scholarly journals A Single Amino Acid Substitution in the Renal Betaine/GABA Transporter Prevents Trafficking to the Plasma Membrane

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Christopher R. Day ◽  
Sashana S. Gordon ◽  
Cherissa L. Vaughn ◽  
Stephen A. Kempson
Keyword(s):  
Plasma Membrane ◽  
Mdck Cells ◽  
Phosphorylation Site ◽  
Renal Medulla ◽  
Hek293 Cells ◽  
Single Amino Acid ◽  
Transport Activity ◽  
Hypertonic Stress ◽  
C Terminus ◽  
Putative Phosphorylation Site

One response to hypertonic stress in the renal medulla and MDCK cells is the upregulation of betaine transporter (BGT1) synthesis, followed by trafficking to the plasma membrane (PM) and an increase in betaine transport. Upregulation of BGT1 was enhanced by inhibitors of phosphatases PP1 and PP2A and was attenuated by inhibitors of protein kinase C, suggesting an important role for phosphorylation reactions. This was tested using mutants of BGT1 tagged with EGFP. The PM trafficking motifs of BGT1 reside near the C terminus, and truncation at lysine560 resulted in a protein that remained intracellular during hypertonic stress. This K560Δ mutant colocalized with endoplasmic reticulum (ER). Substitution of alanine at Thr40, a putative phosphorylation site, also prevented trafficking to the PM during hypertonic stress. Live-cell imaging showed that T40A was not retained in the ER and colocalized with markers for Golgi and endosomes. In contrast, substitution of aspartate or glutamate at Thr40, to mimic phosphorylation, restored normal trafficking to the PM. HEK293 cells transfected with K560Δ or T40A mutants had 10% of the GABA transport activity of native BGT1, but normal transport activity was restored in cells expressing T40E. Normal BGT1 trafficking likely requires phosphorylation at Thr40 in addition to C-terminal motifs.

Subcellular redistribution of the renal betaine transporter during hypertonic stress

2003 ◽  
Vol 285 (5) ◽  
pp. C1091-C1100 ◽  
Author(s):  
Stephen A. Kempson ◽  
Vaibhave Parikh ◽  
Lixuan Xi ◽  
Shaoyou Chu ◽  
Marshall H. Montrose
Keyword(s):  
Plasma Membrane ◽  
Posttranscriptional Regulation ◽  
Fluorescent Protein ◽  
Mdck Cells ◽  
Live Cells ◽  
Hypertonic Medium ◽  
Hypertonic Stress ◽  
Antibody Staining ◽  
Renal Inner Medulla ◽  
Green Fluorescent

The betaine transporter (BGT1) protects cells in the hypertonic renal inner medulla by mediating uptake and accumulation of the osmolyte betaine. Transcriptional regulation plays an essential role in upregulation of BGT1 transport when renal cells are exposed to hypertonic medium for 24 h. Posttranscriptional regulation of the BGT1 protein is largely unexplored. We have investigated the distribution of BGT1 protein in live cells after transfection with BGT1 tagged with enhanced green fluorescent protein (EGFP). Fusion of EGFP to the NH2 terminus of BGT1 produced a fusion protein (EGFP-BGT) with transport properties identical to normal BGT1, as determined by ion dependence, inhibitor sensitivity, and apparent Km for GABA. Confocal microscopy of EGFP-BGT fluorescence in transfected Madin-Darby canine kidney (MDCK) cells showed that hypertonic stress for 24 h induced a shift in subcellular distribution from cytoplasm to plasma membrane. This was confirmed by colocalization with anti-BGT1 antibody staining. In fibroblasts, transfected EGFP-BGT caused increased transport in response to hypertonic stress. The activation of transport was not accompanied by increased expression of EGFP-BGT, as determined by Western blotting. Membrane insertion of EGFP-BGT protein in MDCK cells began within 2-3 h after onset of hypertonic stress and was blocked by cycloheximide. We conclude that posttranscriptional regulation of BGT1 is essential for adaptation to hypertonic stress and that insertion of BGT1 protein to the plasma membrane may require accessory proteins.

scholarly journals Differential phosphorylation signals control endocytosis of GPR15

2017 ◽  
Vol 28 (17) ◽  
pp. 2267-2281 ◽  
Author(s):  
Yukari Okamoto ◽  
Sojin Shikano
Keyword(s):  
Surface Density ◽  
Functional Role ◽  
Control Cell ◽  
Phosphorylation Site ◽  
Hek293 Cells ◽  
C Terminus ◽  
Differential Phosphorylation ◽  
G Protein Coupled

GPR15 is an orphan G protein–coupled receptor (GPCR) that serves for an HIV coreceptor and was also recently found as a novel homing receptor for T-cells implicated in colitis. We show that GPR15 undergoes a constitutive endocytosis in the absence of ligand. The endocytosis was clathrin dependent and partially dependent on β-arrestin in HEK293 cells, and nearly half of the internalized GPR15 receptors were recycled to the plasma membrane. An Ala mutation of the distal C-terminal Arg-354 or Ser-357, which forms a consensus phosphorylation site for basophilic kinases, markedly reduced the endocytosis, whereas phosphomimetic mutation of Ser-357 to Asp did not. Ser-357 was phosphorylated in vitro by multiple kinases, including PKA and PKC, and pharmacological activation of these kinases enhanced both phosphorylation of Ser-357 and endocytosis of GPR15. These results suggested that Ser-357 phosphorylation critically controls the ligand-independent endocytosis of GPR15. The functional role of Ser-357 in endocytosis was distinct from that of a conserved Ser/Thr cluster in the more proximal C-terminus, which was responsible for the β-arrestin– and GPCR kinase–dependent endocytosis of GPR15. Thus phosphorylation signals may differentially control cell surface density of GPR15 through endocytosis.

A nematode gene required for sperm vesicle fusion

1997 ◽  
Vol 110 (9) ◽  
pp. 1073-1081 ◽  
Author(s):  
W.E. Achanzar ◽  
S. Ward
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Sequence Similarity ◽  
Biological Significance ◽  
Single Amino Acid ◽  
Viral Fusion ◽  
Missense Mutations ◽  
Direct Role ◽  
C Terminus ◽  
Human Proteins

During maturation of spermatids to motile spermatozoa in Caenorhabditis elegans, large vesicles called membranous organelles (MOs) fuse with the spermatid plasma membrane. Mutations in the gene fer-1 cause abnormal spermatozoa in which the MOs do not fuse, although they abut the plasma membrane normally. Here we describe the fer-1 gene, which we found to be approximately 8.6 kb in length and to encode a 6.2 kb transcript whose expression is limited to the primary spermatocytes, the cells in which the MOs form. fer-1 is predicted to encode a 235 kDa protein which is highly charged except for a putative transmembrane domain near the C terminus. We identified the mutations associated with five fer-1 alleles, all of which are missense mutations causing single amino acid changes. FER-1 is not similar to any characterized proteins in sequence databases, nor does it contain known functional motifs other than the predicted transmembrane domain. The C-terminal transmembrane domain makes FER-1 resemble some viral fusion proteins, suggesting it may play a direct role in MO-plasma membrane fusion. FER-1 does show significant sequence similarity to several predicted human proteins of unknown function. Two of the identified fer-1 mutations are located in regions of similarity between FER-1 and two of these predicted proteins. This strengthens the biological significance of these similarities and suggests these regions of similarity represent functionally important domains of FER-1 and the human proteins.

scholarly journals The inhibition of monocarboxylate transporter 2 (MCT2) by AR-C155858 is modulated by the associated ancillary protein

2010 ◽  
Vol 431 (2) ◽  
pp. 217-225 ◽  
Author(s):  
Matthew J. Ovens ◽  
Christine Manoharan ◽  
Marieangela C. Wilson ◽  
Clarey M. Murray ◽  
Andrew P. Halestrap
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Monocarboxylate Transporter ◽  
Xenopus Laevis Oocytes ◽  
Transport Activity ◽  
Membrane Expression ◽  
Binding Partner ◽  
Reverse Transcription Pcr ◽  
C Terminus ◽  
Plasma Membrane Expression

In mammalian cells, MCTs (monocarboxylate transporters) require association with an ancillary protein to enable plasma membrane expression of the active transporter. Basigin is the preferred binding partner for MCT1, MCT3 and MCT4, and embigin for MCT2. In rat and rabbit erythrocytes, MCT1 is associated with embigin and basigin respectively, but its sensitivity to inhibition by AR-C155858 was found to be identical. Using RT (reverse transcription)–PCR, we have shown that Xenopus laevis oocytes contain endogenous basigin, but not embigin. Co-expression of exogenous embigin was without effect on either the expression of MCT1 or its inhibition by AR-C155858. In contrast, expression of active MCT2 at the plasma membrane of oocytes was significantly enhanced by co-expression of exogenous embigin. This additional transport activity was insensitive to inhibition by AR-C155858 unlike that by MCT2 expressed with endogenous basigin that was potently inhibited by AR-C155858. Chimaeras and C-terminal truncations of MCT1 and MCT2 were also expressed in oocytes in the presence and absence of exogenous embigin. L-Lactate Km values for these constructs were determined and revealed that the TM (transmembrane) domains of an MCT, most probably TM7–TM12, but not the C-terminus, are the major determinants of L-lactate affinity, whereas the associated ancillary protein has little or no effect. Inhibitor titrations of lactate transport by these constructs indicated that embigin modulates MCT2 sensitivity to AR-C155858 through interactions with both the intracellular C-terminus and TMs 3 and 6 of MCT2. The C-terminus of MCT2 was found to be essential for its expression with endogenous basigin.

scholarly journals A 49-residue sequence motif in the C terminus of Nav1.9 regulates trafficking of the channel to the plasma membrane

2019 ◽  
Vol 295 (4) ◽  
pp. 1077-1090 ◽  
Author(s):  
Daria V. Sizova ◽  
Jianying Huang ◽  
Elizabeth J. Akin ◽  
Mark Estacion ◽  
Carolina Gomis-Perez ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Recombinant Expression ◽  
Critical Role ◽  
Functional Expression ◽  
Hek293 Cells ◽  
Sequence Motif ◽  
Functional Studies ◽  
C Terminus ◽  
Mechanistic Basis ◽  
Heterologous Expression Systems

Genetic and functional studies have confirmed an important role for the voltage-gated sodium channel Nav1.9 in human pain disorders. However, low functional expression of Nav1.9 in heterologous systems (e.g. in human embryonic kidney 293 (HEK293) cells) has hampered studies of its biophysical and pharmacological properties and the development of high-throughput assays for drug development targeting this channel. The mechanistic basis for the low level of Nav1.9 currents in heterologous expression systems is not understood. Here, we implemented a multidisciplinary approach to investigate the mechanisms that govern functional Nav1.9 expression. Recombinant expression of a series of Nav1.9-Nav1.7 C-terminal chimeras in HEK293 cells identified a 49-amino-acid-long motif in the C terminus of the two channels that regulates expression levels of these chimeras. We confirmed the critical role of this motif in the context of a full-length channel chimera, Nav1.9-Ct49aaNav1.7, which displayed significantly increased current density in HEK293 cells while largely retaining the characteristic Nav1.9-gating properties. High-resolution live microscopy indicated that the newly identified C-terminal motif dramatically increases the number of channels on the plasma membrane of HEK293 cells. Molecular modeling results suggested that this motif is exposed on the cytoplasmic face of the folded C terminus, where it might interact with other channel partners. These findings reveal that a 49-residue-long motif in Nav1.9 regulates channel trafficking to the plasma membrane.

scholarly journals Casein Kinase 2 Binds to the C Terminus of Na+/H+ exchanger 3 (NHE3) and Stimulates NHE3 Basal Activity by Phosphorylating a Separate Site in NHE3

2008 ◽  
Vol 19 (9) ◽  
pp. 3859-3870 ◽  
Author(s):  
Rafiquel Sarker ◽  
Mads Grønborg ◽  
Boyoung Cha ◽  
Sachin Mohan ◽  
Yueping Chen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Phosphorylation Site ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
Kinase Assay ◽  
Phosphatase Inhibitors ◽  
C Terminus ◽  
Separate Site ◽  
A Site

Na+/H+ exchanger 3 (NHE3) is the epithelial-brush border isoform responsible for most intestinal and renal Na+ absorption. Its activity is both up- and down-regulated under normal physiological conditions, and it is inhibited in most diarrheal diseases. NHE3 is phosphorylated under basal conditions and Ser/Thr phosphatase inhibitors stimulate basal exchange activity; however, the kinases involved are unknown. To identify kinases that regulate NHE3 under basal conditions, NHE3 was immunoprecipitated; LC-MS/MS of trypsinized NHE3 identified a novel phosphorylation site at S719 of the C terminus, which was predicted to be a casein kinase 2 (CK2) phosphorylation site. This was confirmed by an in vitro kinase assay. The NHE3-S719A mutant but not NHE3-S719D had reduced NHE3 activity due to less plasma membrane NHE3. This was due to reduced exocytosis plus decreased plasma membrane delivery of newly synthesized NHE3. Also, NHE3 activity was inhibited by the CK2 inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole DMAT when wild-type NHE3 was expressed in fibroblasts and Caco-2 cells, but the NHE3-S719 mutant was fully resistant to DMAT. CK2 bound to the NHE3 C-terminal domain, between amino acids 590 and 667, a site different from the site it phosphorylates. CK2 binds to the NHE3 C terminus and stimulates basal NHE3 activity by phosphorylating a separate single site on the NHE3 C terminus (S719), which affects NHE3 trafficking.

scholarly journals A physiologic rise in cytoplasmic calcium ion signal increases pannexin1 channel activity via a C-terminus phosphorylation by CaMKII

2021 ◽  
Vol 118 (32) ◽  
pp. e2108967118
Author(s):  
Ximena López ◽  
Nicolás Palacios-Prado ◽  
Juan Güiza ◽  
Rosalba Escamilla ◽  
Paola Fernández ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Uptake Rate ◽  
Single Channel ◽  
Phosphorylation Site ◽  
Site Directed Mutagenesis ◽  
Membrane Stretch ◽  
C Terminus ◽  
Putative Phosphorylation Site ◽  
In Silico Studies ◽  
Single Channel Currents

Pannexin1 (Panx1) channels are ubiquitously expressed in vertebrate cells and are widely accepted as adenosine triphosphate (ATP)-releasing membrane channels. Activation of Panx1 has been associated with phosphorylation in a specific tyrosine residue or cleavage of its C-terminal domains. In the present work, we identified a residue (S394) as a putative phosphorylation site by Ca2+/calmodulin-dependent kinase II (CaMKII). In HeLa cells transfected with rat Panx1 (rPanx1), membrane stretch (MS)-induced activation—measured by changes in DAPI uptake rate—was drastically reduced by either knockdown of Piezo1 or pharmacological inhibition of calmodulin or CaMKII. By site-directed mutagenesis we generated rPanx1S394A-EGFP (enhanced green fluorescent protein), which lost its sensitivity to MS, and rPanx1S394D-EGFP, mimicking phosphorylation, which shows high DAPI uptake rate without MS stimulation or cleavage of the C terminus. Using whole-cell patch-clamp and outside-out excised patch configurations, we found that rPanx1-EGFP and rPanx1S394D-EGFP channels showed current at all voltages between ±100 mV, similar single channel currents with outward rectification, and unitary conductance (∼30 to 70 pS). However, using cell-attached configuration we found that rPanx1S394D-EGFP channels show increased spontaneous unitary events independent of MS stimulation. In silico studies revealed that phosphorylation of S394 caused conformational changes in the selectivity filter and increased the average volume of lateral tunnels, allowing ATP to be released via these conduits and DAPI uptake directly from the channel mouth to the cytoplasmic space. These results could explain one possible mechanism for activation of rPanx1 upon increase in cytoplasmic Ca2+ signal elicited by diverse physiological conditions in which the C-terminal domain is not cleaved.

scholarly journals Biochemical and Genetic Evidence for Participation of DevR in a Phosphorelay Signal Transduction Pathway Essential for Heterocyst Maturation in Nostoc punctiforme ATCC 29133

1999 ◽  
Vol 181 (14) ◽  
pp. 4430-4434 ◽  
Author(s):  
Kari D. Hagen ◽  
John C. Meeks
Keyword(s):  
Signal Transduction ◽  
Response Regulator ◽  
Phosphorylation Site ◽  
Signal Transduction Pathway ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Insertion Mutant ◽  
Nostoc Punctiforme ◽  
Putative Phosphorylation Site

ABSTRACT In a test of the hypothesis that DevR is a response regulator protein that functions in a phosphorelay signal transduction system involved in heterocyst development in Nostoc punctiformeATCC 29133, purified affinity-tagged DevR was shown to be phosphorylated in vitro by the noncognate sensor kinase EnvZ. Site-directed mutagenesis was used to generate N. punctiforme mutants with single amino acid substitutions at the putative phosphorylation site of DevR. These mutants exhibited a Fox− phenotype like the original devRinsertion mutant UCD 311, consistent with a phosphotransferase role for DevR.

Mechanisms activating latent functions of PIP aquaporin water channels via the interaction between PIP1 and PIP2 proteins

2020 ◽  
Author(s):  
Mineo Shibasaka ◽  
Tomoaki Horie ◽  
Maki Katsuhara
Keyword(s):  
Plasma Membrane ◽  
Water Transport ◽  
Molecular Mechanisms ◽  
Water Channel ◽  
Amino Acid Replacement ◽  
Middle Part ◽  
Single Amino Acid ◽  
Xenopus Laevis Oocytes ◽  
Transport Activity ◽  
Membrane Type

Abstract Plant plasma-membrane type PIP aquaporins are classified into two groups, PIP1s and PIP2s. In this study, we focused on HvPIP1; 2, a PIP1 in barley (Hordeum vulgare), to dissect the molecular mechanisms that evoke HvPIP1-mediated water transport. No HvPIP1; 2 protein was localized to the plasma membrane when expressed alone in Xenopus laevis oocytes. In contrast, a chimeric HvPIP1; 2 protein (HvPIP1; 2_24NC), in which the N- and C-terminal regions were replaced with the corresponding regions from HvPIP2; 4, was found to localize to the plasma membrane of oocytes. However, HvPIP1; 2_24NC showed no water transport activity in swelling assays. These results suggested that the terminal regions of PIP2 proteins direct PIP proteins to the plasma-membrane, but the re-localization of PIP1 proteins was not sufficient to PIP1s functionality as water channel in a membrane. A single amino acid replacement of threonine by methionine in HvPIP2; 4 (HvPIP2; 4T229M) abolished water transport activity. Co-expression of HvPIP1; 2_24NC either with HvPIP2; 4_12NC or HvPIP2; 4TM_12NC, in which the N- and C-terminal regions were replaced with the corresponding regions of HvPIP1; 2, increased the water transport activity in oocytes. These data provided evidence that the HvPIP1; 2 molecule has own water transport activity and an interaction with the middle part of the HvPIP2; 4 protein (except for the N- and C- termini) is required for HvPIP1; 2 functionality as water channel. This molecular mechanism could be applied to other PIP1s and PIP2s in addition to the known mechanism that the terminal regions of some PIP2s lead some PIP1s to the plasma membrane.

Calmodulin disrupts plasma membrane localization of farnesylated KRAS4b by sequestering its lipid moiety

2020 ◽  
Vol 13 (625) ◽  
pp. eaaz0344 ◽  
Author(s):  
Benjamin M. M. Grant ◽  
Masahiro Enomoto ◽  
Sung-In Back ◽  
Ki-Young Lee ◽  
Teklab Gebregiworgis ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Effector Proteins ◽  
Hek293 Cells ◽  
Dependent Manner ◽  
C Terminus ◽  
Guanosine Triphosphatase ◽  
Solution Nuclear Magnetic Resonance ◽  
Dependent Interaction

KRAS4b is a small guanosine triphosphatase (GTPase) protein that regulates several signal transduction pathways that underlie cell proliferation, differentiation, and survival. KRAS4b function requires prenylation of its C terminus and recruitment to the plasma membrane, where KRAS4b activates effector proteins including the RAF family of kinases. The Ca2+-sensing protein calmodulin (CaM) has been suggested to regulate the localization of KRAS4b through direct, Ca2+-dependent interaction, but how CaM and KRAS4b functionally interact is controversial. Here, we determined a crystal structure, which was supported by solution nuclear magnetic resonance (NMR), that revealed the sequestration of the prenyl moiety of KRAS4b in the hydrophobic pocket of the C-terminal lobe of Ca2+-bound CaM. Our engineered fluorescence resonance energy transfer (FRET)–based biosensor probes (CaMeRAS) showed that, upon stimulation of Ca2+ influx by extracellular ligands, KRAS4b reversibly translocated in a Ca2+-CaM–dependent manner from the plasma membrane to the cytoplasm in live HeLa and HEK293 cells. These results reveal a mechanism underlying the inhibition of KRAS4b activity by Ca2+ signaling pathways.

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