Panel A shows two line graphs. The upper graph depicts the dose-response increment in intracellular calcium levels, with the y-axis labeled as Fluo-4 fluorescence times 100 per oocyte and the x-axis labeled as CCK-8 concentration in nanomolar. The lower graph shows the dose-response increment in intracellular cAMP levels, with the y-axis labeled as cAMP in picomoles per oocyte and the x-axis labeled as CCK-8 concentration in nanomolar. Both graphs compare uninjected oocytes and oocytes expressing the rat CCK1R after treatment with CCK-8. Panel B contains Western blots probed with anti-HA and anti-PDI antibodies, showing YPM extracts from oocytes injected with HsAQP12-WT-HA or DrAqp12-WT-HA, with or without CCK1R, and stimulated with increasing doses of CCK-8. The lower panel of B shows a line graph of channel accumulation in the YPs normalized to PDI in response to CCK-8. Panel C displays representative immunoblots of TM and YPM from oocytes expressing CCK1R with HsAQP12-WT-HA, HsAQP12-YPD-HA, DrAqp12-WT-HA, or DrAqp12-YPD-HA, and exposed to CCK-8. Panel D shows representative immunoblots of HsAQP12-WT-HA and DrAqp12-WT-HA in YPM, with corresponding quantitation normalized to PDI, from oocytes treated with vehicle or CCK-8 in the presence or absence of PKC and PKA inhibitors BimII and H89. Panel E presents immunoblots of HsAQP12-WT-HA, RnAQP12-WT-HA, and DrAqp12-WT-HA in YPM, with corresponding quantitation normalized to PDI, from oocytes treated with DMSO, PMA, or FSK.
CCK1R agonist CCK-8 triggers AQP12 YPM targeting via the PKC and PKA signaling pathways in X. laevis oocytes. (A) Dose–response increment in the intracellular levels of Ca2+ ([Ca2+]I; upper panel) and cAMP ([cAMP]I; lower panel) (mean ± SEM; n = 5 independent experiments) in uninjected oocytes and oocytes expressing the rat CCK1R after treatment with the CCK-8 peptide. (B) Anti-HA- and α-PDI–probed western blots of YPM extracts from oocytes injected with HsAQP12-WT-HA (upper blot) or DrAqp12-WT-HA (middle blot), co-expressing or not the CCK1R, and stimulated with increasing doses of CCK-8. The lower panel shows the accumulation of the channels in the YPs normalized to PDI in response to the octapeptide (n = 3 independent experiments). (C) Representative immunoblots of TM and YPM of oocytes expressing the CCK1R together with HsAQP12-WT-HA or HsAQP12-ΔYPD-HA (left blots), or DrAqp12-WT-HA or DrAqp12-ΔYPD-HA (right blots), and exposed to CCK-8. (D) Representative HsAQP12-WT-HA (left) and DrAqp12-WT-HA (right) immunoblots in YPM, and corresponding quantitation normalized to PDI (lower panels), from oocytes expressing the channels and treated with vehicle (0.5% DMSO) or 10 nM of CCK-8, in the presence or absence of 10 μM of PKC and PKA inhibitors BimII and H89, respectively. (E) Representative HsAQP12-WT-HA, HA-tagged rat AQP12 (RnAQP12-WT-HA), and DrAqp12-WT-HA immunoblots in YPM, and corresponding quantitation normalized to PDI (lower panels), from oocytes expressing the channels and treated with DMSO alone or 100 nM of PMA or 100 µM FSK. Data in D and E (mean ± SEM, n = 3 independent experiments) were statistically analyzed by one-way ANOVA, followed by Tukey’s multiple comparisons test. *P < 0.05; **P < 0.01; with respect to oocytes not treated with CCK-8 (D), or PMA or FSK (E), or with respect to oocytes treated with CCK-8 alone (D in parenthesis), or as indicated in brackets (D). Source data are available for this figure: SourceData F6.