This review focuses on the recent advances in functions of spectrins in non-erythroid cells. We discuss new data concerning the commonly known role of the spectrin-based skeleton in control of membrane organization, stability and shape, and tethering protein mosaics to the cellular motors and to all major filament systems. Particular effort has been undertaken to highlight recent advances linking spectrin to cell signaling phenomena and its participation in signal transduction pathways in many cell types.

We use 1,2-diacetylbenzene (1,2-DAB) to probe molecular mechanisms of proximal giant neurofilamentous axonopathy (PGNA), a pathological hallmark of amyotrophic lateral sclerosis. The spinal cord proteome of rodents displaying 1,2-DAB PGNA suggests a reduction in the abundance of α-II spectrin (Spna2), a key protein in the maintenance of axonal integrity. Protein immunoblotting indicates that this reduction is due to Spna2 degradation. We investigated the importance of such degradation in 1,2-DAB PGNA. Spna2 mutant mice lacking a calpain- and/or caspase-sensitive domain (CSD), thus hypothetically resistant to 1,2-DAB, and wild-type littermates, were treated with 1,2-DAB, 35 mg/kg/day, or saline control, for 3 weeks. 1,2-DAB induced motor weakness and PGNA, irrespective of the genotype. Spna2-calpain breakdown products were not detected in mutant mice, which displayed a normal structure of the nervous system under saline treatment. Intriguingly, treatment with 1,2-DAB reduced the abundance of the caspase-specific 120-kDa Spna2 breakdown products. Our findings indicate that degradation of Spna2 by calpain- and/or caspase is not central to the pathogenesis of 1,2-DAB axonopathy. In addition, the Spna2-CSD seems to be not required for the maintenance of the cytoskeleton integrity. Our conceptual framework offers opportunities to study the role of calpain-caspase cross talk, including that of the protease degradomics, in models of axonal degeneration.

The spectrin-based membrane skeleton, a multi-protein scaffold attached to diverse cellular membranes, is presumed to be involved in the stabilization of membranes, the establishment of membrane domains as well as in vesicle trafficking and nuclear functions. Spectrin tetramers made of alpha- and beta-subunits are linked to actin microfilaments, forming a network that binds a multitude of proteins. The most prevalent alpha-spectrin subunit in non-erythroid cells, alphaII-spectrin, contains two particular spectrin repeats in its central region, alpha9 and alpha10, which host an Src homology 3 domain, a tissue-specific spliced sequence of 20 residues, a calmodulin-binding site and major cleavage sites for caspases and calpains. Using yeast two-hybrid screening of kidney libraries, we identified two partners of the alpha9-alpha10 repeats: the potential tumour suppressor Tes, an actin-binding protein mainly located at focal adhesions; and EVL (Ena/vasodilator-stimulated phosphoprotein-like protein), another actin-binding protein, equally recruited at focal adhesions. Interactions between spectrin and overexpressed Tes and EVL were confirmed by co-immunoprecipitation. In vitro studies showed that the interaction between Tes and spectrin is mediated by a LIM (Lin-11, Isl-1 and Mec3) domain of Tes and by the alpha10 repeat of alphaII-spectrin whereas EVL interacts with the Src homology 3 domain located within the alpha9 repeat. Moreover, we describe an in vitro interaction between Tes and EVL, and a co-localization of these two proteins at focal adhesions. These interactions between alphaII-spectrin, Tes and EVL indicate new functions for spectrin in actin dynamics and focal adhesions.

Lutheran (Lu) and Lu(v13), two glycoprotein (gp) isoforms belonging to the immunoglobulin superfamily, represent adhesion molecules that act as erythrocyte receptors for laminin 10/11. These two gps, which differ only by the length of their cytoplasmic tail, carry both Lu blood group and Basal Cell Adhesion Molecule (B-CAM) antigens. Here, analysis of the Triton extractability of recombinant Lu and Lu(v13) gps in K562 transfected cells showed that both gps were mainly associated with the detergent-insoluble material. Patching experiments using Cholera Toxin subunit B indicated that Lu gps were not localized in lipid rafts. Glutathione-S-transferase capture assays showed that the cytoplasmic domain of Lu and Lu(v13) bound to erythroid spectrin, present in a low ionic strength extract from red cell ghosts. Direct interaction with spectrin was confirmed by plasmon resonance assays. Site-directed mutagenesis mapped a major interaction site with spectrin to the RK573-574 motif, located on the cytoplasmic tail of Lu gp, in close vicinity to the inner leaflet of the membrane lipid bilayer. The two Lu adhesion gps represent the first example of a direct link between transmembrane proteins and spectrin in red blood cells. Since Lu gps are low abundant proteins, we speculate that their interaction with spectrin might be critical for signalling and receptor function rather than for participating in the linkage of the lipid bilayer to the red cell skeleton.

The spectrin-actin scaffold underlying the lipid bilayer is considered to participate in cell-shape stabilization and in the organization of specialized membrane subdomains. These structures are dynamic and likely to undergo frequent remodelling during changes in cell shape. Proteolysis of spectrin, which occurs during apoptosis, leads to destabilization of the scaffold. It is also one of the major processes involved in membrane remodelling. Spectrins, the main components of the membrane skeleton, are the targets for two important protease systems: m- and micro-calpains (Ca2+-activated proteases) and caspase-3 (activated during apoptosis). In this paper, we show that caspase-2 also targets spectrin in vitro, and we characterize Ca2+/calmodulin-dependent regulation of spectrin cleavage by caspases. Yeast two-hybrid screening reveals that the large isoform (1/L) of procaspase-2 specifically binds to alphaII-spectrin, while the short isoform does not. Like caspase-3, caspase-2 cleaves alphaII-spectrin in vitro at residue Asp-1185. This study emphasizes a role of executioner caspase for caspase-2. We also demonstrated that the executioner caspase-7 but not caspase-6 cleaves spectrin at residue Asp-1185 in vitro. This spectrin cleavage by caspases 2, 3 and 7 is inhibited by the Ca2+-dependent binding of calmodulin to spectrin. In contrast, calmodulin binding enhances spectrin cleavage by calpain at residue Tyr-1176. These results indicate that alphaII-spectrin cleavage is highly influenced by Ca2+ homoeostasis and calmodulin, which therefore represent potential regulators of the stability and the plasticity of the spectrin-based skeleton.

British Journal of HaematologyVolume 126, Issue 1 Free Access Papers to be published in forthcoming issues. Toward more effective antifungal therapy: the prospects of combination therapy. Myeloma cells can directly contribute to the pool of RANKL in bone bypassing the classic stromal and osteoblast pathway of osteoclast stimulation. Haematogones in the peripheral blood of adults: a four-colour flow cytometry study of 102 patients. Plasma levels of von Willebrand factor regulate ADAMTS-13, its major cleaving protease. Synthesis of osteoprotegerin and RANKL by megakaryocytes is modulated by oestrogen. Direct interaction between the Lu/B-CAM adhesion glycoproteins and erythroid spectrin. Applications of murine and humanized chimeric monoclonal antibodies for red cell phenotyping. Impact of pre-analytical handling on bone marrow mRNA gene expression. Identification of protein Sα gene mutations including four novel mutations in eight unrelated patients with protein S deficiency D. P. Kontoyiannis, D. P. KontoyiannisSearch for more papers by this authorR. E. Lewis, R. E. LewisSearch for more papers by this authorF. P. L. Lai, F. P. L. LaiSearch for more papers by this authorM. Cole-Sinclair, M. Cole-SinclairSearch for more papers by this authorW.-J. Cheng, W.-J. ChengSearch for more papers by this authorJ. M. W. Quinn, J. M. W. QuinnSearch for more papers by this authorM. T. Gillespie, M. T. GillespieSearch for more papers by this authorJ. W. Sentry, J. W. SentrySearch for more papers by this authorH.-G. Schneider, H.-G. SchneiderSearch for more papers by this authorS. H. Kroft, S. H. KroftSearch for more papers by this authorS. L. Asplund, S. L. AsplundSearch for more papers by this authorR. W. McKenna, R. W. McKennaSearch for more papers by this authorN. J. Karandikar, N. J. KarandikarSearch for more papers by this authorP. M. Mannucci, P. M. MannucciSearch for more papers by this authorC. Capoferri, C. CapoferriSearch for more papers by this authorM. T. Canciani, M. T. CancianiSearch for more papers by this authorS. Bord, S. BordSearch for more papers by this authorE. Frith, E. FrithSearch for more papers by this authorD. C. Ireland, D. C. IrelandSearch for more papers by this authorM. A. Scott, M. A. ScottSearch for more papers by this authorJ. I. O. Craig, J. I. O. CraigSearch for more papers by this authorJ. E Compston, J. E CompstonSearch for more papers by this authorY. Kroviarski, Y. KroviarskiSearch for more papers by this authorW. El Nemer, W. El NemerSearch for more papers by this authorP. Gane, P. GaneSearch for more papers by this authorC. Rahuel, C. RahuelSearch for more papers by this authorE. Gauthier, E. GauthierSearch for more papers by this authorM. C. Lecomte, M. C. LecomteSearch for more papers by this authorJ. P. Cartron, J. P. CartronSearch for more papers by this authorY. Colin, Y. ColinSearch for more papers by this authorC. Le Van Kim, C. Le Van KimSearch for more papers by this authorE. Lee, E. LeeSearch for more papers by this authorG. Burgess, G. BurgessSearch for more papers by this authorG. R. Halverson, G. R. HalversonSearch for more papers by this authorT. J. Huang, T. J. HuangSearch for more papers by this authorM. E. Reid, M. E. ReidSearch for more papers by this authorS. Breit, S. BreitSearch for more papers by this authorM. Nees, M. NeesSearch for more papers by this authorU. Schaefer, U. SchaeferSearch for more papers by this authorM. Pfoersich, M. PfoersichSearch for more papers by this authorC. Hagemeier, C. HagemeierSearch for more papers by this authorM. Muckenthaler, M. MuckenthalerSearch for more papers by this authorA. E. Kulozik, A. E. KulozikSearch for more papers by this authorH. Okada, H. OkadaSearch for more papers by this authorA. Takagi, A. TakagiSearch for more papers by this authorT. Murate, T. MurateSearch for more papers by this authorT. Adachi, T. AdachiSearch for more papers by this authorK. Yamamoto, K. YamamotoSearch for more papers by this authorT. Matsushita, T. MatsushitaSearch for more papers by this authorJ. Takamatsu, J. TakamatsuSearch for more papers by this authorK. Sugita, K. SugitaSearch for more papers by this authorM. Sugimoto, M. SugimotoSearch for more papers by this authorA. Yoshioka, A. YoshiokaSearch for more papers by this authorT. Yamazaki, T. YamazakiSearch for more papers by this authorH. Saito, H. SaitoSearch for more papers by this authorT. Kojima, T. KojimaSearch for more papers by this author D. P. Kontoyiannis, D. P. KontoyiannisSearch for more papers by this authorR. E. Lewis, R. E. LewisSearch for more papers by this authorF. P. L. Lai, F. P. L. LaiSearch for more papers by this authorM. Cole-Sinclair, M. Cole-SinclairSearch for more papers by this authorW.-J. Cheng, W.-J. ChengSearch for more papers by this authorJ. M. W. Quinn, J. M. W. QuinnSearch for more papers by this authorM. T. Gillespie, M. T. GillespieSearch for more papers by this authorJ. W. Sentry, J. W. SentrySearch for more papers by this authorH.-G. Schneider, H.-G. SchneiderSearch for more papers by this authorS. H. Kroft, S. H. KroftSearch for more papers by this authorS. L. Asplund, S. L. AsplundSearch for more papers by this authorR. W. McKenna, R. W. McKennaSearch for more papers by this authorN. J. Karandikar, N. J. KarandikarSearch for more papers by this authorP. M. Mannucci, P. M. MannucciSearch for more papers by this authorC. Capoferri, C. CapoferriSearch for more papers by this authorM. T. Canciani, M. T. CancianiSearch for more papers by this authorS. Bord, S. BordSearch for more papers by this authorE. Frith, E. FrithSearch for more papers by this authorD. C. Ireland, D. C. IrelandSearch for more papers by this authorM. A. Scott, M. A. ScottSearch for more papers by this authorJ. I. O. Craig, J. I. O. CraigSearch for more papers by this authorJ. E Compston, J. E CompstonSearch for more papers by this authorY. Kroviarski, Y. KroviarskiSearch for more papers by this authorW. El Nemer, W. El NemerSearch for more papers by this authorP. Gane, P. GaneSearch for more papers by this authorC. Rahuel, C. RahuelSearch for more papers by this authorE. Gauthier, E. GauthierSearch for more papers by this authorM. C. Lecomte, M. C. LecomteSearch for more papers by this authorJ. P. Cartron, J. P. CartronSearch for more papers by this authorY. Colin, Y. ColinSearch for more papers by this authorC. Le Van Kim, C. Le Van KimSearch for more papers by this authorE. Lee, E. LeeSearch for more papers by this authorG. Burgess, G. BurgessSearch for more papers by this authorG. R. Halverson, G. R. HalversonSearch for more papers by this authorT. J. Huang, T. J. HuangSearch for more papers by this authorM. E. Reid, M. E. ReidSearch for more papers by this authorS. Breit, S. BreitSearch for more papers by this authorM. Nees, M. NeesSearch for more papers by this authorU. Schaefer, U. SchaeferSearch for more papers by this authorM. Pfoersich, M. PfoersichSearch for more papers by this authorC. Hagemeier, C. HagemeierSearch for more papers by this authorM. Muckenthaler, M. MuckenthalerSearch for more papers by this authorA. E. Kulozik, A. E. KulozikSearch for more papers by this authorH. Okada, H. OkadaSearch for more papers by this authorA. Takagi, A. TakagiSearch for more papers by this authorT. Murate, T. MurateSearch for more papers by this authorT. Adachi, T. AdachiSearch for more papers by this authorK. Yamamoto, K. YamamotoSearch for more papers by this authorT. Matsushita, T. MatsushitaSearch for more papers by this authorJ. Takamatsu, J. TakamatsuSearch for more papers by this authorK. Sugita, K. SugitaSearch for more papers by this authorM. Sugimoto, M. SugimotoSearch for more papers by this authorA. Yoshioka, A. YoshiokaSearch for more papers by this authorT. Yamazaki, T. YamazakiSearch for more papers by this authorH. Saito, H. SaitoSearch for more papers by this authorT. Kojima, T. KojimaSearch for more papers by this author First published: 14 June 2004 https://doi.org/10.1111/j.1365-2141.2004.005016.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume126, Issue1July 2004 RelatedInformation

The epithelial Na(+) channel (ENaC) is regulated by the ubiquitin-protein ligase Nedd4-2 via interaction with ENaC PY-motifs. These PY-motifs are mutated/deleted in Liddle's syndrome, resulting in elevated Na(+) reabsorption and hypertension explained partly by impaired ENaC-Nedd4-2 interaction. We hypothesized that Nedd4-2 is a susceptibility gene for hypertension and screened 856 renal patients and healthy controls for mutations in a subset of exons of the human Nedd4-2 gene that are relevant for ENaC regulation by PCR/single-strand conformational polymorphism. Several variants were identified, and one nonsynonymous mutation (Nedd4-2-P355L) was further characterized. This mutation next to the 3' donor site of exon 15 does not affect in vitro splicing of Nedd4-2 mRNA. However, in the Xenopus oocyte expression system, Nedd4-2-P355L-dependent ENaC inhibition was weaker compared with the wild type (Nedd4-2-WT), and this difference depended on the presence of intact PY-motifs on ENaC. This could not be explained by the amount of wild type or mutant Nedd4-2 coimmunoprecipitating with ENaC. When the phosphorylation level of human Nedd4-2 Ser(448) (known to be phosphorylated by the Sgk1 kinase) was determined with a specific anti-pSer(448) antibody, we observed stronger basal phosphorylation of Nedd4-2-P355L. Both the phosphorylation level and the accompanying amiloride-sensitive Na(+) currents could be further enhanced to approximately the same levels by coexpressing Sgk1. In addition, the role of the two other putative Sgk1 phosphorylation sites (S342 and T367) appears also to be affected by the P355L mutation. The differential phosphorylation status between wild-type and mutant Nedd4-2 provides an explanation for the different potential to inhibit ENaC activity.

A fine regulation of the amiloride-sensitive Epithelial Sodium Channel (ENaC), made of alpha, beta and gamma subunits, is crucial for maintenance of Na+ balance and blood pressure. Both beta- and gamma-ENaC participate in negative regulation by interacting with Nedd4-2, an E3 ubiquitin-ligase. Disruption of this interaction results in increased ENaC activity (Liddle syndrome). By two-hybrid screenings, we identified new potential partners of alpha-ENaC: WWP1 (E3 ubiquitin-ligase protein), UBC9 and TSG101 (E2 ubiquitin/SUMO-conjugating enzymes) and confirmed these interactions in GST pull-down assays. All these partners are implicated in protein trafficking and could be involved in the regulation of ENaC activity.

Spectrins, components of the membrane skeleton, are implicated in various cellular functions. Understanding the diversity of these functions requires better characterization of the interacting domains of spectrins, such as the SH3 domain. Yeast two-hybrid screening of a kidney cDNA library revealed that the SH3 domain of alphaII-spectrin binds specifically isoform A of low-molecular-weight phosphotyrosine phosphatase (LMW-PTP). The alphaII-spectrin SH3 domain does not interact with LMW-PTP B or C nor does LMW-PTP A interact with the alphaI-spectrin SH3 domain. The interaction of spectrin with LMW-PTP A led us to look for a tyrosine-phosphorylated residue in all-spectrin. Western blotting showed that all-spectrin is tyrosine phosphorylated in vivo. Using mutagenesis on recombinant peptides, we identified the residue Y1176 located in the calpain cleavage site of all-spectrin, near the SH3 domain, as an in vitro substrate for Src kinase and LMW-PTP A. This Y1176 residue is also an in vivo target for kinases and phosphatases in COS cells. Phosphorylation of this residue decreases spectrin sensitivity to calpain in vitro. Similarly, the presence of phosphatase inhibitors in cell culture is associated with the absence of spectrin cleavage products. This suggests that the Y1176 phosphorylation state could modulate spectrin cleavage by calpain and may play an important role during membrane skeleton remodeling.

Spectrins are major components of the membrane skeleton. This protein scaffold is implicated in membrane stabilization, cell spreading, establishment of cell polarity and location of specific membrane proteins. Understanding the diversity of these functions requires to better characterize the complex interactions of these molecules with other cellular proteins through the study of specific modular interacting domains. Spectrin alpha II subunit contains an SH3 domain, the ligands of which have not been clearly identified yet. By yeast two-hybrid screening of rat kidney and human lymphocyte cDNA libraries, we have found that spectrin SH3 domain binds isoform A of the LMW-PTP. Spectrin SH3 domain does not interact with isoforms B and C, although they are present in both tested libraries. LMW-PTP A does not interact with either the SH3 domain of spectrin alpha I subunit or other SH3 domains. Besides, mutations in spectrin SH3 domain abolish the interaction. Although LMW-PTP A is mainly a cytosolic enzyme, we have shown by western blot on a rat kidney cell line (RCCD1) that it is also present in the triton insoluble fraction along with the spectrin based-membrane skeleton. The spectrin SH3 domain-LMW-PTP A interaction may have several functions: (i) Recruitment of this enzyme to its membrane substrates such as PDGF, EGF and insulin receptors. (ii) Dephosphorylation of spectrin itself since tyrosine phosphorylation can occur on spectrin. Using mutagenesis, we have identified one phosphotyrosine residue (Tyr 1176) which is located in the calpain cleavage site, near the SH3 domain. We have shown that this tyrosine is a substrate for the LMW-PTP A and that its phosphorylation can modify the sensitivity of spectrin to calpain. Thus, this suggests that Tyr 1176-phosphorylation state could modulate spectrin cleavage by calpain during membrane skeleton remodeling.

Erythroid spectrin is the main component of the red cell membrane skeleton, which is very important in determining the shape, resistance to mechanical stresses and deformability of red cells. Previously we demonstrated that human erythroid alpha-spectrin is ubiquitinated in vitro and in vivo, and using recombinant peptides we identified on repeat 17 the main ubiquitination site of alpha-spectrin. In order to identify the lysine(s) involved in the ubiquitination process, in the present study we mutated the lysines by site-directed mutagenesis. We found that ubiquitination was dramatically inhibited in peptides carrying the mutation of lysine 27 on repeat 17 (mutants K25,27R and K27R). We also demonstrated that the correct folding of this protein is fundamental for its recognition by the ubiquitin conjugating system. Furthermore, the region flanking lysine 27 showed a 75% similarity with the leucine zipper pattern present in many regulatory proteins. Thus, a new potential ubiquitin recognition motif was identified in alpha-spectrin and may be present in several other proteins.

The spectrin role(s) is (are) very important for the shape and the physical properties of red cells, such as deformability and resistance to mechanical stresses. Moreover a variety of spectrin diseases are known. We have previously demonstrated [Corsi, D., Galluzzi, L., Crinelli, R. & Magnani, M. (1995) J. Biol. Chem. 270, 8928-8935] that human erythroid alpha-spectrin is ubiquitinated in vitro and in vivo. In order to define the ubiquitinated repeats of this long protein and find out a possible function, we have produced recombinant peptides encompassing the alphaIII-, alphaIV-, alphaV- and EF hand domains of alpha-spectrin chain. These peptides were tested in in vitro ubiquitin conjugation assays and two regions susceptibles to ubiquitination were found. The first one, in the alphaIV-domain, includes the repeat 17 and the second one, in the alphaV-domain, includes the repeat 20 and a part of repeat 21. We also demonstrated that the susceptibility to ubiquitination of the alphaV-domain is reduced by interaction with the corresponding portion of beta-spectrin chain (betaIV-domain). Thus, at least ubiquitination of alphaV-domain is susceptible to cytoskeleton assembly and spectrin dimerization.

To the Editor: The erythrocyte skeleton is a complex protein network responsible for the shape and the physical properties of red blood cells (RBCs), such as deformability and resistance to mechanical stress. Spectrin is the major constituent of this membrane skeleton and represents about 25% in

We have examined the properties and interactions of expressed polypeptide fragments from the N-terminus of the α-chain and the C-terminus of the β-chain of human erythroid spectrin. Each polypeptide comprises one complete structural repeating unit, together with the incomplete repeat that interacts with its partner when spectrin tetramers are formed. The shared repeat thus generated is made up of two helices from the C-terminal part of the β-chain and one helix from the N-terminus of the α-chain. Three mutant β-chain fragments with amino acid substitutions in the incomplete terminal repeat were also studied. The α- and β-chain fragments were both substantially monomeric, as shown by sedimentation equilibrium. Circular dichroism analysis and thermal denaturation profiles revealed that the complete repeat present in each fragment had entered the stable tertiary fold. Unexpectedly, the conformational stability of the folded β-chain repeat was found to be grossly perturbed by the mutations, all of them well beyond its C-terminal boundary; possible explanations for this phemomenon are considered. Sedimentation equilibrium showed that in equimolar mixtures the wild-type α- and β-chain peptides formed a 1:1 complex. Mixing curves, observed by circular dichroism, revealed that association was accompanied by an increase in α-helicity. From continuous-variation profiles an association constant in the range 1–2×106 M–1 was inferred. The association was unaffected by the apparently unstructured anionic tail of 54 residues, found at the C-terminus of the spectrin β-chain. Of the three mutations in the β-chain fragment, one (an Ala→Val replacement in the A helix segment of the incomplete repeat) had a relatively small effect on the association with the α-chain fragment, whereas Trp→Arg mutations in the A and in the remote B helix segments were much more deleterious. These observations are consistent with the relative severities of the haemolytic conditions associated with the mutations.

Most of hereditary elliptocytosis (HE) cases are related to a spectrin dimer (SpD) self-association defect. The severity of haemolysis is correlated with the extent of the SpD self-association defect, which itself depends on the location of the mutation regarding the tetramerization site. This site is presumed to involve the first C helix of the alpha chain and the last two helices, A and B, of the beta chain to reconstitute a triple helical structure (A, B and C), as observed along spectrin. Using recombinant peptides, we demonstrated that the first C helix of the alpha chain and the last two helices of the beta chain alone are not sufficient to establish interactions, which only occurred when a complete triple-helical repeat was added to each partner. One adjacent repeat is necessary to stabilize the conformation of both N- and C-terminal structures directly involved in the interaction site and is sufficient to generate a binding affinity similar to that observed in the native molecule. Producing peptides carrying a betaHE mutation, we reproduced the tetramerization defect as observed in patients. Therefore, the betaW2024R and betaW2061R mutations, which replace the invariant tryptophan and a residue located in the hydrophobic core, respectively, affect alpha-beta interactions considerably. In contrast, the betaA2013V mutation, which modifies a residue located outside any presumed interacting regions, has a minor effect on the interaction.

Previously, we demonstrated that alpha-spectrin is a substrate for the ubiquitin system and that this conjugation is a dynamic process (Corsi, D., Galluzzi, L., Crinelli, R., and Magnani, M. (1995) J. Biol. Chem. 270, 8928-8935). In this study, we mapped the sites of ubiquitination on erythrocyte alpha-spectrin. A peptide map of digested alpha-spectrin, previously submitted to in vitro 125I-ubiquitin conjugation, revealed the presence of four distinct labeled bands with Mr 40,000, 36,000, 29,000, and 25,500. Western blotting experiments using antibodies against each alpha-spectrin domain revealed that only IgG anti-alphaIII domain recognized the 125I-labeled ubiquitin peptide of 29 kDa, whereas the IgG anti-alphaV domain recognized the Mr 40,000 125I-ubiquitin-labeled peptide. The other two labeled bands of Mr 36,000 and Mr 25,500 were identified as tetra and tri multiubiquitin chains. Ubiquitination of the alphaIII and alphaV domains was further confirmed by anti-alpha-spectrin domain immunoaffinity chromatography. Endoprotease Lys C-digested spectrin conjugated previously to 125I-ubiquitin was incubated with antibodies against each trypsin-resistant domain of alpha-spectrin. Gamma counting of the radiolabeled antigen-antibody complexes purified by protein A chromatography showed labeling in the IgG anti-alphaIII and anti-alphaV complexes alone. Domain alphaIII is not associated with any known function, whereas domain alphaV contains the nucleation site for the association of the alpha and beta chains. Ubiquitination of the latter domain suggests a role for ubiquitin in the modulation of the stability, deformability, and viscoelastic properties of the erythrocyte membrane.