Abstract
Na/H exchanger regulatory factor-1 (NHERF1) is a cytoplasmic PDZ (postsynaptic density 95/disc large/zona occludens) protein that assembles macromolecular complexes and determines the localization, trafficking, and signaling of select G protein-coupled receptors and other membrane-delimited proteins. The parathyroid hormone receptor (PTHR), which regulates mineral ion homeostasis and bone turnover, is a G protein-coupled receptor harboring a PDZ-binding motif that enables association with NHERF1 and tethering to the actin cytoskeleton. NHERF1 interactions with the PTHR modify its trafficking and signaling. Here, we characterized by live cell imaging the mechanism whereby NHERF1 coordinates the interactions of multiple proteins, as well as the fate of NHERF1 itself upon receptor activation. Upon PTHR stimulation, NHERF1 rapidly dissociates from the receptor and induces receptor aggregation in long lasting clusters that are enriched with the actin-binding protein ezrin and with clathrin. After NHERF1 dissociates from the PTHR, ezrin then directly interacts with the PTHR to stabilize the PTHR at the cell membrane. Recruitment of β-arrestins to the PTHR is delayed until NHERF1 dissociates from the receptor, which is then trafficked to clathrin for internalization. The ability of NHERF to interact dynamically with the PTHR and cognate adapter proteins regulates receptor trafficking and signaling in a spatially and temporally coordinated manner.
Highlights
NHERF tethers the parathyroid hormone receptor (PTHR) at the cell membrane
Recruitment of -arrestins to the PTHR is delayed until Na/H exchanger regulatory factor-1 (NHERF1) dissociates from the receptor, which is trafficked to clathrin for internalization
Inhibition of PTHR internalization by K44A-dynamin did not fully recapitulate the increase in particle size induced by NHERF1. These results suggest that receptor clustering at the cell membrane promoted by NHERF1 is achieved by a combination of inhibition of receptor internalization and by PTHR particle rearrangement
Summary
NHERF tethers the PTHR at the cell membrane. Upon PTHR activation and internalization, the fate of NHERF is uncertain. Results: NHERF interaction with the PTHR promotes ezrin association and delays arrestin recruitment to the receptor. The parathyroid hormone receptor (PTHR), which regulates mineral ion homeostasis and bone turnover, is a G protein-coupled receptor harboring a PDZ-binding motif that enables association with NHERF1 and tethering to the actin cytoskeleton. Recruitment of -arrestins to the PTHR is delayed until NHERF1 dissociates from the receptor, which is trafficked to clathrin for internalization. The PTHR possesses a PDZ recognition domain (ETVM) at its intracellular carboxyl terminus that binds NHERF1 PDZ1 with greater affinity than PDZ2 [13, 14] This interaction and the tethering of NHERF1 to the actin cytoskeleton through the ezrin-binding domain forms a complex that stabilizes the PTHR at the cell membrane, thereby delaying endocytosis and desensitization [15,16,17]. NHERF is no longer linked to the receptor, by promoting association of the PTHR to an ezrin-actin cytoskeletal complex and by delaying -arrestin1/2 recruitment, NHERF1 decreases internalization of the PTHR clusters
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