Endocrine Abstracts

LH receptor (LHR) is a G protein-coupled receptor (GPCR) that plays key roles in reproduction and pregnancy. Endocytic trafficking and sorting of GPCRs to diverse cellular fates represent a key mechanism in defining cellular responses by controlling both the temporal and spatial parameters of cellular signalling. How different processes within the endocytic system are coordinated, however, needs to be mechanistically dissected in more detail. We have recently demonstrated that the interaction of human LHR with the PDZ domain protein GIPC targets the receptor to pre-early endosomes (pre-EEs), specific compartments upstream of the classic early sorting endosome. Targeting of LHR to pre-EEs by GIPC is necessary for receptor sorting to the recycling pathway and for a sustained MAPK signalling profile from pre-EEs. Pre-EEs were also positive for the adaptor protein APPL1 (adaptor protein containing pleckstrin homology PH domain, PTB domain and leucine zipper motif 1). Although APPL1 was not required for LHR endosomal localization, it was essential for receptor recycling, a function not previously ascribed for APPL1 for any membrane cargo. Therefore, this study aimed to characterize the molecular mechanisms mediating LHR recycling. LHR trafficking was visualised via total internal reflection fluorescence (TIRF) microscopy. APPL1 mutants, each lacking one or more C-terminal domains, were tested for their ability to rescue LHR impaired recycling in cells stably expressing shRNA APPL1. Interestingly, mutation of a PKA phoshorylation site, known to interact with the phosphatase OCRL, could not rescue LHR recycling in APPL1 depleted cells. Furthermore, inhibition of PKA dramatically inhibited LHR recycling suggesting a role for LHR signal activation in regulating APPL1 interactions and its sorting from pre-EEs. Overall these findings reveal an unprecedented sorting mechanism for signalling receptors, mediated by APPL1. Diversity in sorting mechanisms raises the possibility that post-endocytic sorting could be programmed for an individual receptor at multiple levels.