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

1994 ◽  
Vol 126 (2) ◽  
pp. 375-389 ◽  
Author(s):  
M Bähler ◽  
R Kroschewski ◽  
H E Stöffler ◽  
T Behrmann
Keyword(s):  
Sequence Analysis ◽  
Light Chain ◽  
Binding Sites ◽  
Adult Brain ◽  
Regulatory Domain ◽  
Tail Domain ◽  
Iq Motif ◽  
Calmodulin Binding ◽  
Myosin I ◽  
Punctate Staining

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.

scholarly journals Identification, characterization and cloning of myr 1, a mammalian myosin-I.

1993 ◽  
Vol 120 (6) ◽  
pp. 1393-1403 ◽  
Author(s):  
C Ruppert ◽  
R Kroschewski ◽  
M Bähler
Keyword(s):  
Amino Acid ◽  
Light Chain ◽  
Brush Border ◽  
Binding Sites ◽  
Neuronal Development ◽  
Amino Acid Sequences ◽  
Dependent Manner ◽  
Tail Domain ◽  
Myosin I ◽  
Splice Forms

We have identified, characterized and cloned a novel mammalian myosin-I motor-molecule, called myr 1 (myosin-I from rat). Myr 1 exists in three alternative splice forms: myr 1a, myr 1b, and myr 1c. These splice forms differ in their numbers of putative calmodulin/light chain binding sites. Myr 1a-c were selectively released by ATP, bound in a nucleotide-dependent manner to F-actin and exhibited amino acid sequences characteristic of myosin-I motor domains. In addition to the motor domain, they contained a regulatory domain with up to six putative calmodulin/light chain binding sites and a tail domain. The tail domain exhibited 47% amino acid sequence identity to the brush border myosin-I tail domain, demonstrating that myr 1 is related to the only other mammalian myosin-I motor molecule that has been characterized so far. In contrast to brush border myosin-I which is expressed in mature enterocytes, myr 1 splice forms were differentially expressed in all tested tissues. Therefore, myr 1 is the first mammalian myosin-I motor molecule with a widespread tissue distribution in neonatal and adult tissues. The myr 1a splice form was preferentially expressed in neuronal tissues. Its expression was developmentally regulated during rat forebrain ontogeny and subcellular fractionation revealed an enrichment in purified growth cone particles, data consistent with a role for myr 1a in neuronal development.

scholarly journals Structure, function, and regulation of myosin 1C.

2005 ◽  
Vol 52 (2) ◽  
pp. 373-380 ◽  
Author(s):  
Barbara Barylko ◽  
Gwanghyun Jung ◽  
Joseph P Albanesi
Keyword(s):  
Plasma Membrane ◽  
Binding Sites ◽  
Subcellular Location ◽  
Lipid Binding ◽  
Tail Domain ◽  
Anionic Lipid ◽  
Myosin I ◽  
Docking Proteins ◽  
Neck Domain ◽  
Anionic Lipids

Myosin 1C, the first mammalian single-headed myosin to be purified, cloned, and sequenced, has been implicated in the translocation of plasma membrane channels and transporters. Like other forms of myosin I (of which eight exist in humans) myosin 1C consists of motor, neck, and tail domains. The neck domain binds calmodulins more tightly in the absence than in the presence of Ca(2+). Release of calmodulins exposes binding sites for anionic lipids, particularly phosphoinositides. The tail domain, which has an isoelectic point of 10.5, interacts with anionic lipid headgroups. When both neck and tail lipid binding sites are engaged, the myosin associates essentially irreversibly with membranes. Despite this tight membrane binding, it is widely believed that myosin 1C docking proteins are necessary for targeting the enzyme to specific subcellular location. The search for these putative myosin 1C receptors is an active area of research.

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

1992 ◽  
Vol 119 (6) ◽  
pp. 1541-1557 ◽  
Author(s):  
E M Espreafico ◽  
R E Cheney ◽  
M Matteoli ◽  
A A Nascimento ◽  
P V De Camilli ◽  
...  
Keyword(s):  
Amino Acid ◽  
Primary Structure ◽  
Binding Sites ◽  
Alpha Helix ◽  
Globular Domain ◽  
Tail Domain ◽  
Myosin V ◽  
Structural Class ◽  
Chicken Brain ◽  
Iq Motif

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)

scholarly journals IQ-motif selectivity in human IQGAP2 and IQGAP3: binding of calmodulin and myosin essential light chain

2011 ◽  
Vol 31 (5) ◽  
pp. 371-379 ◽  
Author(s):  
Erwan Atcheson ◽  
Elaine Hamilton ◽  
Sevvel Pathmanathan ◽  
Brett Greer ◽  
Pat Harriott ◽  
...  
Keyword(s):  
Light Chain ◽  
Binding Sites ◽  
Binding Properties ◽  
Iq Motif ◽  
Binding Partner ◽  
Essential Light Chain ◽  
Calcium Sensing ◽  
Eukaryotic Proteins ◽  
Transient Interactions ◽  
Related Proteins

The IQGAP [IQ-motif-containing GAP (GTPase-activating protein)] family members are eukaryotic proteins that act at the interface between cellular signalling and the cytoskeleton. As such they collect numerous inputs from a variety of signalling pathways. A key binding partner is the calcium-sensing protein CaM (calmodulin). This protein binds mainly through a series of IQ-motifs which are located towards the middle of the primary sequence of the IQGAPs. In some IQGAPs, these motifs also provide binding sites for CaM-like proteins such as myosin essential light chain and S100B. Using synthetic peptides and native gel electrophoresis, the binding properties of the IQ-motifs from human IQGAP2 and IQGAP3 have been mapped. The second and third IQ-motifs in IQGAP2 and all four of the IQ-motifs of IQGAP3 interacted with CaM in the presence of calcium ions. However, there were differences in the type of interaction: while some IQ-motifs were able to form complexes with CaM which were stable under the conditions of the experiment, others formed more transient interactions. The first IQ-motifs from IQGAP2 and IQGAP3 formed transient interactions with CaM in the absence of calcium and the first motif from IQGAP3 formed a transient interaction with the myosin essential light chain Mlc1sa. None of these IQ-motifs interacted with S100B. Molecular modelling suggested that all of the IQ-motifs, except the first one from IQGAP2 formed α-helices in solution. These results extend our knowledge of the selectivity of IQ-motifs for CaM and related proteins.

scholarly journals A novel mammalian myosin I from rat with an SH3 domain localizes to Con A-inducible, F-actin-rich structures at cell-cell contacts.

1995 ◽  
Vol 129 (3) ◽  
pp. 819-830 ◽  
Author(s):  
H E Stöffler ◽  
C Ruppert ◽  
J Reinhard ◽  
M Bähler
Keyword(s):  
Light Chain ◽  
Sh3 Domain ◽  
Actin Binding ◽  
Cell Contact ◽  
Tail Domain ◽  
Con A ◽  
Cell Contacts ◽  
Head Domain ◽  
Myosin I ◽  
Cell Cell

In an effort to determine diversity and function of mammalian myosin I molecules, we report here the cloning and characterization of myr 3 (third unconventional myosin from rat), a novel mammalian myosin I from rat tissues that is related to myosin I molecules from protozoa. Like the protozoan myosin I molecules, myr 3 consists of a myosin head domain, a single light chain binding motif, and a tail region that includes a COOH-terminal SH3 domain. However, myr 3 lacks the regulatory phosphorylation site present in the head domain of protozoan myosin I molecules. Evidence was obtained that the COOH terminus of the tail domain is involved in regulating F-actin binding activity of the NH2-terminal head domain. The light chain of myr 3 was identified as the Ca(2+)-binding protein calmodulin. Northern blot and immunoblot analyses revealed that myr 3 is expressed in many tissues and cell lines. Immunofluorescence studies with anti-myr 3 antibodies in NRK cells demonstrated that myr 3 is localized in the cytoplasm and in elongated structures at regions of cell-cell contact. These elongated structures contained F-actin and alpha-actinin but were devoid of vinculin. Incubation of NRK cells with Con A stimulated the formation of myr 3-containing structures along cell-cell contacts. These results suggest for myr 3 a function mediated by cell-cell contact.

scholarly journals Localization of Antibody-Binding Sites by Sequence Analysis of Cloned Pilin Genes from Neisseria Gonorrhoeae

Microbiology ◽  
1987 ◽  
Vol 133 (4) ◽  
pp. 825-833 ◽  
Author(s):  
I. J. Nicolson ◽  
A. C. F. Perry ◽  
M. Virji ◽  
J. E. Heckels ◽  
J. R. Saunders
Keyword(s):  
Sequence Analysis ◽  
Neisseria Gonorrhoeae ◽  
Binding Sites ◽  
Antibody Binding

scholarly journals Isolation of the native form of chicken gizzard myosin light-chain kinase

1984 ◽  
Vol 218 (3) ◽  
pp. 863-870 ◽  
Author(s):  
P K Ngai ◽  
C A Carruthers ◽  
M P Walsh
Keyword(s):  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Binding Protein ◽  
Cross Reactivity ◽  
Native Form ◽  
Chicken Gizzard ◽  
Calmodulin Binding ◽  
Chymotryptic Peptide ◽  
Dependent Protein

A simple and rapid procedure for the purification of the native form of chicken gizzard myosin light-chain kinase (Mr 136000) is described which eliminates problems of proteolysis previously encountered. During this procedure, a calmodulin-binding protein of Mr 141000, which previously co-purified with the myosin light-chain kinase, is removed and shown to be a distinct protein on the basis of lack of kinase activity, different chymotryptic peptide maps, lack of cross-reactivity with a monoclonal antibody to turkey gizzard myosin light-chain kinase, and lack of phosphorylation by the purified catalytic subunit of cyclic AMP-dependent protein kinase. This Mr-141000 calmodulin-binding protein is identified as caldesmon on the basis of Ca2+-dependent interaction with calmodulin, subunit Mr, Ca2+-independent interaction with skeletal-muscle F-actin, Ca2+-dependent competition between calmodulin and F-actin for caldesmon, and tissue content.

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