Endocrine Abstracts

The LH receptor (LHR) is essential for mediating multiple functions in reproduction and pregnancy. LHR belongs to the superfamily of G-protein-coupled receptors (GPCRs) that impacts nearly every aspect of human physiology and pathophysiology. Membrane trafficking is a critical mechanism for cells to decode complex signalling networks into specific downstream responses, including the signalling pathways activated by GPCRs. A detailed molecular description of GPCR trafficking is indispensable for our basic understanding of cellular regulation and how aberrant signalling can result in disease.

We have recently identified a novel endosomal compartment critical for the sorting and signalling of distinct GPCRs, we term the very early endosome (VEE). Using our model GPCR for the VEE, LHR, we provide evidence for an unprecedented role for the adaptor protein APPL1 in regulated sorting from the VEE driven by LHR signalling. Employing high resolution total internal reflected fluorescence microscopy (TIR-FM), in combination with a pH sensitive GFP-tagged LHR to directly visualise individual LHR recycling events at the plasma membrane, we unveil detailed mechanistic information about LHR recycling and the role of PKA and APPL1 in this process. Altering LHR trafficking fate through the use of chemical inhibitors, siRNA and a conformational biosensor we revealed differential activation of the signalling pathways either from the plasma membrane and/or from VEEs. Interestingly, whilst Gαq/calcium pathway was totally dependent on LHR internalization, two waves of Gαs/cAMP pathway activation occurred, from the plasma membrane and from VEEs.

Overall these findings propose a novel mechanism for the post-endocytic sorting of GPCRs and a system where receptor activity could be reprogrammed by altering location of receptors. Physiologically, this may be a key adaptive mechanism for GPCRs, however, this could be exploited therapeutically in order to provide important insight to perturbed LHR activity under pathophysiological conditions.