Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1988 Nov 1;107(5):1749–1757. doi: 10.1083/jcb.107.5.1749

Structural and immunological characterization of the myosin-like 110-kD subunit of the intestinal microvillar 110K-calmodulin complex: evidence for discrete myosin head and calmodulin-binding domains

PMCID: PMC2115315  PMID: 2460467

Abstract

The actin bundle within each microvillus of the intestinal brush border is tethered laterally to the membrane by spirally arranged bridges. These bridges are thought to be composed of a protein complex consisting of a 110-kD subunit and multiple molecules of bound calmodulin (CM). Recent studies indicate that this complex, termed 110K- CM, is myosin-like with respect to its actin binding and ATPase properties. In this study, possible structural similarity between the 110-kD subunit and myosin was examined using two sets of mAbs; one was generated against Acanthamoeba myosin II and the other against the 110- kD subunit of avian 110K-CM. The myosin II mAbs had been shown previously to be cross-reactive with skeletal muscle myosin, with the epitope(s) localized to the 50-kD tryptic fragment of the subfragment-1 (S1) domain. The 110K mAbs (CX 1-5) reacted with the 110-kD subunit as well as with the heavy chain of skeletal but not with that of smooth or brush border myosin. All five of these 110K mAbs reacted with the 25- kD, NH2-terminal tryptic fragment of chicken skeletal S1, which contains the ATP-binding site of myosin. Similar tryptic digestion of 110K-CM revealed that these five mAbs all reacted with a 36-kD fragment of 110K (as well as larger 90- and 54-kD fragments) which by photoaffinity labeling was shown to contain the ATP-binding site(s) of the 110K subunit. CM binding to these same tryptic digests of 110K-CM revealed that only the 90-kD fragment retained both ATP- and CM-binding domains. CM binding was observed to several tryptic fragments of 60, 40, 29, and 18 kD, none of which contain the myosin head epitopes. These results suggest structural similarity between the 110K and myosin S1, including those domains involved in ATP- and actin binding, and provide additional evidence that 110K-CM is a myosin. These studies also support the results of Coluccio and Bretscher (1988. J. Cell Biol. 106:367-373) that the calmodulin-binding site(s) and the myosin head region of the 110-kD subunit lie in discrete functional domains of the molecule.

Full Text

The Full Text of this article is available as a PDF (2.2 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Carlin R. K., Grab D. J., Siekevitz P. The binding of radio-iodinated calmodulin to proteins on denaturing gels. Ann N Y Acad Sci. 1980;356:73–74. doi: 10.1111/j.1749-6632.1980.tb29600.x. [DOI] [PubMed] [Google Scholar]
  2. Collins J. H., Borysenko C. W. The 110,000-dalton actin- and calmodulin-binding protein from intestinal brush border is a myosin-like ATPase. J Biol Chem. 1984 Nov 25;259(22):14128–14135. [PubMed] [Google Scholar]
  3. Coluccio L. M., Bretscher A. Calcium-regulated cooperative binding of the microvillar 110K-calmodulin complex to F-actin: formation of decorated filaments. J Cell Biol. 1987 Jul;105(1):325–333. doi: 10.1083/jcb.105.1.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Coluccio L. M., Bretscher A. Mapping of the microvillar 110K-calmodulin complex: calmodulin-associated or -free fragments of the 110-kD polypeptide bind F-actin and retain ATPase activity. J Cell Biol. 1988 Feb;106(2):367–373. doi: 10.1083/jcb.106.2.367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Conzelman K. A., Mooseker M. S. The 110-kD protein-calmodulin complex of the intestinal microvillus is an actin-activated MgATPase. J Cell Biol. 1987 Jul;105(1):313–324. doi: 10.1083/jcb.105.1.313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Glenney J. R., Jr, Glenney P. Comparison of Ca++-regulated events in the intestinal brush border. J Cell Biol. 1985 Mar;100(3):754–763. doi: 10.1083/jcb.100.3.754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Glenney J. R., Jr, Glenney P. The microvillus 110K cytoskeletal protein is an integral membrane protein. Cell. 1984 Jul;37(3):743–751. doi: 10.1016/0092-8674(84)90410-0. [DOI] [PubMed] [Google Scholar]
  8. Hoshimaru M., Nakanishi S. Identification of a new type of mammalian myosin heavy chain by molecular cloning. Overlap of its mRNA with preprotachykinin B mRNA. J Biol Chem. 1987 Oct 25;262(30):14625–14632. [PubMed] [Google Scholar]
  9. Howe C. L., Keller T. C., 3rd, Mooseker M. S., Wasserman R. H. Analysis of cytoskeletal proteins and Ca2+-dependent regulation of structure in intestinal brush borders from rachitic chicks. Proc Natl Acad Sci U S A. 1982 Feb;79(4):1134–1138. doi: 10.1073/pnas.79.4.1134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Howe C. L., Mooseker M. S. Characterization of the 110-kdalton actin-calmodulin-, and membrane-binding protein from microvilli of intestinal epithelial cells. J Cell Biol. 1983 Oct;97(4):974–985. doi: 10.1083/jcb.97.4.974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Keller T. C., 3rd, Mooseker M. S. Ca++-calmodulin-dependent phosphorylation of myosin, and its role in brush border contraction in vitro. J Cell Biol. 1982 Dec;95(3):943–959. doi: 10.1083/jcb.95.3.943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kiehart D. P., Kaiser D. A., Pollard T. D. Monoclonal antibodies demonstrate limited structural homology between myosin isozymes from Acanthamoeba. J Cell Biol. 1984 Sep;99(3):1002–1014. doi: 10.1083/jcb.99.3.1002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Korn E. D., Atkinson M. A., Brzeska H., Hammer J. A., 3rd, Jung G., Lynch T. J. Structure-function studies on Acanthamoeba myosins IA, IB, and II. J Cell Biochem. 1988 Jan;36(1):37–50. doi: 10.1002/jcb.240360105. [DOI] [PubMed] [Google Scholar]
  14. Korn E. D., Hammer J. A., 3rd Myosins of nonmuscle cells. Annu Rev Biophys Biophys Chem. 1988;17:23–45. doi: 10.1146/annurev.bb.17.060188.000323. [DOI] [PubMed] [Google Scholar]
  15. Krizek J., Coluccio L. M., Bretscher A. ATPase activity of the microvillar 110 kDa polypeptide-calmodulin complex is activated in Mg2+ and inhibited in K+-EDTA by F-actin. FEBS Lett. 1987 Dec 10;225(1-2):269–272. doi: 10.1016/0014-5793(87)81172-9. [DOI] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Margossian S. S., Lowey S. Preparation of myosin and its subfragments from rabbit skeletal muscle. Methods Enzymol. 1982;85(Pt B):55–71. doi: 10.1016/0076-6879(82)85009-x. [DOI] [PubMed] [Google Scholar]
  18. Maruta H., Korn E. D. Direct photoaffinity labeling by nucleotides of the apparent catalytic site on the heavy chains of smooth muscle and Acanthamoeba myosins. J Biol Chem. 1981 Jan 10;256(1):499–502. [PubMed] [Google Scholar]
  19. Matsudaira P. T., Burgess D. R. Organization of the cross-filaments in intestinal microvilli. J Cell Biol. 1982 Mar;92(3):657–664. doi: 10.1083/jcb.92.3.657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Matsudaira P. T., Burgess D. R. SDS microslab linear gradient polyacrylamide gel electrophoresis. Anal Biochem. 1978 Jul 1;87(2):386–396. doi: 10.1016/0003-2697(78)90688-7. [DOI] [PubMed] [Google Scholar]
  21. Mooseker M. S., Bonder E. M., Conzelman K. A., Fishkind D. J., Howe C. L., Keller T. C., 3rd The cytoskeletal apparatus of the intestinal brush border. Kroc Found Ser. 1984;17:287–307. [PubMed] [Google Scholar]
  22. Mooseker M. S., Coleman T. R., Conzelman K. A. Calcium and the regulation of cytoskeletal assembly, structure and contractility. Ciba Found Symp. 1986;122:232–249. doi: 10.1002/9780470513347.ch14. [DOI] [PubMed] [Google Scholar]
  23. Mooseker M. S. Organization, chemistry, and assembly of the cytoskeletal apparatus of the intestinal brush border. Annu Rev Cell Biol. 1985;1:209–241. doi: 10.1146/annurev.cb.01.110185.001233. [DOI] [PubMed] [Google Scholar]
  24. Mooseker M. S., Tilney L. G. Organization of an actin filament-membrane complex. Filament polarity and membrane attachment in the microvilli of intestinal epithelial cells. J Cell Biol. 1975 Dec;67(3):725–743. doi: 10.1083/jcb.67.3.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mornet D., Bertrand R., Pantel P., Audemard E., Kassab R. Structure of the actin-myosin interface. Nature. 1981 Jul 23;292(5821):301–306. doi: 10.1038/292301a0. [DOI] [PubMed] [Google Scholar]
  26. Rochette-Egly C., Haffen K. Developmental pattern of calmodulin-binding proteins in rat jejunal epithelial cells. Differentiation. 1987;35(3):219–227. doi: 10.1111/j.1432-0436.1987.tb00172.x. [DOI] [PubMed] [Google Scholar]
  27. Sellers J. R., Pato M. D., Adelstein R. S. Reversible phosphorylation of smooth muscle myosin, heavy meromyosin, and platelet myosin. J Biol Chem. 1981 Dec 25;256(24):13137–13142. [PubMed] [Google Scholar]
  28. Sheetz M. P., Spudich J. A. Movement of myosin-coated fluorescent beads on actin cables in vitro. Nature. 1983 May 5;303(5912):31–35. doi: 10.1038/303031a0. [DOI] [PubMed] [Google Scholar]
  29. Shibayama T., Carboni J. M., Mooseker M. S. Assembly of the intestinal brush border: appearance and redistribution of microvillar core proteins in developing chick enterocytes. J Cell Biol. 1987 Jul;105(1):335–344. doi: 10.1083/jcb.105.1.335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sobieszek A., Bremel R. D. Preparation and properties of vertebrate smooth-muscle myofibrils and actomyosin. Eur J Biochem. 1975 Jun 16;55(1):49–60. doi: 10.1111/j.1432-1033.1975.tb02137.x. [DOI] [PubMed] [Google Scholar]
  31. Swanljung-Collins H., Montibeller J., Collins J. H. Purification and characterization of the 110-kDa actin- and calmodulin-binding protein from intestinal brush border: a myosin-like ATPase. Methods Enzymol. 1987;139:137–148. doi: 10.1016/0076-6879(87)39081-0. [DOI] [PubMed] [Google Scholar]
  32. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Verner K., Bretscher A. Microvillus 110K-calmodulin: effects of nucleotides on isolated cytoskeletons and the interaction of the purified complex with F-actin. J Cell Biol. 1985 May;100(5):1455–1465. doi: 10.1083/jcb.100.5.1455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Walker J. E., Saraste M., Runswick M. J., Gay N. J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1(8):945–951. doi: 10.1002/j.1460-2075.1982.tb01276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Warrick H. M., Spudich J. A. Myosin structure and function in cell motility. Annu Rev Cell Biol. 1987;3:379–421. doi: 10.1146/annurev.cb.03.110187.002115. [DOI] [PubMed] [Google Scholar]
  36. Weeds A. G., Pope B. Studies on the chymotryptic digestion of myosin. Effects of divalent cations on proteolytic susceptibility. J Mol Biol. 1977 Apr;111(2):129–157. doi: 10.1016/s0022-2836(77)80119-8. [DOI] [PubMed] [Google Scholar]
  37. Weeds A. G., Taylor R. S. Separation of subfragment-1 isoenzymes from rabbit skeletal muscle myosin. Nature. 1975 Sep 4;257(5521):54–56. doi: 10.1038/257054a0. [DOI] [PubMed] [Google Scholar]
  38. Winkelmann D. A., Lowey S. Probing myosin head structure with monoclonal antibodies. J Mol Biol. 1986 Apr 20;188(4):595–612. doi: 10.1016/s0022-2836(86)80009-2. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES