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Adducin: a Physical Model with Implications for Function in Assembly of Spectrin-Actin Complexes

1995; Elsevier BV; Volume: 270; Issue: 32 Linguagem: Inglês

10.1074/jbc.270.32.18990

1083-351X

Christine A. Hughes, Vann Bennett,

Enzyme Structure and Function

Adducin binds to spectrin-actin complexes, promotes association of spectrin with actin, and is subject to regulation by calmodulin as well as protein kinases A and C. Adducin is a heteromer comprised of homologous α- and β-subunits with an NH2-terminal protease-resistant head domain, connected by a neck region to a COOH-terminal hydrophilic, protease-sensitive region. This study provides evidence that adducin in solution is a mixture of heterodimers and tetramers. CD spectroscopy of COOH-terminal domains of α- and β-adducin bacterial recombinants provides direct evidence for an unstructured random coil configuration. Cross-linking, proteolysis, and blot-binding experiments suggest a model for the adducin tetramer in which four head domains contact one another to form a globular core with extended interacting α- and β-adducin tails. The site for binding to spectrin-actin complexes on adducin was identified as the COOH-terminal tail of both the α- and β-adducin subunits. The capacity of native adducin to recruit spectrin to actin filaments is similar to that of adducin tail domains. Thus, adducin tail domains alone are sufficient to interact with F-actin and a single spectrin and to recruit additional spectrin molecules to the ternary complex. Adducin binds to spectrin-actin complexes, promotes association of spectrin with actin, and is subject to regulation by calmodulin as well as protein kinases A and C. Adducin is a heteromer comprised of homologous α- and β-subunits with an NH2-terminal protease-resistant head domain, connected by a neck region to a COOH-terminal hydrophilic, protease-sensitive region. This study provides evidence that adducin in solution is a mixture of heterodimers and tetramers. CD spectroscopy of COOH-terminal domains of α- and β-adducin bacterial recombinants provides direct evidence for an unstructured random coil configuration. Cross-linking, proteolysis, and blot-binding experiments suggest a model for the adducin tetramer in which four head domains contact one another to form a globular core with extended interacting α- and β-adducin tails. The site for binding to spectrin-actin complexes on adducin was identified as the COOH-terminal tail of both the α- and β-adducin subunits. The capacity of native adducin to recruit spectrin to actin filaments is similar to that of adducin tail domains. Thus, adducin tail domains alone are sufficient to interact with F-actin and a single spectrin and to recruit additional spectrin molecules to the ternary complex. The spectrin-based membrane skeleton was first isolated as a meshwork of integral and peripheral membrane proteins when erythrocytes were extracted with non-ionic detergents(1Yu J. Fischman D.A. Steck T.L. J. Supramol. Struct. 1973; 1: 233-248Crossref PubMed Scopus (444) Google Scholar). Electron micrographs of the stretched membrane skeleton reveal a regular lattice-like organization of 5-6 spectrin molecules linked in a polygonal array (2Byers T.J. Branton D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6153-6157Crossref PubMed Scopus (310) Google Scholar). The vertices of this lattice are formed by short actin filaments of 12-14 monomers in length. Accessory proteins are found at the junctions where spectrin and actin interact: these include adducin, protein 4.1, dematin (protein 4.9), tropomyosin, and tropomodulin. These proteins are thought to be important in stabilizing spectrin-actin complexes, forming membrane associations, and assembling the membrane skeleton(3Bennett V. Gilligan D.M. Annu. Rev. Cell Biol. 1993; 9: 27-66Crossref PubMed Scopus (442) Google Scholar). This network of proteins was first defined in the red blood cell; however, many of the proteins present in the red cell membrane skeleton have closely related isoforms expressed in non-erythroid cells. The spectrin-actin lattice has thus far been visualized only in erythrocytes, and the organization of spectrin in different cells has yet to be defined. Adducin (adducere: to bring together) was first identified as a calmodulin-binding protein associated with the red cell membrane skeleton(4Gardner K. Bennett V. J. Biol. Chem. 1986; 261: 1339-1348Abstract Full Text PDF PubMed Google Scholar). Adducin binds to spectrin-actin complexes, is able to recruit a second spectrin molecule to this ternary complex(5Gardner K. Bennett V. Nature. 1987; 328: 359-362Crossref PubMed Scopus (197) Google Scholar), and bundles actin filaments at high concentrations(6Mische S.M. Mooseker M.S. Morrow J.S. J. Cell Biol. 1987; 105: 2837-2845Crossref PubMed Scopus (146) Google Scholar, 7Taylor K.A. Taylor D.W. J. Mol. Biol. 1993; 230: 196-205Crossref PubMed Scopus (55) Google Scholar). Association of adducin with spectrin-actin complexes in in vitro assays has been interpreted as occurring through an ordered pathway with the following steps: spectrin and actin bind, adducin associates with the spectrin-actin complex, and then an additional spectrin molecule associates with the adducin-spectrin-actin ternary complex(5Gardner K. Bennett V. Nature. 1987; 328: 359-362Crossref PubMed Scopus (197) Google Scholar, 8Gardner K. Bennett V. Satir P. Condeelis J. Lazarides E. Signal Transduction in Cytoplasmic Organization. Alan R. Liss, Inc., New York1988: 293-311Google Scholar). This protein is a substrate for protein kinases A and C(9Bennett V. Gardner K. Steiner J.P. J. Biol. Chem. 1988; 263: 5860-5869Abstract Full Text PDF PubMed Google Scholar, 10Ling E. Gardner K. Bennett V. J. Biol. Chem. 1986; 261: 13875-13878Abstract Full Text PDF PubMed Google Scholar, 11Waseem A. Palfrey H.C. Eur. J. Biochem. 1988; 178: 563-573Crossref PubMed Scopus (30) Google Scholar), and consists of two subunits, α (molecular mass = 81 kDa) and β (molecular mass = 80 kDa), which are 49% identical and 66% similar in amino acid sequence(12Joshi R. Gilligan D.M. Otto E. McLaughlin T. Bennett V. J. Cell Biol. 1991; 115: 665-675Crossref PubMed Scopus (121) Google Scholar). Adducin isoforms are expressed in many tissues including brain, kidney, lung, liver, and lens based on immunoblots and Northern blot analysis(4Gardner K. Bennett V. J. Biol. Chem. 1986; 261: 1339-1348Abstract Full Text PDF PubMed Google Scholar, 12Joshi R. Gilligan D.M. Otto E. McLaughlin T. Bennett V. J. Cell Biol. 1991; 115: 665-675Crossref PubMed Scopus (121) Google Scholar). Adducin has been functionally characterized from bovine brain as well as erythrocytes (9Bennett V. Gardner K. Steiner J.P. J. Biol. Chem. 1988; 263: 5860-5869Abstract Full Text PDF PubMed Google Scholar). In addition, several alternatively spliced isoforms have been described for both the α (13Taylor S.A.M. Snell R.G. Buckler A. Ambrose C. Duyao M. Church D. Lin C.S. Altherr M. Bates G.P. Groot N. Barnes G. Shaw D.J. Lehrach H. Wasmuth J.J. Harper P.S. Housman D.E. MacDonald M.E. Gusella J. Nature Genet. 1992; 2: 223-227Crossref PubMed Scopus (37) Google Scholar) and β (12Joshi R. Gilligan D.M. Otto E. McLaughlin T. Bennett V. J. Cell Biol. 1991; 115: 665-675Crossref PubMed Scopus (121) Google Scholar, 14Gilligan D.M. Bennett V. J. Cell Biol. 1991; 115 (abstr.): 42Google Scholar, 15Tripodi G. Piscone A. Borsani G. Tisminetzky S. Salardi S. Sidoli A. James P. Pongor S. Bianchi G. Baralle F.E. Biochem. Biophys. Res. Commun. 1991; 177: 939-947Crossref PubMed Scopus (41) Google Scholar, 16Chapline C. Ramsay K. Klauck T. Jaken S. J. Biol. Chem. 1993; 268: 6858-6861Abstract Full Text PDF PubMed Google Scholar) adducin genes. In vitro activities of adducin suggest a function in constructing the spectrin-actin lattice. The pattern of expression of adducin is also consistent with a role in assembly of spectrin-actin complexes. Adducin is expressed early in erythroid differentiation at the proerythroblast stage and is present at the time when the spectrin skeleton is assembled(17Nehls V. Drenckhahn D. Joshi R. Bennett V. Blood. 1991; 78: 1692-1696Crossref PubMed Google Scholar). Adducin is localized at cell-cell contacts with spectrin and actin in cultured epithelial cells (18Kaiser H.W. O'Keefe E. Bennett V. J. Cell Biol. 1989; 109: 557-569Crossref PubMed Scopus (120) Google Scholar) and no longer localizes to the plasma membrane of epithelial cells when β-spectrin is disrupted(19Hu R.-J. Moorthy S. Bennett V. J. Cell Biol. 1995; 128: 1069-1080Crossref PubMed Scopus (57) Google Scholar). An adducin-like protein is associated with the hts phenotype of defective oocyte development in Drosophila(20Yue L. Spradling A.C. Genes & Dev. 1992; 6: 2443-2454Crossref PubMed Scopus (222) Google Scholar). The hts gene product is localized to ring canals, which are abundant in actin filaments and represent a specialized region of cell-cell contact. It has been speculated that mutant hts could affect ring canal formation either by disrupting actin filament binding directly, or indirectly by interfering with spectrin organization(21Robinson D.N. Cant K. Cooley L. Development. 1994; 120: 2015-2025Crossref PubMed Google Scholar). Adducin thus may be involved with actin and spectrin in forming specialized contact sites in organisms as diverse as Drosophila and mammals. Adducin subunits are comprised of structurally distinct domains: an NH2-terminal 40 kDa globular head domain, an 8 kDa neck domain, and a COOH-terminal domain that has been predicted to be an extended tail(12Joshi R. Gilligan D.M. Otto E. McLaughlin T. Bennett V. J. Cell Biol. 1991; 115: 665-675Crossref PubMed Scopus (121) Google Scholar, 22Joshi R. Bennett V. J. Biol. Chem. 1990; 265: 13130-13136Abstract Full Text PDF PubMed Google Scholar). COOH-terminal regions of adducin subunits have not been isolated from native adducin due to their protease sensitivity. These tail regions are difficult to visualize by electron microscopy and have been referred to as “fairy tails” due to their elusive properties. This report characterizes COOH-terminal domains expressed in bacteria, presents evidence for an extended configuration, and demonstrates that the tail domains of adducin are responsible for association with spectrin-actin complexes. In addition, domains involved in intersubunit contact between α- and β-subunits in an adducin tetramer are defined, and a model for organization of subunits in adducin tetramers is proposed. 125I-Bolton-Hunter reagent, bovine serum albumin, HEPES, and sucrose were obtained from ICN. Carrier-free Na125I was from Amersham Corp. Nitrocellulose and electrophoresis reagents were from Bio-Rad. Diispropyl fluorophosphate, leupeptin, pepstatin A, dithiothreitol, phenylmethylsulfonyl fluoride, EDTA, EGTA, sodium bromide, sodium fluoride, sodium phosphate, sodium azide, Tween 20, and Triton X-100 were from Sigma. V8 protease and ethylene glycol bis(succinimidylsuccinate) were purchased from Pierce. The pGEMEX vector was from Promega. S-Sepharose resin, Mono Q and Mono S columns, and protein A were obtained from Pharmacia. Determination of protein concentration was performed by the procedure of Bradford (23Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211664) Google Scholar) using bovine serum albumin as a standard. SDS-polyacrylamide gel electrophoresis was performed using 0.2% SDS with the buffers of Fairbanks et al.(24Fairbanks G. Steck T. Wallach D. Biochemistry. 1971; 10: 2606-2617Crossref PubMed Scopus (6156) Google Scholar) on 1.5-mm thick 3.5-17% exponential gradient slab gels. Immunoblotting using 125I-labeled protein A to visualize antibodies was performed as described(25Davis J. Bennett V. J. Biol. Chem. 1983; 258: 7757-7766Abstract Full Text PDF PubMed Google Scholar). Adducin, spectrin, and recombinant adducin polypeptides were iodinated with Bolton-Hunter reagent(26Bennett V. Methods. Enzymol. 1983; 96: 313-324Crossref PubMed Scopus (157) Google Scholar). Actin was isolated from an acetone powder of rabbit skeletal muscle as described (27Pardee J.D. Spudich J.A. Methods Cell Biol. 1982; 24: 271-289Crossref PubMed Scopus (336) Google Scholar) and was further purified by gel filtration on a Superose 12 column. Spectrin was isolated from high salt extracts of bovine brain membranes according to the procedure of Bennett(26Bennett V. Methods. Enzymol. 1983; 96: 313-324Crossref PubMed Scopus (157) Google Scholar). Human donor blood (4 units of whole blood) was obtained from the American Red Cross. Adducin was purified from the low salt extract of human erythrocyte ghosts (26Bennett V. Methods. Enzymol. 1983; 96: 313-324Crossref PubMed Scopus (157) Google Scholar) with some modifications. The low salt extract was loaded onto an S-Sepharose column, washed with 0.2 M NaCl, and eluted with 0.5 M NaCl. The S-Sepharose fractions were dialyzed against 10 mM sodium phosphate, 1 mM EDTA, 1 mM DTT, 1The abbreviations used are: DTTdithiothreitolPAGEpolyacrylamide gel electrophoresiscpmcounts/minute. 0.05% Tween 20, and 1 mM NaN3, pH 7.4, and loaded onto a Mono Q column. This column was then washed with 0.10 M NaBr for 20 min. Adducin was then eluted with a linear gradient of 0.10-0.5 M NaBr in the dialysis buffer over 40 min. Peak adducin fractions eluted at 0.15 M NaBr. The NH2-terminal adducin core domains were digested with trypsin as described(22Joshi R. Bennett V. J. Biol. Chem. 1990; 265: 13130-13136Abstract Full Text PDF PubMed Google Scholar). Calmodulin was purified from bovine brain as described(28Gopalakrishna R. Anderson W. Biochem. Biophys. Res. Commun. 1982; 104: 830-836Crossref PubMed Scopus (717) Google Scholar). The MARCKS protein was a gift from Dr. Perry Blackshear, Duke University Medical Center. dithiothreitol polyacrylamide gel electrophoresis counts/minute. The Stokes radii (Rs) of native adducin, cross-linked adducin, native core domain, cross-linked V8 fragment, and α- and β-adducin tail constructs were estimated by gel filtration (29Siegel L.M. Monty K.J. Biochim. Biophys. Acta. 1966; 112: 346-362Crossref PubMed Scopus (1542) Google Scholar) on Superose 6 and 12 columns equilibrated with 10 mM sodium phosphate, 100 mM NaCl, 1 mM DTT, 0.05% Tween 20, 1 mM NaN3, pH 7.4, and calibrated with protein standards: catalase (Rs= 5.2 nm), aldolase (Rs = 4.8 nm), bovine serum albumin (Rs = 3.5 nm), ovalbumin (Rs = 2.84 nm), and cytochrome c (Rs = 2.0 nm). Sedimentation coefficients were estimated by rate-zonal sedimentation on 5-20% linear sucrose gradients (30Martin R.G. Ames B.N. J. Biol. Chem. 1961; 236: 1372-1379Abstract Full Text PDF PubMed Google Scholar) dissolved in 10 mM sodium phosphate, 100 mM NaCl, 1 mM NaN3, 1 mM DTT, pH 7.4, and calibrated with standard proteins: catalase (11.3 S), aldolase (7.3 S), ovalbumin (3.5 S), bovine serum albumin (4.6 S), and cytochrome c (1.75 S). Circular dichroism experiments were performed on an Aviv 62 DS instrument. The samples were dialyzed into a buffer containing 5 mM sodium phosphate, 100 mM NaF, and 0.5 mM NaN3, pH 7.4. Wavelength scans were performed from 190 to 280 nm at 4°C. The equation used to estimate percent helicity was taken from (31Chen Y. Yang J. Biochem. Biophys. Res. Commun. 1971; 44: 1285-1291Crossref PubMed Scopus (325) Google Scholar) ([θ]222 = −30300fH- 2340, where fH represents helical fraction). Spectra for thermal melts were collected over the indicated temperature range at 222 nm. The rate of heating in these experiments was 1.0°C/0.5 min. The cDNAs encoding the COOH-terminal domains of the α- and β-adducin gene were cloned into the pGEMEX expression vector, a pET plasmid with a T7 promoter(32Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorf J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (5980) Google Scholar). A unique NheI restriction site was generated by polymerase chain reaction. Constructs were inserted into the NheI site immediately 3′ to the AUG codon so that both expressed polypeptides have the additional amino acid sequence MAS on the amino terminus(33Davis L.H. Bennett V. J. Biol. Chem. 1994; 269: 4409-4416Abstract Full Text PDF PubMed Google Scholar). The recombinant plasmids were first transformed into a non-expressor JM109 strain. Plasmids purified from the JM109 strain were later transformed into an expressor strain of E. coli (BL21 pLysS (DE3)). Expression of recombinant polypeptides was induced by addition of 1 mM isopropyl-1-thio-β-D-galactopyranoside. The bacteria were harvested at 4-h post-induction. Subsequent lysis and centrifugation revealed both the α- and β-adducin tail polypeptides to be in the soluble supernatant fractions. Since these constructs are heat stable, the 1% Triton X-100 bacterial lysates were further purified by heating to 70°C for 20 min and centrifuged at 140,000 × g. The soluble fraction was loaded onto a Mono S column and eluted with a linear 0-0.5 M NaBr gradient over 78 min. Both α- and β-adducin tail fractions eluted at 0.35-0.4 M NaBr. Typically, 15-20 mg of pure adducin tail construct can be purified/liter bacterial culture. The α-tail construct contains residues 430-737 and the β-tail construct encompasses residues 409-726 of the α- and β-adducin genes(12Joshi R. Gilligan D.M. Otto E. McLaughlin T. Bennett V. J. Cell Biol. 1991; 115: 665-675Crossref PubMed Scopus (121) Google Scholar). Initial characterization of this protein had shown that the α- and β-subunits of adducin are present in a 1:1 stoichiometry, with an apparent molecular mass of 200 kDa which was interpreted as an adducin heterodimer(4Gardner K. Bennett V. J. Biol. Chem. 1986; 261: 1339-1348Abstract Full Text PDF PubMed Google Scholar). Subsequent analysis of the α- and β-adducin genes demonstrated that the molecular mass of the subunits was actually 81 and 80 kDa, respectively, instead of the apparent molecular masses of 103 and 97 kDa estimated from migration on SDS-electrophoresis(12Joshi R. Gilligan D.M. Otto E. McLaughlin T. Bennett V. J. Cell Biol. 1991; 115: 665-675Crossref PubMed Scopus (121) Google Scholar). Thus, a dimer of adducin would have an actual molecular mass of 160 kDa. Chemical cross-linking experiments showed that adducin quantitatively cross-links to a tetramer (Fig. 1). This cross-linking to higher forms had initially been noted in a previous study(9Bennett V. Gardner K. Steiner J.P. J. Biol. Chem. 1988; 263: 5860-5869Abstract Full Text PDF PubMed Google Scholar). The major form of cross-linked adducin migrated at an estimated 350 kDa on SDS-PAGE, which would be a reasonable size for an adducin tetramer (Fig. 1). Cross-linked adducin contained α- and β-subunits as determined by immunoblots with subunit specific antibodies. The cross-linking reaction was sensitive to urea and high salt (Fig. 1, lanes 4-8), indicating that cross-linked oligomers involved specific protein-protein interactions and not merely nonspecific aggregation or collisions in solution. To assess the oligomeric state of adducin, physical properties of the covalently linked tetramer were determined in conjunction with those of the native uncross-linked molecule (Table1). The Stokes radius for both species was estimated by gel filtration. The native adducin peak is broad, giving an average Rs of 6.9 nm. The cross-linked adducin tetramer peak on the Superose 6 column is much sharper, giving a Stokes radius of 8.0 nm and falls within the leading edge of the uncross-linked adducin profile (Fig. 2). These data suggest that native adducin contains a mixture of dimers and tetramers either in a static state or involved in a dimer-tetramer equilibrium. The nearly quantitative formation of tetramer by cross-linking implies that during the time of the cross-linking reaction (15-30 min) essentially all dimers associate to form tetramers. Attempts were made to isolate a cross-linked dimer form of adducin but were not successful.TABLE I Open table in a new tab Molecular masses of cross-linked and native adducin were calculated to be 232 and 211 kDa, respectively, based on the Stokes radii from gel filtration and sedimentation coefficients determined by rate zonal sedimentation on sucrose gradients (Table1). The apparent molecular mass of cross-linked adducin is significantly lower than expected for a tetramer of 322 kDa and suggests an incorrect value for either the sedimentation coefficient or the Stokes radius. The most likely source of error is Stokes radius determined by gel filtration, with lower than actual values resulting from interaction of adducin with the gel matrix, or from anomalous behavior observed with other asymmetric proteins(37Nozaki Y. Schecter N.M. Reynolds J.A. Tanford C. Biochemistry. 1976; 15: 3884-3890Crossref PubMed Scopus (143) Google Scholar). The tail domain of adducin is the source of aberrant behavior in SDS-electrophoresis as well as gel filtration (see below). The COOH-terminal region of both the α- and β-adducin subunits is hydrophilic and highly protease sensitive, in contrast to the stable globular NH2 terminus (Fig. 3). Due to protease sensitivity, we were unable to purify this region from limited proteolytic digests of native adducin. Therefore, COOH-terminal domains of α- and β-adducin were expressed as bacterial recombinant proteins (see “Experimental Procedures”). Isolated COOH-terminal constructs migrated at an apparent 60 kDa, substantially higher than predicted molecular mass of 34 and 33 kDa based on amino acid sequence. These domains thus are likely to be responsible for anomalous migration of native adducin on SDS-gels, which migrates at an apparent molecular mass of 20 kDa higher than the actual size based on amino acid sequence. Circular dichroism indicated that COOH-terminal tail regions of α- and β-adducin are unstructured, with no evidence for α-helix or β-sheet based on signals at 208 and 222 nm, and a negative peak at 200 nm which is indicative of a random coil (Fig. 4B). Spectra of the COOH-terminal domains are in contrast to those of intact erythrocyte adducin and the NH2-terminal core region of adducin which contain approximately 10-15%α-helix as well as β-sheet secondary structure (Fig. 4A). A thermal melt was performed on both the adducin core and the α-adducin tail construct at 222 nm (Fig. 5, A and B). Loss of secondary structure in the NH2-terminal core can be seen at a midpoint of 59°C with decreasing ellipticity at 222 nm. The α-adducin tail appears to be heat stable, and the spectrum at 222 nm does not change over the temperature range of 25-71°C.Figure 5:The COOH-terminal adducin tails are extended, unstructured molecules and are heat-stable. The red cell NH2-terminal adducin core loses secondary structure cooperatively at 222 nm at 59°C, whereas the ellipticity at 222 nm does not change over the temperature range for α-adducin tail. Thermal melts were monitored at 222 nm by circular dichroism. A, erythrocyte adducin core. B, α-adducin tail.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Circular dichroism data support the idea of an unstructured COOH terminus, and the protease sensitivity of this domain implies an extended, non-globular structure. Molecular masses of 55 and 48 kDa calculated from the Stokes radii and sedimentation coefficients indicate that α- and β-adducin tails (34 kDa based on amino acid sequence) are either a mixture of monomers and dimers, or that the Stokes radius has been underestimated by gel filtration (see above) and the domains actually are dimers with a true molecular mass of 68 kDa (Table1). The frictional ratio for the tail constructs is estimated at about 1.3 using a molecular mass of 55 kDa, which was lower than expected. However a corrected molecular mass of 68 kDa yields a frictional ratio of 1.6, which is closer to the value anticipated from an extended conformation. The calculated molecular mass of 232 kDa for cross-linked adducin also is smaller than expected for a 320-kDa tetramer (see above). The globular head domain, in contrast to native adducin and COOH-terminal tail domains, behaves as expected on gel filtration and SDS-electrophoresis (22Joshi R. Bennett V. J. Biol. Chem. 1990; 265: 13130-13136Abstract Full Text PDF PubMed Google Scholar) (Table1). Association of domains in the adducin molecule had not been previously ascertained. Cross-linking, proteolysis and blot-binding experiments were performed to address this question. Adducin was cross-linked and subjected to limited V8 proteolysis. A protease-resistant 160-kDa fragment was generated after 10-20 min digestion of cross-linked adducin with V8 protease, while similar digestion of uncross-linked adducin resulted in a 40-kDa polypeptide. The 160-kDa cross-linked fragment (Fig. 6, lanes 7-9) did not react with an affinity-purified polyclonal antibody against the COOH-terminal tail region of red cell adducin, unlike the intact tetramer (Fig. 6, lane 6). This indicated that the 160-kDa fragment contained NH2-terminal adducin sequence. The size of this fragment was the correct size for a covalently cross-linked tetramer of four NH2-terminal head domains. Sucrose gradients and gel filtration were performed in parallel on the 160-kDa cross-linked V8 fragment and the native NH2-terminal core fragment. Both displayed similar sedimentation coefficients and Stokes radii; the frictional ratio was calculated at 1.2, indicative of a globular molecule. A molecular mass of 122 kDa was calculated from hydrodynamic values for native and cross-linked head domains (Table1). As further evidence for head domain contacts, the isolated NH2-terminal fragment was able to cross-link to trimers and tetramers(22Joshi R. Bennett V. J. Biol. Chem. 1990; 265: 13130-13136Abstract Full Text PDF PubMed Google Scholar). Radiolabeled recombinant α- and β-adducin tail constructs were used as ligands to evaluate possible interactions with adducin and NH2- and COOH-terminal domains immobilized on nitrocellulose paper (Fig. 7, panel A). α- and β-adducin tail domains associated with tails of both subunits, as well as the intact erythrocyte adducin protein (panels B and D). This binding is specific, as evidenced by the displacement of binding when 1.3 μM unlabeled α- or β-adducin tail is included in the incubation (panels C and E). Individual subunit tails were able to form α-α, α-β, and β-β contacts. Neither the α- nor β-tails bound to the NH2-terminal core. The binding site on adducin for participation in the ternary complex of spectrin-actin-adducin was mapped to the COOH terminus of both the α- and the β-subunit based on cosedimentation assays with spectrin, F-actin, and various adducin domains (Fig. 8). Spectrin-dependent binding of 125I-labeled α- and β-adducin tails to F-actin in these experiments shows that the adducin tails bind to spectrin-actin complexes with a KD of 180 and 210 nM, respectively, about 5-6-fold lower affinity than native adducin but with a similar capacity. Hill coefficients for the α- and β-adducin tail binding data are 1.4, indicating positive cooperativity. NH2-terminal core domains were inactive in these assays. Adducin has been seen to promote the binding of spectrin to F-actin (5, Fig. 9A). Fig. 9B shows that both the α- and β-adducin tails can also stimulate binding of spectrin to actin. The double-reciprocal plot for this experiment estimated a KD of 100 nM for the red cell adducin; the adducin tails displayed a 2-3-fold lower affinity than the native adducin in this assay (KD = 270 nM). The capacity of the native adducin-dependent binding of spectrin to actin was similar to that of the recombinant adducin tail constructs (Fig. 9C). The NH2-terminal 48-kDa core-neck and 40-kDa core domain fragments did not show any activity over background (spectrin binding to actin alone) in this assay (data not shown). The extreme COOH terminus of both the α- and β-adducin subunits contains a highly polybasic region of 22 amino acids that has 41% identity to the MARCKS protein(12Joshi R. Gilligan D.M. Otto E. McLaughlin T. Bennett V. J. Cell Biol. 1991; 115: 665-675Crossref PubMed Scopus (121) Google Scholar, 38Graff J.M. Stumpo D.J. Blackshear P.J. J. Biol. Chem. 1989; 264: 11912-11919Abstract Full Text PDF PubMed Google Scholar). The MARCKS protein was also analyzed using this assay but did not show any spectrin-dependent binding to F-actin (data not shown). The polybasic region at the extreme COOH terminus of the α- and β-adducin subunits, which is similar to MARCKS, therefore is not sufficient to bind spectrin-actin complexes. This report characterizes the adducin molecule in terms of oligomeric structure, folding of domains, and definition of domains involved in contact with spectrin-actin complexes. Experiments with cross-linked and native adducin indicate that adducin in solution is a mixture of heterodimers and tetramers. Circular dichroism spectroscopy of COOH-terminal domains of α- and β-adducin expressed in bacteria provides direct evidence for an unstructured random coil configuration for the COOH-terminal region. Some predictions for a model of the adducin molecule can be made based upon the cross-linking, physical properties, proteolysis, and blot binding data (Fig. 10). Four amino-terminal head domains contact one another, eliminating a model where two antiparallel dimers come together, to form a tetramer. Exact orientation of the tails, whether two or four α-α, α-β, or β-β interactions, cannot be assessed at the level of these experiments. The flexible, extended tails would provide asymmetry to the molecule; intramolecular tail contacts may also occur. Presumably these extended, protease-sensitive tail regions are stabilized by their binding to spectrin and actin filaments. The site on adducin for binding to spectrin-actin complexes was identified as the COOH-terminal tail of both the α- and β-adducin subunits based on activities of expressed domains and lack of activity of the NH2-terminal head domain. Adducin had previously been characterized as an α/β heterodimer of 200 kDa, based upon migration on SDS-PAGE and physical properties(4Gardner K. Bennett V. J. Biol. Chem. 1986; 261: 1339-1348Abstract Full Text PDF PubMed Google Scholar). Properties of cross-linked adducin tetramer and of COOH-terminal domains determined in this study provide evidence that the tail domains have anomolous behavior on gel filtration and SDS-polyacrylamide electrophoresis leading to underestimates of the Stokes radius of the intact molecule and overestimates of monomer molecular mass. An additional complicating factor is that adducin exists in solution as a mixture of dimers and tetramers. Adducin tetramers were inferred by previous electron microscopy studies, in which the diameter of adducin and NH2-terminal protease-resistant domains were identical and similar to predicted dimensions of a 160-kDa globular protein(12Joshi R. Gilligan D.M. Otto E. McLaughlin T. Bennett V. J. Cell Biol. 1991; 115: 665-675Crossref PubMed Scopus (121) Google Scholar). The question of whether adducin engages in a dimer-tetramer equilibrium or exists as a static mixture of dimers and tetramers needs to be addressed in future experiments. Adducin binds Ca2+/calmodulin and accepts phosphate from protein kinases A and C. Ca2+/calmodulin does not affect the profile of native adducin on gel filtration (data not shown). However, the effects of phosphorylation on the oligomeric state of adducin have yet to be tested. Although adducin in erythrocytes contains α- and β-subunits in a 1:1 ratio, only α-adducin has been detected in keratinocytes (18Kaiser H.W. O'Keefe E. Bennett V. J. Cell Biol. 1989; 109: 557-569Crossref PubMed Scopus (120) Google Scholar) and fibroblasts(39Waseem A. Cho K. Palfrey H.C. FASEB J. 1988; 2: A349Google Scholar). It is pertinent in this regard that the site for adducin binding to spectrin-actin complexes has been mapped to the COOH-terminal region of both α- and β-subunits, and that both α- and β-COOH-terminal domains can self-associate (Fig. 7). Individual subunits should therefore be capable of interacting with spectrin-actin complexes and at least of forming homodimers. It will be of interest to determine if assembly of adducin subunits to tetramers requires both α- and β-subunits and to evaluate activities of α-adducin in the absence of β-adducin. The COOH-terminal domain of adducin, although unstructured, is an essential portion of the molecule. Activities in addition to spectrin-actin binding, recruitment of additional spectrin molecules to F-actin, and self-association include the site for Ca2+/calmodulin binding on β-adducin(40Scaramuzzino D.A. Morrow J.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3398-3402Crossref PubMed Scopus (25) Google Scholar), and a binding site for stomatin (band 7.2b)(41Sinard J.H. Stewart G.H. Argent A.C. Morrow J.S. Mol. Biol. Cell. 1994; 5 (abstr.): 421Google Scholar). The COOH-terminal domain also is likely to contribute to functional diversity among adducin isoforms, since alternatively spliced variants have conserved NH2-terminal sequences and variable COOH termini(13Taylor S.A.M. Snell R.G. Buckler A. Ambrose C. Duyao M. Church D. Lin C.S. Altherr M. Bates G.P. Groot N. Barnes G. Shaw D.J. Lehrach H. Wasmuth J.J. Harper P.S. Housman D.E. MacDonald M.E. Gusella J. Nature Genet. 1992; 2: 223-227Crossref PubMed Scopus (37) Google Scholar, 14Gilligan D.M. Bennett V. J. Cell Biol. 1991; 115 (abstr.): 42Google Scholar, 15Tripodi G. Piscone A. Borsani G. Tisminetzky S. Salardi S. Sidoli A. James P. Pongor S. Bianchi G. Baralle F.E. Biochem. Biophys. Res. Commun. 1991; 177: 939-947Crossref PubMed Scopus (41) Google Scholar, 16Chapline C. Ramsay K. Klauck T. Jaken S. J. Biol. Chem. 1993; 268: 6858-6861Abstract Full Text PDF PubMed Google Scholar). The NH2-terminal head domain, in contrast, is the most conserved region of adducin but has no known role other than its role in stabilizing the adducin tetramer. Determining the function(s) of the NH2-terminal domain will be an interesting challenge for the future. We thank Diana Gilligan for the full-length α- and β-adducin cDNA clones.