Formation of a Ternary Complex among NHERF1, β-Arrestin, and Parathyroid Hormone Receptor

Abstract

␤-Arrestins are crucial regulators of G-protein coupled receptor (GPCR) signaling, desensitization, and internalization. Despite the long-standing paradigm that agonist-promoted receptor phosphorylation is required for ␤-arrestin2 recruitment, emerging evidence suggests that phosphorylation-independent mechanisms play a role in ␤-arrestin2 recruitment by GPCRs. Several PDZ proteins are known to interact with GPCRs and serve as cytosolic adaptors to modulate receptor signaling and trafficking. Na ؉ /H ؉ exchange regulatory factors (NHERFs) exert a major role in GPCR signaling. By combining imaging and biochemical and biophysical methods we investigated the interplay among NHERF1, ␤-arrestin2, and the parathyroid hormone receptor type 1 (PTHR). We show that NHERF1 and ␤-arrestin2 can independently bind to the PTHR and form a ternary complex in cultured human embryonic kidney cells and Chinese hamster ovary cells. Although NHERF1 interacts constitutively with the PTHR, ␤-arrestin2 binding is promoted by receptor activation. NHERF1 interacts directly with ␤-arrestin2 without using the PTHR as an interface. Fluorescence resonance energy transfer studies revealed that the kinetics of PTHR and ␤-arrestin2 interactions were modulated by NHERF1. These findings suggest a model in which NHERF1 may serve as an adaptor, bringing ␤-arrestin2 into close proximity to the PTHR, thereby facilitating ␤-arrestin2 recruitment after receptor activation.Scaffold proteins play an increasingly recognized role in the regulation and the signal transduction of G protein coupled receptors (GPCRs).2 The interaction of GPCRs with ␤-arrestins results in several distinct effects: (i) signal termination by homologous receptor desensitization, (ii) recruitment of elements of the internalization machinery thereby promoting GPCR endocytosis, and (iii) switching GPCR signaling to nonclassical pathways such as the mitogen-activated protein kinase pathway (1-3). Despite their limited sequence homology, nearly all GPCRs bind to ␤-arrestins, and ␤-arrestin recruitment is considered to be a universal mechanism for GPCR regulation. Rather than binding to a specific motif, ␤-arrestins appear to interact with several parts of the receptor involving the second and third intracellular loops and the C terminus (4 -6). It is generally thought that ␤-arrestin-receptor interaction involves an initial weak interaction of cytosolic ␤-arrestins with an active and phosphorylated receptor conformation, followed by conformational rearrangements of ␤-arrestins that further stabilize the receptor⅐arrestin complex (7,8).The receptor for parathyroid hormone (PTH) and PTH-related protein is involved in regulating calcium homeostasis and bone remodeling (9). Agonist occupancy of the PTH/PTH-related protein receptor (PTHR) leads to G s -mediated stimulation of adenylyl cyclase, resulting in cAMP production and PKA activation, and G q/11 -mediated phosphatidylinositol-specific phospholipase C␤ stimulation, leading to inositol 1,4,5-trisphosphate formation, calcium mobilization...

Dynamic Na+-H+ Exchanger Regulatory Factor-1 Association and Dissociation Regulate Parathyroid Hormone Receptor Trafficking at Membrane Microdomains

“…However, the fate of NHERF1 and the subsequent events triggered by the activation of the PTHR remain unknown. In contrast to the static model of PTHR-NHERF1 interaction previously proposed (23), we now show dynamic interactions between NHERF1 and the PTHR that determine and coordinate the series of events preceding receptor internalization. Upon PTH stimulation, NHERF1 rapidly dissociates from the PTHR, resulting in receptor redistribution in membrane-retained clusters.…”

“…Such an interaction may transiently occur when ␤-arrestins are recruited to the receptor and NHERF1 is being released. This brief interaction could be stabilized by crosslinking agents (23). In our hands, after PTH stimulation, fulllength HA-tagged PTHR showed negligible coimmunoprecipitation with ␤-arrestin in the presence of NHERF compared with strong interaction in the absence of NHERF (16).…”

“…Thus, trafficking of ␤-arrestins to the receptor and consequent receptor internalization may be impeded, whereas NHERF1 or the ezrinactin cytoskeleton complex is bound to the receptor. However, a ternary complex of PTHR-NHERF1 and ␤-Arrestin has been described (23). Such an interaction may transiently occur when ␤-arrestins are recruited to the receptor and NHERF1 is being released.…”

Regulation of G Protein-Coupled Receptor Function by Na+/H+Exchange Regulatory Factors

“…However, an alternative model has been suggested in which NHERF1 may serve as an adaptor, bringing ␤-arrestin-2 into proximity of the PTH1R. This model would facilitate ␤-arrestin-2 recruitment after receptor activation by stabilizing a ternary complex (Klenk et al, 2010). Such interactions may briefly occur when ␤-arrestins are recruited to NHERF1-bound PTH1R.…”

Drug Transporters and Na+/H+Exchange Regulatory Factor PSD-95/Drosophila Discs Large/ZO-1 Proteins

“…NHERF1 and NHERF2 contain two tandem nonidentical PDZ domains and an EBD, whereas NHERF3 and NHERF4 have four PDZ domains but lack an EBD (Seidler et al, 2009). NHERF proteins play an important role in organizing signaling complexes, controlling apical membrane trafficking, and coupling apical membrane transporters and receptors with other PDZ targets (Wang et al, 2007;Klenk et al, 2010;Georgescu et al, 2014). Although NHERF isoforms share these common functional characteristics, they perform distinct physiologic functions.…”