Further characterization of a fodrin-containing transmembrane complex from mouse T-lymphoma cells

A transmembrane complex containing fodrin (an actin-binding protein) and a major surface glycoprotein (GP 180) was previously isolated from mouse T-lymphoma cells by the complementary techniques of non-ionic detergent extraction and sucrose gradient centrifugation (Bourguignon et al. (1985) J. Cell Biol. 101, 477-487). The analysis of this complex has been extended to verify the structural association and further define the interaction between fodrin and GP 180. The association between fodrin and GP 180 has been confirmed by the following evidence: co-sedimentation of fodrin and GP 180 in a single peak on a sucrose gradient with a sedimentation coefficient of 20 S; a constant ratio of fodrin and GP 180 across the 20 S peak; the specific co-precipitation of GP 180 with fodrin from the 20 S peak using anti-fodrin antibody; and the colocalization of fodrin and GP 180 from the 20 S peak on actin filaments using an immuno-electron microscopic technique. Furthermore, this fodrin-GP 180 complex can be readily dissociated and reassembled in the presence and absence of 0.6 M NaCl, respectively. The fact that this fodrin-GP 180 complex displays actin-binding ability indicates that this transmembrane complex may play an important role in the linking event between receptors and the cytoskeleton during lymphocyte patching and capping.     

The lymphoma transmembrane glycoprotein GP85 (CD44) is a novel guanine nucleotide-binding protein which regulates GP85 (CD44)-ankyrin interaction.

In this study, we have used photoaffinity labeling by [32P]azido-GTP as well as [32P]ADP-ribosylation by pertussis toxin (PT) and cholera toxin (CT) to identify GTP-binding proteins associated with mouse T-lymphoma plasma membranes. Our results indicate that GP85 (CD44) can be photoaffinity labeled by [32P] azido-GTP and [32P]ADP-ribosylated by both PT and CT. Using purified GP85 (CD44) obtained by Triton X-100 extraction, wheat germ agglutinin-Sepharose, and anti-GP85 (CD44) antibody affinity chromatographies, we have further characterized GP85 (CD44) as a GTP-binding protein. GP85 (CD44) is found to bind guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) in a time- and dose-dependent manner with a dissociation constant of 0.83 nM. Importantly, GP85 (CD44) appears to display a GTPase activity which hydrolyzes [gamma-32P]GTP at a rate of 0.011 mol of Pi released/mol of GP85 (CD44)/min. This GTPase activity can be readily inhibited by PT- or CT-mediated ribosylation of GP85 (CD44). Most interestingly, GTP binding significantly enhances the interaction of purified GP85 (CD44) with ankyrin, whereas ADP-ribosylation of GP85 (CD44) by PT or CT inhibits the GTP-induced increase in ankyrin binding to GP85 (CD44). In addition to GP85 (CD44) being the first reported transmembrane GTP-binding protein, these results suggest that GTP plays an important role in promoting the interaction between GP85 (CD44) and its underlying membrane cytoskeleton through ankyrin.     

A lymphoma plasma membrane-associated protein with ankyrin-like properties

In this study we have used several complementary techniques to isolate and characterize a 72-kD polypeptide that is tightly associated with a major mouse T-lymphoma membrane glycoprotein, gp 85 (a wheat germ agglutinin-binding protein), in a 16 S complex. These two proteins do not separate in the presence of high salt but can be dissociated by treatment with 2 M urea. Further analysis indicates that the 72-kD protein has ankyrin-like properties based on the following criteria: (a) it cross-reacts with specific antibodies raised against erythrocyte and brain ankyrin; (b) it displays a peptide mapping pattern and a pI (between 6.5 and 6.8) similar to that of the 72-kD proteolytic fragment of erythrocyte ankyrin; (c) it competes with erythrocyte ghost membranes (spectrin-depleted preparations) for spectrin binding; and (d) it binds to purified spectrin and fodrin molecules. Most importantly, in intact lymphoma cells this ankyrin-like protein is localized directly underneath the plasma membrane and is found to be preferentially accumulated beneath receptor cap structures as well as associated with a membrane-cytoskeleton complex preparation. It is proposed that the ankyrin-like 72-kD protein may play an important role in linking certain surface glycoprotein(s) to fodrin which, in turn, binds to actin filaments required for lymphocyte cap formation.     

A T-lymphoma transmembrane glycoprotein (gp180) is linked to the cytoskeletal protein, fodrin

A major mouse T-lymphoma surface glycoprotein (gp180) has been identified by labeling cells with 125I and [3H]glucosamine. After ligand-induced receptor patching and/or capping, the amount of gp 180 in the membrane-associated cytoskeleton fraction increases in direct proportion to the percentage of patched/capped cells. There is a parallel increase in the amount of fodrin in the membrane-associated cytoskeleton fraction. Evidence is presented that gp180 is the same as or very similar to the T-lymphocyte-specific glycoprotein T-200. An immunobinding assay of Nonidet P-40-solubilized plasma membrane selectively co-isolates gp180 and fodrin. After induction of receptor rearrangement, double-label immunofluorescence reveals that fodrin accumulated directly beneath gp180 patches and caps. Membrane extraction with Triton X-114 followed by sucrose gradient centrifugation permits isolation of a gp180-fodrin complex with a 1:1 molar ratio and sedimentation coefficient(s) of approximately 20. This complex remains stable during isoelectric focusing and exhibits a pl in the range of 5.2-5.7. On the basis of our results we conclude that gp180, an integral membrane glycoprotein, and fodrin, a component of the membrane-associated cytoskeleton, are closely associated into a complex. Furthermore, we contend that, through fodrin's association with actin, this complex is of functional significance in ligand-induced patching and capping of gp180. We also propose that, through lateral interactions in the plane of the membrane, the gp180-fodrin complex might be responsible for linking other surface receptors to the intracellular microfilament network during lymphocyte patching and capping.     

Comparison of spectrin isolated from erythroid and non-erythroid sources

Spectrin from erythrocytes and two other tissues (brain and intestine) were isolated from two distant species, pig and chicken; some structural and functional properties were compared. A quantitative antibody inhibition assay was used to determine that antibodies to mammalian red cell spectrin cross-react very poorly, if at all, with their non-erythroid (brain) counterpart and similarly antibodies to pig brain spectrin (fodrin) cross-react very weakly with erythroid spectrin. By contrast, antibodies which were directed against the 240000-Mr subunit of avian fodrin were completely inhibited with avian spectrin and vice versa. To analyze the structural relatedness of these molecules further we compared the chymotryptic iodinated peptide maps generated from each individual subunit. Consistent with the antibody results, we find little (less than 10%) homology between peptides derived from mammalian fodrin and spectrin, but complete homology (100%) of the peptides derived from the 240000-Mr subunits of chicken fodrin, spectrin and another related molecule from intestine, TW260/240. Whereas the peptide maps of fodrin (brain spectrin) revealed striking similarity between divergent species, suggesting a high degree of structural conservation, the peptide maps of erythrocyte spectrin was highly variable between species, indicating that it has diverged considerably in mammalian evolution. In addition we have compared a functional activity of mammalian spectrins, the ability to bind calmodulin, using two different assays. Both results show that, whereas fodrin-calmodulin interaction can be readily demonstrated, the binding to mammalian erythroid spectrin is negligible. This suggests that the high-affinity calmodulin site present on fodrin has been lost from spectrin in mammalian evolution.     

Membrane interaction of erythroid spectrin: surface-density-dependent high-affinity binding to phosphatidylethanolamine

Density-dependent spectrin binding to dimyristoylphosphatidylcholine/dimyristoylphosphatidylethanolamine (DMPC/DMPE) small uni-lamellar vesicles (SUVs) has been directly evaluated in this work from the increase in the extent of quenching of the tryptophan fluorescence of spectrin at two different temperatures, above and below the main phase transition temperatures (Tm). Results from the binding studies of spectrin to phospholipid SUVs indicated that the binding dissociation constant Kd, increased from 45 +/- 7 nM in pure DMPC SUVs to 219 +/- 20 nM in DMPC/DMPE (50:50) SUVs, both in the gel and liquid crystalline phase. However, in pure DMPE SUVs the Kd decreased drastically to 0.7 +/- 0.2 nM in the gel phase at 18 degrees C and to 2.6 +/- 0.7 nM in the fluid phase at 55 degrees C indicating a high affinity binding of spectrin for the bilayer-forming DMPE. The maximum extent of phospholipid-induced quenching and the number of spectrin molecules associated with one SUV particle, evaluated in the present work, led to a model in DMPC/DMPE bilayer membranes indicating the PE-binding site of spectrin to localize at one of the terminal domains of the dimeric spectrin. A direct evidence of the localization of the PE-binding site at one of the terminal ends of the spectrin dimer also came from electron microscopic observation in fluid membranes made of bovine brain PE.     

Demonstration that the leukocyte common antigen CD45 is a protein tyrosine phosphatase

It has been proposed on the basis of amino acid sequence homology that the leukocyte common antigen CD45 represents a family of catalytically active, receptor-linked protein tyrosine phosphatases [Charbonneau, H., Tonks, N. K., Walsh, K. A., & Fischer, E. H. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7182-7186]. The present study confirms that CD45 possesses intrinsic protein tyrosine phosphatase (PTPase) activity. First, a mouse monoclonal antibody to CD45 (mAb 9.4) specifically eliminated, by precipitation, PTPase activity from a high Mr fraction containing CD45, prepared by gel filtration (Sephacryl S200) of a Triton X-100 extract of human spleen. Second, PTPase activity was demonstrated in a highly purified preparation of CD45 that was eluted with a high pH buffer from an affinity column, constructed from the same antibody. Third, on sucrose density gradient centrifugation, PTPase activity was only found in those fractions that contained CD45 as determined by Western analysis. When CD45 was caused to aggregate, first by reacting it with mAb 9.4 and then adding a secondary, cross-linking anti-mouse mAb, the PTPase activity shifted to the same higher Mr fractions that contained CD45. No shift in CD45 or PTPase was observed following addition of a control IgG2a. On this basis, it is concluded that CD45 is a protein tyrosine phosphatase.     

Identification of the calmodulin binding domain of alpha-fodrin and implications for folding

A cDNA clone producing a protein that binds calmodulin has been isolated from a mouse macrophage library. The cDNA was sequenced and identified as coding for fodrin. By deleting part of the sequence, the calmodulin binding domain was located. The site is situated on repeat 11 of fodrin probably on its extra arm. This part of the sequence exhibits great similarity to other calmodulin binding proteins. Analysis of the sequence and spatial structure of calmodulin revealed a domain which is quite complementary to the sequence identified on fodrin. These results provide a new insight into the structure of fodrin and consequently into the structure of proteins of the spectrin family. A model for the general folding of these molecules is proposed, involving a simple three-layer folding. The structure was further corroborated by analysis of charge distribution in the vicinity of the calmodulin binding site. The folding we propose is in good agreement with digestion experiments and explains observations in diseases resulting from mutations of human spectrin.     

Preliminary characterization of phosphotyrosine phosphatase activities in human peripheral blood lymphocytes: Identification of CD45 as a phosphotyrosine phosphatase

A preliminary characterization of the protein phosphotyrosine phosphatase (PT-Pase) activity in human peripheral blood lymphocytes (PBL) has been made using two tyrosine-containing peptides and the epidermal growth factor receptor from A-431 cells as substrates. High PTPase activity with a pH optimum near 7.4 was observed in both the membrane and the cytosolic fractions. At least three distinct fractions with PTPase activity were separated on DEAE cellulose columns, indicating that the enzyme is heterogeneous. Vanadate, molybdate, and salts of zinc, copper, and mercury were all effective enzyme inhibitors, although the inhibition was generally incomplete and showed some variation with the enzyme preparation. The difficulty in completely inhibiting PTPase activity in lymphocytes may help explain the variation between laboratories in studies of tyrosine phosphorylation in these cells. Studies with highly purified T lymphocytes obtained by filtration of PBL through nylon wool columns indicated that the activity is present in T cells. Absorption with agarose containing anti-HLe-I, a mouse monoclonal lgGi antibody specific for the leukocyte-specific surface protein T-200 (CD45), removed up to 40% of the PTPase activity. Enzyme activity was recovered on the immunoadsorbent after extensive washing, confirming that the enzyme was being bound to the beads. Immunoabsorbents containing other mouse lgGi antibodies failed to bind PTPase activity, indicating that the binding to beads with anti-HLe-I antibody is specific. Further characterization of the CD45 and PTPase activities in lymphocytes may provide a better under standing of the role of protein tyrosine phosphorylation in the regulation of proliferation and differentiation in these cells.     

Phospholipid binding by proteins of the spectrin family: a comparative study

Erythroid and neuronal spectrin (fodrin) are both known to interact strongly with the aminophospholipids that occur in the inner leaflet of plasma membranes. In erythroid spectrin the positions of the binding sites within the constituent (alphaI and betaI) polypeptide chains have been defined, and also the importance of the lipid interaction in regulating the properties of the membrane. Here we report the locations of the corresponding binding sites in the alphaII and betaII chains that make up the fodrin molecule. Of the 10 lipid-binding repeats in the erythroid spectrin chains 5 are conserved in fodrin; one cluster of 3 consecutive structural repeating units in alphaI erythroid spectrin (repeats 8-10) is displaced by one repeat in alphaII fodrin (repeats 9-11). Fodrin also contains one binding site at the N-terminus of the alphaII chain, not present in the erythroid protein. The regions of the two spectrins containing equivalent lipid-binding sites show a much higher degree of sequence identity than corresponding repeats that do not share this property. The evolutionary conservation of the distribution of a large proportion of strong lipid-binding sites in the polypeptide chains of these two proteins of disparate character argues for a specific function of fodrin-phospholipid interactions in the neuron.     

Analysis of interleukin 5 receptors on murine eosinophils: a comparison with receptors on B13 cells

The interleukin 5 receptor (IL-5R) on murine eosinophils and a mouse B cell line (B13) was investigated using iodinated murine IL-5 produced in the baculovirus system. Electrophoretic analysis of this recombinant protein identified a range of bands Mr 26,000 to 32,000 resulting from differential glycosylation. The specific activity and binding kinetics of the iodinated IL-5 (125I-IL-5) were essentially identical to unlabeled material. Both high-affinity (Kd approximately 50 pM) and low-affinity (Kd approximately 1 nM) receptor populations were identified on murine eosinophils. Approximately 50 high-affinity receptors and 10,000 low-affinity receptors were present. This was compared with approximately 2,000 high-affinity (Kd approximately 80 pM) and about 8,000 low-affinity (Kd approximately 3 nM) sites on B13 cells. An antibody that inhibits IL-5 binding to, and proliferation of, B13 cells (R52.120) was also shown to inhibit eosinophil proliferation, suggesting that eosinophils and B cells bear the equivalent IL-5 binding proteins.     

Binding of brain spectrin to the 70-kDa neurofilament subunit protein

Brain spectrin, or fodrin, a major protein of the subaxolemmal cytoskeleton, associates specifically in in vitro assays with the 70-kDa neurofilament subunit (NF-L) and with glial filaments from pig spinal cord. As an initial approach to the identification of the fodrin-binding proteins, a crude preparation of neurofilaments was resolved by electrophoresis on SDS/polyacrylamide gels and then transferred to nitrocellulose paper, which was 'blotted' with 125I-fodrin. A significant binding of fodrin was observed on polypeptides of 70 kDa, 52 kDa and 20 kDa. These polypeptides were further purified and identified respectively as the NF-L subunit of neurofilaments, the glial fibrillary acidic protein (GFP) and the myelin basic protein. The binding of fodrin to NF-L was reversible and concentration-dependent. The ability of the pure NF-L and GFP to form filaments was used to quantify their association with fodrin. a) The binding of fodrin to reassembled NF-L was saturable with a stoichiometry of 1 mol fodrin bound/50 +/- 10 mol NF-L and an apparent dissociation constant Kd = 4.3 x 10(-7) M. b) The binding involved the N-terminal domain of the polypeptide chain derived from the [2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine] cleavage of NF-L. c) Binding occurred optimally at physiological pH (6.8-7.2) and salt concentrations (50 mM). d) Interestingly, calmodulin, a Ca2+-binding protein, which has been shown to bind to fodrin, was found to reinforce the binding of fodrin to the NF-L, at Ca2+ physiological concentrations. The binding of fodrin to pure neurofilaments was not affected by the presence of the 200-kDa (NF-H) and the 160-kDa (NF-M) subunits. The apparent dissociation constant for the binding of fodrin to NF-L in the pure NF was 1.0 x 10(-6) M with 1 mol fodrin bound/80 +/- 10 mol NF-L. Moreover, the binding of fodrin to GFP, demonstrated in blot assays, was confirmed by cosedimentation experiments. The apparent dissociation constant Kd for the fodrin binding was 2.8 x 10(-7) M and the maximum binding was 1 mol fodrin/55 +/- 10 mol GFP.     

The calmodulin-binding site in alpha-fodrin is near the calcium-dependent protease-I cleavage site

Fodrin (brain spectrin) binds calmodulin and is susceptible to proteolysis by calcium-dependent protease I (CDP-I, calcium-activated neutral protease I, or calpain I). Both events involve the central region of the alpha-fodrin subunit, and calmodulin binding enhances the sensitivity of fodrin to CDP-I mediated proteolysis. Fragments of fodrin, generated chemically or proteolytically, which retain calmodulin binding activity have been identified and analyzed by two-dimensional peptide mapping and by direct protein sequencing. Both CDP-I and calmodulin interact with the terminal portion of the eleventh repetitive unit in fodrin, which is at the center of the molecule. CDP-I cleavage occurs between Tyr104 and Gly105 and preserves the calmodulin binding activity of the carboxyl-terminal fragment. In contrast, chymotryptic cleavage at Trp120 reduces the ability of this fragment to bind calmodulin, and tryptic cleavage beyond Trp120 completely eliminates calmodulin binding activity. It is concluded that Ser-Lys-Thr-Ala-Ser-Pro-Trp-Lys-Ser-Ala-Arg-Leu-Met-Val-His-Thr-Val-Ala- Thr- Phe-Asn-Ser-Ile-Lys, a 24-residue peptide which bridges repeats 11 and 12 of brain alpha spectrin contains the high affinity calmodulin binding domain.     

Fodrin CaM-binding domain cleavage by Pet from enteroaggregative Escherichia coli leads to actin cytoskeletal disruption

We have previously shown that the plasmid-encoded toxin (Pet) of enteroaggregative Escherichia coli produces cytotoxic and enterotoxic effects. Pet-intoxicated epithelial cells reveal contraction of the cytoskeleton and loss of actin stress fibres. Pet effects require its internalization into epithelial cells. We have also shown that Pet degrades erythroid spectrin. Pet delivery within the intestine suggests that Pet may degrade epithelial fodrin (non-erythroid spectrin). Here we demonstrate that Pet has affinity for alpha-fodrin (formally named alphaII spectrin) in vitro and in vivo and cleaves epithelial fodrin, causing its redistribution within the cells. When Pet has produced its cytoskeletal effects, fodrin is found in intracellular aggregates as membrane blebs. Pet cleaves recombinant GST-fodrin, generating two breakdown products of 37 and 72 kDa. Sequencing of the 37 kDa fragment demonstrated that the cleavage site occurred within fodrin's 11th repetitive unit between M1198 and V1199, in the calmodulin binding domain. Site-directed mutagenesis of these amino acids prevented fodrin degradation by Pet. Pet also cleaves epithelial fodrin from cultured Pet-treated cells. A mutant in the Pet serine protease motif was unable to cause fodrin redistribution or to cleave GST-fodrin. This is the first report showing cleavage of alpha-fodrin by a bacterial protease. Cleavage occurs in the middle of the calmodulin binding domain, which leads to cytoskeleton disruption.     

Thermodynamic characterization of allosteric glycogen phosphorylase inhibitors

Glycogen phosphorylase (GP) is a validated target for the treatment of type 2 diabetes. Here we describe highly potent GP inhibitors, AVE5688, AVE2865, and AVE9423. The first two compounds are optimized members of the acyl urea series. The latter represents a novel quinolone class of GP inhibitors, which is introduced in this study. In the enzyme assay, both inhibitor types compete with the physiological activator AMP and act synergistically with glucose. Isothermal titration calorimetry (ITC) shows that the compounds strongly bind to nonphosphorylated, inactive GP (GPb). Binding to phosphorylated, active GP (GPa) is substantially weaker, and the thermodynamic profile reflects a coupled transition to the inactive (tense) conformation. Crystal structures confirm that the three inhibitors bind to the AMP site of tense state GP. These data provide the first direct evidence that acyl urea and quinolone compounds are allosteric inhibitors that selectively bind to and stabilize the inactive conformation of the enzyme. Furthermore, ITC reveals markedly different thermodynamic contributions to inhibitor potency that can be related to the binding modes observed in the cocrystal structures. For AVE5688, which occupies only the lower part of the bifurcated AMP site, binding to GPb (Kd = 170 nM) is exclusively enthalpic (Delta H = -9.0 kcal/mol, TDelta S = 0.3 kcal/mol). The inhibitors AVE2865 (Kd = 9 nM, Delta H = -6.8 kcal/mol, TDelta S = 4.2 kcal/mol) and AVE9423 (Kd = 24 nM, Delta H = -5.9 kcal/mol, TDelta S = 4.6 kcal/mol) fully exploit the volume of the binding pocket. Their pronounced binding entropy can be attributed to the extensive displacement of solvent molecules as well as to ionic interactions with the phosphate recognition site.     

Evidence that binding to the carboxyl-terminal heparin-binding domain (Hep II) dominates the interaction between plasma fibronectin and heparin

We assessed the participation of the three known heparin-binding domains of PFn (Hep I, Hep II, Hep III) in their interaction with heparin by making a quantitative comparison of the fluid-phase heparin affinities of PFn and PFn fragments under physiologic pH and ionic strength conditions. Using a fluorescence polarization binding assay that employed a PFn affinity-purified fluorescein-labeled heparin preparation, we found that greater than 98% of the total PFn heparin-binding sites exhibit a Kd in the 118-217 nM range. We also identified a minor (less than 2%) class of binding sites exhibiting very high affinity (Kd approximately 1 nM) in PFn and the carboxyl-terminal 190/170 and 150/136 kDa PFn fragments. This latter activity probably reflects multivalent inter- or intramolecular heparin-binding activity. Amino-terminal PFn fragments containing Hep I (72 and 29 kDa) exhibited low affinity for heparin under physiologic buffer conditions (Kd approximately 30,000 mM). PFn fragments (190/170 and 150/136 kDa) containing both the carboxyl-terminal Hep II and central Hep III domains retained most of the heparin-binding activity of native PFn (Kd = 278-492 nM). The isolated Hep II domain (33-kDa fragment) exhibited appreciable, but somewhat lower (2-5-fold), heparin affinity compared to the 190/170-kDa PFn fragment. Heparin binding to the 100-kDa PFn fragment containing Hep III was barely detectable (Kd greater than 30,000 nM). From these observations, we conclude that PFn contains only one major functional heparin-binding site per subunit, Hep II, that dominates the interaction between heparin and PFn.     

Interaction of calmodulin with the red cell and its membrane skeleton and with spectrin

The binding of calmodulin to red cell membrane cytoskeletons and to purified spectrin from red cells and bovine brain spectrin (fodrin) has been examined. Under physiological solvent conditions binding can be measured by ultracentrifugal pelleting assays. The membrane cytoskeletons contained a single class of binding sites, with a concentration similar to that of spectrin dimers and an association constant of 1.5 X 10(5) M-1. Binding is calcium dependent and is suppressed by the calmodulin inhibitor trifluoperazine. The binding showed a marked dependence on ionic strength, with a maximum at 0.05 M, and a steep dependence on pH, with a maximum at pH 6.5. It was unaffected by 5 mM magnesium. An azidocalmodulin derivative, under the conditions of our experiments, did not label the spectrin-containing complex, although it could be used to demonstrate binding to fodrin. Binding of calmodulin to spectrin tetramers and fodrin in solution could be demonstrated by a pelleting assay after addition of F-actin. Calculations (which are necessarily rough) suggest that at the free calcium concentration prevailing in a normal red cell about 1 in 20 of the calmodulin binding sites in spectrin will be occupied; this proportion will rise rapidly with increasing intracellular calcium. To determine whether inhibition of calmodulin binding to red cell proteins disturbs the control of cell shape, as has been suggested, calcium ions were removed from the cell by addition of an ionophore and of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to the external medium. This did not affect the discoid shape. Trifluoperazine still induced stomatocytosis, exactly as in untreated cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

The spectrin super-family

The review is focused on recent data on the primary sequences of erythroid and non-erythroid spectrins. As in other fields, the techniques of molecular genetics have allowed great advances in our knowledge of the structure and the genetic story of these molecules. Comparison of alpha-chains sequences of the non-erythroid (fodrin) and erythroid spectrin demonstrated that the fodrin alpha-genes are strictly conserved across species, while the mammalian spectrin genes have diverged rapidly. Spectrin and fodrin alpha-chains are largely composed of homologous 106-amino-acid repeat units. Spectrin alpha-chain is lacking a 37 amino-acid sequence which bears the calmodulin-binding site of the fodrin alpha-chain. The highest degree of homology between the spectrin alpha-chain and the fodrin alpha-chain lies in a central atypical segment unrelated to the canonical repeat sequence. This region is closely related to the N-terminal segment of several src-tyrosine kinases and to a domain of phospholipase C. Like the spectrin alpha-chain, the major central part of the spectrin beta-chain is made up of repeat units of 106 amino-acids. The N-terminal domain of the beta-chain, and especially the actin binding site, is the region of greatest homology among members of the spectrin super-family, including Drosophila spectrin beta-chain, dystrophin and alpha-actinin. The C-terminal extremity of the erythroid beta-chain is also of great interest, since tissue-specific differential processing of 3'beta-spectrin gene pre-mRNA generates a beta spectrin-isoform with a unique C-terminus in human skeletal muscle.     

The catalytic activity of the CD45 membrane-proximal phosphatase domain is required for TCR signaling and regulation.

Cell surface expression of CD45, a receptor-like protein tyrosine phosphatase (PTPase), is required for T cell antigen receptor (TCR)-mediated signal transduction. Like the majority of transmembrane PTPases, CD45 contains two cytoplasmic phosphatase domains, whose relative in vivo function is not known. Site-directed mutagenesis of the individual catalytic residues of the two CD45 phosphatase domains indicates that the catalytic activity of the membrane-proximal domain is both necessary and sufficient for restoration of TCR signal transduction in a CD45-deficient cell. The putative catalytic activity of the distal phosphatase domain is not required for proximal TCR-mediated signaling events. Moreover, in the context of a chimeric PTPase receptor, the putative catalytic activity of the distal phosphatase domain is not required for ligand-induced negative regulation of PTPase function. We also demonstrate that the phosphorylation of the C-terminal tyrosine of Lck, a site of negative regulation, is reduced only when CD45 mutants with demonstrable in vitro phosphatase activity are introduced into the CD45-deficient cells. These results demonstrate that the phosphatase activity of CD45 is critical for TCR signaling, and for regulating the levels of C-terminal phosphorylated Lck molecules.