Clathrin Heavy Chain Knockdown Impacts CXCR4 Signaling and Post-translational Modification

DeNies, Maxwell S.; Rosselli‐Murai, Luciana K.; Schnell, Santiago; Liu, Allen

doi:10.3389/fcell.2019.00077

Cited by 10 publications

(32 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To investigate this, we examined the localization of CXCR4 PTM during receptor signaling. As previously observed 33 , upon CXCL12 addition UMB2 detection was drastically reduced at both plasma membrane and intracellular compartments, suggesting that both plasma membrane and internal pools of CXCR4 are post-translationally modified ( Fig. 2a).…”

Section: Introductionsupporting

confidence: 82%

“…WT CXCR4 was generated as previously described 33 . K3R and K3R/Q mutant receptors were generated by PCR mutagenesis of WT CXCR4 in the pLVX plasmid using the NEB Quick-change mutagenesis kit.…”

Section: Dna Constructs and Stable Cell Linesmentioning

confidence: 99%

“…Flow cytometry experiments for plasma membrane receptor labeling were conducted as previously described 33 . For intracellular staining, cells were first disassociated using 50 µM EDTA in Ca 2+ -free PBS and fix for 10 min in 4% paraformaldehyde at room temperature.…”

Section: Flow Cytometry Experimentsmentioning

confidence: 99%

“…To do so, we needed a strategy to robustly detect CXCR4 PTM as well as a method to modulate receptor localization. We previously established the use of a monoclonal CXCR4 antibody (UMB2) as a robust tool to study CXCR4 PTM 33 . This commercially available antibody is raised against the C-terminus of the receptor, and upon CXCL12 stimulus quickly loses its ability to detect CXCR4 due to receptor PTM 33 .…”

Section: Introductionmentioning

confidence: 99%

“…1a). We chose RPE cells to study CXCR4 overexpression because they do not have appreciable endogenous CXCR4 expression, are unresponsive to CXCL12, and were previously established as a cell culture model to study CXCR4 biology 33 . We found that by mutating C-terminal lysine residues to arginine (K3R), CXCR4 plasma membrane localization was reduced by more than 50% (Fig.…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

β-arrestin mediates communication between plasma membrane and intracellular GPCRs to regulate signaling

DeNies

Smrcka

Schnell

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

It has become increasingly apparent that G protein-coupled receptor (GPCR) localization is a master regulator of cell signaling. However, the molecular mechanisms involved in this process are not well understood. To date, observations of intracellular GPCR activation can be organized into two categories: a dependence on OCT3 cationic channelpermeable ligands or the necessity of endocytic trafficking. Using CXC chemokine receptor 4 (CXCR4) as a model, we identified a third mechanism of intracellular GPCR signaling. We show that independent of membrane permeable ligands and endocytosis, upon stimulation, plasma membrane and internal pools of CXCR4 are post-translationally modified and collectively regulate EGR1 transcription. We found that β-arrestin-1 (arrestin 2) is necessary to mediate communication between plasma membrane and internal pools of CXCR4. Notably, these observations may explain that while CXCR4 overexpression is highly correlated with cancer metastasis and mortality, plasma membrane localization is not. Together these data support a model were a small initial pool of plasma membrane-localized GPCRs are capable of activating internal receptordependent signaling events.3 While extracellular inputs, cell membrane receptors, and resulting transcriptional programs are diverse, many receptor-signaling events converge to a reduced number of signaling hubs. Cellular mechanisms that mediate this process as well as strategies to control these actions remain outstanding questions. Over the last decade, we have learned that GPCR spatiotemporal signaling is one mechanism used by cells to translate diverse environmental information into actionable intracellular decisions while using seemingly redundant signaling cascades 1 . Extensive research has illustrated that GPCRs elicit distinct signaling events at different plasma membrane micro-domains as well as endocytic compartments that are important for cell physiology and disease pathogenesis [1][2][3][4][5][6][7][8][9] . These studies support a model where the location, in addition to magnitude, of a signaling event is important for cellular decision-making. Others have shown that GPCR site-specific post-translational modifications (PTMs) modulate adaptor protein recruitment, GPCR localization, and consequently receptor signaling events 10-12 .Together these observations motivated us to reexamine some confounding observations pertaining to the relationship of receptor localization, PTM, and signaling for CXCR4.CXCR4 is a type 1 GPCR that regulates a variety of biological processes such as cell migration, embryogenesis, and immune cell homeostasis 10,[13][14][15][16] . It is deregulated in 23 different cancers and overexpression is often correlated with metastasis and mortality 17-21 . However, surprisingly, plasma membrane expression is not correlated with metastasis 21 and in some cancer tumor specimens as well as cell culture models, samples with poor CXCR4 plasma membrane localization remain responsive to CXCR4 agonist 22-26 . CXCR4 is activated by a highly rec...

show abstract

Section: Introductionsupporting

confidence: 82%

Section: Dna Constructs and Stable Cell Linesmentioning

confidence: 99%

Section: Flow Cytometry Experimentsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations