Jaw1/LRMP increases Ca2+ influx upon GPCR stimulation with heterogeneous effect on the activity of each ITPR subtype

Abstract

Ca2+ influx upon G protein-coupled receptor (GPCR) stimulation is observed as a cytosolic Ca2+ concentration oscillation crucial to initiating downstream responses including cell proliferation, differentiation, and cell–cell communication. Although Jaw1 is known to interact with inositol 1,4,5-triphosphate receptor (ITPRs), Ca2+ channels on the endoplasmic reticulum, the function of Jaw1 in the Ca2+ dynamics with physiological stimulation remains unclear. In this study, using inducible Jaw1-expressing HEK293 cells, we showed that Jaw1 increases Ca2+ influx by GPCR stimulation via changing the Ca2+ influx oscillation pattern. Furthermore, we showed that Jaw1 increases the Ca2+ release activity of all ITPR subtypes in a subtly different manner. It is well known that the Ca2+ influx oscillation pattern varies from cell type to cell type, therefore these findings provide an insight into the relationship between the heterogeneous Ca2+ dynamics and the specific ITPR and Jaw1 expression patterns.

Cleavage of the Jaw1 C-terminal region enhances its augmentative effect on the Ca2+release via inositol 1,4,5-trisphosphate receptors

“…S3). Jaw1 has an additional role to increase the Ca 2+ release from the ER via interaction with IP 3 Rs upon GPCR stimulation, as we recently reported (Okumura et al ., 2022). Therefore, we measured Ca 2+ flux in Jaw1 KO Flp-In T-REx HEK293 (Jaw1 KO), Jaw1 IE, and AASS IE cells via stimulation with ATP.…”

“…We previously reported that Jaw1 interacts with IP 3 Rs via its coiled-coil domain, and a mutant lacking the coiled-coil domain, has no augmentative effect of Jaw1 on the Ca 2+ signaling, which indicates that Jaw1 directly increases the Ca 2+ release activity of IP 3 Rs (Okumura et al ., 2022). In this study, the augmentative effect of the AASS mutation on the Ca 2+ release from the ER was slightly lower than that of wild-type Jaw1 (Fig.…”

“…The plasmids, pcDNA5 FRT/TO HA FLAG Ms Jaw1 (Kozono et al ., 2018), pcDNA5 FRT/TO HA FLAG Hu Jaw1 (Kozono et al ., 2021), pcDNA3.1 (+) Hu Jaw1 (Kozono et al ., 2021), and pcDNA5 FRT/TO Hu Jaw1 (Okumura et al ., 2022), were produced as described in previous studies. The primer sets used to produce the plasmids are shown in Table S1.…”

“…Jaw1 KO Flp-In T-REx HEK293 (Jaw1 KO) cells and Jaw1 KO Flp-In T-REx HEK293 Hu Jaw1 (Jaw1 IE) cells was produced as previously described (Okumura et al ., 2022). Flp-In T-REx HEK293 FLAG Ms Jaw1, FLAG Ms Jaw1 K1-K5, Hu Jaw1, Hu Jaw1 opsin, and Hu AASS were produced by co-transfection of pOG44 Flp-Recombinase Expression Vector (#V600520; Thermo Fisher Scientific, Waltham, MA, USA) with pcDNA5 FRT/TO HA FLAG Ms Jaw1, pcDNA5 FRT/TO HA FLAG Ms Jaw1 K1-K5, pcDNA5 FRT/TO Hu Jaw1, pcDNA5 FRT/TO Hu Jaw1 opsin and pcDNA5 FRT/TO Hu AASS, respectively, into the Flp-In™ T-REx™ HEK293 cell line or Jaw1 KO cells (#R78007; Thermo Fisher Scientific, Waltham, MA, USA).…”

“…However, the cleavage site, the enzymes responsible for cleavage, and the physiological significance of the cleavage remain unknown. Jaw1 is involved in Ca 2+ release from the ER into the cytosol upon the stimulation of G protein-coupled receptor (GPCR) through the interaction with inositol 1,4,5-trisphosphate receptors (IP 3 Rs), calcium channels on the ER membrane (Chang et al ., 2021; Okumura et al ., 2022; Prüschenk et al ., 2021; Shindo et al ., 2010). Thus, Jaw1 is a bifunctional protein as a KASH protein and as an IP 3 R regulator; however, the underlying mechanisms of these functions remain unclear.…”