Actin-Sorting Nexin 27 (SNX27)-Retromer Complex Mediates Rapid Parathyroid Hormone Receptor Recycling

Abstract

The G protein-coupled parathyroid hormone receptor (PTHR) regulates mineral-ion homeostasis and bone remodeling. Upon parathyroid hormone (PTH) stimulation, the PTHR internalizes into early endosomes and subsequently traffics to the retromer complex, a sorting platform on early endosomes that promotes recycling of surface receptors. The C terminus of the PTHR contains a type I PDZ ligand that binds PDZ domaincontaining proteins. Mass spectrometry identified sorting nexin 27 (SNX27) in isolated endosomes as a PTHR binding partner. PTH treatment enriched endosomal PTHR. SNX27 contains a PDZ domain and serves as a cargo selector for the retromer complex. VPS26, VPS29, and VPS35 retromer subunits were isolated with PTHR in endosomes from cells stimulated with PTH. Molecular dynamics and protein binding studies establish that PTHR and SNX27 interactions depend on the PDZ recognition motif in PTHR and the PDZ domain of SNX27. Depletion of either SNX27 or VPS35 or actin depolymerization decreased the rate of PTHR recycling following agonist stimulation. Mutating the PDZ ligand of PTHR abolished the interaction with SNX27 but did not affect the overall rate of recycling, suggesting that PTHR may directly engage the retromer complex. Coimmunoprecipitation and overlay experiments show that both intact and mutated PTHR bind retromer through the VPS26 protomer and sequentially assemble a ternary complex with PTHR and SNX27. SNX27-independent recycling may involve N-ethylmaleimide-sensitive factor, which binds both PDZ intact and mutant PTHRs. We conclude that PTHR recycles rapidly through at least two pathways, one involving the ASRT complex of actin, SNX27, and retromer and another possibly involving N-ethylmaleimide-sensitive factor.

GPCR Signaling and Trafficking: The Long and Short of It

“…This PDZ ligand assumes a Type I consensus sequence (E-T-V-M) that is compatible with the canonical recognition signature of the SNX27-PDZ domain. Parallel studies recently confirmed this engagement, thereby providing a direct molecular link to the ASRT complex [50, 63]. First, a PDZ-dependent interaction was demonstrated between PTHR and SNX27 by combining coimmunoprecipitation studies in HEK293 cells with isothermal titration calorimetry (ITC), the later titrating a series of native and mutant PTHR-PDZ-ligand peptides against the SNX27-PDZ domain.…”

Retromer- and WASH-dependent sorting of nutrient transporters requires a multivalent interaction network with ANKRD50

“…SNX27 is directly bound to the retromer subunit VPS26 and simultaneously binds to a multitude of PDZ-and tyrosine-based sorting motifs in the cytosolic tail of its cargo proteins, thereby driving their recycling back to the cell surface (Steinberg et al, 2013;Gallon et al, 2014;Ghai et al, 2013). Retromer and SNX27 have been shown to recycle a wide range of surface molecules, ranging from signalling receptors like the β2-adrenergic receptor (Lauffer et al, 2010) or the parathyroid hormone receptor (PTHR) (Chan et al, 2016;McGarvey et al, 2016), and nutrient transporters (Steinberg et al, 2013) to neuronal glutamate receptors (Wang et al, 2013;Loo et al, 2014;Hussain et al, 2014). Although loss of retromer as well as of the WASH complex is lethal early in embryogenesis (Wen et al, 2011;Gomez et al, 2012), loss of SNX27 is lethal shortly upon birth of genetically modified mice (Cai et al, 2011), highlighting the physiological relevance of SNX27-, retromer-and/or WASH-driven recycling processes.…”

β2-adrenergic receptor control of endosomal PTH receptor signaling via Gβγ

“…Can other proteins also contribute to the synergistic action of the β 2 AR on endosomal cAMP signaling mediated by PTH? For example, sorting nexin 27 (Snx27) regulating β 2 AR and PTHR recycling 1,23,24 might be sequestered by activated β 2 AR, thus allowing extended endosomal PTHR signaling. Does the mechanism shown in this study apply to other bone cells such as chondrocytes and osteocytes, which also express these two receptors?…”