Poster Presentation Annual Meetings of the Endocrine Society of Australia and Society for Reproductive Biology and Australia and New Zealand Bone and Mineral Society 2016

The extreme C-terminus of PTHR encodes the key molecular determinants for PTHR-SNX27 interaction and trafficking (#269)

Audrey S Chan 1 , Thomas Clairfeuille 2 , Brett M Collins 2 , Nathan Pavlos 1
  1. Cellular Orthopaedic Laboratory, School of Surgery, University of Western Australia, Perth, WA, Australia
  2. Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD, Australia

Parathyroid hormone receptor (PTHR) is a prototypical GPCR critical for the regulation of bone development and mineral ion metabolism.  Upon stimulation, PTHR is internalised at the plasma membrane (PM) into endosomes where ‘non-canonical’ signalling is maintained. Signal termination and post-endocytic sorting of PTHR is driven by protein trafficking machineries which recognise short linear binding motifs encoded within the cytosolic tail of the receptor. We recently identified the endocytic protein Sorting Nexin 27 (SNX27) as a novel regulator of PTHR trafficking and demonstrated that SNX27 acts as an adaptor to couple the receptor to the retromer recycling complex. PTHR-SNX27 interaction is dictated by a unique PDZ-domain housed within SNX27, engaging the PDZ-binding motif (PDZbm) found within the PTHR C-terminus. While the PTHR-PDZbm is critical for SNX27 cargo-recognition, the relative contribution of individual amino acid residues within this motif remains unclear. To define the key molecular determinants required for PTHR-SNX27 interaction we generated a series of PTHR mutants with single alanine substitutions across the entire PDZbm (EWETVM; denoted A1-A7) and compared (i) their relative binding affinities to the SNX27-PDZ domain by isothermal titration calorimetry (ITC) and (ii) co-occupancy with SNX27 on endosomes by confocal microscopy.  Alanine substitution at either the -3 and/or -5 position of the PTHR-PDZbm (predicted to form electrostatic bonds with Arg58 of SNX27) abolished PTHR-SNX27 binding and co-localisation on endosomes.  Addition of a single alanine (+1) to the PTHR-PDZbm or truncation of the entire PDZbm (ΔPDZbm) similarly impaired SNX27 association(s). Interestingly, while alanine substitutions did not influence PM targeting or receptor internalization, they increased the propensity of PTHR to mis-traffic towards degradative late-endo-lysosomal pathways. Together, these findings unveil the minimal molecular determinants of PTHR-SNX27 interaction and support the view that a major function of SNX27 is to ferry receptors away from degradative pathways and towards retromer tubules for recycling.