scholarly journals Molecular dissection of radixin: distinct and interdependent functions of the amino- and carboxy-terminal domains.

1995 ◽  
Vol 129 (4) ◽  
pp. 1007-1022 ◽  
Author(s):  
M D Henry ◽  
C Gonzalez Agosti ◽  
F Solomon
Keyword(s):  
Full Length ◽  
Detectable Effect ◽  
Limiting Factor ◽  
Cell Physiology ◽  
Erm Proteins ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Terminal Domains

The ERM proteins--ezrin, radixin, and moesin--occur in particular cortical cytoskeletal structures. Several lines of evidence suggest that they interact with both cytoskeletal elements and plasma membrane components. Here we described the properties of full-length and truncated radixin polypeptides expressed in transfected cells. In stable transfectants, exogenous full-length radixin behaves much like endogenous ERM proteins, localizing to the same cortical structures. However, the presence of full-length radixin or its carboxy-terminal domain in cortical structures correlates with greatly diminished staining of endogenous moesin in those structures, suggesting that radixin and moesin compete for a limiting factor required for normal associations in the cell. The results also reveal distinct roles for the amino- and carboxy-terminal domains. At low levels relative to endogenous radixin, the carboxy-terminal polypeptide is associated with most of the correct cortical targets except cleavage furrows. In contrast, the amino-terminal polypeptide is diffusely localized throughout the cell. Low level expression of full-length radixin or either of the truncated polypeptides has no detectable effect on cell physiology. However, high level expression of the carboxy-terminal domain dramatically disrupts normal cytoskeletal structures and functions. At these high levels, the amino-terminal polypeptide does localize to cortical structures, but does not affect the cells. We conclude that the behavior of radixin in cells depends upon activities contributed by separate domains of the protein, but also requires modulating interactions between those domains.

Disruption of dynamic cell surface architecture of NIH3T3 fibroblasts by the N-terminal domains of moesin and ezrin: in vivo imaging with GFP fusion proteins

1999 ◽  
Vol 112 (1) ◽  
pp. 111-125 ◽  
Author(s):  
M.R. Amieva ◽  
P. Litman ◽  
L. Huang ◽  
E. Ichimaru ◽  
H. Furthmayr
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Full Length ◽  
Cell Behavior ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal

Lamellipodia, filopodia, microspikes and retraction fibers are characteristic features of a dynamic and continuously changing cell surface architecture and moesin, ezrin and radixin are thought to function in these microextensions as reversible links between plasma membrane proteins and actin microfilaments. Full-length and truncated domains of the three proteins were fused to green fluorescent protein (GFP), expressed in NIH3T3 cells, and distribution and behaviour of cells were analysed by using digitally enhanced differential interference contrast (DIC) and fluorescence video microscopy. The amino-terminal (N-)domains of all three proteins localize to the plasma membrane and fluorescence recordings parallel the dynamic changes in cell surface morphology observed by DIC microscopy of cultured cells. Expression of this domain, however, significantly affects cell surface architecture by the formation of abnormally long and fragile filopodia that poorly attach and retract abnormally. Even more striking are abundant irregular, branched and motionless membraneous structures that accumulate during retraction of lamellipodia. These are devoid of actin, endogenous moesin, ezrin and radixin, but contain the GFP-labeled domain. While a large proportion of endogenous proteins can be extracted with non-ionic detergents as in untransfected control cells, >90% of N-moesin and >60% of N-ezrin and N-radixin remain insoluble. The minimal size of the domain of moesin required for membrane localization and change in behavior includes residues 1–320. Deletions of amino acid residues from either end result in diffuse intracellular distribution, but also in normal cell behavior. Expression of GFP-fusions of full-length moesin or its carboxy-terminal domain has no effect on cell behavior during the observation period of 6–8 hours. The data suggest that, in the absence of the carboxy-terminal domain, N-moesin, -ezrin and -radixin interact tightly with the plasma membrane and interfere with normal functions of endogeneous proteins mainly during retraction.

The Amino-Terminal 1−185 Domain of ApoE Promotes the Clearance of Lipoprotein Remnants in Vivo.The Carboxy-Terminal Domain Is Required for Induction of Hyperlipidemia in Normal and ApoE-Deficient Mice†

Biochemistry ◽  
2001 ◽  
Vol 40 (20) ◽  
pp. 6027-6035 ◽  
Author(s):  
Kyriakos E. Kypreos ◽  
Pamela Morani ◽  
Ko Willems van Dijk ◽  
Louis M. Havekes ◽  
Vassilis I. Zannis
Keyword(s):  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Deficient Mice

scholarly journals A Major Determinant for Membrane Protein Interaction Localizes to the Carboxy-Terminal Domain of the Mouse Coronavirus Nucleocapsid Protein

2005 ◽  
Vol 79 (21) ◽  
pp. 13285-13297 ◽  
Author(s):  
Kelley R. Hurst ◽  
Lili Kuo ◽  
Cheri A. Koetzner ◽  
Rong Ye ◽  
Bilan Hsue ◽  
...  
Keyword(s):  
Viral Envelope ◽  
M Protein ◽  
N Protein ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Dominant Negative Mutation ◽  
Carboxy Terminal Domain ◽  
Domain 3 ◽  
Positive Strand Rna ◽  
Carboxy Terminal

ABSTRACT The two major constituents of coronavirus virions are the membrane (M) and nucleocapsid (N) proteins. The M protein is anchored in the viral envelope by three transmembrane segments flanked by a short amino-terminal ectodomain and a large carboxy-terminal endodomain. The M endodomain interacts with the viral nucleocapsid, which consists of the positive-strand RNA genome helically encapsidated by N protein monomers. In previous work with the coronavirus mouse hepatitis virus (MHV), a highly defective M protein mutant, MΔ2, was constructed. This mutant contained a 2-amino-acid carboxy-terminal truncation of the M protein. Analysis of second-site revertants of MΔ2 revealed mutations in the carboxy-terminal region of the N protein that compensated for the defect in the M protein. To seek further genetic evidence corroborating this interaction, we generated a comprehensive set of clustered charged-to-alanine mutants in the carboxy-terminal domain 3 of N protein. One of these mutants, CCA4, had a highly defective phenotype similar to that of MΔ2. Transfer of the CCA4 mutation into a partially diploid MHV genome showed that CCA4 was a loss-of-function mutation rather than a dominant-negative mutation. Analysis of multiple second-site revertants of CCA4 revealed mutations in both the M protein and the N protein that could compensate for the original lesion in N. These data more precisely define the region of the N protein that interacts with the M protein. Further, we found that fusion of domain 3 of the N protein to the carboxy terminus of a heterologous protein caused it to be incorporated into MHV virions.

scholarly journals 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.

1989 ◽  
Vol 109 (6) ◽  
pp. 2895-2903 ◽  
Author(s):  
A Garcia ◽  
E Coudrier ◽  
J Carboni ◽  
J Anderson ◽  
J Vandekerkhove ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Heavy Chain ◽  
Brush Border ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Head Domain ◽  
Myosin I ◽  
Chicken Tissue ◽  
Carboxy Terminal

The actin bundle within each microvillus of the intestinal brush border is laterally tethered to the membrane by bridges composed of the protein complex, 110-kD-calmodulin. Previous studies have shown that avian 110-kD-calmodulin shares many properties with myosins including mechanochemical activity. In the present study, a cDNA molecule encoding 1,000 amino acids of the 110-kD protein has been sequenced, providing direct evidence that this protein is a vertebrate homologue of the tail-less, single-headed myosin I first described in amoeboid cells. The primary structure of the 110-kD protein (or brush border myosin I heavy chain) consists of two domains, an amino-terminal "head" domain and a 35-kD carboxy-terminal "tail" domain. The head domain is homologous to the S1 domain of other known myosins, with highest homology observed between that of Acanthamoeba myosin IB and the S1 domain of the protein encoded by bovine myosin I heavy chain gene (MIHC; Hoshimaru, M., and S. Nakanishi. 1987. J. Biol. Chem. 262:14625-14632). The carboxy-terminal domain shows no significant homology with any other known myosins except that of the bovine MIHC. This demonstrates that the bovine MIHC gene most probably encodes the heavy chain of bovine brush border myosin I (BBMI). A bacterially expressed fusion protein encoded by the brush border 110-kD cDNA binds calmodulin. Proteolytic removal of the carboxy-terminal domain of the fusion protein results in loss of calmodulin binding activity, a result consistent with previous studies on the domain structure of the 110-kD protein. No hydrophobic sequence is present in the molecule indicating that chicken BBMI heavy chain is probably not an integral membrane protein. Northern blot analysis of various chicken tissue indicates that BBMI heavy chain is preferentially expressed in the intestine.

scholarly journals THE STRUCTURAL BASIS FOR BINDING OF COMPLEMENT BY IMMUNOGLOBULIN M

1974 ◽  
Vol 140 (4) ◽  
pp. 1117-1121 ◽  
Author(s):  
Mary M. Hurst ◽  
John E. Volanakis ◽  
Raymond B. Hester ◽  
Robert M. Stroud ◽  
J. Claude Bennett
Keyword(s):  
Amino Acid ◽  
Structural Features ◽  
Immunoglobulin M ◽  
Structural Basis ◽  
Amino Acid Residues ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Insight Into

An insight into the structural features of human IgM that are responsible for its capacity to bind the first component of complement (C) has been obtained by examining the ability of IgM subfragments to bind active C1 (C1). The smallest two fragments found to bind C1 were the major CNBr fragment of the Fc portion of IgM and the CH4 fragment of the carboxy-terminal domain. The smallest fragment which fixes C1 has a disaggregated mol wt of 6,800, consists of 60 residues, and contains no carbohydrate. Structural considerations and sequence overlaps suggest that the amino-terminal side of the CH4 domain (24 amino acid residues) might be responsible for fixing C1.

scholarly journals Ezrin contains cytoskeleton and membrane binding domains accounting for its proposed role as a membrane-cytoskeletal linker.

1993 ◽  
Vol 120 (1) ◽  
pp. 129-139 ◽  
Author(s):  
M Algrain ◽  
O Turunen ◽  
A Vaheri ◽  
D Louvard ◽  
M Arpin
Keyword(s):  
Plasma Membrane ◽  
Tyrosine Kinases ◽  
Actin Filaments ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Binding Domains ◽  
Carboxy Terminal Domain ◽  
Cell Surface Structures ◽  
Carboxy Terminal ◽  
Amino Terminal Domain

Ezrin, a widespread protein present in actin-containing cell-surface structures, is a substrate of some protein tyrosine kinases. Based on its primary and secondary structure similarities with talin and band 4.1 it has been suggested that this protein could play a role in linking the cytoskeleton to the plasma membrane (Gould, K.L., A. Bretscher, F.S. Esch, and T. Hunter. 1989. EMBO (Eur. Mol. Biol. Organ.), J. 8:4133-4142; Turunen, O., R. Winqvist, R. Pakkanen, K.-H. Grzeschik, T. Wahlström, and A. Vaheri. 1989. J. Biol. Chem. 264:16727-16732). To test this hypothesis, we transiently expressed the complete human ezrin cDNA, or truncated cDNAs encoding the amino- and carboxy-terminal domains of the protein, in CV-1 cells. Protein epitope tagging was used to unambiguously determine the subcellular distribution of the protein encoded by the transfected cDNA. We show that this protein is concentrated underneath the dorsal plasma membrane in all actin-containing structures and is partially detergent insoluble. The amino-terminal domain displays the same localization but is readily extractable by nonionic detergent. The carboxy-terminal domain colocalizes with microvillar actin filaments as well as with stress fibers and remains associated with actin filaments after detergent extraction, and with disorganized actin structures after cytochalasin D treatment. Our results clearly demonstrate that ezrin interacts with membrane-associated components via its amino-terminal domain, and with the cytoskeleton via its carboxy-terminal domain. The amino-terminal domain could include the main determinant that restricts the entire protein to the cortical cytoskeleton in contact with the dorsal plasma membrane and its specialized microdomains such as microvilli, microspikes and lamellipodia.

scholarly journals The Carboxy-Terminal Domain of Hsc70 Provides Binding Sites for a Distinct Set of Chaperone Cofactors

1998 ◽  
Vol 18 (4) ◽  
pp. 2023-2028 ◽  
Author(s):  
Jens Demand ◽  
Jens Lüders ◽  
Jörg Höhfeld
Keyword(s):  
Binding Sites ◽  
Mammalian Cells ◽  
Chaperone Activity ◽  
Structural Basis ◽  
Atpase Domain ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Chaperone Protein ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal

ABSTRACT The modulation of the chaperone activity of the heat shock cognate Hsc70 protein in mammalian cells involves cooperation with chaperone cofactors, such as Hsp40; BAG-1; the Hsc70-interacting protein, Hip; and the Hsc70-Hsp90-organizing protein, Hop. By employing the yeast two-hybrid system and in vitro interaction assays, we have provided insight into the structural basis that underlies Hsc70’s cooperation with different cofactors. The carboxy-terminal domain of Hsc70, previously shown to form a lid over the peptide binding pocket of the chaperone protein, mediates the interaction of Hsc70 with Hsp40 and Hop. Remarkably, the two cofactors bind to the carboxy terminus of Hsc70 in a noncompetitive manner, revealing the existence of distinct binding sites for Hsp40 and Hop within this domain. In contrast, Hip interacts exclusively with the amino-terminal ATPase domain of Hsc70. Hence, Hsc70 possesses separate nonoverlapping binding sites for Hsp40, Hip, and Hop. This appears to enable the chaperone protein to cooperate simultaneously with multiple cofactors. On the other hand, BAG-1 and Hip have recently been shown to compete in binding to the ATPase domain. Our data thus establish the existence of a network of cooperating and competing cofactors regulating the chaperone activity of Hsc70 in the mammalian cell.

scholarly journals Residues near the Amino Terminus of Rns Are Essential for Positive Autoregulation and DNA Binding

2008 ◽  
Vol 190 (7) ◽  
pp. 2279-2285 ◽  
Author(s):  
Georgeta N. Basturea ◽  
Maria D. Bodero ◽  
Mario E. Moreno ◽  
George P. Munson
Keyword(s):  
Dna Binding ◽  
Amino Terminus ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Amino Terminal Domain ◽  
Regulated Promoters

ABSTRACT Most members of the AraC/XylS family contain a conserved carboxy-terminal DNA binding domain and a less conserved amino-terminal domain involved in binding small-molecule effectors and dimerization. However, there is no evidence that Rns, a regulator of enterotoxigenic Escherichia coli virulence genes, responds to an effector ligand, and in this study we found that the amino-terminal domain of Rns does not form homodimers in vivo. Exposure of Rns to the chemical cross-linker glutaraldehyde revealed that the full-length protein is also a monomer in vitro. Nevertheless, deletion analysis of Rns demonstrated that the first 60 amino acids of the protein are essential for the activation and repression of Rns-regulated promoters in vivo. Amino-terminal truncation of Rns abolished DNA binding in vitro, and two randomly generated mutations, I14T and N16D, that independently abolished Rns autoregulation were isolated. Further analysis of these mutations revealed that they have disparate effects at other Rns-regulated promoters and suggest that they may be involved in an interaction with the carboxy-terminal domain of Rns. Thus, evolution may have preserved the amino terminus of Rns because it is essential for the regulator's activity even though it apparently lacks the two functions, dimerization and ligand binding, usually associated with the amino-terminal domains of AraC/XylS family members.

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