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The anchorage of spectrin to biological membranes is mediated by protein and phosphoinositol phospholipid interactions. In epithelial cells, a nascent spectrin skeleton assembles in regions of cadherin-mediated cell-cell contact, and conversely, cytoskeletal assembly is required to complete the cell-adhesion process. The molecular interactions guiding these processes remain incompletely understood. We have examined the interaction of spectrin with alpha-catenin, a component of the adhesion complex. Spectrin (alphaIIbetaII) and alpha-catenin coprecipitate from extracts of confluent Madin-Darby canine kidney, HT29, and Clone A cells and from solutions of purified spectrin and alpha-catenin in vitro. By surface plasmon resonance and in vitro binding assays, we find that alpha-catenin binds alphaIIbetaII spectrin with an apparent K(d) of approximately 20-100 nm. By gel-overlay assay, alpha-catenin binds recombinant betaII-spectrin peptides that include the first 313 residues of spectrin but not to peptides that lack this region. Similarly, the binding activity of alpha-catenin is fully accounted for in recombinant peptides encompassing the NH(2)-terminal 228 amino acid region of alpha-catenin. An in vivo role for the interaction of spectrin with alpha-catenin is suggested by the impaired membrane assembly of spectrin and its enhanced detergent solubility in Clone A cells that harbor a defective alpha-catenin. Transfection of these cells with wild-type alpha-catenin reestablishes alpha-catenin at the plasma membrane and coincidentally recruits spectrin to the membrane. We propose that ankyrin-independent interactions of modest affinity between alpha-catenin and the amino-terminal domain of beta-spectrin augment the interaction between alpha-catenin and actin, and together they provide a polyvalent linkage directing the topographic assembly of a nascent spectrin-actin skeleton to membrane regions enriched in E-cadherin.

Affinity purification of a phage-displayed library, expressing random peptide 12-mers at the N terminus of protein III, has identified 10 distinct novel sequences which bind troponin C specifically. The troponin C-selected peptides yield a consensus binding sequence of (V/L)(D/E)XLKXXLXXLA. Sequence comparison revealed as much as a 62.5% similarity between phiT5, the peptide sequence of the phage clone with the highest level of binding to troponin C, and the N-terminal region of troponin I isoforms. Biotinylated peptides corresponding to library-derived sequences and similar sequences from various isoforms of troponin I were synthesized shown to bind troponin C specifically. Alkaline phosphatase fusion proteins of two of the phage clone sequences bound troponin C specifically, and were specifically competed by both library-derived and native troponin I peptides. Measurement of equilibrium dissociation constants of the peptides by surface plasmon resonance yielded dissociation constants for troponin C as low as 0.43 microM for pT5; in contrast, dissociation constants for calmodulin were greater than 6 microM for all peptides studied. Nondenaturing polyacrylamide gel electrophoresis demonstrated that pT5 formed a stable complex with troponin C in the presence of calcium. We also found that the pT5 peptide inhibited the maximal calcium-activated tension of rabbit psoas muscle fibers.

In addition to reducing hemoglobin-O2 affinity, 2,3-diphosphoglycerate (DPG) is known to modulate the mechanical properties of the erythrocyte membrane. By fluorescence spectroscopy and differential scanning calorimetry, we demonstrate that DPG binds the cytoplasmic domain of erythrocyte membrane band 3 in two stages characterized by apparent KD values of approximately approximately 2 and 12 mM. DPG was also shown to perturb the stability of ankyrin, protein 4.1, and protein 4.2 in situ and to directly bind to protein 4.1. In studies of membrane-skeleton interactions, DPG was observed to inhibit the fast and slow phases of ankyrin binding to band 3 and to reduce both the number of ankyrin sites and affinity of ankyrin for each class of site. The inhibition was biphasic, similar to the band 3-DPG binding isotherm; however, at physiological DPG concentrations a reduction in only 15% of the ankyrin sites was observed. In contrast, inhibition of protein 4.1 binding to the membrane reached 65% at physiological DPG concentrations (approximately approximately 5.9 mM); at more elevated concentrations, blockade was nearly quantitative, affecting glycophorin and band 3 sites alike. Taken together with previous observations, these data suggest that DPG's effect on O2 delivery may extend beyond its well recognized impact on hemoglobin-O2 affinity.

To determine why deletion of the nine amino acids joining the membrane and cytoplasmic domains of band 3 from Southeast Asian ovalocytes (SAO) renders the erythrocytes rigid, we compared the structural and functional properties of SAO and normal band 3. Calorimetric data, inhibitor binding studies, and anion transport assays all reveal that the membrane-spanning domain of SAO band 3 is denatured, while proteolysis studies and circular dichroism spectroscopy suggest the mutant domain retains much secondary structure. It is concluded that the transmembrane helices of SAO band 3 are dissociated and randomized but not unfolded. The cytoplasmic domain of SAO band 3 was shown to be structurally and functionally normal based on (i) calorimetric properties, (ii) native conformational change, (iii) ability to form an intersubunit disulfide bond, (iv) affinity and capacity for binding ankyrin and protein 4.1, and (v) kinetics of association with ankyrin. However, both normal and mutant isoforms of band 3 in SAO cells were found to adhere nonspecifically to the spectrin skeleton. Further, when SAO cells were osmotically swollen, the detergent extractability of band 3 became normal. We propose that much of band 3 is nonspecifically entrapped in the spectrin network in SAO cells and that this nonspecific adhesion may be responsible for the rigidity of the SAO erythrocyte.

Of the several proteins that bind along the cytoplasmic domain of erythrocyte membrane band 3, only the sites of interaction of proteins 4.1 and 4.2 remain to be at least partially localized. Using five independent techniques, we have undertaken to map and characterize the binding site of band 4.1 on band 3. First, transfer of a radioactive cross-linker (125I-2-(p-azido-salicylamido)ethyl-1-3-dithiopropionate) from purified band 4.1 to its binding sites on stripped inside-out erythrocyte membrane vesicles (stripped IOVs) revealed major labeling of band 3, glycophorin C, and glycophorin A. Proteolytic mapping of the stripped IOVs then demonstrated that the label on band 3 was confined largely to a fragment comprising residues 1-201. Second, competitive binding experiments with Fab fragments of monoclonal and peptide-specific polyclonal antibodies to numerous epitopes along the cytoplasmic domain of band 3 displayed stoichiometric competition only with Fabs to epitopes between residues 1 and 91 of band 3. Weak competition was also observed with Fabs to a sequence of the cytoplasmic domain directly adjacent to the membrane-spanning domain, but only at 50-100-fold excess of Fab. Third, band 4.1 protected band 3 from chymotryptic hydrolysis at tyrosine 46 and to a much lesser extent at a site within the junctional peptide connecting the membrane-spanning and cytoplasmic domains of band 3. Fourth, ankyrin, which has been previously shown to interact with band 3 both near a putative central hinge and at the N terminus competed with band 4.1 for band 3 in stripped IOVs. Since band 4.1 does not associate with band 3 near the flexible central hinge, the competition with ankyrin can be assumed to derive from a mutual association with the N terminus. Finally, a synthetic peptide corresponding to residues 1-15 of band 3 was found to mildly inhibit band 4.1 binding to stripped IOVs. Taken together, these data suggest that band 4.1 binds band 3 predominantly near the N terminus, with a possible secondary site near the junction of the cytoplasmic domain and the membrane.