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

Abstract

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.

Labyrinthin: A distinct pan-adenocarcinoma diagnostic and immunotherapeutic tumor specific antigen

“…Specifically, labyrinthin is missing ER-targeting RK motifs found in ASPH and junctate [ 1 ]. ASPH is further subject to ER localization due to the presence of six RR a.a. motifs that maintain type II membrane proteins in the ER [ 76 ] plus five RK and four KR motifs important for ER trafficking [ 77 , 78 , 79 , 80 ]. Various protein structural computer analyses agree that labyrinthin is predominantly extracellular and oriented as a type II protein [ 1 ] and Figure 2 ).…”

Topology and adenocarcinoma cell localization dataset on the labyrinthin biomarker

“…ASPH is further subject to ER localization due to the presence of six RR a.a. motifs that maintain type II membrane proteins in the ER (Schutze et al, 1994) plus five RK and four KR motifs important for ER trafficking (Giraudo and Maccioni, 2003; Keller et al, 2001; O’kelly et al, 2002; Rangel-Garcia et al, 2021). Various protein structural computer analyses agree that labyrinthin is predominantly extracellular and oriented as a type II protein (Babich et al, 2021) and Figure 2).…”

“…Though antibody cross-reactivity is likely, localization of labyrinthin (cell surface) would help distinguish antibody signal vs. intracellular junctate and ASPH. Indeed, the essential endoplasmic reticulum (ER)-targeting RK/KR and KKXX motifs, and RR motifs that maintain type II proteins in the ER [13, 14, 15], are only present in ASPH and junctate (Figs. 1B and 1C).…”

“…(B) & (C) Epitopes for mouse monoclonal anti-labyrinthin antibody MCA 44-3A6 [12] and the widely used FB-50 [10,11] are shown in red and blue, respectively. RK/KR and KKXX motifs essential for ER targeting of proteins, and RR motifs that maintain type II proteins in the ER [13,14,15] The presence or absence of labyrinthin on non-permeabilized human A549 lung adenocarcinoma and normal cells is shown. Because adenocarcinomas are neoplasia of epithelial tissue that have glandular origin, normal primary cultures (Cambrex/Lonza Bioscience) of human astrocytes, renal proximal tubule epithelial cells, and small airway epithelial cells were selected as counter models of study.…”

“…(B) & (C) Epitopes for mouse monoclonal anti-labyrinthin antibody MCA 44-3A6[12] and the widely used FB-50[10,11] are shown in red and blue, respectively. RK/KR and KKXX motifs essential for ER targeting of proteins, and RR motifs that maintain type II proteins in the ER[13,14,15] are underlined (bars). *Partial sequence identified…”