Phosphorylation of a tyrosine at the N‐terminus regulates the surface expression of GIRK5 homomultimers

Mora, Sílvia; Escobar, Luis A.

doi:10.1016/j.febslet.2005.04.056

Cited by 6 publications

(11 citation statements)

References 38 publications

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“…Since we previously determined that Y16 is a key residue that determines if GIRK5 is transported to the plasma membrane [15], we investigated how the localization of the phospho-null GIRK5 (EGFP-Y/A) changed over the course of six days. The first day after injection, a faint expression was observed across the animal pole (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The supernatant was mixed with 2 µl of Laemmli buffer per oocyte, heated to 95°C following the procedure described before [15].…”

Section: Methodsmentioning

confidence: 99%

“…Whole-cell currents were recorded two to four days after mRNA injection with a twoelectrode voltage clamp amplifier (Geneclamp 500) and a Digidata 1322A interface with pClamp8 software (Axon Instruments) at room temperature (22–24°C), as described previously [15]. The recording solution contained: 118 mM KCl, 1 mM CaCl2, 2 mM MgCl2, and 5 mM HEPES, pH 7.4.…”

Section: Methodsmentioning

confidence: 99%

“…Previously, we found that within this N-terminus, the dephosphorylation of Y16 determines whether GIRK5 is transported to the plasma membrane [15]. Since GIRK5 surface expression occurred only one hour after incubation with a Protein Tyrosine Kinase inhibitor, we hypothesized then that GIRK5 was retained in a subcellular compartment.…”

Section: Introductionmentioning

confidence: 97%

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The Polarization of the G-Protein Activated Potassium Channel GIRK5 to the Vegetal Pole of Xenopus laevis Oocytes Is Driven by a Di-Leucine Motif

et al. 2013

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The G protein-coupled inwardly-rectifying potassium channels (known as GIRK or Kir3) form functional heterotetramers gated by G-βγ subunits. GIRK channels participate in heart rate modulation and neuronal postsynaptic inhibition in mammals. In Xenopus laevis oocytes, GIRK5 is a functional homomultimer. Previously, we found that phosphorylation of a tyrosine (Y16) at its N-terminus downregulates the surface expression of GIRK5. In this work, we elucidated the subcellular localization and trafficking of GIRK5 in oocytes. Several EGFP-GIRK5 chimeras were produced and an ECFP construct was used to identify the endoplasmic reticulum (ER). Whereas GIRK5-WT was retained in the ER at the animal pole, the phospho-null GIRK5-Y16A was localized to the vegetal pole. Interestingly, a construct with an N-terminal Δ25 deletion produced an even distribution of the channel in the whole oocyte. Through an alanine-scan, we identified an acidic cluster/di-leucine sorting-signal recognition motif between E17 and I22. We quantified the effect of each amino acid residue within this di-leucine motif in determining the distribution of GIRK5 to the animal and vegetal poles. We found that Y16 and I22 contributed to functional expression and were dominant in the polarization of GIRK5. We thus conclude that the N-terminal acidic di-leucine motif of GIRK5 determines its retention and polarized trafficking within Xl oocytes.

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Section: Resultsmentioning

confidence: 99%

“…The supernatant was mixed with 2 µl of Laemmli buffer per oocyte, heated to 95°C following the procedure described before [15].…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 97%

See 2 more Smart Citations

The Polarization of the G-Protein Activated Potassium Channel GIRK5 to the Vegetal Pole of Xenopus laevis Oocytes Is Driven by a Di-Leucine Motif

et al. 2013

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“…For example, protein kinase C-mediated phosphorylation of serine 185 on K ir 3.1 reduces open-state probability (4), and phosphorylation of tyrosine 16 on K ir 3.5 results in reduced functional expression of the channel (5). Research from our group has shown that after heterologous gene expression of the K ir 3.1 subunits and the TrkB receptor in Xenopus oocytes, activation of the receptor-tyrosine kinase with BDNF reduced channel current.…”

mentioning

confidence: 99%

Tyrosine Phosphorylation of Kir3 following κ-Opioid Receptor Activation of p38 MAPK Causes Heterologous Desensitization

Clayton

Chavkin

2009

Journal of Biological Chemistry

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Prior studies showed that tyrosine 12 phosphorylation in the N-terminal, cytoplasmic domain of the G-protein-gated inwardly rectifying potassium channel, K ir 3.1 facilitates channel deactivation by increasing intrinsic GTPase activity of the channel. Using a phosphoselective antibody directed against this residue (pY12), we now report that partial sciatic nerve ligation increased pY12-K ir 3.1-immunoreactivity (ir) in the ipsilateral dorsal horn of wildtype mice, but not in mice lacking the -opioid receptor (KOR) or lacking the G-protein receptor kinase 3 (GRK3) genes. Treatment of AtT-20 cells stably expressing KOR-GFP with the selective KOR agonist U50,488 increased both phospho-p38-ir and pY12-K ir 3.1-ir. The U50,488-induced increase in pY12-K ir 3.1-ir was blocked by the p38 inhibitor SB203580. Cells expressing KOR(S369A)-GFP did not increase either phospho-p38-ir or pY12-K ir 3.1-ir following U50,488 treatment. Whole cell voltage clamp of AtT-20 cells expressing KOR-GFP demonstrated that p38 activation by U50,488 reduced somatostatin-evoked K ir 3 currents. This heterologous desensitization was blocked by SB203580 and was not evident in cells expressing KOR(S369A)-GFP. Tyrosine phosphorylation of K ir 3.1 was likely mediated by p38 MAPK activation of Src kinase. U50,488 also increased (pY418)Src-ir; this increase was blocked by SB203580 and not evident in KOR(S369A)-GFP expressing AtT20 cells; the Src inhibitor PP2 blocked the U50,488-induced increase in pY12-K ir 3.1-ir; and the heterologous desensitization of K ir 3 currents was blocked by PP2. These results suggest that KOR causes phosphorylation of Y12-K ir 3.1 and channel inhibition through a GRK3-, p38 MAPK-and Src-dependent mechanism. Reduced inward potassium current following nerve ligation would increase dorsal horn neuronal excitability and may contribute to the neuropathic pain response.

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Identification of a unique endoplasmic retention motif in the Xenopus GIRK5 channel and its contribution to oocyte maturation

et al. 2021

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G-protein activated inward-rectifying potassium (K +) channels (Kir3/GIRK) participate in cell excitability. The GIRK5 channel is present in Xenopus laevis oocytes. In an attempt to investigate the physiological role of GIRK5, we identified a non-canonical di-arginine endoplasmic reticulum (ER) retention motif (KRXY). This retention motif is located at the N-terminal region of GIRK5, coded by two small exons found only in X. laevis and X. tropicalis. These novel exons are expressed through use of an alternative transcription start site. Mutations in the sequence KRXY produced functional channels and induced progesterone-independent oocyte meiotic progression. The chimeric proteins EGFP-GIRK5-WT and EGFP-GIRK5K13AR14A were localized to the ER and the plasma membrane of the vegetal pole of the oocyte, respectively. Silencing of GIRK5 or blocking of this channel by external barium prevented progesterone-induced meiotic progression. The endogenous level of GIRK5 protein decreased through oocyte stages in prophase I augmenting by progesterone. In conclusion, we have identified a unique mechanism by which the expression pattern of a K + channel evolved to control Xenopus oocyte maturation.

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Phosphorylation of a tyrosine at the N‐terminus regulates the surface expression of GIRK5 homomultimers

Cited by 6 publications

References 38 publications

The Polarization of the G-Protein Activated Potassium Channel GIRK5 to the Vegetal Pole of Xenopus laevis Oocytes Is Driven by a Di-Leucine Motif

The Polarization of the G-Protein Activated Potassium Channel GIRK5 to the Vegetal Pole of Xenopus laevis Oocytes Is Driven by a Di-Leucine Motif

Tyrosine Phosphorylation of Kir3 following κ-Opioid Receptor Activation of p38 MAPK Causes Heterologous Desensitization

Identification of a unique endoplasmic retention motif in the Xenopus GIRK5 channel and its contribution to oocyte maturation

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