The parathyroid hormone receptor type 1 (PTHR) is a prototypical GPCR critical for the regulation of postnatal bone development, remodelling and mineral ion metabolism. Upon stimulation, surface-resident PTHR is internalised 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 have 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 complex1. The PTHR-SNX27 interaction is mediated by the SNX27-PDZ domain and the C-terminal PDZ-binding motif (PDZbm) of PTHR. 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).
By combining isothermal titration calorimetry (ITC) and confocal microscopy, we assessed the binding affinities and endosomal co-occupancy of wildtype and mutant PTHR with SNX27.
Alanine substitutions at either the (-3) and/or (-5) position of the PTHR-PDZbm (predicted to form electrostatic bonds with Arg58 of SNX27-PDZ) abolished both PTHR-SNX27 binding and endosomal co-localisation. Similarly, the addition of a single alanine (+1) to the PTHR-PDZbm or truncation (ΔPDZbm) impaired association(s) with SNX272. Interestingly, whereas the alanine substitutions did not influence PM targeting or receptor internalisation, they increased the propensity of PTHR to mis-traffic towards degradative late-endolysosomal pathways.
Together, these findings unveil the minimal molecular determinants of PTHR-SNX27 interaction and support the view that SNX27 functions to ferry receptors away from degradative pathways and towards retromer tubules for recycling.