scholarly journals CD63 interacts with the carboxy terminus of the colonic H+-K+-ATPase to increase plasma membrane localization and86Rb+uptake

2005 ◽  
Vol 288 (6) ◽  
pp. C1279-C1286 ◽  
Author(s):  
Juan Codina ◽  
Jian Li ◽  
Thomas D. DuBose
Keyword(s):  
Plasma Membrane ◽  
Protein Interactions ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Carboxy Terminus ◽  
Protein Protein Interactions ◽  
Α Subunit ◽  
Hek 293 ◽  
And Function

The carboxy terminus (CT) of the colonic H+-K+-ATPase is required for stable assembly with the β-subunit, translocation to the plasma membrane, and efficient function of the transporter. To identify protein-protein interactions involved in the localization and function of HKα2, we selected 84 amino acids in the CT of the α-subunit of mouse colonic H+-K+-ATPase (CT-HKα2) as the bait in a yeast two-hybrid screen of a mouse kidney cDNA library. The longest identified clone was CD63. To characterize the interaction of CT-HKα2with CD63, recombinant CT-HKα2and CD63 were synthesized in vitro and incubated, and complexes were immunoprecipitated. CT-HKα2protein (but not CT-HKα1) coprecipitated with CD63, confirming stable assembly of HKα2with CD63. In HEK-293 transfected with HKα2plus β1-Na+-K+-ATPase, suppression of CD63 by RNA interference increased cell surface expression of HKα2/NKβ1and86Rb+uptake. These studies demonstrate that CD63 participates in the regulation of the abundance of the HKα2-NKβ1complex in the cell membrane.

Several adaptor proteins promote intracellular localisation of the transporter MRP4/ABCC4 in platelets and haematopoietic cells

2017 ◽  
Vol 117 (01) ◽  
pp. 105-115 ◽  
Author(s):  
Yvonne Schaletzki ◽  
Marie-Luise Kromrey ◽  
Susanne Bröderdorf ◽  
Elke Hammer ◽  
Markus Grube ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Interactions ◽  
Pdz Domain ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Apical Plasma Membrane ◽  
Dense Granules ◽  
Haematopoietic Cells ◽  
And Function

SummaryThe multidrug resistance protein 4 (MRP4/ABCC4) has been identified as an important transporter for signalling molecules including cyclic nucleotides and several lipid mediators in platelets and may thus represent a novel target to interfere with platelet function. Besides its localisation in the plasma membrane, MRP4 has been also detected in the membrane of dense granules in resting platelets. In polarised cells it is localised at the basolateral or apical plasma membrane. To date, the mechanism of MRP4 trafficking has not been elucidated; protein interactions may regulate both the localisation and function of this transporter. We approached this issue by searching for interacting proteins by in vitro binding assays, followed by immunoblotting and mass spectrometry, and by visualising their co-localisation in platelets and haematopoietic cells. We identified the PDZ domain containing scaffold proteins ezrin-binding protein 50 (EBP50/NHERF1), postsynaptic density protein 95 (PSD95), and sorting nexin 27 (SNX27), but also the adaptor protein complex 3 subunit β3A (AP3B1) and the heat shock protein HSP90 as putative interaction partners of MRP4. The knockdown of SNX27, PSD95, and AP3B1 by siRNA in megakaryoblastic leuk aemia cells led to a redistribution of MRP4 from intracellular structures to the plasma membrane. Inhibition of HSP90 led to a diminished expression and retention of MRP4 in the endoplasmic reticulum. These results indicate that MRP4 localisation and function are regulated by multiple protein interactions. Changes in the adaptor proteins can hence lead to altered localisation and function of the transporter.Supplementary Material to this article is available at www.thrombosis-online.com.

scholarly journals Critical roles for p22phox in the structural maturation and subcellular targeting of Nox3

2007 ◽  
Vol 403 (1) ◽  
pp. 97-108 ◽  
Author(s):  
Yoko Nakano ◽  
Botond Banfi ◽  
Algirdas J. Jesaitis ◽  
Mary C. Dinauer ◽  
Lee-Ann H. Allen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Subcellular Targeting ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Regulatory Subunits ◽  
Hek 293 Cells ◽  
293 Cells ◽  
A Subunit ◽  
And Function

Otoconia are small biominerals in the inner ear that are indispensable for the normal perception of gravity and motion. Normal otoconia biogenesis requires Nox3, a Nox (NADPH oxidase) highly expressed in the vestibular system. In HEK-293 cells (human embryonic kidney cells) transfected with the Nox regulatory subunits NoxO1 (Nox organizer 1) and NoxA1 (Nox activator 1), functional murine Nox3 was expressed in the plasma membrane and exhibited a haem spectrum identical with that of Nox2, the electron transferase of the phagocyte Nox. In vitro Nox3 cDNA expressed an ∼50 kDa primary translation product that underwent N-linked glycosylation in the presence of canine microsomes. RNAi (RNA interference)-mediated reduction of endogenous p22phox, a subunit essential for stabilization of Nox2 in phagocytes, decreased Nox3 activity in reconstituted HEK-293 cells. p22phox co-precipitated not only with Nox3 and NoxO1 from transfectants expressing all three proteins, but also with NoxO1 in the absence of Nox3, indicating that p22phox physically associated with both Nox3 and with NoxO1. The plasma membrane localization of Nox3 but not of NoxO1 required p22phox. Moreover, the glycosylation and maturation of Nox3 required p22phox expression, suggesting that p22phox was required for the proper biosynthesis and function of Nox3. Taken together, these studies demonstrate critical roles for p22phox at several distinct points in the maturation and assembly of a functionally competent Nox3 in the plasma membrane.

scholarly journals A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function.

1995 ◽  
Vol 15 (10) ◽  
pp. 5214-5225 ◽  
Author(s):  
A D Catling ◽  
H J Schaeffer ◽  
C W Reuter ◽  
G R Reddy ◽  
M J Weber
Keyword(s):  
Protein Interactions ◽  
Critical Role ◽  
Phosphorylation Site ◽  
Specific Protein ◽  
Protein Protein Interactions ◽  
Serum Stimulation ◽  
And Function

Mammalian MEK1 and MEK2 contain a proline-rich (PR) sequence that is absent both from the yeast homologs Ste7 and Byr1 and from a recently cloned activator of the JNK/stress-activated protein kinases, SEK1/MKK4. Since this PR sequence occurs in MEKs that are regulated by Raf family enzymes but is missing from MEKs and SEKs activated independently of Raf, we sought to investigate the role of this sequence in MEK1 and MEK2 regulation and function. Deletion of the PR sequence from MEK1 blocked the ability of MEK1 to associate with members of the Raf family and markedly attenuated activation of the protein in vivo following growth factor stimulation. In addition, this sequence was necessary for efficient activation of MEK1 in vitro by B-Raf but dispensable for activation by a novel MEK1 activator which we have previously detected in fractionated fibroblast extracts. Furthermore, we found that a phosphorylation site within the PR sequence of MEK1 was required for sustained MEK1 activity in response to serum stimulation of quiescent fibroblasts. Consistent with this observation, we observed that MEK2, which lacks a phosphorylation site at the corresponding position, was activated only transiently following serum stimulation. Finally, we found that deletion of the PR sequence from a constitutively activated MEK1 mutant rendered the protein nontransforming in Rat1 fibroblasts. These observations indicate a critical role for the PR sequence in directing specific protein-protein interactions important for the activation, inactivation, and downstream functioning of the MEKs.

scholarly journals Low HLA-C expression at cell surfaces correlates with increased turnover of heavy chain mRNA.

1995 ◽  
Vol 181 (6) ◽  
pp. 2085-2095 ◽  
Author(s):  
J A McCutcheon ◽  
J Gumperz ◽  
K D Smith ◽  
C T Lutz ◽  
P Parham
Keyword(s):  
Cell Surface ◽  
Heavy Chain ◽  
Protein Interactions ◽  
Stop Codon ◽  
Mrna Level ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Protein Protein Interactions ◽  
Peptide Binding Groove ◽  
Beta 2 Microglobulin

In comparison with HLA-A and -B, the protein products of the HLA-C locus are poorly characterized, in part because of their low level of expression at the cell surface. Here, we examine how protein-protein interactions during assembly and regulation of the mRNA level affect cell surface expression of HLA-C. We find that intrinsic properties of the HLA-C heavy chain proteins do not correlate with low cell surface expression: HLA-C heavy chains associate and dissociate with beta 2-microglobulin (beta 2m) at rates comparable to those found for HLA-A and -B, and increased competition for beta 2m does not alter the surface expression of HLA-C. From studies of chimeric genes spliced from the HLA-B7 and -Cw3 genes, we find that chimeric proteins containing the B7 peptide-binding groove can have low cell surface expression, suggesting that inefficiency in binding peptides is not the cause of low cell surface expression for HLA-C. The surface levels of HLA-A, -B, or -C in cells transfected with cDNA can be similar, implicating noncoding regions of HLA-C heavy chain genes in the regulation of surface expression. We find that HLA-C mRNA is expressed at lower levels than HLA-B mRNA and that this difference results from faster degradation of the HLA-C message. Experiments examining chimeric B7/Cw3 and B7/Cw6 genes suggest that a region determining low expression of HLA-C is to be found between the 3' end of exon 3 and a site in the 3' untranslated region, approximately 600 bases downstream of the translation stop codon.

scholarly journals Label-free methods for optical in vitro characterization of protein–protein interactions

2021 ◽  
Author(s):  
Fabian Soltermann ◽  
Weston B. Struwe ◽  
Philipp Kukura
Keyword(s):  
Protein Interactions ◽  
Label Free ◽  
Protein Protein Interactions ◽  
Cellular Processes ◽  
P Protein ◽  
In Vitro Characterization ◽  
And Function

Protein–protein interactions are involved in the regulation and function of the majority of cellular processes.

scholarly journals Mechanisms of Melanocortin-2 Receptor (MC2R) Internalization and Recycling in Human Embryonic Kidney (HEK) Cells: Identification of Key Ser/Thr (S/T) Amino Acids

2011 ◽  
Vol 25 (11) ◽  
pp. 1961-1977 ◽  
Author(s):  
Simon Roy ◽  
Sébastien Jean Roy ◽  
Sandra Pinard ◽  
Louis-Daniel Taillefer ◽  
Mohamed Rached ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Molecular Mechanisms ◽  
Surface Expression ◽  
Receptor Internalization ◽  
Cell Surface Expression ◽  
Target Cells ◽  
Camp Accumulation ◽  
Human Embryonic Kidney ◽  
And Function

Abstract ACTH is the most important stimulus of the adrenal cortex. The precise molecular mechanisms underlying the ACTH response are not yet clarified. The functional ACTH receptor includes melanocortin-2 receptor (MC2R) and MC2R accessory proteins (MRAP). In human embryonic kidney 293/Flp recombinase target cells expressing MC2R, MRAP1 isoforms, and MRAP2, we found that ACTH induced a concentration-dependent and arrestin-, clathrin-, and dynamin-dependent MC2R/MRAP1 internalization, followed by intracellular colocalization with Rab (Ras-like small guanosine triphosphate enzyme)4-, Rab5-, and Rab11-positive recycling endosomes. Preincubation of cells with monensin and brefeldin A revealed that 28% of the internalized receptors were recycled back to the plasma membrane and participated in total accumulation of cAMP. Moreover, certain intracellular Ser and Thr (S/T) residues of MC2R were found to play important roles not only in plasma membrane targeting and function but also in promoting receptor internalization. The S/T residues T131, S140, T204, and S280 were involved in MRAP1-independent cell-surface MC2R expression. Other mutants (S140A, S208A, and S202D) had lower cell-surface expressions in absence of MRAPβ. In addition, T143A and T147D drastically impaired cell-surface expression and function, whereas T131A, T131D, and S280D abrogated MC2R internalization. Thus, the modification of MC2R intracellular S/T residues may positively or negatively regulate its plasma membrane expression and the capacity of ACTH to induce cAMP accumulation. Mutations of T131, T143, T147, and S280 into either A or D had major repercussions on cell-surface expression, cAMP accumulation, and/or internalization parameters, pointing mostly to the second intracellular loop as being crucial for MC2R expression and functional regulation.

scholarly journals p53 Forms Redox-Dependent Protein–Protein Interactions through Cysteine 277

Antioxidants ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 1578
Author(s):  
Tao Shi ◽  
Paulien E. Polderman ◽  
Marc Pagès-Gallego ◽  
Robert M. van Es ◽  
Harmjan R. Vos ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Fine Tuning ◽  
Protein Protein Interactions ◽  
Post Translational Modification ◽  
Cysteine Oxidation ◽  
Interaction Partners ◽  
Dependent Protein ◽  
And Function

Reversible cysteine oxidation plays an essential role in redox signaling by reversibly altering protein structure and function. Cysteine oxidation may lead to intra- and intermolecular disulfide formation, and the latter can drastically stabilize protein–protein interactions in a more oxidizing milieu. The activity of the tumor suppressor p53 is regulated at multiple levels, including various post-translational modification (PTM) and protein–protein interactions. In the past few decades, p53 has been shown to be a redox-sensitive protein, and undergoes reversible cysteine oxidation both in vitro and in vivo. It is not clear, however, whether p53 also forms intermolecular disulfides with interacting proteins and whether these redox-dependent interactions contribute to the regulation of p53. In the present study, by combining (co-)immunoprecipitation, quantitative mass spectrometry and Western blot we found that p53 forms disulfide-dependent interactions with several proteins under oxidizing conditions. Cysteine 277 is required for most of the disulfide-dependent interactions of p53, including those with 14-3-3q and 53BP1. These interaction partners may play a role in fine-tuning p53 activity under oxidizing conditions.

scholarly journals Protein interaction landscapes revealed by advanced in vivo cross-linking–mass spectrometry

2021 ◽  
Vol 118 (32) ◽  
pp. e2023360118
Author(s):  
Andrew Wheat ◽  
Clinton Yu ◽  
Xiaorong Wang ◽  
Anthony M. Burke ◽  
Ilan E. Chemmama ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Cross Linking ◽  
Technological Advancement ◽  
Protein Protein Interactions ◽  
Hek 293 ◽  
293 Cells ◽  
And Function

Defining protein–protein interactions (PPIs) in their native environment is crucial to understanding protein structure and function. Cross-linking–mass spectrometry (XL-MS) has proven effective in capturing PPIs in living cells; however, the proteome coverage remains limited. Here, we have developed a robust in vivo XL-MS platform to facilitate in-depth PPI mapping by integrating a multifunctional MS-cleavable cross-linker with sample preparation strategies and high-resolution MS. The advancement of click chemistry–based enrichment significantly enhanced the detection of cross-linked peptides for proteome-wide analyses. This platform enabled the identification of 13,904 unique lysine–lysine linkages from in vivo cross-linked HEK 293 cells, permitting construction of the largest in vivo PPI network to date, comprising 6,439 interactions among 2,484 proteins. These results allowed us to generate a highly detailed yet panoramic portrait of human interactomes associated with diverse cellular pathways. The strategy presented here signifies a technological advancement for in vivo PPI mapping at the systems level and can be generalized for charting protein interaction landscapes in any organisms.

scholarly journals Modulation of hippocampal network oscillation by PICK1-dependent cell surface expression of mGlu3 receptors

2021 ◽  
Author(s):  
Pola Tuduri ◽  
Nathalie Bouquier ◽  
Benoit Girard ◽  
Enora Moutin ◽  
Maxime Thouaye ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Hippocampal Slices ◽  
Pdz Domain ◽  
Surface Expression ◽  
Theta Oscillations ◽  
Cell Surface Expression ◽  
Hippocampal Theta

mGlu3 receptors control the sleep/wake architecture which plays a role in the glutamatergic pathophysiology of schizophrenia. Interestingly, mGlu3 receptors expression is decreased in the brain of schizophrenic patients. However, little is known about the molecular mechanisms regulating mGlu3 receptors at the cell membrane. Subcellular receptor localization is strongly dependent on protein-protein interactions. Here we show that mGlu3 interacts with PICK1 and that their binding is important for receptor surface expression and function. Disruption of their interaction via an mGlu3 C-terminal mimicking peptide or an inhibitor of the PDZ domain of PICK1 altered the functional expression of mGlu3 receptors. Consequently, we investigated whether disruption of the mGlu3-PICK1 interaction affects hippocampal theta oscillations in vitro and in vivo. We found a decreased frequency of theta oscillations in organotypic hippocampal slices, similar to what previously observed in mGlu3 -/- mice. In addition, hippocampal theta power was reduced during REM sleep, NREM sleep and wake states after intra-ventricular administration of the mGlu3 C-terminal mimicking peptide. Targeting the mGlu3-PICK1 complex could thus be relevant to the pathophysiology of schizophrenia.

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