Characterisation of the molecular mechanisms regulating luteinizing hormone receptor

Abstract

G protein coupled receptors (GPCRs) are the major family of membrane proteins transducing extracellular stimuli into intracellular signals. The luteinizing hormone receptor (LHR) is a GPCR expressed in gonadal and extragonadal tissues where it plays pivotal roles in reproduction and pregnancy. Endocytic trafficking of GPCRs represents a key mechanism in defining cellular responses by controlling signalling at both temporal and spatial level. After ligand activation, LHR is internalised into very early endosomes (VEEs), small vesicles (~400nm in diameter) close to the plasma membrane and distinct from the classic early endosome. Our laboratory identified the Adaptor Protein containing pleckstrin homology (PH) domain, PTB domain and Leucine zipper motif 1 (APPL1) as a marker of ~50% of VEEs where LHR is internalised to. My aims were to characterise the VEE by identifying the mechanisms dictating LHR post-endocytic sorting from this novel compartment, the role of APPL1 and how this impacts receptor signalling. Imaging LHR recycling in real time at single event resolution, enabled me to identify the kinetics and the machinery involved. While APPL1 was not required for LHR localisation in VEEs, it is essential for receptor recycling and negative regulation of cAMP production; two functions never ascribed before to APPL1 for any membrane cargo. These two functions of APPL1 are regulated in an opposing manner by phosphorylation of Ser410 of APPL1, achieved by activation of the Gαs-cAMP-PKA pathway, highlighting the mutual regulation of GPCR trafficking and signalling. I also demonstrated that both Gαs-cAMP and Gαq/11-calcium signalling require LHR internalisation, that endosomal signalling and recycling involves distinct adenylate cyclases and that Gαs activation is restricted to microdomains of the VEE. Finally, LHR recycling and cAMP signalling are also regulated by APPL1 in primary human endometrial stromal cells, where LHR trafficking and signalling could be perturbed in pathological conditions such as PCOS.