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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.

Interactions of the developmentally regulated chondroitin sulfate proteoglycan NG2 with human plasminogen and kringle domain-containing plasminogen fragments have been analyzed by solid-phase immunoassays and by surface plasmon resonance. In immunoassays, the core protein of NG2 binds specifically and saturably to plasminogen, which consists of five kringle domains and a serine protease domain, and to angiostatin, which contains plasminogen kringle domains 1-3. Apparent dissociation constants for these interactions range from 12 to 75 nm. Additional evidence for NG2 interaction with kringle domains comes from its binding to plasminogen kringle domain 4 and to miniplasminogen (kringle domain 5 plus the protease domain) with apparent dissociation constants in the 18-71 nm range. Inhibition of plasminogen and angiostatin binding to NG2 by 6-aminohexanoic acid suggests that lysine binding sites are involved in kringle interaction with NG2. The interaction of NG2 with plasminogen and angiostatin has very interesting functional consequences. 1) Soluble NG2 significantly enhances the activation of plasminogen by urokinase type plasminogen activator. 2) The antagonistic effect of angiostatin on endothelial cell proliferation is inhibited by soluble NG2. Both of these effects of NG2 should make the proteoglycan a positive regulator of the cell migration and proliferation required for angiogenesis.

Focal adhesion kinase (FAK) and paxillin are focal adhesion-associated, phosphotyrosine-containing proteins that physically interact. A previous study has demonstrated that paxillin contains two binding sites for FAK. We have further characterized these two binding sites and have demonstrated that the binding affinity of the carboxyl-terminal domain of FAK is the same for each of the two binding sites. The presence of both binding sites increases the affinity for FAK by 5-10-fold. A conserved paxillin sequence called the LD motif has been implicated in FAK binding. We show that mutations in the LD motifs in both FAK-binding sites are required to dramatically impair FAK binding in vitro. A paxillin mutant containing point mutations in both FAK-binding sites was characterized. The mutant exhibited reduced levels of phosphotyrosine relative to wild type paxillin in subconfluent cells growing in culture, following cell adhesion to fibronectin and in src-transformed fibroblasts. These results suggest that paxillin must bind FAK for maximal phosphorylation in response to cell adhesion and that FAK may function to direct tyrosine phosphorylation of paxillin in the process of transformation by the src oncogene.

Drosophila Rrp1 is a DNA repair nuclease whose C-terminal region shares extensive homology with Escherichia coli exonuclease III, has nuclease activity, and provides resistance to oxidative and alkylating agents in repair-deficient E. coli strains. The N-terminal 421 amino acid region of Rrp1, which binds and renatures homologous single-stranded DNA, does not share homology with any known protein. Proteolysis by endoproteinase Glu-C (protease V8) reduces the Rrp1 protein to a single, cleavage-resistant peptide. The peptide (referred to as Rrp1-C274) begins with the sequence TKTTV, corresponding to cleavage between Glu-405 and Thr-406 of Rrp1. We determined that nuclease activity is intrinsic to Rrp1-C274 although altered when compared with Rrp1; 3'-exonuclease activity is reduced 210-fold, 3'-phosphodiesterase activity is reduced 6.8-fold, and no difference in apurinic/apyrimidinic endonuclease activity is observed. Rrp1 and Rrp1-C274 are both monomers with frictional coefficients of 2.2 and 1.4, respectively. Circular dichroism results indicate that Rrp1-C274 is predominantly alpha-helical, while the N-terminal 399 amino acids is predominantly random coil. These results suggest that Rrp1 may have a bipartite structural organization; a highly organized, globular C-terminal domain; and an asymmetric, protease-sensitive random coil-enriched N-terminal region. A shape model for this bipartite structure is proposed and discussed.

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.

Syntrophins, a family of intracellular peripheral membrane proteins of the dystrophin-associated protein complex (DAPC), each contain a single PDZ domain. Syntrophin PDZ domains bind C-terminal peptide sequences with the consensus R/K-E-S/T-X-V-COOH, an interaction that mediates association of skeletal muscle sodium channels with the DAPC. Here, we have isolated cyclic peptide ligands for syntrophin PDZ domains from a library of combinatorial peptides displayed at the N terminus of protein III of bacteriophage M13. Affinity selection from a library of X10C peptides yielded ligands with the consensus X-(R/K)-E-T-C-L/M-A-G-X-Psi-C, where Psi represents any hydrophobic amino acid. These peptides contain residues (underlined) similar to the C-terminal consensus sequence for binding to syntrophin PDZ domains and bind to the same site on syntrophin PDZ domains as C-terminal peptides, but do not bind to other closely related PDZ domains. PDZ binding is dependent on the formation of an intramolecular disulfide bond in the peptides, since treatment with dithiothreitol, or substitution of either of the two cysteines with alanines, eliminated this activity. Furthermore, amino acid replacements revealed that most residues in the phage-selected peptides are required for binding. Our results define a new mode of binding to PDZ domains and suggest that proteins containing similar conformationally constrained sequences may be ligands for PDZ domains.

The major anion exchanger in type A intercalated cells of the cortical and medullary collecting ducts of the human kidney is a truncated isoform of erythrocyte band 3 (AE1) that lacks the N-terminal 65 residues. Because this missing sequence has been implicated in the binding of ankyrin, protein 4.1, several glycolytic enzymes, hemoglobin, and hemichromes in erythrocytes, we have undertaken examination of the structure and peripheral protein interactions of this kidney isoform. The cytoplasmic domain of kidney band 3, kidney CDB3, was expressed in Escherichia coli and purified to homogeneity. The kidney isoform exhibited a circular dichroism spectrum and Stokes radius similar to its larger erythrocyte counterpart. Kidney CDB3 was also observed to engage in the same conformational equilibrium characteristic of erythrocyte CDB3. In contrast, the tryptophan and cysteine clusters of kidney CDB3 behaved very differently from erythrocyte CDB3 in response to pH changes and oxidizing conditions. Furthermore, kidney CDB3 did not bind ankyrin, protein 4.1, or aldolase, and expression of erythrocyte CDB3 was toxic to its bacterial host, whereas expression of kidney CDB3 was not. Taken together, these data suggest that the absence of the N-terminal 65 amino acids in kidney CDB3 eliminates the major function currently ascribed to CDB3 in erythrocytes, i.e. that of peripheral protein binding. The primary function of residues 66-379 found in kidney CDB3 thus remains to be elucidated.

Central to spectrin's function is its association with the plasma membrane. The linking proteins ankyrin and protein 4.1 partly mediate this association, and their interactions with spectrin are well understood. Both beta I (erythrocyte) and beta II (fodrin, beta G) spectrin also associate with unknown protein receptors in crude membrane preparations by ankyrin and protein 4.1 independent mechanisms. As a first step to understanding this interaction, kinetic and equilibrium assays have been used to monitor which regions of beta I and beta II spectrin inhibit the binding of purified 125I-labeled bovine brain spectrin to demyelinated and NaOH-stripped bovine brain membranes. A series of 19 recombinant proteins spanning the entire sequence of beta II spectrin, including an alternatively spliced NH2-terminal isoform (beta II epsilon 2 spectrin), were prepared as glutathione S-transferase fusion proteins. Also prepared were peptides representing the alternatively spliced COOH-terminal domain found in beta I epsilon 2 spectrin ("muscle spectrin"). Two distinct sequence motifs inhibited the binding of native brain spectrin. Membrane association domain 1 (MAD1) was represented in all fusion peptides that included spectrin repeat 1. These peptides slowed the kinetics of brain spectrin binding and inhibited up to 46% of the maximal binding under the conditions of these assays (apparent Ki < or = 0.2 microM). Peptides representative of repeats 2-17 of beta II spectrin were devoid of inhibitory activity. The second membrane association domain (MAD2) was identified in penultimate COOH-terminal sequences (domain III) of both beta II and beta I epsilon 2 spectrin. These sequences were absent in beta I epsilon 1 (erythrocyte) spectrin. MAD2 competitively inhibited over 80% of brain spectrin binding in these assays, with an apparent Ki < or = 0.1 microM. Direct binding studies confirmed that both MAD1 and MAD2 peptides associated with membranes with affinities comparable to their inhibition constants. Sequence comparisons suggest that MAD1 is created by the insertion of two non-homologous sequence motifs into repeat 1, extending it from 106 to 122 amino acids. Similarly, MAD2 encompasses a putative site of beta gamma-heterotrimeric G-protein binding called the pleckstrin homology domain, and MAD2 may in fact be the pleckstrin homology domain although this has not been rigorously proven. Collectively these studies identify two novel functional motifs in spectrin that mediate ankyrin independent association with membranes. We hypothesize that these motifs and their still to be discovered ligands play a primary role in the nascent assembly and stabilization of an ordered and polarized spectrin skeleton.

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.