Endocrine Abstracts

G protein-coupled receptors (GPCRs) are one of the largest families of the mammalian genome and represent the single most common therapeutic target. The glycoprotein hormones; LH and human chorionic gonadotrophin, act at a single GPCR the LH/hCGR, whose roles in reproductive function and pregnancy are well known. Endocytic trafficking of GPCRs represents a key mechanism in defining cellular responses in complex signaling pathways by controlling both the temporal and spatial parameters of cellular signalling. Studies on LH/hCGR signalling have unveiled unexpected and diverse facets to its regulation via endocytic trafficking. The early endosome (EE) has traditionally been thought of as the earliest primary site of protein sorting in the endocytic pathway. Using the LH/hCGR as a model receptor, we identify a biochemically distinct compartment, preceding the EE that mediates the post-endocytic sorting of the LH/hCGR. We show that these very early sorting endosomes (VESEs) contain the adaptor protein APPL1 (adapter protein containing PH domain, PTB domain and leucine zipper motif) yet do not contain the EE markers Rab5 and EEA1. Receptors are directed to and sorted from this compartment to the regulated recycling pathway, through interactions with the PDZ protein GAIP-interacting protein (GIPC, C terminus). The C-terminal tail of the LH/hCGR was both necessary and sufficient for interacting with GIPC and targeting to the VESE. Loss of cellular GIPC, via siRNA-mediated knockdown, reroutes receptors to EEs and prevents sorting to the recycling pathway. Total-internal reflection microscopy revealed that GIPC is recruited to LH/hCGR in clathrin-coated pits. Importantly, altering the trafficking of receptors from the VESEs to the EEs, or enriching cargo in the VESE reprograms LH/hCGR signalling. These findings reveal an unprecedented heterogeneity in the endocytic platforms involved in cargo sorting. Further, that endosomal signalling from VESEs provides acute spatial compartmentalization for potentially diverse receptor signalling systems.

Declaration of funding

This work was supported by the Wellcome Trust (WT085099MA) and Genesis Research Trust (P15844).