Retromer in Osteoblasts Interacts With Protein Phosphatase 1 Regulator Subunit 14C, Terminates Parathyroid Hormone's Signaling, and Promotes Its Catabolic Response

Abstract

Parathyroid hormone (PTH) plays critical, but distinct, roles in bone remodeling, including bone formation (anabolic response) and resorption (catabolic response). Although its signaling and function have been extensively investigated, it just began to be understood how distinct functions are induced by PTH activating a common receptor, the PTH type 1 receptor (PTH1R), and how PTH1R signaling is terminated. Here, we provide evidence for vacuolar protein sorting 35 (VPS35), a major component of retromer, in regulating PTH1R trafficking, turning off PTH signaling, and promoting its catabolic function. VPS35 is expressed in osteoblast (OB)-lineage cells. VPS35-deficiency in OBs impaired PTH(1–34)-promoted PTH1R translocation to the trans-Golgi network, enhanced PTH(1–34)-driven signaling, and reduced PTH(1–34)'s catabolic response in culture and in mice. Further mechanical studies revealed that VPS35 interacts with not only PTH1R, but also protein phosphatase 1 regulatory subunit 14C (PPP1R14C), an inhibitory subunit of PP1 phosphatase. PPP1R14C also interacts with PTH1R, which is necessary for the increased endosomal PTH1R signaling and decreased PTH(1–34)'s catabolic response in VPS35-deficient OB-lineage cells. Taken together, these results suggest that VPS35 deregulates PTH1R-signaling likely by its interaction with PTH1R and PPP1R14C. This event is critical for the control of PTH(1–34)-signaling dynamics, which may underlie PTH-induced catabolic response and adequate bone remodeling.

GPCR Signaling and Trafficking: The Long and Short of It

“…β 2 AR), knockdown of SNX27 or retromer expression by short-interfering hairpin RNAs led to reduced PTHR recycling and/or cell surface availability. Paradoxically, despite receptor down-regulation, loss of either ASRT component coincided with elevated endosomal cAMP-signaling in HEK293 cells [43] and osteoblastic cells derived from the bones of SNX27 knockout [50], and haplodeficient Vps35 mice [64]. Together, these studies infer that the ASRT complex functions as a ‘molecular brake’ to restrict PTHR-signaling at the endosomes.…”

“…Additional considerations, however, including potential alterations in auxiliary proteins that intersect with the ASRT complex and impinge on receptor entry into endosomal tubules, need also be considered. The protein phosphatase 1 regulatory subunit 14C (PPP1R14C), an inhibitory subunit of PP1 phosphatase, which binds directly to Vps35, for example, was recently shown to promote PTHR-endosomal, but not cell surface, signaling in osteoblastic cells [64]. How PPP1R14C interacts with Vps35 and modulates endosomal PTHR signaling remains poorly understood but may relate to delayed entry into ASRT endosomal tubules, resulting in prolonged endosomal residence time, enhanced intracellular PTHR signaling and eventual misdirection into degradative lysosomes as observed in this study [65].…”

“…The protein phosphatase 1 regulatory subunit 14C (PPP1R14C), an inhibitory subunit of PP1 phosphatase, which binds directly to Vps35, for example, was recently shown to promote PTHR-endosomal, but not cell surface, signaling in osteoblastic cells [64]. How PPP1R14C interacts with Vps35 and modulates endosomal PTHR signaling remains poorly understood but may relate to delayed entry into ASRT endosomal tubules, resulting in prolonged endosomal residence time, enhanced intracellular PTHR signaling and eventual misdirection into degradative lysosomes as observed in this study [65]. In fact, this mechanism has been recently shown to underscore the ability of arrestin domain-containing protein 3 (ARRDC3), a member of the β-arrestin family of visual/β-arrestins and Vps26-related proteins [66] to prolong on the endosomal occupancy and associated cAMP-signaling of β 2 AR [67].…”

“…β 2 AR). By mapping the subcellular distribution of exogenously expressed PTHR in osteoblast-like cells (MC3T3), recent work by Xiong et al not only confirmed the existence of this indirect recycling route, but also suggested that retromer (Vps35) governs this pathway [64]. Of note however, in this study, the PTHR was monitored as a C-terminal fusion protein (PTHR-mCherry), which would occlude its PDZ-recognition motif and thereby its interaction with SNX27.…”

“…monocyte/macrophages). More recently, a follow up study by the same group, using specific Vps35 knockout mice in the osteoblast-lineage (osteocalcin-Cre), revealed a mild low bone mass phenotype associated with altered PTH-driven PTHR-signaling in osteoblasts [64]. Consequently, treatment with PTH(1–34) promoted exaggerated PTH-induced anabolic responses during bone remodeling (turnover) which was paralleled by a decrease in the catabolic activity of osteoclasts, resulting in a net elevation in trabecular (primary) bone mass.…”