Parathyroid hormone (PTH) regulates extracellular calcium homeostasis through the type 1 PTH receptor (PTH1R) expressed in kidney and bone. The PTH1R undergoes ␤-arrestin/dynamin-mediated endocytosis in response to the biologically active forms of PTH, PTH-(1-34), and PTH-(1-84). We now show that amino-truncated forms of PTH that do not activate the PTH1R nonetheless induce PTH1R internalization in a cell-specific pattern. Activation-independent PTH1R endocytosis proceeds through a distinct arrestin-independent mechanism that is operative in cells lacking the adaptor protein Na/H exchange regulatory factor 1 (NHERF1) (ezrin-binding protein 50). Using a combination of radioligand binding experiments and quantitative, live cell confocal microscopy of fluorescently tagged PTH1Rs, we show that in kidney distal tubule cells and rat osteosarcoma cells, which lack NHERF1, the synthetic antagonist PTH-(7-34) and naturally circulating PTH-(7-84) induce internalization of PTH1R in a ␤-arrestin-independent but dynamindependent manner. Expression of NHERF1 in these cells inhibited antagonist-induced endocytosis. Conversely, expression of dominant-negative forms of NHERF1 conferred internalization sensitivity to PTH-(7-34) in cells expressing NHERF1. Mutation of the PTH1R PDZ-binding motif abrogated interaction of the receptor with NHERF1. These mutated receptors were fully functional but were now internalized in response to PTH-(7-34) even in NHERF1-expressing cells. Removing the NHERF1 ERM domain or inhibiting actin polymerization allowed otherwise inactive ligands to internalize the PTH1R. These results demonstrate that NHERF1 acts as a molecular switch that legislates the conditional efficacy of PTH fragments. Distinct endocytic pathways are determined by NHERF1 that are operative for the PTH1R in kidney and bone cells.Extracellular calcium homeostasis in vertebrate animals is primarily under the endocrine control of the parathyroid hormone (PTH) 1 /type I PTH receptor (PTH1R). The PTH1R, predominantly expressed in kidney and bone cells, belongs to class B of the large superfamily of G protein-coupled receptors (GPCRs) that consists of receptors for peptide hormones and neuropeptides (1). Class B GPCRs are characterized by a common topology and by their ability to couple to multiple signaling pathways via distinct G proteins.PTH is synthesized by the parathyroid glands as a mature peptide of 84 amino acids that is stored in secretory vesicles and dense core granules. Reductions of extracellular calcium levels are detected by the calcium-sensing receptor on parathyroid chief cells and promote the release of PTH, which acts on bone (to increase resorption) and kidney (to augment reabsorption), thereby restoring serum calcium levels. PTH-(1-84) is usually the major form of PTH secreted by the parathyroid glands. However, recent analyses reveal that PTH fragments that are likely to be PTH-(7-84) are also secreted by the parathyroid glands and generated by peripheral metabolism (2, 3). These PTH fragments or their synthetic a...

The sodium-hydrogen exchange regulatory factor 1 (NHERF-1/EBP50) interacts with the C terminus of several G proteincoupled receptors (GPCRs). We examined the role of NHERF-1 and the cytoskeleton on the distribution, dynamics, and trafficking of the ␤ 2 -adrenergic receptor (␤ 2 AR; a type A receptor), the parathyroid hormone receptor (PTH1R; type B), and the calcium-sensing receptor (CaSR; type C) using fluorescence recovery after photobleaching, total internal reflection fluorescence, and image correlation spectroscopy. ␤ 2 AR bundles were observed only in cells that expressed NHERF-1, whereas the PTH1R was localized to bundles that parallel stress fibers independently of NHERF-1. The CaSR was never observed in bundles. NHERF-1 reduced the diffusion of the ␤ 2 AR and the PTH1R. The addition of ligand increased the diffusion coefficient and the mobile fraction of the PTH1R. Isoproterenol decreased the immobile fraction but did not affect the diffusion coefficient of the ␤ 2 AR. The diffusion of the CaSR was unaffected by NHERF-1 or the addition of calcium. NHERF-1 reduced the rate of ligand-induced internalization of the PTH1R. This phenomenon was accompanied by a reduction of the rate of arrestin binding to PTH1R in ligand-exposed cells. We conclude that some GPCRs, such as the ␤ 2 AR, are attached to the cytoskeleton primarily via the binding of NHERF-1. Others, such as the PTH1R, bind the cytoskeleton via several interacting proteins, one of which is NHERF-1. Finally, receptors such as the CaSR do not interact with the cytoskeleton in any significant manner. These interactions, or the lack thereof, govern the dynamics and trafficking of the receptor.Cell membranes are highly heterogeneous structures consisting of an ensemble of fluctuating microdomains with distinct lipid and protein compositions. These microdomains play important functions in signal transduction processes by increasing the rate and efficiency of coupling of key intermolecular interactions involved in specific signaling processes (1-3). The cytoskeleton has also been implicated in the regulation of signal transduction processes by serving as a substrate for the anchoring of specific proteins (4), regulating traffic (5), and partitioning the cell membrane into microdomains through the formation of effective barriers to the diffusion of lipids and proteins present in the bulk of the plasma membrane (6, 7). However, this general model of the cytoskeleton's role in the regulation of signaling processes depicts a somewhat passive picture. Most studies of the relationships among cytoskeletal structures and signaling processes focused on the effects of extracellular signals on cytoskeletal reorganization rather than on the effects of the cytoskeleton upon signaling pathways. This view has been recently challenged by the discovery of a family of proteins containing N-terminal postsynaptic density protein (PSD95)/Drosophila disc large tumor suppressor (DlgA)/Zo-1 protein (PDZ) 2 domains (which interact with a variety of signaling molecules) and a C-te...

Na ؉ -H ؉ exchanger regulatory factor-1 (NHERF1) is a PDZ protein that scaffolds membrane proteins, including sodiumphosphate co-transport protein 2A (NPT2A) at the plasma membrane. NHERF1 is a phosphoprotein with 40 Ser and Thr residues. Here, using tandem MS analysis, we characterized the sites of parathyroid hormone (PTH)-induced NHERF1 phosphorylation and identified 10 high-confidence phosphorylation sites. did not affect phosphate uptake, but S290A substitution abolished PTH-dependent phosphate transport. Unexpectedly, Ser 290 was rapidly dephosphorylated and rephosphorylated after PTH stimulation, and we found that protein phosphatase 1␣ (PP1␣), which binds NHERF1 through a conserved VxF/W PP1 motif, dephosphorylates Ser 290 . Mutating 257 VPF 259 eliminated PP1 binding and blunted dephosphorylation. Tautomycetin blocked PP1 activity and abrogated PTH-sensitive phosphate transport. Using fluorescence lifetime imaging (FLIM), we observed that PTH paradoxically and transiently elevates intracellular phosphate. Added phosphate blocked PP1␣-mediated Ser 290 dephosphorylation of recombinant NHERF1. Hydrogen-deuterium exchange MS revealed that ␤-sheets in NHERF1's PDZ2 domain display lower deuterium uptake than those in the structurally . 4 The abbreviations used are: PTH, parathyroid hormone; PTHR, PTH receptor; NHERF1, Na ϩ /H ϩ -exchanger regulatory factor 1; GnTI Ϫ , N-acetylglucosaminyltransferase-deficient HEK-293S cells; TAP-NHERF1, tandem affinity purification-tagged NHERF1; FLIM, fluorescence lifetime imaging; HDX, hydrogen/deuterium exchange mass spectrometry; pSer 290 , phosphorylated Ser 290 ; NP40, Nonidet P-40; TAMRA, carboxytetramethylrhodamine; SILAC, stable isotope labeling of amino acids in cell culture; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; ND, nondeuterated; FD, fully deuterated; EBD, ezrin-binding domain; PDB, Protein Data Bank; 2Me-4OMe-TM, 7-hydroxy-5,5-dimethyl-10-(4-methoxy-2-methylphenyl)-dibenzo-[b,e]-silin-3(5H)-one; ANOVA, analysis of variance; ID, intrinsically disordered; TTN, tautomycetin; RPTEC, renal proximal tubule epithelial cell; pen/strep, penicillin and streptomycin; CFP, cyan fluorescent protein; FERM, Ezrin-Radixin-Moesin; MD, molecular dynamics.

Bone growth and remodeling depend upon the opposing rates of bone formation and resorption. These functions are regulated by intrinsic seven transmembrane-spanning receptors, the parathyroid hormone receptor (PTH1R) and frizzled (FZD), through their respective ligands, parathyroid hormone (PTH) and Wnt. FZD activation of canonical ␤-catenin signaling requires the adapter protein Dishevelled (Dvl). We identified a Dvl-binding motif in the PTH1R. Here, we report that the PTH1R activates the ␤-catenin pathway by directly recruiting Dvl, independent of Wnt or LRP5/6. PTH1R coimmunoprecipitated with Dvl. Deleting the carboxyl-terminal PTH1R PDZrecognition domain did not abrogate PTH1R-Dvl interactions; nor did truncating the receptor at position 480. However, further deletion eliminating the putative Dvl recognition domain abolished PTH1R interactions with Dvl. PTH activated ␤-catenin in a time-and concentration-dependent manner and translocated ␤-catenin to the nucleus. ␤-Catenin activation was inhibited by Dvl2 dominant negatives and by short hairpin RNA sequences targeted against Dvl2. PTH-induced osteoclastogenesis was also inhibited by Dvl2 dominant negative mutants. These findings demonstrate that G protein-coupled receptors other than FZD directly activate ␤-catenin signaling, thereby mimicking many of the functions of the canonical Wnt-FZD pathway. The distinct modes whereby FZD and PTH1R activate ␤-catenin control convergent or divergent effects on osteoblast differentiation, and osteoclastogenesis may arise from PTH1R-induced second messenger phosphorylation.Wnts are secreted lipid-modified glycoproteins that act as ligands to stimulate signal transduction pathways through FZD (frizzled) receptors and LRP5/6 (lipoprotein receptor-related protein 5/6) co-receptors. Canonical and non-canonical Wnt signaling pathways have been described (1). In the canonical pathway, in the absence of Wnt ligands, ␤-catenin is targeted to a destruction complex with APC (adenomatous polyposis coli), CK1 (casein kinase 1), GSK3␤ (glycogen synthase kinase 3␤), and axin. Amino-terminal phosphorylation by CK1 and GSK3␤ followed by subsequent ubiquitination targets ␤-catenin for proteasomal degradation (2). Wnt binding to cognate FZD receptors and LRP5/6 causes recruitment of the PDZ (PSD-95, Discs-large, and ZO-1) protein Dvl (Dishevelled) to the plasma membrane by direct interaction with FZD receptors. The recruitment of Dvl to the plasma membrane results in the formation of Dvl oligomers, which interact with axin. Parallel phosphorylation of the co-receptors LRP5 and LRP6 induces binding to axin, resulting in disruption of the destruction complex. As a consequence, ␤-catenin escapes proteasomal degradation and translocates to the nucleus, where it regulates the activity of the transcription factors TCF (T cell factor) and Lef (lymphocyte enhancer-binding factor).Ten FZD receptors constitute a distinct family of seven transmembrane-spanning receptors (3). Whether or not they couple to G proteins remains controversial (4). Notably,...

G protein-coupled receptors (GPCRs) mediate the action of many hormones, cytokines, and sensory and chemical signals. It is generally thought that receptor desensitization and internalization require occupancy and activation of the GPCR. PTH and PTHrP receptor (PTH1R) belongs to GPCR class B and is the major regulator of extracellular calcium homeostasis. Using kidney distal convoluted tubule cells transfected with a human PTH1R/enhanced green fluorescent protein fusion protein, quantitative, real-time fluorescence microscopy was used to analyze receptor internalization. In these cells, which are the target of the calcium-sparing action of PTH, PTH(1-34) activated adenylyl cyclase (AC) and phospholipase C (PLC) and PTH1R endocytosis. PTH(1-31), however, stimulated AC and PLC but not PTH1R endocytosis. Conversely, PTH(7-34) rapidly stimulated PTH1R internalization without activating AC or PLC. PTH(2-34) and (3-34) caused PTH1R internalization intermediate between PTH(1-34) and (7-34). PTH1R sequestration occurred in a dynamin- and clathrin-dependent manner. Directly activating AC inhibited PTH1R internalization in response to PTH(7-34). PTH1R endocytosis was sensitive to protein kinase C inhibition. PTH(1-34), (7-34), and (1-31) evoked PTH1R phosphorylation. Removal of most of the C terminus of the PTH1R eliminated receptor phosphorylation and the cAMP/protein kinase C sensitivity of internalization. PTH(1-34) and (7-34) internalized the truncated PTH1R with identical kinetics, and the response was unaffected by forskolin. Thus, the PTH1R C terminus contains regulatory sequences that are involved in, but not required for, PTH1R internalization. The results demonstrate that receptor activation and internalization can be selectively dissociated.