, we have employed selectively modified heparin oligosaccharides to elucidate the dependence of the binding on different sulfate groups and on fragment length. The shortest PDGF-AL binding domain consists of 6-8 monosaccharide units. Studies using selectively desulfated heparins and heparin fragments suggest that N-, 2-O-, and 6-O-sulfate groups all contribute to the interaction. Structural comparison of heparan sulfate oligosaccharides separated by affinity chromatography on immobilized PDGF-AL showed that the bound pool was enriched in -IdceA(2-OSO3)-GlcNSO3(6-OSO3)- disaccharide units. Furthermore, analogous separation of a partially O-desulfated heparin decamer preparation, using a highly selective nitrocellulose filter-trapping system, yielded a PDGF-AL-bound fraction in which more than half of the disaccharide units had the structure -IdceA(2-OSO3)-GlcNSO3(6-OSO3)-. Our results suggest that the interaction between PDGF-AL and heparin/heparan sulfate is mediated via N-sulfated saccharide domains containing both 2-O- and 6-O-sulfate groups.     

Syndecans, heparan sulfate proteoglycans, maintain the proteolytic balance of acute wound fluids

An imbalance between proteases and antiproteases is thought to play a role in the inflammatory injury that regulates wound healing. The activities of some proteases and antiproteases found in inflammatory fluids can be modified in vitro by heparin, a mast cell-derived glycosaminoglycan. Because syndecans, a family of cell surface heparan sulfate proteoglycans, are the major cellular source of heparin-like glycosaminoglycan, we asked whether syndecans modify protease activities in vivo. Syndecan-1 and syndecan-4 ectodomains are shed into acute human dermal wound fluids (Subramanian, S. V., Fitzgerald, M. L., and Bernfield, M. (1997) J. Biol. Chem. 272, 14713-14720). Moreover, purified syndecan-1 ectodomain binds cathepsin G (Kd = 56 nM) and elastase (Kd = 35 nM) tightly and reduces the affinity of these proteases for their physiological inhibitors. Purified syndecan-1 ectodomain protects cathepsin G from inhibition by alpha1-antichymotrypsin and squamous cell carcinoma antigen 2 and elastase from inhibition by alpha1-proteinase inhibitor by decreasing second order rate constants for protease-antiprotease associations (kass) by 3700-, 32-, and 60-fold, respectively. Both enzymatic degradation of heparan sulfate and immunodepletion of the syndecan-1 and -4 in wound fluid reduce these proteolytic activities in the fluid, indicating that the proteases in the wound environment are regulated by interactions with syndecan ectodomains. Thus, syndecans are shed into acute wound fluids, where they can modify the proteolytic balance of the fluid. This suggests a novel physiological role for these soluble heparan sulfate proteoglycans.     

A peptide sequence of heparin/heparan sulfate (HP/HS)-interacting protein supports selective, high affinity binding of HP/HS and cell attachment

We previously have identified a novel cell surface heparan sulfate/heparin (HS/HP)-interacting protein (HIP) found in human uterine epithelia and a variety of other human epithelial and endothelial cells and cell lines (Liu, S., Smith, S. E., Julian, J., Rohde, L. H., Karin, N. J., and Carson, D. D. (1996) J. Biol. Chem. 271, 11817-11823; Rohde, L. H., Julian, J., Babaknia, A., and Carson, D. D. (1996) J. Biol. Chem. 271, 11824-11830). The amino acid sequence predicted for HIP revealed a potential HS/HP-binding motif. In the present studies, a synthetic peptide corresponding to this putative HS/HP-binding motif, HIP peptide, was synthesized and examined with regard to its HS/HP binding and cell attachment promoting activity. Results using solid phase binding assays demonstrate that HIP peptide binds HS/HP with high selectivity and has high affinity for bulk HP (50% saturation congruent with 300 nM) and even higher affinity for a subset of polysaccharides found in commercial [3H]HP (half-saturation congruent with 10 nM). Moreover, HIP peptide binds subsets of cell and extracellular matrix-associated HS and dermatan sulfate expressed by RL95 cells, a human uterine adenocarcinoma cell line. HIP peptide also binds a similar fraction of HS as well as dermatan sulfate expressed by JAR cells, a human choriocarcinoma cell line. In contrast to binding of cell- or extracellular matrix-associated HS, HIP peptide does not bind secreted or released forms of HS or DS from either RL95 or JAR cells to a significant extent. HS species that bind to HIP peptide are generally larger, have a higher negative charge density, and have a larger proportion of di- and trisulfated disaccharide units than HS species that do not bind to HIP peptide, demonstrating structural differences among these polysaccharides. This same peptide supports HS-dependent JAR cell attachment. Collectively, these data demonstrate that a linear peptide sequence found within HIP can account, at least in part, for the HS/HP binding and cell adhesion promoting activities of this protein.     

Identification of the protein 4.1 binding interface on glycophorin C and p55, a homologue of the Drosophila discs-large tumor suppressor protein

Protein 4.1 is the prototype of a family of proteins that include ezrin, talin, brain tumor suppressor merlin, and tyrosine phosphatases. All members of the protein 4.1 superfamily share a highly conserved N-terminal 30-kDa domain whose biological function is poorly understood. It is believed that the attachment of the cytoskeleton to the membrane may be mediated via this 30-kDa domain, a function that requires formation of multiprotein complexes at the plasma membrane. In this investigation, synthetically tagged peptides and bacterially expressed proteins were used to map the protein 4.1 binding site on human erythroid glycophorin C, a transmembrane glycoprotein, and on human erythroid p55, a palmitoylated peripheral membrane phosphoprotein. The results show that the 30-kDa domain of protein 4.1 binds to a 12-amino acid segment within the cytoplasmic domain of glycophorin C and to a positively charged, 39-amino acid motif in p55. Sequences similar to this charged motif are conserved in other members of the p55 superfamily, including the Drosophila discs-large tumor suppressor protein. Our data provide new insights into how protein 4.1, glycophorin C, p55, and their non-erythroid homologues, interact with the cytoskeleton to exert their physiological effects.