scholarly journals Analysis of the interactions between the C-terminal cytoplasmic domains of KCNQ1 and KCNE1 channel subunits

2010 ◽  
Vol 428 (1) ◽  
pp. 75-84 ◽  
Author(s):  
Renjian Zheng ◽  
Keith Thompson ◽  
Edmond Obeng-Gyimah ◽  
Dana Alessi ◽  
Jerri Chen ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Proximal Region ◽  
Physical Interaction ◽  
Interaction Domain ◽  
Α Subunit ◽  
C Terminus ◽  
Tetramerization Domain ◽  
K Current ◽  
Membrane Spanning ◽  
Lqt Syndrome

Ion channel subunits encoded by KCNQ1 and KCNE1 produce the slowly activating K+ current (IKs) that plays a central role in myocardial repolarization. The KCNQ1 α-subunit and the KCNE1 β-subunit assemble with their membrane-spanning segments interacting, resulting in transformation of channel activation kinetics. We recently reported a functional interaction involving C-terminal portions of the two subunits with ensuing regulation of channel deactivation. In the present study, we provide evidence characterizing a physical interaction between the KCNQ1-CT (KCNE1 C-terminus) and the KCNE1-CT (KCNE1 C-terminus). When expressed in cultured cells, the KCNE1-CT co-localized with KCNQ1, co-immunoprecipitated with KCNQ1 and perturbed deactivation kinetics of the KCNQ1 currents. Purified KCNQ1-CT and KCNE1-CT physically interacted in pull-down experiments, indicating a direct association. Deletion analysis of KCNQ1-CT indicated that the KCNE1-CT binds to a KCNQ1 region just after the last transmembrane segment, but N-terminal to the tetramerization domain. SPR (surface plasmon resonance) corroborated the pull-down results, showing that the most proximal region (KCNQ1 amino acids 349–438) contributed most to the bimolecular interaction with a dissociation constant of ~4 μM. LQT (long QT) mutants of the KCNE1-CT, D76N and W87F, retained binding to the KCNQ1-CT with comparable affinity, indicating that these disease-causing mutations do not alter channel behaviour by disruption of the association. Several LQT mutations involving the KCNQ1-CT, however, showed various effects on KCNQ1/KCNE1 association. Our results indicate that the KCNQ1-CT and the KCNE1-CT comprise an independent interaction domain that may play a role in IKs channel regulation that is potentially affected in some LQTS (LQT syndrome) mutations.

scholarly journals Post-translational incorporation of the antiproliferative agent azatyrosine into the C-terminus of α-tubulin

2003 ◽  
Vol 375 (1) ◽  
pp. 121-129 ◽  
Author(s):  
Silvia A. PURRO ◽  
C. Gastón BISIG ◽  
María A. CONTIN ◽  
Héctor S. BARRA ◽  
Carlos A. ARCE
Keyword(s):  
Cultured Cells ◽  
In Vivo Studies ◽  
Brain Extract ◽  
Similar Degree ◽  
C6 Cells ◽  
Post Translational Modification ◽  
Α Subunit ◽  
C Terminus

Detyrosination/tyrosination of tubulin is a post-translational modification that occurs at the C-terminus of the α-subunit, giving rise to microtubules rich in either tyrosinated or detyrosinated tubulin which coexist in the cell. We hereby report that the tyrosine analogue, azatyrosine, can be incorporated into the C-terminus of α-tubulin instead of tyrosine. Azatyrosine is structurally identical to tyrosine except that a nitrogen atom replaces carbon-2 of the phenolic group. Azatyrosine competitively excluded incorporation of [14C]tyrosine into tubulin of soluble brain extract. A newly developed rabbit antibody specific to C-terminal azatyrosine was used to study incorporation of azatyrosine in cultured cells. When added to the culture medium (Ham's F12K), azatyrosine was incorporated into tubulin of glioma-derived C6 cells. This incorporation was reversible, i.e. after withdrawal of azatyrosine, tubulin lost azatyrosine and reincorporated tyrosine. Azatyrosinated tubulin self-assembled into microtubules to a similar degree as total tubulin both in vitro and in vivo. Studies by other groups have shown that treatment of certain types of cultured cancer cells with azatyrosine leads to reversion of phenotype to normal, and that administration of azatyrosine into animals harbouring human proto-oncogenic c-Ha-ras prevents tumour formation. These interesting observations led us to study this phenomenon in relation to tubulin status. Under conditions in which tubulin was mostly azatyrosinated, C6 cells remained viable but did not proliferate. After 7–10 days under these conditions, morphology changed from a fused, elongated shape to a rounded soma with thin processes. Incorporation of azatyrosine into the C-terminus of α-tubulin is proposed as one possible cause of reversion of the malignant phenotype.

scholarly journals Lysosome-associated protein transmembrane 4α (LAPTM4α) requires two tandemly arranged tyrosine-based signals for sorting to lysosomes

2002 ◽  
Vol 365 (3) ◽  
pp. 721-730 ◽  
Author(s):  
Douglas L. HOGUE ◽  
Colin NASH ◽  
Victor LING ◽  
Tom C. HOBMAN
Keyword(s):  
Cultured Cells ◽  
Chemotherapeutic Agents ◽  
P Glycoprotein ◽  
C Terminus ◽  
Sorting Signals ◽  
Suitable Target ◽  
Membrane Spanning ◽  
High Level ◽  
Membrane Spanning Domains ◽  
Intracellular Compartmentalization

Lysosome-associated protein transmembrane 4α (LAPTM4α) and homologues comprise a family of conserved proteins, which are found in mammals, insects and nematodes. LAPTM4α functions to regulate the intracellular compartmentalization of amphipathic solutes and possibly the sensitivity of cells toward anthracyclines, antibiotics, ionophores, nucleobases and organic cations. This is similar to the multidrug-resistance phenotype exhibited by cells synthesizing high levels of P-glycoprotein. Accordingly, it is possible that LAPTM4α may be a suitable target for development of novel chemotherapeutic agents. LAPTM4α contains four putative membrane-spanning domains and a 55 amino acid C-terminal region that faces the cytoplasm. Localization of LAPTM4α to endosomes and lysosomes appears to be tightly controlled as transient high-level expression of LAPTM4α in cultured cells resulted in no detectable protein on the cell surface. Mutagenic analysis of the C-terminus of LAPTM4α indicated that two tandomly arranged tyrosine-containing motifs in the cytoplasmic domain are required for efficient localization of LAPTM4α to vesicles containing the lysosomal marker lysosomal glycoprotein 120. Although a number of membrane proteins that localize to endosomes/lysosomes contain more than one independently functioning sorting signal, to our knowledge, LAPTM4α is the first example of a membrane protein that requires two tandemly arranged tyrosine-based sorting signals for efficient localization in these compartments.

scholarly journals Interaction Between the SNARE SYP121 and the Plasma Membrane Aquaporin PIP2;7 Involves Different Protein Domains

2021 ◽  
Vol 11 ◽  
Author(s):  
Timothée Laloux ◽  
Irwin Matyjaszczyk ◽  
Simon Beaudelot ◽  
Charles Hachez ◽  
François Chaumont
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Interaction Mechanism ◽  
Bimolecular Fluorescence Complementation ◽  
Physical Interaction ◽  
Amino Acid Residues ◽  
Interaction Domain ◽  
C Terminus ◽  
Full Interaction ◽  
Plasma Membrane Intrinsic Proteins

Plasma membrane intrinsic proteins (PIPs) are channels facilitating the passive diffusion of water and small solutes. Arabidopsis PIP2;7 trafficking occurs through physical interaction with SNARE proteins including the syntaxin SYP121, a plasma membrane Qa-SNARE involved in membrane fusion. To better understand the interaction mechanism, we aimed at identifying the interaction motifs in SYP121 and PIP2;7 using ratiometric bimolecular fluorescence complementation assays in Nicotiana benthamiana. SYP121 consists of four regions, N, H, Q, and C, and sequential deletions revealed that the C region, containing the transmembrane domain, as well as the H and Q regions, containing the Habc and Qa-SNARE functional domains, interact with PIP2;7. Neither the linker between the Habc and the Qa-SNARE domains nor the H or Q regions alone could fully restore the interaction with PIP2;7, suggesting that the interacting motif depends on the conformation taken by the HQ region. When investigating the interacting motif(s) in PIP2;7, we observed that deletion of the cytosolic N- and/or C- terminus led to a significant decrease in the interaction with SYP121. Shorter deletions revealed that at the N-terminal amino acid residues 18–26 were involved in the interaction. Domain swapping experiments between PIP2;7 and PIP2;6, a PIP isoform that does not interact with SYP121, showed that PIP2;7 N-terminal part up to the loop C was required to restore the full interaction signal, suggesting that, as it is the case for SYP121, the interaction motif(s) in PIP2;7 depend on the protein conformation. Finally, we also showed that PIP2;7 physically interacted with other Arabidopsis SYP1s and SYP121 orthologs.

Dystroglycan regulates proper expression, submembranous localization and subsequent phosphorylation of Dp71 through physical interaction

2020 ◽  
Vol 29 (19) ◽  
pp. 3312-3326
Author(s):  
Takahiro Fujimoto ◽  
Takeshi Yaoi ◽  
Hidekazu Tanaka ◽  
Kyoko Itoh
Keyword(s):  
Structural Integrity ◽  
Cultured Cells ◽  
Point Mutations ◽  
Proximal Region ◽  
Molecular Characteristics ◽  
Physical Interaction ◽  
Amino Acid Residues ◽  
Ef Hand ◽  
Short Product ◽  
Hand Region

Abstract Dystrophin–dystroglycan complex (DGC) plays important roles for structural integrity and cell signaling, and its defects cause progressive muscular degeneration and intellectual disability. Dystrophin short product, Dp71, is abundantly expressed in multiple tissues other than muscle and is suspected of contributing to cognitive functions; however, its molecular characteristics and relation to dystroglycan (DG) remain unknown. Here, we report that DG physically interacts with Dp71 in cultured cells. Intriguingly, DG expression positively and DG knockdown negatively affected the steady-state expression, submembranous localization and subsequent phosphorylation of Dp71. Mechanistically, two EF-hand regions along with a ZZ motif of Dp71 mediate its association with the transmembrane proximal region, amino acid residues 788–806, of DG cytoplasmic domain. Most importantly, the pathogenic point mutations of Dp71, C272Y in the ZZ motif or L170del in the second EF-hand region, impaired its binding to DG, submembranous localization and phosphorylation of Dp71, indicating the relevance of DG-dependent Dp71 regulatory mechanism to pathophysiological conditions. Since Dp140, another dystrophin product, was also regulated by DG in the same manner as Dp71, our results uncovered a tight molecular relation between DG and dystrophin, which has broad implications for understanding the DGC-related cellular physiology and pathophysiology.

scholarly journals Physical Interaction of Calmodulin with the 5-Hydroxytryptamine2C Receptor C-Terminus Is Essential for G Protein-independent, Arrestin-dependent Receptor Signaling

2008 ◽  
Vol 19 (11) ◽  
pp. 4640-4650 ◽  
Author(s):  
Marilyne Labasque ◽  
Eric Reiter ◽  
Carine Becamel ◽  
Joël Bockaert ◽  
Philippe Marin
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Cortical Neurons ◽  
Receptor Activation ◽  
Proximal Region ◽  
Dominant Negative ◽  
Binding Motif ◽  
Physical Interaction ◽  
Receptor Signaling ◽  
C Terminus

The serotonin (5-hydroxytryptamine; 5-HT)2C receptor is a G protein-coupled receptor (GPCR) exclusively expressed in CNS that has been implicated in numerous brain disorders, including anxio-depressive states. Like many GPCRs, 5-HT2C receptors physically interact with a variety of intracellular proteins in addition to G proteins. Here, we show that calmodulin (CaM) binds to a prototypic Ca2+-dependent “1-10” CaM-binding motif located in the proximal region of the 5-HT2C receptor C-terminus upon receptor activation by 5-HT. Mutation of this motif inhibited both β-arrestin recruitment by 5-HT2C receptor and receptor-operated extracellular signal-regulated kinase (ERK) 1,2 signaling in human embryonic kidney-293 cells, which was independent of G proteins and dependent on β-arrestins. A similar inhibition was observed in cells expressing a dominant-negative CaM or depleted of CaM by RNA interference. Expression of the CaM mutant also prevented receptor-mediated ERK1,2 phosphorylation in cultured cortical neurons and choroid plexus epithelial cells that endogenously express 5-HT2C receptors. Collectively, these findings demonstrate that physical interaction of CaM with recombinant and native 5-HT2C receptors is critical for G protein-independent, arrestin-dependent receptor signaling. This signaling pathway might be involved in neurogenesis induced by chronic treatment with 5-HT2C receptor agonists and their antidepressant-like activity.

scholarly journals Functionally Active T1-T1 Interfaces Revealed by the Accessibility of Intracellular Thiolate Groups in Kv4 Channels

2005 ◽  
Vol 126 (1) ◽  
pp. 55-69 ◽  
Author(s):  
Guangyu Wang ◽  
Mohammad Shahidullah ◽  
Carmen A. Rocha ◽  
Candace Strang ◽  
Paul J. Pfaffinger ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Α Subunit ◽  
Irreversible Inhibition ◽  
Tetramerization Domain ◽  
Activated State ◽  
Kv4 Channels ◽  
Voltage Dependent ◽  
Membrane Spanning ◽  
Modification Rate

Gating of voltage-dependent K+ channels involves movements of membrane-spanning regions that control the opening of the pore. Much less is known, however, about the contributions of large intracellular channel domains to the conformational changes that underlie gating. Here, we investigated the functional role of intracellular regions in Kv4 channels by probing relevant cysteines with thiol-specific reagents. We find that reagent application to the intracellular side of inside-out patches results in time-dependent irreversible inhibition of Kv4.1 and Kv4.3 currents. In the absence or presence of Kv4-specific auxiliary subunits, mutational and electrophysiological analyses showed that none of the 14 intracellular cysteines is essential for channel gating. C110, C131, and C132 in the intersubunit interface of the tetramerization domain (T1) are targets responsible for the irreversible inhibition by a methanethiosulfonate derivative (MTSET). This result is surprising because structural studies of Kv4-T1 crystals predicted protection of the targeted thiolate groups by constitutive high-affinity Zn2+ coordination. Also, added Zn2+ or a potent Zn2+ chelator (TPEN) does not significantly modulate the accessibility of MTSET to C110, C131, or C132; and furthermore, when the three critical cysteines remained as possible targets, the MTSET modification rate of the activated state is ∼200-fold faster than that of the resting state. Biochemical experiments confirmed the chemical modification of the intact α-subunit and the purified tetrameric T1 domain by MTS reagents. These results conclusively demonstrate that the T1–T1 interface of Kv4 channels is functionally active and dynamic, and that critical reactive thiolate groups in this interface may not be protected by Zn2+ binding.

scholarly journals The two C-terminal tyrosines stabilize occluded Na/K pump conformations containing Na or K ions

2010 ◽  
Vol 136 (1) ◽  
pp. 63-82 ◽  
Author(s):  
Natascia Vedovato ◽  
David C. Gadsby
Keyword(s):  
Point Mutations ◽  
Pump Current ◽  
Binding Pocket ◽  
Inhibitory Influence ◽  
Electrical Measurements ◽  
Α Subunit ◽  
Apparent Affinity ◽  
C Terminus ◽  
Na Ions ◽  
Almost All

Interactions of the three transported Na ions with the Na/K pump remain incompletely understood. Na/K pump crystal structures show that the extended C terminus of the Na,K–adenosine triphosphatase (ATPase) α subunit directly contacts transmembrane helices. Deletion of the last five residues (KETYY in almost all Na/K pumps) markedly lowered the apparent affinity for Na activation of pump phosphorylation from ATP, a reflection of cytoplasmic Na affinity for forming the occluded E1P(Na3) conformation. ATPase assays further suggested that C-terminal truncations also interfere with low affinity Na interactions, which are attributable to extracellular effects. Because extracellular Na ions traverse part of the membrane’s electric field to reach their binding sites in the Na/K pump, their movements generate currents that can be monitored with high resolution. We report here electrical measurements to examine how Na/K pump interactions with extracellular Na ions are influenced by C-terminal truncations. We deleted the last two (YY) or five (KESYY) residues in Xenopus laevis α1 Na/K pumps made ouabain resistant by either of two kinds of point mutations and measured their currents as 10-mM ouabain–sensitive currents in Xenopus oocytes after silencing endogenous Xenopus Na/K pumps with 1 µM ouabain. We found the low affinity inhibitory influence of extracellular Na on outward Na/K pump current at negative voltages to be impaired in all of the C-terminally truncated pumps. Correspondingly, voltage jump–induced transient charge movements that reflect pump interactions with extracellular Na ions were strongly shifted to more negative potentials; this signals a several-fold reduction of the apparent affinity for extracellular Na in the truncated pumps. Parallel lowering of Na affinity on both sides of the membrane argues that the C-terminal contacts provide important stabilization of the occluded E1P(Na3) conformation, regardless of the route of Na ion entry into the binding pocket. Gating measurements of palytoxin-opened Na/K pump channels additionally imply that the C-terminal contacts also help stabilize pump conformations with occluded K ions.

scholarly journals The C Terminus of Foamy Retrovirus Gag Contains Determinants for Encapsidation of Pol Protein into Virions

2008 ◽  
Vol 82 (21) ◽  
pp. 10803-10810 ◽  
Author(s):  
Eun-Gyung Lee ◽  
Maxine L. Linial
Keyword(s):  
Site Directed Mutagenesis ◽  
Rna Packaging ◽  
Interaction Domain ◽  
Virus Particles ◽  
Charged Amino Acids ◽  
C Terminus ◽  
Foamy Viruses ◽  
Substitution Mutations ◽  
Gag Assembly ◽  
Positively Charged

ABSTRACT Foamy viruses (FV) differ from orthoretroviruses in many aspects of their replication cycle. A major difference is in the mode of Pol expression, regulation, and encapsidation into virions. Orthoretroviruses synthesize Pol as a Gag-Pol fusion protein so that Pol is encapsidated into virus particles through Gag assembly domains. However, as FV express Pol independently of Gag from a spliced mRNA, packaging occurs through a distinct mechanism. FV genomic RNA contains cis-acting sequences that are required for Pol packaging, suggesting that Pol binds to RNA for its encapsidation. However, it is not known whether Gag is directly involved in Pol packaging. Previously our laboratory showed that sequences flanking the three glycine-arginine-rich (GR) boxes at the C terminus of FV Gag contain domains important for RNA packaging and Pol expression, cleavage, and packaging. We have now shown that both deletion and substitution mutations in the first GR box (GR1) prevented neither the assembly of particles with wild-type density nor packaging of RNA genomes but led to a defect in Pol packaging. Site-directed mutagenesis of GR1 indicated that the clustered positively charged amino acids in GR1 play important roles in Pol packaging. Our results suggest that GR1 contains a Pol interaction domain and that a Gag-Pol complex is formed and binds to RNA for incorporation into virions.

Transmembrane prolyl 4-hydroxylase is a fourth prolyl 4-hydroxylase regulating EPO production and erythropoiesis

Blood ◽  
2012 ◽  
Vol 120 (16) ◽  
pp. 3336-3344 ◽  
Author(s):  
Anu Laitala ◽  
Ellinoora Aro ◽  
Gail Walkinshaw ◽  
Joni M. Mäki ◽  
Maarit Rossi ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Mrna Level ◽  
Hypoxia Inducible Factor ◽  
Mrna Levels ◽  
Wild Type ◽  
Α Subunit ◽  
Hepcidin Expression ◽  
In The Wild ◽  
Hepcidin Mrna

AbstractAn endoplasmic reticulum transmembrane prolyl 4-hydroxylase (P4H-TM) is able to hydroxylate the α subunit of the hypoxia-inducible factor (HIF) in vitro and in cultured cells, but nothing is known about its roles in mammalian erythropoiesis. We studied such roles here by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm−/− mice. This caused larger increases in serum Epo concentration and kidney but not liver Hif-1α and Hif-2α protein and Epo mRNA levels than in wild-type mice, while the liver Hepcidin mRNA level was lower in the P4h-tm−/− mice than in the wild-type. Similar, but not identical, differences were also seen between FG-4497–treated Hif-p4h-2 hypomorphic (Hif-p4h-2gt/gt) and Hif-p4h-3−/− mice versus wild-type mice. FG-4497 administration increased hemoglobin and hematocrit values similarly in the P4h-tm−/− and wild-type mice, but caused higher increases in both values in the Hif-p4h-2gt/gt mice and in hematocrit value in the Hif-p4h-3−/− mice than in the wild-type. Hif-p4h-2gt/gt/P4h-tm−/− double gene-modified mice nevertheless had increased hemoglobin and hematocrit values without any FG-4497 administration, although no such abnormalities were seen in the Hif-p4h-2gt/gt or P4h-tm−/− mice. Our data thus indicate that P4H-TM plays a role in the regulation of EPO production, hepcidin expression, and erythropoiesis.

IRON MAN interacts with BRUTUS to maintain iron homeostasis in Arabidopsis

2021 ◽  
Vol 118 (39) ◽  
pp. e2109063118
Author(s):  
Yang Li ◽  
Cheng Kai Lu ◽  
Chen Yang Li ◽  
Ri Hua Lei ◽  
Meng Na Pu ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Transcription Factors ◽  
Iron Homeostasis ◽  
Fe Deficiency ◽  
Small Peptides ◽  
Interaction Domain ◽  
C Terminus ◽  
Positive Regulators ◽  
Fe Homeostasis ◽  
And Function

IRON MAN (IMA) peptides, a family of small peptides, control iron (Fe) transport in plants, but their roles in Fe signaling remain unclear. BRUTUS (BTS) is a potential Fe sensor that negatively regulates Fe homeostasis by promoting the ubiquitin-mediated degradation of bHLH105 and bHLH115, two positive regulators of the Fe deficiency response. Here, we show that IMA peptides interact with BTS. The C-terminal parts of IMA peptides contain a conserved BTS interaction domain (BID) that is responsible for their interaction with the C terminus of BTS. Arabidopsis thaliana plants constitutively expressing IMA genes phenocopy the bts-2 mutant. Moreover, IMA peptides are ubiquitinated and degraded by BTS. bHLH105 and bHLH115 also share a BID, which accounts for their interaction with BTS. IMA peptides compete with bHLH105/bHLH115 for interaction with BTS, thereby inhibiting the degradation of these transcription factors by BTS. Genetic analyses suggest that bHLH105/bHLH115 and IMA3 have additive roles and function downstream of BTS. Moreover, the transcription of both BTS and IMA3 is activated directly by bHLH105 and bHLH115 under Fe-deficient conditions. Our findings provide a conceptual framework for understanding the regulation of Fe homeostasis: IMA peptides protect bHLH105/bHLH115 from degradation by sequestering BTS, thereby activating the Fe deficiency response.

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