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 thromboxane A 2 receptor (TP) is a G protein-coupled receptor that is expressed as two alternatively spliced isoforms, ␣ (343 residues) and ␤ (407 residues) that share the first 328 residues. We have previously shown that TP␤, but not TP␣, undergoes agonist-induced internalization in a dynamin-, GRK-, and arrestindependent manner. In the present report, we demonstrate that TP␤, but not TP␣, also undergoes tonic internalization. Tonic internalization of TP␤ was temperature-and dynamin-dependent and was inhibited by sucrose and NH 4 Cl treatment but unaffected by wildtype or dominant-negative GRKs or arrestins. Truncation and site-directed mutagenesis revealed that a YX 3 motif (where X is any residue and is a bulky hydrophobic residue) found in the proximal portion of the carboxyl-terminal tail of TP␤ was critical for tonic internalization but had no role in agonist-induced internalization. Interestingly, introduction of either a YX 2 or YX 3 motif in the carboxyl-terminal tail of TP␣ induced tonic internalization of this receptor. Additional analysis revealed that tonically internalized TP␤ undergoes recycling back to the cell surface suggesting that tonic internalization may play a role in maintaining an intracellular pool of TP␤. Our data demonstrate the presence of distinct signals for tonic and agonist-induced internalization of TP␤ and represent the first report of a YX 3 motif involved in tonic internalization of a cell surface receptor.Cell surface receptors provide a primary mechanism by which cells perceive their environment. Many cell surface receptors are dynamically regulated and often undergo a process of endocytic sorting (1). For some receptors (e.g. G proteincoupled and growth factor), sorting is often initiated by hormone binding, whereas for others (e.g. low density lipoprotein and transferrin), the receptors undergo continuous or tonic internalization and recycling. Recent studies have demonstrated that several GPCRs 1 including the CXCR4, thyrotropin, M 2 muscarinic, and thrombin receptors also undergo tonic internalization (2-5). Although no particular motif responsible for tonic internalization of GPCRs has been identified, tyrosine-containing (YXX and NPXY) and dileucine motifs have been shown to be determinants for a number of other receptor types (1). Various studies have demonstrated direct interaction between YXX motifs and the chain of the clathrin-associated proteins AP-1, AP-2 (Ref. 6 and references therein), and AP-3 (7, 8), allowing the efficient targeting of transmembrane proteins containing these motifs to clathrin-coated vesicles.Thromboxane has been implicated in a number of cardiovascular, bronchial, and kidney diseases (9, 10). It is produced by the sequential metabolism of arachidonic acid by cyclooxygenase and thromboxane synthase following activation of a variety of cell types including platelets, macrophages, and vascular smooth muscle cells (11). Thromboxane is a strong activator of platelet aggregation and smooth muscle cell proliferation and mediates its effects v...

We evaluated the ability of different trypsin-revealed tethered ligand (TL) sequences of rat proteinase-activated receptor 2 (rPAR 2 ) and the corresponding soluble TL-derived agonist peptides to trigger agonist-biased signaling. To do so, we mutated the proteolytically revealed TL sequence of rPAR 2 and examined the impact on stimulating intracellular calcium transients and mitogen-activated protein (MAP) Ser 38 constructs recruited ␤-arrestins-1 or -2 in response to trypsin stimulation, whereas both ␤-arrestins were recruited to these mutants by SLIGRL-NH 2 . The lack of trypsin-triggered ␤-arrestin interactions correlated with impaired trypsin-activated TL-mutant receptor internalization. Trypsinstimulated MAP kinase activation by the TL-mutated receptors was not blocked by inhibitors of G␣ i (pertussis toxin),, or the epidermal growth factor (EGF) receptor [4-(3Ј-chloroanilino)-6,7-dimethoxy-quinazoline (AG1478)], but was inhibited by the Rhokinase inhibitor (R)-(ϩ)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, 2HCl (Y27362). The data indicate that the proteolytically revealed TL sequence(s) and the mode of its presentation to the receptor (tethered versus soluble) can confer biased signaling by PAR 2 , its arrestin recruitment, and its internalization. Thus, PAR 2 can signal to multiple pathways that are differentially triggered by distinct proteinase-revealed TLs or by synthetic signal-selective activating peptides.Proteinase-activated receptors (PARs) are unique members of the G-protein-coupled superfamily of receptors (GPCRs), modeled as seven transmembrane domain cell-surface receptors that mediate diverse signaling events in response to proteolytic exposure of an N-terminal tethered ligand (TL) sequence. PAR 2 , the second member of this family to be cloned (Nystedt et al., 1994;Bohm et al., 1996) ABBREVIATIONS: PAR, proteinase-activated receptor; A23187, calcimycin; AG1478, 4-(3Ј-chloroanilino)-6,7-dimethoxy-quinazoline; BRET, bioluminescence resonance energy transfer; DKO, double-knockout ␤-arrestin-deficient mouse embryo-derived fibroblasts; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; GP2A,pyrazole-3-carboxamide; GPCR, G-protein-coupled receptor; H1152, (S)-(ϩ)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride; HEK, human embryonic kidney; KNRK, normal rat kidney cell line transformed by Kirsten murine sarcoma virus; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; MEF, mouse embryo-derived fibroblasts; MMP, matrix metalloproteinase; PAR 2 or wt-rPAR 2 , wild-type rat proteinase-activated receptor-2 having the trypsin-revealed tethered ligand sequence SLIGRL-; PAR 2 -Ala 37-38 , mutated rat PAR 2 with a trypsin-revealed tethered ligand sequence AAIGRL-; PAR 2 -Ala 39 -42 , mutated rat PAR 2 with a trypsin-revealed tethered ligand sequence SLAAAA-; PAR 2 -Leu 37 Ser 38 , mutated rat PAR 2 with a trypsin-revealed tethered ligand sequence LSIGRL...

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The most widely studied pathway underlying agonist-promoted internalization of G protein-coupled receptors (GPCRs) involves ␤-arrestin and clathrin-coated pits. However, both ␤-arrestin-and clathrin-independent processes have also been reported. Classically, the endocytic routes are characterized using pharmacological inhibitors and various dominant negative mutants, resulting sometimes in conflicting results and interpretational difficulties. Here, taking advantage of the fact that ␤-arrestin binding to the ␤2 subunit of the clathrin adaptor AP-2 (␤2-adaptin) is needed for the ␤-arrestin-mediated targeting of GPCRs to clathrin-coated pits, we developed a bioluminescence resonance energy transfer-based approach directly assessing the molecular steps involved in the endocytosis of GPCRs in living cells. For 10 of the 12 receptors tested, including some that were previously suggested to internalize via clathrinindependent pathways, agonist stimulation promoted ␤-arrestin 1 and 2 interaction with ␤2-adaptin, indicating a ␤-arrestinand clathrin-dependent endocytic process. Detailed analyses of ␤-arrestin interactions with both the receptor and ␤2-adaptin also allowed us to demonstrate that recruitment of ␤-arrestins to the receptor and the ensuing conformational changes are the leading events preceding AP-2 engagement and subsequent clathrin-mediated endocytosis. Among the receptors tested, only the endothelin A and B receptors failed to promote interaction between ␤-arrestins and ␤2-adaptin. However, both receptors recruited ␤-arrestins upon agonist stimulation, suggesting a ␤-arrestin-dependent but clathrin-independent route of internalization for these two receptors. In addition to providing a new tool to dissect the molecular events involved in GPCR endocytosis, the bioluminescence resonance energy transfer-based ␤-arrestin/␤2-adaptin interaction assay represents a novel biosensor to assess receptor activation. G protein-coupled receptors (GPCRs)7 are seven transmembrane domain receptors that constitute the largest family of cell surface proteins involved in signal transduction. In humans, it is estimated that GPCRs are encoded by ϳ800 distinct genes that control a variety of important physiological responses (1). Following agonist binding, GPCRs undergo conformational changes that regulate the activity of downstream effector systems to mediate various cellular responses. The extent and duration of GPCR signaling is tightly regulated by mechanisms that terminate the initial signaling and later re-establish the capacity of the receptors to respond to new agonist exposure. The removal of GPCRs from the cell surface, also known as internalization or endocytosis, plays an important role in these processes (2, 3). For most GPCRs, rapid feedback desensitization is initiated by G protein-coupled receptor kinases that phosphorylate agonist-occupied GPCRs to create high affinity binding sites for ␤-arrestins, which in turn uncouple the receptor from its cognate G protein (reviewed in Ref. 4). ␤-Arrestins also target rece...

†These authors contributed equally to the work.Activated human neuropeptide Y Y 1 receptors rapidly desensitize and internalize through clathrin-coated pits and recycle from early and recycling endosomes, unlike Y 2 receptors that neither internalize nor desensitize. To identify motifs implicated in Y 1 receptor desensitization and trafficking, mutants with varying C-terminal truncations or a substituted Y 2 C-terminus were constructed. Point mutations of key putative residues were made in athat we have identified and in the second intracellular i2 loop. Receptors were analyzed by functional assays, spectrofluorimetric measurements on living cells, flow cytometry, confocal imaging and bioluminescence resonance energy transfer assays for b-arrestin activation and adaptor protein (AP-2) complex recruitment. Inhibitory GTP-binding protein-dependent signaling of Y 1 receptors to adenylyl cyclase and desensitization was unaffected by C-terminal truncations or mutations, while C-terminal deletion mutants of 42 and 61 amino acids no longer internalized. Substitutions of Thr357, Asp358, Ser360 and Thr362 by Ala in the C-terminus abolished both internalization and b-arrestin activation but not desensitization. A Pro145 substitution by His in an i2 consensus motif reported to mediate phosphorylation-independent recruitment of b-arrestins affected neither desensitization, internalization or recycling kinetics of activated Y 1 receptors nor b-arrestin activation. Interestingly, combining Pro145 substitution by His and C-terminal substitutions significantly attenuates Y 1 desensitization. In the Y 2 receptor, replacement of His155 with Pro at this position in the i2 loop motif promotes agonist-mediated desensitization, b-arrestin activation, internalization and recycling. Overall, our results indicate that b-arrestin-mediated desensitization and internalization of Y 1 and Y 2 receptors are differentially regulated by the C-terminal motif and the i2 loop consensus motif.

Although ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a PDZ domain-containing protein known to bind to various channels, receptors, cytoskeletal elements, and cytoplasmic proteins, there is still very little evidence for a role of EBP50 in the regulation of receptor signal transduction. In this report, we show that EBP50 inhibits the phospholipase C (PLC)-␤-mediated inositol phosphate production of a G␣ q -coupled receptor as well as PLC-␤ activation by the constitutively active G␣ q -R183C mutant. Coimmunoprecipitation experiments revealed that EBP50 interacts with G␣ q and to a greater extent with G␣ q -R183C. Agonist stimulation of the thromboxane A 2 receptor (TP receptor) resulted in an increased interaction between EBP50 and G␣ q , suggesting that EBP50 preferentially interacts with activated G␣ q . We also demonstrate that EBP50 inhibits G␣ q signaling by preventing the interaction between G␣ q and the TP receptor and between activated G␣ q and PLC-␤1. Investigation of the EBP50 regions involved in G␣ q binding indicated that its two PDZ domains are responsible for this interaction. This study constitutes the first demonstration of an interaction between a G protein ␣ subunit and another protein through a PDZ domain, with broad implications in the regulation of diverse physiological systems. EBP501 (also known as NHERF1), a 55-kDa phosphoprotein, was first identified as a cofactor essential for protein kinase A-mediated inhibition of Na ϩ /H ϩ exchanger isoform 3 (NHE3) (1). EBP50 contains two PDZ domains (PDZ1 and PDZ2) implicated in multiple protein-protein interactions, and an ERM domain, which binds to the actin-associated ERM proteins (ezrin, radixin, moesin, and merlin) (2, 3). EBP50 was also found to interact with a small number of transmembrane proteins such as the cystic fibrosis transmembrane conductance regulator (CFTR) (4), the P2Y1 purinergic receptor (5), the platelet-derived growth factor receptor (6), the ␤ 2 -adrenergic receptor (5), the B1 subunit of the H ϩ -ATPase (7), and the type IIa sodium phosphate cotransporter (8). EBP50 also associates with the phospholipases C (PLC)-␤1/␤2, and with the TRP4 and TRP5 calcium channels to form a PLC-␤1/2-TRP4/5-EBP50 protein complex (9). The physiological role of this interaction on the regulation of PLC-␤1/2 remains undefined. The EBP50 protein can also bind through its PDZ domains to various intracellular proteins, including GRK6A (10), EPI64 (11), and Yes-associated protein 65 (12). A close relative of EBP50 has been identified and is known as E3KARP (13), SIP-1 (14), and NHERF2 (15). EBP50 and NHERF2 share 52% amino acid identity and a conserved domain architecture (13). It has been shown recently that the PDZ domains of EBP50 can homooligomerize and also hetero-oligomerize with the PDZ domains of NHERF2 (16). Despite the growing evidence suggesting the role of EBP50 as a scaffolding protein involved in the formation of signaling complexes, there is still little evidence for a role of EBP50 in the regulation of transmembrane recep...

G protein-coupled receptors (GPCRs) represent a vast family of transmembrane proteins involved in the regulation of several physiological responses. The thromboxane A2 receptor (present as two isoforms: TP␣ and TP␤) is a GPCR displaying diverse pharmacological effects. As seen for many other GPCRs, TP␤ is regulated by agonistinduced internalization. In the present study, we report the identification by yeast two-hybrid screening of Nm23-H2, a nucleoside diphosphate kinase, as a new interacting molecular partner with the C-terminal tail of TP␤. This interaction was confirmed in a cellular context when Nm23-H2 was co-immunoprecipitated with TP␤ in HEK293 cells, a process dependent on agonist stimulation of the receptor. We observed that agonist-induced internalization of TP␤ was regulated by Nm23-H2 through modulation of Rac1 signaling. Immunofluorescence microscopy in HEK293 cells revealed that Nm23-H2 had a cytoplasmic and nuclear localization but was induced to translocate to the plasma membrane upon stimulation of TP␤ to show extensive co-localization with the receptor. Our findings represent the first demonstration of an interaction of an Nm23 protein with a membrane receptor and constitute a novel molecular regulatory mechanism of GPCR endocytosis.