Endocrine Abstracts

Membrane trafficking of G-protein coupled receptors (GPCRs) is a critical mechanism by which cells mediate complex signaling pathways. Endosomes are increasingly recognized as platforms for GPCR signaling. The VEE was first identified using the LHR, a GPCR that is key in reproduction. We have shown that recycling of the LHR is driven by receptor mediated Gαs cAMP signaling from the VEE and PKA-dependent phosphorylation of the adaptor protein, APPL1, while APPL1 negatively regulates LH-activated VEE signaling in a PKA-independent manner. In the ovary, LHR also activates Gαq-signaling during the LH surge, a signal that is important in ovulation. The role of the VEE and APPL1 on the Gαq-pathway are unknown. Here we investigate if LHR-mediated Gαq-signaling is spatially regulated via APPL1 and how LHR-Gαs/Gαq spatially directed signaling may be integrated. Pre-treatment with Dyngo-4a in HEK293s expressing LHR reduced LH-dependent IP1 levels, indicating the Gαq-pathway requires receptor internalization. Depletion of APPL1 had no effect on IP1-levels, suggesting APPL1 doesn’t impact Gαq-signaling despite its ability to negatively regulate Gαs-signaling. Potential G-protein crosstalk was investigated in HEK293 GΔs and GΔq CRISPR-Cas9 knockouts. GΔs resulted in a complete loss of Gαs-cAMP while, GΔq resulted in increased cAMP compared to WT. However, pre-treatment with a Gαq chemical inhibitor in WT HEK293 showed no change in cAMP-levels. Depletion of APPL1 in GΔq resulted in loss of sensitivity to APPL1 via Gαs-cAMP in contrast to the negative regulatory effects that were previously observed. Depletion of APPL1 in GΔs cells resulted in a decrease of Gαq-IP1. These data combined indicate regulatory roles for the G-proteins where their physical presence mediate responses within a signaling-complex of proteins in which APPL1 also plays a regulatory role, thereby having a synergistic effect in propagating LHR signaling to modulate activity at the level of the VEE and APPL1.