Abstract P803: Pituitary Adenylate Cyclase-Activating Polypeptide via Cyclic-AMP Inhibits Nogo-A by Internalizing Its Receptor NgR1

Introduction: After a stroke, axonal regeneration is inhibited by diverse axonal growth inhibitors, such as Nogo-A. They bind to the Nogo-A receptor 1 (NgR1) and induce the collapse of growth cones and inhibit neurite outgrowth. Since NgR1 is the receptor for a variety of axonal growth inhibitors, it is a crucial target for the prevention of axonal growth inhibition. Pituitary adenylate cyclase-activating polypeptide (PACAP) has neuroprotective and neurotrophic activities and increases neuritogenesis and synaptic plasticity. It enhances functional recovery after stroke in various animal models. Methods: Neuroscreen-1 (NS-1) cells were selected for this study as they produce rapid and robust neurite outgrowth with NGF. Cell surface NgR1 was detected using the indirect immunofluorescence method. The internalization of NgR1 was quantitated using the biotinylation method and Western immunoblotting. Results: Using the indirect immunofluorescence method, we found that PACAP (PACAP-38) induced a rapid decrease in the cell surface expression of NgR1 in NS-1 cells. The biotinylation method revealed that PACAP induced the internalization of NgR1. This internalization of NgR1 was blocked by pretreatment of NS-1 cells with SQ 22536, an inhibitor for adenylate cyclase, suggesting that cAMP plays a crucial role in the internalization of NgR1. The protein kinase A (PKA)-specific inhibitor KT5720 did not block PACAP-induced NgR1 internalization, whereas the exchange protein directly activated by cAMP (Epac)-specific inhibitor ESI-09 blocked this internalization. Collectively, this data suggests that PACAP-induced NgR1 internalization is independent of PKA but is dependent on Epac. The PACAP-induced decrease in cell surface expression of NgR1 and its internalization desensitized NS-1 cells to Nogo-66-induced growth cone collapse and enhanced neuritogenesis. Conclusion: Cyclic-AMP and Epac are involved in the PACAP-induced desensitization of neuronal cells to Nogo-A and increase in neuritogenesis. Since PACAP crosses the blood-brain barrier, it may be a useful therapeutic agent to overcome axonal growth inhibitors and enhance functional recovery after stroke.

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Abstract WP141: Cyclic-AMP Induces Nogo-A Receptor NgR1 Internalization and Inhibits Nogo-A-Mediated Collapse of Growth Cone

Stroke ◽

10.1161/str.51.suppl_1.wp141 ◽

2020 ◽

Vol 51 (Suppl_1) ◽

Author(s):

Rayudu Gopalakrishna ◽

Angela Zhu ◽

Andrew Oh ◽

Julie Nguyen ◽

Charlotte Lin ◽

...

Keyword(s):

Cell Surface ◽

Growth Cone ◽

Axonal Regeneration ◽

Axonal Growth ◽

Surface Expression ◽

Cell Surface Expression ◽

Intracellular Camp ◽

Growth Inhibitors ◽

Intracellular Compartment ◽

Growth Cone Collapse

Recovery of stroke and neuronal injuries requires the promotion of axonal regeneration from the remaining neurons. However, axonal regeneration is inhibited by diverse axonal growth inhibitors, such as Nogo-A. C-terminal domain of Nogo-A, Nogo-66 binds to the Nogo-A receptor 1 (NgR1) and induces the collapse of growth cones and inhibition of neurite outgrowth. NgR1 is also a receptor for additional axonal growth inhibitors. In this study, by using indirect immunofluorescence and biotinylation method, we have found that a cell-permeable cAMP analog (dibutyryl-cAMP) and other intracellular cAMP-elevating agents, such as forskolin, which directly activates adenylyl cyclase, and rolipram, which inhibits cyclic nucleotide phosphodiesterase, all induced rapid internalization of the cell surface NgR1 in Neuroscreen-1 (NS-1) cells. This endocytosis of NgR1 is lipid raft mediated. These cAMP-elevating agents induced a reversible distribution of NgR1 between the cell surface and intracellular compartment; NgR1 distributed to the cell surface at low levels of cAMP and distributed to an intracellular compartment at high levels of cAMP. Using pharmacological activators and inhibitors of protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac), we found that NgR1 internalization is independent of PKA but dependent on Epac. There is a correlation between the decrease in cell surface expression of NgR1 decreased sensitivity of NS-1 cells to Nogo-66-induced growth cone collapse. Therefore, besides axonal growth inhibitors affecting neurons, neurons by themselves self-regulate their own sensitivity to extracellular cues such as axonal growth inhibitors. This normal cellular regulatory mechanism may be therapeutically applied to overcome axonal growth inhibitors and enhance functional recovery after stroke and neuronal injuries.

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Obesity-Linked Variants of Melanocortin-4 Receptor Are Misfolded in the Endoplasmic Reticulum and Can Be Rescued to the Cell Surface by a Chemical Chaperone

Endocrine Reviews ◽

10.1210/edrv.31.4.9985 ◽

2010 ◽

Vol 31 (4) ◽

pp. 605-605

Author(s):

Susana Granell ◽

Sameer Mohammad ◽

Ramanagouda Ramanagoudr-Bhojappa ◽

Giulia Baldini

Keyword(s):

Endoplasmic Reticulum ◽

Cell Surface ◽

Er Stress ◽

Binding Protein ◽

Intracellular Localization ◽

Specific Inhibitor ◽

Surface Expression ◽

Cell Surface Expression ◽

Chemical Chaperone ◽

Melanocortin 4 Receptor

Abstract Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor expressed in the brain where it controls food intake. Many obesity-linked MC4R variants are poorly expressed at the plasma membrane and are retained intracellularly. We have studied the intracellular localization of four obesity-linked MC4R variants, P78L, R165W, I316S, and I317T, in immortalized neurons. We find that these variants are all retained in the endoplasmic reticulum (ER), are ubiquitinated to a greater extent than the wild-type (wt) receptor, and induce ER stress with increased levels of ER chaperones as compared with wt-MC4R and appearance of CCAAT/enhancer-binding protein homologous protein. Expression of the X-box-binding-protein-1 with selective activation of a protective branch of the unfolded protein response did not have any effect on the cell surface expression of MC4R-I316S. Conversely, the pharmacological chaperone 4-phenyl butyric acid (PBA) increased the cell surface expression of wt-MC4R, MC4R-I316S, and I317T by more than 40%. PBA decreased ubiquitination of MC4R-I316S and prevented ER stress induced by expression of the mutant, suggesting that the drug functions to promote MC4R folding. MC4R-I316S rescued to the cell surface is functional, with a 52% increase in agonist-induced cAMP production, as compared with untreated cells. Also direct inhibition of wt-MC4R and MC4R-I316S ubiquitination by a specific inhibitor of the ubiquitin-activating enzyme 1 increased by approximately 40% the expression of the receptors at the cell surface, and the effects of PBA and ubiquitin-activating enzyme 1 were additive. These data offer a cell-based rationale that drugs that improve MC4R folding or decrease ER-associated degradation of the receptor may function to treat some forms of hereditary obesity.

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Cyclic AMP Increases Cell Surface Expression of Functional Na,K-ATPase Units in Mammalian Cortical Collecting Duct Principal Cells

Molecular Biology of the Cell ◽

10.1091/mbc.12.2.255 ◽

2001 ◽

Vol 12 (2) ◽

pp. 255-264 ◽

Cited By ~ 56

Author(s):

Sandrine Gonin ◽

Georges Deschênes ◽

Frank Roger ◽

Marcelle Bens ◽

Pierre-Yves Martin ◽

...

Keyword(s):

Cyclic Amp ◽

Cell Surface ◽

Atpase Activity ◽

Cultured Cells ◽

Collecting Duct ◽

Brefeldin A ◽

Surface Expression ◽

Cell Surface Expression ◽

Cortical Collecting Duct ◽

Expression And Activity

Cyclic AMP (cAMP) stimulates the transport of Na+ and Na,K-ATPase activity in the renal cortical collecting duct (CCD). The aim of this study was to investigate the mechanism whereby cAMP stimulates the Na,K-ATPase activity in microdissected rat CCDs and cultured mouse mpkCCDc14 collecting duct cells. db-cAMP (10−3 M) stimulated by 2-fold the activity of Na,K-ATPase from rat CCDs as well as the ouabain-sensitive component of 86Rb+ uptake by rat CCDs (1.7-fold) and cultured mouse CCD cells (1.5-fold). Pretreatment of rat CCDs with saponin increased the total Na,K-ATPase activity without further stimulation by db-cAMP. Western blotting performed after a biotinylation procedure revealed that db-cAMP increased the amount of Na,K-ATPase at the cell surface in both intact rat CCDs (1.7-fold) and cultured cells (1.3-fold), and that this increase was not related to changes in Na,K-ATPase internalization. Brefeldin A and low temperature (20°C) prevented both the db-cAMP-dependent increase in cell surface expression and activity of Na,K-ATPase in both intact rat CCDs and cultured cells. Pretreatment with the intracellular Ca2+chelator bis-(o-aminophenoxy)-N,N,N′,N′-tetraacetic acid also blunted the increment in cell surface expression and activity of Na,K-ATPase caused by db-cAMP. In conclusion, these results strongly suggest that the cAMP-dependent stimulation of Na,K-ATPase activity in CCD results from the translocation of active pump units from an intracellular compartment to the plasma membrane.

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Faculty Opinions recommendation of Assembly and cell surface expression of TAP-independent, chloroquine-sensitive and interferon-gamma-inducible class I MHC complexes in transformed fibroblast cell lines are regulated by tapasin.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1009214.121258 ◽

2002 ◽

Author(s):

Tim Elliott

Keyword(s):

Cell Surface ◽

Cell Lines ◽

Interferon Gamma ◽

Fibroblast Cell ◽

Surface Expression ◽

Class I ◽

Cell Surface Expression ◽

Class I Mhc ◽

Fibroblast Cell Lines

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Faculty Opinions recommendation of Mechanism of cell surface expression of the Streptococcus mitis platelet binding proteins PblA and PblB.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1082761.535676 ◽

2007 ◽

Author(s):

Tim Mitchell

Keyword(s):

Cell Surface ◽

Binding Proteins ◽

Surface Expression ◽

Cell Surface Expression ◽

Streptococcus Mitis ◽

Platelet Binding

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Persistent HIV Transcription Induces Sialyl-Lewis-X Glyco-Antigen Cell-Surface Expression

SSRN Electronic Journal ◽

10.2139/ssrn.3565035 ◽

2020 ◽

Author(s):

Florent Colomb ◽

Leila B. Giron ◽

Leticia Kuri Cervantes ◽

Tongcui Ma ◽

Samson Adeniji ◽

...

Keyword(s):

Cell Surface ◽

Surface Expression ◽

Sialyl Lewis X ◽

Cell Surface Expression ◽

Lewis X ◽

Hiv Transcription ◽

Sialyl Lewis

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Influence of β-D-mannuronic acid, as a new member of non-steroidal anti-inflammatory drugs family, on expression pattern of chemokines and their receptors in rheumatoid arthritis

Current Drug Discovery Technologies ◽

10.2174/1570163816666191023103118 ◽

2019 ◽

Vol 16 ◽

Author(s):

Mona Aslani ◽

Arman Ahmadzadeh ◽

Zahra Aghazadeh ◽

Majid Zaki-Dizaji ◽

Laleh Sharifi ◽

...

Keyword(s):

Cell Surface ◽

Mrna Expression ◽

Surface Expression ◽

Phase Iii ◽

Cell Surface Expression ◽

Optimum Dose ◽

High Dose ◽

Mannuronic Acid ◽

Anti Inflammatory ◽

High Doses

Background: : Based on the encouraging results of phase III clinical trial of β-D-mannuronic acid (M2000) (as a new anti-inflammatory drug) in patients with RA, in this study, we aimed to evaluate the effects of this drug on the expression of chemokines and their receptors in PBMCs of RA patients. Methods:: PBMCs of RA patients and healthy controls were separated and the patients' cells were treated with low, moderate and high doses (5, 25 and 50 μg/mL) of M2000 and optimum dose (1 μg/mL) of diclofenac, as a control in RPMI-1640 medium. Real-time PCR was used for evaluating the mRNA expression of CXCR3, CXCR4, CCR2, CCR5 and CCL2/MCP-1. Cell surface expression of CCR2 was investigated using flow cytometry. Results:: CCR5 mRNA expression reduced significantly, after treatment of the patients' cells with all three doses of M2000 and optimum dose of diclofenac. CXCR3 mRNA expression down-regulated significantly followed by treatment of these cells with moderate and high doses of M2000 and optimum dose of diclofenac. CXCR4 mRNA expression declined significantly after treatment of these cells with moderate and high doses of M2000. CCL2 mRNA expression significantly reduced only followed by treatment of these cells with high dose of M2000, whereas, mRNA and cell surface expressions of CCR2 diminished significantly followed by treatment of these cells with high dose of M2000 and optimum dose of diclofenac. Conclusion:: According to our results, M2000 through the down-regulation of chemokines and their receptors may restrict the infiltration of immune cells into the synovium.

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