Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1994 Jul 2;126(2):375–389. doi: 10.1083/jcb.126.2.375

Rat myr 4 defines a novel subclass of myosin I: identification, distribution, localization, and mapping of calmodulin-binding sites with differential calcium sensitivity

PMCID: PMC2200021  PMID: 8034741

Abstract

We report the identification and characterization of myr 4 (myosin from rat), the first mammalian myosin I that is not closely related to brush border myosin I. Myr 4 contains a myosin head (motor) domain, a regulatory domain with light chain binding sites and a tail domain. Sequence analysis of myosin I head (motor) domains suggested that myr 4 defines a novel subclass of myosin I's. This subclass is clearly different from the vertebrate brush border myosin I subclass (which includes myr 1) and the myosin I subclass(es) identified from Acanthamoeba castellanii and Dictyostelium discoideum. In accordance with this notion, a detailed sequence analysis of all myosin I tail domains revealed that the myr 4 tail is unique, except for a newly identified myosin I tail homology motif detected in all myosin I tail sequences. The Ca(2+)-binding protein calmodulin was demonstrated to be associated with myr 4. Calmodulin binding activity of myr 4 was mapped by gel overlay assays to the two consecutive light chain binding motifs (IQ motifs) present in the regulatory domain. These two binding sites differed in their Ca2+ requirements for optimal calmodulin binding. The NH2-terminal IQ motif bound calmodulin in the absence of free Ca2+, whereas the COOH-terminal IQ motif bound calmodulin in the presence of free Ca2+. A further Ca(2+)-dependent calmodulin binding site was mapped to amino acids 776-874 in the myr 4 tail domain. These results demonstrate a differential Ca2+ sensitivity for calmodulin binding by IQ motifs, and they suggest that myr 4 activity might be regulated by Ca2+/calmodulin. Myr 4 was demonstrated to be expressed in many cell lines and rat tissues with the highest level of expression in adult brain tissue. Its expression was developmentally regulated during rat brain ontogeny, rising 2-3 wk postnatally, and being maximal in adult brain. Immunofluorescence localization demonstrated that myr 4 is expressed in subpopulations of neurons. In these neurons, prominent punctate staining was detected in cell bodies and apical dendrites. A punctate staining that did not obviously colocalize with the bulk of F- actin was also observed in C6 rat glioma cells. The observed punctate staining for myr 4 is reminiscent of a membranous localization.

Full Text

The Full Text of this article is available as a PDF (3.5 MB).

Selected References

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

  1. Adams R. J., Pollard T. D. Binding of myosin I to membrane lipids. Nature. 1989 Aug 17;340(6234):565–568. doi: 10.1038/340565a0. [DOI] [PubMed] [Google Scholar]
  2. Baines I. C., Brzeska H., Korn E. D. Differential localization of Acanthamoeba myosin I isoforms. J Cell Biol. 1992 Dec;119(5):1193–1203. doi: 10.1083/jcb.119.5.1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baines I. C., Korn E. D. Localization of myosin IC and myosin II in Acanthamoeba castellanii by indirect immunofluorescence and immunogold electron microscopy. J Cell Biol. 1990 Nov;111(5 Pt 1):1895–1904. doi: 10.1083/jcb.111.5.1895. [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. 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]
  6. 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]
  7. Cheney R. E., Riley M. A., Mooseker M. S. Phylogenetic analysis of the myosin superfamily. Cell Motil Cytoskeleton. 1993;24(4):215–223. doi: 10.1002/cm.970240402. [DOI] [PubMed] [Google Scholar]
  8. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [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. 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]
  11. Coluccio L. M., Bretscher A. Reassociation of microvillar core proteins: making a microvillar core in vitro. J Cell Biol. 1989 Feb;108(2):495–502. doi: 10.1083/jcb.108.2.495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Coluccio L. M., Conaty C. Myosin-I in mammalian liver. Cell Motil Cytoskeleton. 1993;24(3):189–199. doi: 10.1002/cm.970240306. [DOI] [PubMed] [Google Scholar]
  13. Conrad P. A., Giuliano K. A., Fisher G., Collins K., Matsudaira P. T., Taylor D. L. Relative distribution of actin, myosin I, and myosin II during the wound healing response of fibroblasts. J Cell Biol. 1993 Mar;120(6):1381–1391. doi: 10.1083/jcb.120.6.1381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. De Lozanne A., Spudich J. A. Disruption of the Dictyostelium myosin heavy chain gene by homologous recombination. Science. 1987 May 29;236(4805):1086–1091. doi: 10.1126/science.3576222. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Doberstein S. K., Pollard T. D. Localization and specificity of the phospholipid and actin binding sites on the tail of Acanthamoeba myosin IC. J Cell Biol. 1992 Jun;117(6):1241–1249. doi: 10.1083/jcb.117.6.1241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Drenckhahn D., Dermietzel R. Organization of the actin filament cytoskeleton in the intestinal brush border: a quantitative and qualitative immunoelectron microscope study. J Cell Biol. 1988 Sep;107(3):1037–1048. doi: 10.1083/jcb.107.3.1037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Eshel Y., Shai Y., Vorherr T., Carafoli E., Salomon Y. Synthetic peptides corresponding to the calmodulin-binding domains of skeletal muscle myosin light chain kinase and human erythrocyte Ca2+ pump interact with and permeabilize liposomes and cell membranes. Biochemistry. 1993 Jul 6;32(26):6721–6728. doi: 10.1021/bi00077a027. [DOI] [PubMed] [Google Scholar]
  20. Fath K. R., Burgess D. R. Golgi-derived vesicles from developing epithelial cells bind actin filaments and possess myosin-I as a cytoplasmically oriented peripheral membrane protein. J Cell Biol. 1993 Jan;120(1):117–127. doi: 10.1083/jcb.120.1.117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Filoteo A. G., Enyedi A., Penniston J. T. The lipid-binding peptide from the plasma membrane Ca2+ pump binds calmodulin, and the primary calmodulin-binding domain interacts with lipid. J Biol Chem. 1992 Jun 15;267(17):11800–11805. [PubMed] [Google Scholar]
  22. Fukui Y., Lynch T. J., Brzeska H., Korn E. D. Myosin I is located at the leading edges of locomoting Dictyostelium amoebae. Nature. 1989 Sep 28;341(6240):328–331. doi: 10.1038/341328a0. [DOI] [PubMed] [Google Scholar]
  23. Gadasi H., Korn E. D. Evidence for differential intracellular localization of the Acanthamoeba myosin isoenzymes. Nature. 1980 Jul 31;286(5772):452–456. doi: 10.1038/286452a0. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Glenney J. R., Jr, Weber K. Calmodulin-binding proteins of the microfilaments present in isolated brush borders and microvilli of intestinal epithelial cells. J Biol Chem. 1980 Nov 25;255(22):10551–10554. [PubMed] [Google Scholar]
  26. Goodson H. V., Spudich J. A. Molecular evolution of the myosin family: relationships derived from comparisons of amino acid sequences. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):659–663. doi: 10.1073/pnas.90.2.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Green N., Alexander H., Olson A., Alexander S., Shinnick T. M., Sutcliffe J. G., Lerner R. A. Immunogenic structure of the influenza virus hemagglutinin. Cell. 1982 Mar;28(3):477–487. doi: 10.1016/0092-8674(82)90202-1. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Henikoff S. Unidirectional digestion with exonuclease III in DNA sequence analysis. Methods Enzymol. 1987;155:156–165. doi: 10.1016/0076-6879(87)55014-5. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. Huxley H. E. The mechanism of muscular contraction. Science. 1969 Jun 20;164(3886):1356–1365. doi: 10.1126/science.164.3886.1356. [DOI] [PubMed] [Google Scholar]
  34. Jung G., Fukui Y., Martin B., Hammer J. A., 3rd Sequence, expression pattern, intracellular localization, and targeted disruption of the Dictyostelium myosin ID heavy chain isoform. J Biol Chem. 1993 Jul 15;268(20):14981–14990. [PubMed] [Google Scholar]
  35. Jung G., Korn E. D., Hammer J. A., 3rd The heavy chain of Acanthamoeba myosin IB is a fusion of myosin-like and non-myosin-like sequences. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6720–6724. doi: 10.1073/pnas.84.19.6720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Jung G., Saxe C. L., 3rd, Kimmel A. R., Hammer J. A., 3rd Dictyostelium discoideum contains a gene encoding a myosin I heavy chain. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6186–6190. doi: 10.1073/pnas.86.16.6186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Jung G., Schmidt C. J., Hammer J. A., 3rd Myosin I heavy-chain genes of Acanthamoeba castellanii: cloning of a second gene and evidence for the existence of a third isoform. Gene. 1989 Oct 30;82(2):269–280. doi: 10.1016/0378-1119(89)90052-8. [DOI] [PubMed] [Google Scholar]
  38. Knecht D. A., Loomis W. F. Antisense RNA inactivation of myosin heavy chain gene expression in Dictyostelium discoideum. Science. 1987 May 29;236(4805):1081–1086. doi: 10.1126/science.3576221. [DOI] [PubMed] [Google Scholar]
  39. Koslovsky J. S., Qian C., Jiang X., Mercer J. A. Molecular cloning of a mouse myosin I expressed in brain. FEBS Lett. 1993 Apr 5;320(2):121–124. doi: 10.1016/0014-5793(93)80075-6. [DOI] [PubMed] [Google Scholar]
  40. Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987 Oct 26;15(20):8125–8148. doi: 10.1093/nar/15.20.8125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Lee C., Levin A., Branton D. Copper staining: a five-minute protein stain for sodium dodecyl sulfate-polyacrylamide gels. Anal Biochem. 1987 Nov 1;166(2):308–312. doi: 10.1016/0003-2697(87)90579-3. [DOI] [PubMed] [Google Scholar]
  42. Li D., Chantler P. D. Evidence for a new member of the myosin I family from mammalian brain. J Neurochem. 1992 Oct;59(4):1344–1351. doi: 10.1111/j.1471-4159.1992.tb08446.x. [DOI] [PubMed] [Google Scholar]
  43. Lynch T. J., Albanesi J. P., Korn E. D., Robinson E. A., Bowers B., Fujisaki H. ATPase activities and actin-binding properties of subfragments of Acanthamoeba myosin IA. J Biol Chem. 1986 Dec 25;261(36):17156–17162. [PubMed] [Google Scholar]
  44. Lynch T. J., Brzeska H., Miyata H., Korn E. D. Purification and characterization of a third isoform of myosin I from Acanthamoeba castellanii. J Biol Chem. 1989 Nov 15;264(32):19333–19339. [PubMed] [Google Scholar]
  45. Matsudaira P. T., Burgess D. R. Identification and organization of the components in the isolated microvillus cytoskeleton. J Cell Biol. 1979 Dec;83(3):667–673. doi: 10.1083/jcb.83.3.667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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]
  47. Miyata H., Bowers B., Korn E. D. Plasma membrane association of Acanthamoeba myosin I. J Cell Biol. 1989 Oct;109(4 Pt 1):1519–1528. doi: 10.1083/jcb.109.4.1519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Pardee J. D., Spudich J. A. Purification of muscle actin. Methods Cell Biol. 1982;24:271–289. doi: 10.1016/s0091-679x(08)60661-5. [DOI] [PubMed] [Google Scholar]
  49. Pasternak C., Spudich J. A., Elson E. L. Capping of surface receptors and concomitant cortical tension are generated by conventional myosin. Nature. 1989 Oct 12;341(6242):549–551. doi: 10.1038/341549a0. [DOI] [PubMed] [Google Scholar]
  50. 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]
  51. Pollard T. D., Korn E. D. Acanthamoeba myosin. I. Isolation from Acanthamoeba castellanii of an enzyme similar to muscle myosin. J Biol Chem. 1973 Jul 10;248(13):4682–4690. [PubMed] [Google Scholar]
  52. Porter J. A., Yu M., Doberstein S. K., Pollard T. D., Montell C. Dependence of calmodulin localization in the retina on the NINAC unconventional myosin. Science. 1993 Nov 12;262(5136):1038–1042. doi: 10.1126/science.8235618. [DOI] [PubMed] [Google Scholar]
  53. Ruppert C., Kroschewski R., Bähler M. Identification, characterization and cloning of myr 1, a mammalian myosin-I. J Cell Biol. 1993 Mar;120(6):1393–1403. doi: 10.1083/jcb.120.6.1393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. 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]
  55. Sherr E. H., Joyce M. P., Greene L. A. Mammalian myosin I alpha, I beta, and I gamma: new widely expressed genes of the myosin I family. J Cell Biol. 1993 Mar;120(6):1405–1416. doi: 10.1083/jcb.120.6.1405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Slaughter G. R., Means A. R. Use of the 125I-labeled protein gel overlay technique to study calmodulin-binding proteins. Methods Enzymol. 1987;139:433–444. doi: 10.1016/0076-6879(87)39104-9. [DOI] [PubMed] [Google Scholar]
  57. 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]
  58. Titus M. A., Warrick H. M., Spudich J. A. Multiple actin-based motor genes in Dictyostelium. Cell Regul. 1989 Nov;1(1):55–63. doi: 10.1091/mbc.1.1.55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Urrutia R. A., Jung G., Hammer J. A., 3rd The Dictyostelium myosin IE heavy chain gene encodes a truncated isoform that lacks sequences corresponding to the actin binding site in the tail. Biochim Biophys Acta. 1993 May 28;1173(2):225–229. doi: 10.1016/0167-4781(93)90185-g. [DOI] [PubMed] [Google Scholar]
  60. Wagner M. C., Barylko B., Albanesi J. P. Tissue distribution and subcellular localization of mammalian myosin I. J Cell Biol. 1992 Oct;119(1):163–170. doi: 10.1083/jcb.119.1.163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Yonemura S., Pollard T. D. The localization of myosin I and myosin II in Acanthamoeba by fluorescence microscopy. J Cell Sci. 1992 Jul;102(Pt 3):629–642. doi: 10.1242/jcs.102.3.629. [DOI] [PubMed] [Google Scholar]
  62. Zozulya S., Stryer L. Calcium-myristoyl protein switch. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11569–11573. doi: 10.1073/pnas.89.23.11569. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES