GEC1 Interacts with the κ Opioid Receptor and Enhances Expression of the Receptor

Chen, Chongguang; Li, Jianguo; Chen, Yong; Huang, Peng; Wang, Yulin; Liu‐Chen, Lee‐Yuan

doi:10.1074/jbc.m509805200

Cited by 85 publications

(106 citation statements)

References 53 publications

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“…Interestingly, some escort proteins are responsible for increasing the cell surface localization of GPCRs by increasing the mature form of the receptors. For example, the glycosylation state of CRLR varies depending on its interaction with different RAMPs (29), GEC1 increases the mature form and the localization of the κ-opioid receptor in the plasma membrane (40), and MRAP2 increases the cell surface presence and maturation of the melanocortin-2 receptor (41). It has also been shown that the pseudosignal peptide of CRF 2α R inhibits the trafficking of the receptor to the plasma membrane by allowing its interaction with calnexin (25,42).…”

Section: Discussionmentioning

confidence: 99%

CRF binding protein facilitates the presence of CRF type 2α receptor on the cell surface

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Cerda

Pereira

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Corticotropin releasing factor binding protein (CRF-BP) was originally recognized as CRF sequestering protein. However, its differential subcellular localization in different brain nuclei suggests that CRF-BP may have additional functions. There is evidence that CRF-BP potentiates CRF and urocortin 1 actions through CRF type 2 receptors (CRF 2 R). CRF 2 R is a G protein-coupled receptor (GPCR) that is found mainly intracellularly as most GPCRs. The access of GPCRs to the cell surface is tightly regulated by escort proteins. We hypothesized that CRF-BP binds to CRF 2 R, exerting an escort protein role. We analyzed the colocalization of CRF-BP and CRF 2 R in cultured rat mesencephalic neurons, and the localization and interaction of heterologous expressed CRF-BP and CRF 2α R in yeast, human embryonic kidney 293, and rat pheochromocytoma 12 cells. Our results showed that CRF-BP and CRF 2 R naturally colocalize in the neurites of cultured mesencephalic neurons. Heterologous expression of each protein showed that CRF-BP was localized mainly in secretory granules and CRF 2α R in the endoplasmic reticulum. In contrast, CRF-BP and CRF 2α R colocalized when both proteins are coexpressed. Here we show that CRF-BP physically interacts with the CRF 2α R but not the CRF 2β R isoform, increasing CRF 2α R on the cell surface. Thus, CRF-BP emerges as a GPCR escort protein increasing the understanding of GPCR trafficking.T he corticotropin releasing factor (CRF) system plays a key role in the response and adaptation to stressful stimuli (1, 2) and in the interaction between stress and addiction (3). The CRF system acts on the hypothalamic-pituitary-adrenal axis (4, 5) and in different brain regions (1, 6). The CRF system includes four peptides, CRF type 1 (CRF 1 R) and type 2 (CRF 2 R), G protein-coupled receptors (GPCRs) (6-8), and CRF binding protein (CRF-BP) (9). CRF-BP was described as a circulating polypeptide in pregnant women (10, 11). CRF-BP binds CRF and urocortin with high affinity (12). Different functions have been proposed for CRF-BP (13). On one hand, CRF-BP exerts an inhibitory role by sequestering CRF peptide (9, 14-16). On other hand, a facilitatory role of CRF-BP on CRF-dependent neuronal plasticity depending on CRF 2 R in the rat ventral tegmental area (VTA) has been described (17, 18). Recently, it has been shown that CRF-BP and CRF 2 R are important for ethanol binge drinking behavior (19). The anatomical evidence showing that CRF-BP has different subcellular distribution depending on the neuronal context (20) further supports several roles for CRF-BP.Three isoforms of CRF 2 R have been reported, the α isoform being the most expressed in the brain (21). CRF 2α R is localized intracellularly in neurons of the rat dorsal raphe nucleus and that exposure to acute (22) and repeated stress (23) increases its presence in the plasma membrane. CRF 2α R overexpressed in human embryonic kidney (HEK293T) cells is associated with the endoplasmic reticulum (ER) (24). Schulz et al. (25) showed that CRF 2α R is retained in...

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Section: Discussionmentioning

confidence: 99%

CRF binding protein facilitates the presence of CRF type 2α receptor on the cell surface

Slater

Cerda

Pereira

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Transfection with luciferase siRNA, used as the control, did not affect the hKOPR level compared with no transfection (data not shown). Our previous data showed that FLAG-hKOR migrated as two bands of M r 55 (mature receptor) and 45 (immature receptor) (7). Therefore, 14-3-3 may be involved in regulation of export of hKOPR along the ER-Golgi-plasma membranes in N2A-FLAG-hKOPR cells.…”

Section: Knockdown Of 14-3-3 Decreased Expression Of the Hkopr In N2amentioning

confidence: 99%

“…Experiments were performed using our previously published procedure (7). Cells were washed with DMEM without L-Met and L-Cys and preincubated in the same medium for 1 h at 37°C.…”

Section: S]met/cys Labeling Of the Hkopr And Pulse-chase Experimentsmentioning

confidence: 99%

“…Immunoblotting was performed to examine the expression of FLAG-hKOPR proteins as we described previously (7,26). Cells were solubilized with 2ϫ Laemmli sample buffer (4% SDS, 0.1 M DTT, 20% glycerol, 62.5 mM Tris, and bromphenol blue, pH6.8), subjected to 8% Tricine-SDS-PAGE, and transferred onto PDVF Immobilon membranes.…”

Section: Immunoblotting Of Flag-hkoprmentioning

confidence: 99%

“…4). We have demonstrated that the human KOPR (hKOPR) interacts with NHERF-1/EBP50 (5,6) and GEC1 (7) and that these interactions play important roles in signal transduction and trafficking of the KOPR in the internalization and export pathways, respectively. Using proteomic analyses, we found that 14-3-3 was one of the proteins co-immunoprecipitated with FLAG-tagged hKOPR from the extract of Neuro2a (N2A) cells stably expressing the FLAG-hKOPR (N2A-FLAGhKOPR cells).…”

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confidence: 99%

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14-3-3ζ Protein Regulates Anterograde Transport of the Human κ-Opioid Receptor (hKOPR)

Chen

Huang

et al. 2012

Journal of Biological Chemistry

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Structural analysis of the human cannabinoid receptor one carboxyl‐terminus identifies two amphipathic helices

et al. 2009

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Recent research has implicated the C-terminus of G-protein coupled receptors in key events such as receptor activation and subsequent intracellular sorting, yet obtaining structural information of the entire C-tail has proven a formidable task. Here, a peptide corresponding to the full-length C-tail of the human CB1 receptor (residues 400–472) was expressed in E.coli and purified in a soluble form. Circular dichroism (CD) spectroscopy revealed that the peptide adopts an α-helical conformation in negatively charged and zwitterionic detergents (48–51% and 36–38%, respectively), whereas it exhibited the CD signature of unordered structure at low concentration in aqueous solution. Interestingly, 27% helicity was displayed at high peptide concentration suggesting that self-association induces helix formation in the absence of a membrane mimetic. NMR spectroscopy of the doubly labeled (15N- and 13C-) C-terminus in dodecylphosphocholine (DPC) identified two amphipathic α-helical domains. The first domain, S401-F412, corresponds to the helix 8 common to G protein-coupled receptors while the second domain, A440-M461, is a newly identified structural motif in the distal region of the carboxyl-terminus of the receptor. Molecular modeling of the C-tail in DPC indicates that both helices lie parallel to the plane of the membrane with their hydrophobic and hydrophilic faces poised for critical interactions.

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GEC1 Interacts with the κ Opioid Receptor and Enhances Expression of the Receptor

Cited by 85 publications

References 53 publications

CRF binding protein facilitates the presence of CRF type 2α receptor on the cell surface

CRF binding protein facilitates the presence of CRF type 2α receptor on the cell surface

14-3-3ζ Protein Regulates Anterograde Transport of the Human κ-Opioid Receptor (hKOPR)

Structural analysis of the human cannabinoid receptor one carboxyl‐terminus identifies two amphipathic helices

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