ADP-ribosylation factor 6 regulates endothelin-1-induced lipolysis in adipocytes

Journal of Biological Chemistry

2014

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Background: The subcellular localization and cellular functions of EFA6R are unknown.Results: EFA6R requires dual targeting through the PH and CC domains to localize to the plasma membrane and function as a GEF.Conclusion: Plasma membrane-localized EFA6R stimulates actin reorganization through ARF6 activation.Significance: This study provides insight into the localization and cellular functions of EFA6R.

Section: Methodsmentioning

confidence: 99%

Exchange Factor EFA6R Requires C-terminal Targeting to the Plasma Membrane to Promote Cytoskeletal Rearrangement through the Activation of ADP-ribosylation Factor 6 (ARF6)

Journal of Biological Chemistry

2014

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“…Membranes were washed and then incubated with the HRP-conjugated anti-mouse secondary antibody (diluted 1:2500 in blocking buffer) for 1 h at room temperature. Membranes were then incubated in ECL select substrate and bands visualised using the ChemiDocTM XRS system (Bio-Rad)3435. Blots probed with the anti-GFP mouse antibody were stripped with western blot stripping buffer (Thermo Scientific) and reprobed with the anti-VSVG rabbit antibody (diluted 1:1000) in blocking buffer) and the HRP-conjugated anti-rabbit secondary antibody (diluted 1:2500 in blocking buffer) as described above.…”

Section: Methodsmentioning

confidence: 99%

“…Mutation of the SP (Ala21Arg) to prevent its cleavage has been shown to result in retention of the GLP-1R within the ER. Further, a mutation of Glu34 was shown to facilitate GLP-1R cell surface expression when the SP was deleted14. The aa sequence following the SP in the GLP-1R, Gly27-Trp 39 , is relatively hydrophobic and it has previously been suggested that this region may be recognised by the SRP for synthesis of the receptor1415.…”

mentioning

confidence: 99%

The regions within the N-terminus critical for human glucagon like peptide-1 receptor (hGLP-1R) cell Surface expression

Thompson

2014

Sci Rep

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The hGLP-1R is a target for the treatment of type 2 diabetes and belongs to the class B family of GPCRs. Like other class B GPCRs, the GLP-1R contains an N-terminal signal peptide (SP) and undergoes N-linked glycosylation, which are important for its trafficking and maturation. This study analysed the role of the SP, the hydrophobic region after the SP (HRASP), glycosylation and the conserved residues within the N-terminus in GLP-1R trafficking. HGLP-1R targeted to the cell surface showed no SP, and the SP deleted mutant, but not the mutants defective in SP cleavage, showed cell surface expression, demonstrating the importance of SP cleavage for hGLP-1R cell surface expression. The N-terminal deletions of hGLP-1R revealed that the HRASP, not the SP, is essential for cell surface expression of GLP-1R. Further, inhibition of hGLP-1R glycosylation prevented cell surface expression of the receptor. Mutation of Trp39, Tyr69 and Tyr88, which are required for agonist binding, in the GLP-1R abolished cell surface expression of the receptor independent of the SP cleavage or N-linked glycosylation. In conclusion, the N-terminus of hGLP-1R regulates receptor trafficking and maturation. Therefore this study provides insight into the role of hGLP-1R N-terminus on the receptor cell surface expression.

“…Myr-ARF6p, but not Myr-ARF1p, treatment increased cAMP accumulation over basal to a similar level in both groups, suggesting that there is no alteration in LHCGR internalisation in GCs from PCOS ( Figure 4A). ARF6 function depends on its activation by GEFs such as cytohesins (Davies et al, 2014a, Davies et al, 2014b). We and others have shown the involvement of cytohesin2, through ARF6 activation, in agonist-induced internalisation of LHCGR (Hunzicker-Dunn et al, 2002, Kanamarlapudi et al, 2012b.…”

Section: Lhcgr Protein Expression In Gcs From Normal and Pcos Women Wmentioning

confidence: 99%

Luteinizing hormone/chorionic gonadotrophin receptor overexpressed in granulosa cells from polycystic ovary syndrome ovaries is functionally active

Reproductive BioMedicine Online

Gordon

Bernal

2016

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General rightsThis document is made available in accordance with publisher policies. Please cite only the published version using the reference above. AbstractPolycystic ovarian syndrome (PCOS) is associated with anovulatory infertility. Luteinizing hormone/chorionic gonadotropin receptor (LHCGR), which is critical for ovulation, has been suggested to be expressed prematurely in the ovarian follicles of women with PCOS. The objective of this study was to analyse the expression and activity of LHCGR in ovarian granulosa cells from PCOS patients and the involvement of ARF6 small GTPase in LHCGR internalisation. Granulosa cells (GCs) isolated from follicular fluid collected during oocyte retrieval from normal women (n=19) and women with PCOS (n=17) were used to study differences in LHCGR protein expression and activity between normal and PCOS patients.LHCGR expression is up-regulated in GCs from PCOS women. LHCGR in PCOS GCs is functionally active as evidenced by increased cAMP production upon human gonadotrophin (HCG)-stimulation. Moreover, ARF6 is highly expressed in GCs from PCOS patients and HCG-stimulation increases the levels of active ARF6. The inhibition of ARF6 activation attenuates HCG-induced LHCGR internalisation in both normal and PCOS GCs, indicating that there are no alterations in LHCGR internalisation in GCs from PCOS. In conclusion, the expression and activation of LHCGR and ARF6 are up-regulated in GCs from PCOS women but the mechanism of agonist-induced LHCGR internalisation is unaltered.