Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1992 Dec 2;119(6):1541–1557. doi: 10.1083/jcb.119.6.1541

Primary structure and cellular localization of chicken brain myosin-V (p190), an unconventional myosin with calmodulin light chains

PMCID: PMC2289763  PMID: 1469047

Abstract

Recent biochemical studies of p190, a calmodulin (CM)-binding protein purified from vertebrate brain, have demonstrated that this protein, purified as a complex with bound CM, shares a number of properties with myosins (Espindola, F. S., E. M. Espreafico, M. V. Coelho, A. R. Martins, F. R. C. Costa, M. S. Mooseker, and R. E. Larson. 1992. J. Cell Biol. 118:359-368). To determine whether or not p190 was a member of the myosin family of proteins, a set of overlapping cDNAs encoding the full-length protein sequence of chicken brain p190 was isolated and sequenced. Verification that the deduced primary structure was that of p190 was demonstrated through microsequence analysis of a cyanogen bromide peptide generated from chick brain p190. The deduced primary structure of chicken brain p190 revealed that this 1,830-amino acid (aa) 212,509-D) protein is a member of a novel structural class of unconventional myosins that includes the gene products encoded by the dilute locus of mouse and the MYO2 gene of Saccharomyces cerevisiae. We have named the p190-CM complex "myosin-V" based on the results of a detailed sequence comparison of the head domains of 29 myosin heavy chains (hc), which has revealed that this myosin, based on head structure, is the fifth of six distinct structural classes of myosin to be described thus far. Like the presumed products of the mouse dilute and yeast MYO2 genes, the head domain of chicken myosin-V hc (aa 1-764) is linked to a "neck" domain (aa 765-909) consisting of six tandem repeats of an approximately 23-aa "IQ-motif." All known myosins contain at least one such motif at their head-tail junctions; these IQ-motifs may function as calmodulin or light chain binding sites. The tail domain of chicken myosin-V consists of an initial 511 aa predicted to form several segments of coiled-coil alpha helix followed by a terminal 410-aa globular domain (aa, 1,421-1,830). Interestingly, a portion of the tail domain (aa, 1,094-1,830) shares 58% amino acid sequence identity with a 723-aa protein from mouse brain reported to be a glutamic acid decarboxylase. The neck region of chicken myosin-V, which contains the IQ-motifs, was demonstrated to contain the binding sites for CM by analyzing CM binding to bacterially expressed fusion proteins containing the head, neck, and tail domains. Immunolocalization of myosin-V in brain and in cultured cells revealed an unusual distribution for this myosin in both neurons and nonneuronal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Full Text

The Full Text of this article is available as a PDF (3.7 MB).

Selected References

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

  1. Baekkeskov S., Aanstoot H. J., Christgau S., Reetz A., Solimena M., Cascalho M., Folli F., Richter-Olesen H., De Camilli P., Camilli P. D. Identification of the 64K autoantigen in insulin-dependent diabetes as the GABA-synthesizing enzyme glutamic acid decarboxylase. Nature. 1990 Sep 13;347(6289):151–156. doi: 10.1038/347151a0. [DOI] [PubMed] [Google Scholar]
  2. Banker G. A., Cowan W. M. Rat hippocampal neurons in dispersed cell culture. Brain Res. 1977 May 13;126(3):397–342. doi: 10.1016/0006-8993(77)90594-7. [DOI] [PubMed] [Google Scholar]
  3. Bartlett W. P., Banker G. A. An electron microscopic study of the development of axons and dendrites by hippocampal neurons in culture. I. Cells which develop without intercellular contacts. J Neurosci. 1984 Aug;4(8):1944–1953. doi: 10.1523/JNEUROSCI.04-08-01944.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barylko B., Wagner M. C., Reizes O., Albanesi J. P. Purification and characterization of a mammalian myosin I. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):490–494. doi: 10.1073/pnas.89.2.490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carboni J. M., Conzelman K. A., Adams R. A., Kaiser D. A., Pollard T. D., Mooseker M. S. 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. J Cell Biol. 1988 Nov;107(5):1749–1757. doi: 10.1083/jcb.107.5.1749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carlin R. K., Grab D. J., Siekevitz P. Function of a calmodulin in postsynaptic densities. III. Calmodulin-binding proteins of the postsynaptic density. J Cell Biol. 1981 Jun;89(3):449–455. doi: 10.1083/jcb.89.3.449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chapman E. R., Au D., Alexander K. A., Nicolson T. A., Storm D. R. Characterization of the calmodulin binding domain of neuromodulin. Functional significance of serine 41 and phenylalanine 42. J Biol Chem. 1991 Jan 5;266(1):207–213. [PubMed] [Google Scholar]
  8. Cheney R. E., Mooseker M. S. Unconventional myosins. Curr Opin Cell Biol. 1992 Feb;4(1):27–35. doi: 10.1016/0955-0674(92)90055-h. [DOI] [PubMed] [Google Scholar]
  9. Collins K., Sellers J. R., Matsudaira P. Calmodulin dissociation regulates brush border myosin I (110-kD-calmodulin) mechanochemical activity in vitro. J Cell Biol. 1990 Apr;110(4):1137–1147. doi: 10.1083/jcb.110.4.1137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Coluccio L. M. Identification of the microvillar 110-kDa calmodulin complex (myosin-1) in kidney. Eur J Cell Biol. 1991 Dec;56(2):286–294. [PubMed] [Google Scholar]
  12. De Camilli P., Cameron R., Greengard P. Synapsin I (protein I), a nerve terminal-specific phosphoprotein. I. Its general distribution in synapses of the central and peripheral nervous system demonstrated by immunofluorescence in frozen and plastic sections. J Cell Biol. 1983 May;96(5):1337–1354. doi: 10.1083/jcb.96.5.1337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Erlander M. G., Tillakaratne N. J., Feldblum S., Patel N., Tobin A. J. Two genes encode distinct glutamate decarboxylases. Neuron. 1991 Jul;7(1):91–100. doi: 10.1016/0896-6273(91)90077-d. [DOI] [PubMed] [Google Scholar]
  15. Espindola F. S., Espreafico E. M., Coelho M. V., Martins A. R., Costa F. R., Mooseker M. S., Larson R. E. Biochemical and immunological characterization of p190-calmodulin complex from vertebrate brain: a novel calmodulin-binding myosin. J Cell Biol. 1992 Jul;118(2):359–368. doi: 10.1083/jcb.118.2.359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Garcia A., Coudrier E., Carboni J., Anderson J., Vandekerkhove J., Mooseker M., Louvard D., Arpin M. Partial deduced sequence of the 110-kD-calmodulin complex of the avian intestinal microvillus shows that this mechanoenzyme is a member of the myosin I family. J Cell Biol. 1989 Dec;109(6 Pt 1):2895–2903. doi: 10.1083/jcb.109.6.2895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Halsall D. J., Hammer J. A., 3rd A second isoform of chicken brush border myosin I contains a 29-residue inserted sequence that binds calmodulin. FEBS Lett. 1990 Jul 2;267(1):126–130. doi: 10.1016/0014-5793(90)80305-3. [DOI] [PubMed] [Google Scholar]
  18. Hammer J. A. Novel myosins. Trends Cell Biol. 1991 Aug;1(2-3):50–56. doi: 10.1016/0962-8924(91)90089-r. [DOI] [PubMed] [Google Scholar]
  19. Hayden S. M., Wolenski J. S., Mooseker M. S. Binding of brush border myosin I to phospholipid vesicles. J Cell Biol. 1990 Aug;111(2):443–451. doi: 10.1083/jcb.111.2.443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Higgins D. G., Bleasby A. J., Fuchs R. CLUSTAL V: improved software for multiple sequence alignment. Comput Appl Biosci. 1992 Apr;8(2):189–191. doi: 10.1093/bioinformatics/8.2.189. [DOI] [PubMed] [Google Scholar]
  21. Horowitz J. A., Hammer J. A., 3rd A new Acanthamoeba myosin heavy chain. Cloning of the gene and immunological identification of the polypeptide. J Biol Chem. 1990 Nov 25;265(33):20646–20652. [PubMed] [Google Scholar]
  22. 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]
  23. Huang W. M., Reed-Fourquet L., Wu E., Wu J. Y. Molecular cloning and amino acid sequence of brain L-glutamate decarboxylase. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8491–8495. doi: 10.1073/pnas.87.21.8491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hunkapiller M. W., Lujan E., Ostrander F., Hood L. E. Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis. Methods Enzymol. 1983;91:227–236. doi: 10.1016/s0076-6879(83)91019-4. [DOI] [PubMed] [Google Scholar]
  25. Johnston G. C., Prendergast J. A., Singer R. A. The Saccharomyces cerevisiae MYO2 gene encodes an essential myosin for vectorial transport of vesicles. J Cell Biol. 1991 May;113(3):539–551. doi: 10.1083/jcb.113.3.539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Karlsen A. E., Hagopian W. A., Grubin C. E., Dube S., Disteche C. M., Adler D. A., Bärmeier H., Mathewes S., Grant F. J., Foster D. Cloning and primary structure of a human islet isoform of glutamic acid decarboxylase from chromosome 10. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8337–8341. doi: 10.1073/pnas.88.19.8337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kobayashi Y., Kaufman D. L., Tobin A. J. Glutamic acid decarboxylase cDNA: nucleotide sequence encoding an enzymatically active fusion protein. J Neurosci. 1987 Sep;7(9):2768–2772. doi: 10.1523/JNEUROSCI.07-09-02768.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kretsinger R. H. Structure and evolution of calcium-modulated proteins. CRC Crit Rev Biochem. 1980;8(2):119–174. doi: 10.3109/10409238009105467. [DOI] [PubMed] [Google Scholar]
  29. Kuznetsov S. A., Langford G. M., Weiss D. G. Actin-dependent organelle movement in squid axoplasm. Nature. 1992 Apr 23;356(6371):722–725. doi: 10.1038/356722a0. [DOI] [PubMed] [Google Scholar]
  30. Larson R. E., Espindola F. S., Espreafico E. M. Calmodulin-binding proteins and calcium/calmodulin-regulated enzyme activities associated with brain actomyosin. J Neurochem. 1990 Apr;54(4):1288–1294. doi: 10.1111/j.1471-4159.1990.tb01961.x. [DOI] [PubMed] [Google Scholar]
  31. Larson R. E., Pitta D. E., Ferro J. A. A novel 190 kDa calmodulin-binding protein associated with brain actomyosin. Braz J Med Biol Res. 1988;21(2):213–217. [PubMed] [Google Scholar]
  32. Lillie S. H., Brown S. S. Suppression of a myosin defect by a kinesin-related gene. Nature. 1992 Mar 26;356(6367):358–361. doi: 10.1038/356358a0. [DOI] [PubMed] [Google Scholar]
  33. Lupas A., Van Dyke M., Stock J. Predicting coiled coils from protein sequences. Science. 1991 May 24;252(5009):1162–1164. doi: 10.1126/science.252.5009.1162. [DOI] [PubMed] [Google Scholar]
  34. Matsudaira P. Limited N-terminal sequence analysis. Methods Enzymol. 1990;182:602–613. doi: 10.1016/0076-6879(90)82047-6. [DOI] [PubMed] [Google Scholar]
  35. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  36. Matteoli M., Takei K., Cameron R., Hurlbut P., Johnston P. A., Südhof T. C., Jahn R., De Camilli P. Association of Rab3A with synaptic vesicles at late stages of the secretory pathway. J Cell Biol. 1991 Nov;115(3):625–633. doi: 10.1083/jcb.115.3.625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. McNally E. M., Bravo-Zehnder M. M., Leinwand L. A. Identification of sequences necessary for the association of cardiac myosin subunits. J Cell Biol. 1991 May;113(3):585–590. doi: 10.1083/jcb.113.3.585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Mercer J. A., Seperack P. K., Strobel M. C., Copeland N. G., Jenkins N. A. Novel myosin heavy chain encoded by murine dilute coat colour locus. Nature. 1991 Feb 21;349(6311):709–713. doi: 10.1038/349709a0. [DOI] [PubMed] [Google Scholar]
  39. Mitchell E. J., Karn J., Brown D. M., Newman A., Jakes R., Kendrick-Jones J. Regulatory and essential light-chain-binding sites in myosin heavy chain subfragment-1 mapped by site-directed mutagenesis. J Mol Biol. 1989 Jul 5;208(1):199–205. doi: 10.1016/0022-2836(89)90096-x. [DOI] [PubMed] [Google Scholar]
  40. Montell C., Rubin G. M. The Drosophila ninaC locus encodes two photoreceptor cell specific proteins with domains homologous to protein kinases and the myosin heavy chain head. Cell. 1988 Mar 11;52(5):757–772. doi: 10.1016/0092-8674(88)90413-8. [DOI] [PubMed] [Google Scholar]
  41. Moore K. J., Swing D. A., Copeland N. G., Jenkins N. A. Interaction of the murine dilute suppressor gene (dsu) with fourteen coat color mutations. Genetics. 1990 Jun;125(2):421–430. doi: 10.1093/genetics/125.2.421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Nyitray L., Goodwin E. B., Szent-Györgyi A. G. Complete primary structure of a scallop striated muscle myosin heavy chain. Sequence comparison with other heavy chains reveals regions that might be critical for regulation. J Biol Chem. 1991 Oct 5;266(28):18469–18476. [PubMed] [Google Scholar]
  43. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Pollard T. D., Doberstein S. K., Zot H. G. Myosin-I. Annu Rev Physiol. 1991;53:653–681. doi: 10.1146/annurev.ph.53.030191.003253. [DOI] [PubMed] [Google Scholar]
  45. Rechsteiner M. PEST sequences are signals for rapid intracellular proteolysis. Semin Cell Biol. 1990 Dec;1(6):433–440. [PubMed] [Google Scholar]
  46. Saitou N., Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987 Jul;4(4):406–425. doi: 10.1093/oxfordjournals.molbev.a040454. [DOI] [PubMed] [Google Scholar]
  47. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Settleman J., Narasimhan V., Foster L. C., Weinberg R. A. Molecular cloning of cDNAs encoding the GAP-associated protein p190: implications for a signaling pathway from ras to the nucleus. Cell. 1992 May 1;69(3):539–549. doi: 10.1016/0092-8674(92)90454-k. [DOI] [PubMed] [Google Scholar]
  49. Swanljung-Collins H., Collins J. H. Ca2+ stimulates the Mg2(+)-ATPase activity of brush border myosin I with three or four calmodulin light chains but inhibits with less than two bound. J Biol Chem. 1991 Jan 15;266(2):1312–1319. [PubMed] [Google Scholar]
  50. Swanljung-Collins H., Collins J. H. Phosphorylation of brush border myosin I by protein kinase C is regulated by Ca(2+)-stimulated binding of myosin I to phosphatidylserine concerted with calmodulin dissociation. J Biol Chem. 1992 Feb 15;267(5):3445–3454. [PubMed] [Google Scholar]
  51. 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]
  52. Tuszynski G. P., Knight L., Piperno J. R., Walsh P. N. A rapid method for removal of [125I]iodide following iodination of protein solutions. Anal Biochem. 1980 Jul 15;106(1):118–122. doi: 10.1016/0003-2697(80)90126-8. [DOI] [PubMed] [Google Scholar]
  53. 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]

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

RESOURCES