Many G-protein-coupled receptors carry C-terminal ligand motifs for PSD-95/discs large/ZO-1 (PDZ) domains; via interaction with PDZ domain-containing scaffold proteins, this allows for integration of ...receptors into signaling complexes. However, the presence of PDZ domain proteins attached to intracellular membranes suggests that PDZ-type interactions may also contribute to subcellular sorting of receptors. The protein interacting specifically with Tc10 (PIST; also known as GOPC) is a trans-Golgi-associated protein that interacts through its single PDZ domain with a variety of cell surface receptors. Here we show that PIST controls trafficking of the interacting β1-adrenergic receptor both in the anterograde, biosynthetic pathway and during postendocytic recycling. Overexpression and knockdown experiments show that PIST leads to retention of the receptor in the trans-Golgi network (TGN), to the effect that overexpressed PIST reduces activation of the MAPK pathway by β1-adrenergic receptor (β1AR) agonists. Receptors can be released from retention in the TGN by coexpression of the plasma membrane-associated scaffold PSD-95, which allows for transport of receptors to the plasma membrane. Stimulation of β1 receptors and activation of the cAMP pathway lead to relocation of PIST from the TGN to an endosome-like compartment. Here PIST colocalizes with SNX1 and the internalized β1AR and protects endocytosed receptors from lysosomal degradation. In agreement, β1AR levels are decreased in hippocampi of PIST-deficient mice. Our data suggest that PIST contributes to the fine-tuning of β1AR sorting both during biosynthetic and postendocytic trafficking.
Background: PIST/GOPC is a Golgi-associated protein that interacts with several G-protein-coupled receptors via its single PDZ domain.
Results: PIST retains β1-adrenergic receptors in intracellular compartments and interferes with receptor degradation after endocytosis.
Conclusion: PIST stabilizes the receptor in an intracellular compartment.
Significance: PDZ proteins associated with intracellular membranes confer specific features to the subcellular targeting of interacting receptors.
The gentamicin drug product is a complex mixture of numerous components, many of which have not individually undergone safety and efficacy assessments. This is in contrast to the majority of ...medicines that require rigorous characterizations of trace impurities and are dosed as single components. In gentamicin, four components, known as gentamicin congeners C1, C1a, C2, and C2a, comprise the majority of the mixture. A liquid chromatography-mass spectroscopy analysis revealed that the relative abundances of each gentamicin congener in commercial formulations can vary up to 1.9-fold depending on the commercial source of the gentamicin. To determine if the gentamicin used for antibiotic susceptibility testing (AST) would be predictive of the microbiological activity of the product used to dose patients, the relative abundances of the four congeners contained on commercial AST disks were measured. It was found that the congener abundances on the commercial AST disks varied up to 4.1-fold. After purification of the four gentamicin congeners, similar potencies against bacterial strains lacking aminoglycoside-modifying enzymes (AMEs) were observed. However, the potency of the congeners against strains harboring a common AME differed up to 128-fold. Nephrotoxicity of the individual gentamicin congeners also differed significantly in cell-based and repeat-dose rat nephrotoxicity studies. Variations in the composition of commercial gentamicin products combined with toxicity differences between gentamicin congeners suggest that some gentamicin formulations may be more nephrotoxic. Our results also raise the concern that gentamicin susceptibility test results may not be predictive of patient outcomes and could lead to unexpected clinical treatment failures.
A fundamental and still largely unresolved question is how neurons achieve rapid delivery of selected signaling receptors throughout the elaborate dendritic arbor. Here we show that this requires a ...conserved sorting machinery called retromer. Retromer-associated endosomes are distributed within dendrites in ∼2 μm intervals and supply frequent membrane fusion events into the dendritic shaft domain immediately adjacent to (<300 nm from) the donor endosome and typically without full endosome discharge. Retromer-associated endosomes contain β-adrenergic receptors as well as ionotropic glutamate receptors, and retromer knockdown reduces extrasynaptic insertion of adrenergic receptors as well as functional expression of AMPA and NMDA receptors at synapses. We propose that retromer supports a broadly distributed network of plasma membrane delivery to dendrites, organized in micron-scale axial territories to render essentially all regions of the postsynaptic surface within rapid diffusion distance of a local exocytic event.
Clathrin, a cytosolic protein composed of heavy and light chain subunits, assembles into a vesicle coat, controlling receptor-mediated endocytosis. To establish clathrin light chain (CLC) function in ...vivo, we engineered mice lacking CLCa, the major CLC isoform in B lymphocytes, generating animals with CLC-deficient B cells. In CLCa-null mice, the germinal centers have fewer B cells, and they are enriched for IgA-producing cells. This enhanced switch to IgA production in the absence of CLCa was attributable to increased transforming growth factor β receptor 2 (TGFβR2) signaling resulting from defective endocytosis. Internalization of C-X-C chemokine receptor 4 (CXCR4), but not CXCR5, was affected in CLCa-null B cells, and CLC depletion from cell lines affected endocytosis of the δ-opioid receptor, but not the β2-adrenergic receptor, defining a role for CLCs in the uptake of a subset of signaling receptors. This instance of clathrin subunit deletion in vertebrates demonstrates that CLCs contribute to clathrin’s role in vivo by influencing cargo selectivity, a function previously assigned exclusively to adaptor molecules.
Synaptic activity is modulated by the activation of neuromodulator receptors present in dendrites of neurons. The majority of neuromodulator receptors are G protein coupled receptors (GPCRs), in ...which membrane trafficking regulates their activities. Membrane trafficking of neuromodulator receptors and their signaling occurs on a rapid time scale and emerging studies indicate that neuromodulator receptors function not just from the plasma membrane but also from the endocytic compartments. Here, we describe a live cell imaging approach using spinning disk confocal microscopy to investigate the effect of neuromodulator receptor activation on synaptic activity by measuring calcium dynamics in primary rat striatal neurons. The advantages of spinning disk confocal microscopy and recent improvements in the genetically encoded calcium sensor, GCaMP6, provide an imaging approach to image both the receptor membrane trafficking to endocytic compartments, and calcium dynamics at a high spatial and temporal resolution. We believe this approach of imaging both the neuromodulator receptor membrane trafficking and synaptic activity using GCaMP6 is a powerful tool to address many questions regarding possible roles of membrane trafficking of neuromodulator receptors in synaptic activity.
AbstractAn extra-adrenal paraganglioma is an uncommon tumour that arises from the paraganglia associated with the autonomous nervous system. A paraganglioma arising in the sensory-somatic nervous ...system is extremely rare and clinically is easily confused with other neurogenic tumours. We describe a paraganglioma that arose in the median nerve of a 22-year-old woman.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The human genome contains genes encoding for over 40 different types of kinesin and kinesin-like proteins. Of these, the functions of 13 kinesins remain uncharacterized. In this study, we constructed ...a plasmid containing the ORF of KIF18B and revealed that the KIF18B message of approximately 3
kb is expressed in a tissue- and cell type-specific manners. A polypeptide of 842 amino acids was deduced from the ORF sequence. We identified another form of 873 amino acids which arises from alternative splicing at the C-terminal end. We also generated an anti-KIF18B antibody which detects a protein band of 120
kDa. Western analyses showed that the protein level of KIF18B is elevated at late G
2 through metaphase, very similar to cyclin B1. Immunocytochemical staining revealed that the KIF18B protein is present predominantly in the nucleus and to a lesser extent in the cytoplasm of interphase cells. During mitosis, most KIF18B was found to be closely associated with astral microtubules emanating from the spindle pole during prometaphase and metaphase. Meanwhile, KIF18B was not detected at anaphase and telophase, consistent with the Western blotting data. The nuclear localization signal was roughly determined by using several EGFP-tagged deletion mutants of KIF18B. Together, the expression of KIF18B is regulated in a cell cycle-dependent manner and therefore may play an important role(s) in cell division.
Intracellular transport is essential for cytoplasm organization, but mechanisms regulating transport are mostly unknown. In Xenopus melanophores, melanosome transport is regulated by cAMP-dependent ...protein kinase A (PKA) 1. Melanosome aggregation is triggered by melatonin, whereas dispersion is induced by melanocyte-stimulating hormone (MSH) 2. The action of hormones is mediated by cAMP: High cAMP in MSH-treated cells stimulates PKA, whereas low cAMP in melatonin-treated cells inhibits it. PKA activity is typically restricted to specific cell compartments by A-kinase anchoring proteins (AKAPs) 3. Recently, Rab32 has been implicated in protein trafficking to melanosomes 4 and shown to function as an AKAP on mitochondria 5. Here, we tested the hypothesis that Rab32 is involved in regulation of melanosome transport by PKA. We demonstrated that Rab32 is localized to the surface of melanosomes in a GTP-dependent manner and binds to the regulatory subunit RIIα of PKA. Both RIIα and Cβ subunits of PKA are required for transport regulation and are recruited to melanosomes by Rab32. Overexpression of wild-type Rab32, but not mutants unable to bind PKA or melanosomes, inhibits melanosome aggregation by melatonin. Therefore, in melanophores, Rab32 is a melanosome-specific AKAP that is essential for regulation of melanosome transport.
A nurse's character directly affects care quality. This study aimed to identify the concept of nurses' care-tailored character in clinical settings and to ascertain its attributes. A concept analysis ...was performed using the hybrid model. In the results, nurses' care-tailored character in clinical settings was defined as the conduct, behavior, and attitude toward performing as an expert and creating better interpersonal relationships for high-quality care. The attributes were divided into two dimensions. The professional dimension comprised responsibility, diligence, and temperance. The interpersonal dimension comprised trustworthiness, caring interactions, respect, and fairness. These findings can contribute to nurses modifying inappropriate behaviors and improving her/his own character for better patient care.
Intracellular transport is an essential for proper organization of the cytoplasm in every eukaryotic cell, but the mechanisms regulating transport are mostly unknown. In Xenopus melanophores, ...melanosome transport is regulated by cAMP-dependent protein kinase A (PKA). Melanosome transport toward the cell center is triggered by melatonin whereas melanosome dispersion throughout the cytoplasm is induced by melanocyte-stimulating hormone (MSH). The activity of hormones is mediated by cAMP: high cAMP in MSH-treated cells stimulates PKA, while low cAMP in melatonin-treated cells inhibits PKA. This work focuses on the understanding how PKA regulates melanosome transport in Xenopus melanophores. This study examines two questions. Firstly, how is a multi-functioning kinase, PKA, able to specifically regulate melanosome transport in Xenopus melanophores? PKA is involved in many cellular functions. Thus for PKA to specifically regulate melanosome transport, it requires mechanism that enhance its specificity. This study showed that PKA specificity is enhanced first by use of a specific isoform, Cβ/RIIα., Furthermore, Rab32 binding compartmentalizes PKA to melanosomes, thereby restricting its activity to the level of the melanosome. Secondly, how does PKA activation cause melanosome dispersion? To clarify the mechanism, we identified PKA substrates by two-dimensional gel analysis coupled to anti-phospho-PKA substrate immunoblots and tandem mass spectrometry (MS/MS). Among the identified PKA substrates, melanophilin, a part of tripartite complex on melanosomes that binds to Rab27a and recruits actin dependent motor myosin Va, was determined to be phospho-PKA substrate by Western blotting with phospho-PKA substrate specific antibody. By a combination of mutational and rescue assay, we showed that phosphomimic form of melanophilin, S205D, disrupts melanosome transport, whereas the non-phosphorylable form of melanophilin does not. The research presented here contributes to the understanding how PKA regulates melanosomes transport: first, by exploring the mechanism that enhance PKA specificity during melanosome transport; second, by identifying melanophilin as a PKA substrate involved in melanosome transport that activates dispersion.
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