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

Abstract

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.

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

“…To compare the localization of GIRK5 and the KR double-alanine mutant, their EGFP chimeras were produced, injected in oocytes and observed by confocal microscopy. Previously, we found EGFP-GIRK5 WT localized to the nucleus, perinuclear space and ER in the animal pole of X. laevis oocytes [15]. As expected, K13R14A EGFPconstruct was not retained in the ER but observed distributed throughout the cytoplasm (most probably in vesicles) and plasma membrane of the vegetal pole (Fig.…”

“…The GIRK5 cDNAs and EGFP chimera constructs were subcloned into the pRSSP6013A3‐UWE vector (pBF) and sequenced. The ER‐enhanced cyan fluorescent protein ECFP‐ER cDNA to identify the ER [15] was donated by Luis Vaca (Inst. Fisiol.…”

“…Previously, we observed that recombinant GIRK5 was retained in the ER of X. laevis oocytes [15]; this retention was linked to phosphorylation of a tyrosine Fig. 1.…”

“…Among their mammalian homologues, GIRK5 contains a long N-terminal sequence (Fig. 1); however, few studies have been performed about the intracellular trafficking of GIRK5 [15].…”

A Novel Di-Leucine Motif at the N-Terminus of Human Organic Solute Transporter Beta Is Essential for Protein Association and Membrane Localization

“…There are multiple mechanisms underlying protein interaction and cellular trafficking, some of which depend on dileucine motifs on their cytoplasmic carboxyl- and /or amino-terminal fragments [ 23 – 26 ]. Vergarajauregui et al have identified two separate dileucine-type motifs that co-operate to regulate the transport of mucolipin-1 to lysosomes [ 27 ].…”