Internalization Determinants of the Parathyroid Hormone Receptor Differentially Regulate β-Arrestin/Receptor Association
2002; Elsevier BV; Volume: 277; Issue: 10 Linguagem: Inglês
10.1074/jbc.m110433200
ISSN1083-351X
AutoresJean‐Pierre Vilardaga, Cornelius Krasel, Stéphanie Chauvin, Tom Bambino, Martin J. Lohse, Robert A. Nissenson,
Tópico(s)Cellular transport and secretion
Resumoβ-Arrestins have been implicated in regulating internalization of the parathyroid hormone receptor (PTHR), but the structural features in the receptor required for this effect are unknown. In the present study performed in HEK-293 cells, we demonstrated that different topological domains of PTHR are implicated in agonist-dependent receptor internalization; truncation of the cytoplasmic tail (PTHR-TR), selective mutations of the cytoplasmic tail to remove the sites of parathyroid hormone (PTH)-stimulated phosphorylation (PTHR-PD), and mutations in the third transmembrane helix (N289A) or in the third cytoplasmic loop (K382A) resulted in a 30–60% reduction in 125I-PTH-related protein internalization. To better define the role of these internalization determinants, we have tested the ability of these mutant PTHRs to associate with β-arrestins by using three different methodological approaches: 1) ability of overexpression of β-arrestins to restore the internalization of125I-PTH-related protein for the mutant PTHRs; 2) visualization of PTH-mediated trafficking of β-arrestin1 and -2 fused to the green fluorescent protein with receptors by confocal microscopy; 3) quantification of β-arrestin1-green fluorescent protein translocation by Western blot. Our data reveal that the receptor' cytoplasmic tail contains determinants of β-arrestin interaction that are distinct from the phosphorylation sites and are sufficient for transient association of β-arrestin2, but stable association requires receptor phosphorylation. Determinants in the receptor's core (Asn-289 and Lys-382) appear to regulate internalization of the receptor/β-arrestin complex toward early endocytic endosomes during the initial step of endocytosis. β-Arrestins have been implicated in regulating internalization of the parathyroid hormone receptor (PTHR), but the structural features in the receptor required for this effect are unknown. In the present study performed in HEK-293 cells, we demonstrated that different topological domains of PTHR are implicated in agonist-dependent receptor internalization; truncation of the cytoplasmic tail (PTHR-TR), selective mutations of the cytoplasmic tail to remove the sites of parathyroid hormone (PTH)-stimulated phosphorylation (PTHR-PD), and mutations in the third transmembrane helix (N289A) or in the third cytoplasmic loop (K382A) resulted in a 30–60% reduction in 125I-PTH-related protein internalization. To better define the role of these internalization determinants, we have tested the ability of these mutant PTHRs to associate with β-arrestins by using three different methodological approaches: 1) ability of overexpression of β-arrestins to restore the internalization of125I-PTH-related protein for the mutant PTHRs; 2) visualization of PTH-mediated trafficking of β-arrestin1 and -2 fused to the green fluorescent protein with receptors by confocal microscopy; 3) quantification of β-arrestin1-green fluorescent protein translocation by Western blot. Our data reveal that the receptor' cytoplasmic tail contains determinants of β-arrestin interaction that are distinct from the phosphorylation sites and are sufficient for transient association of β-arrestin2, but stable association requires receptor phosphorylation. Determinants in the receptor's core (Asn-289 and Lys-382) appear to regulate internalization of the receptor/β-arrestin complex toward early endocytic endosomes during the initial step of endocytosis. G protein-coupled receptor G protein-coupled receptor kinase parathyroid hormone parathyroid hormone-related protein parathyroid hormone/parathyroid hormone-related protein receptor green fluorescent protein phosphate-buffered saline serum-free Dulbecco's modified Eagle's medium containing HEPES and bovine serum albumin truncation phosphorylation-deficient Agonist binding to most G protein-coupled receptors (GPCRs)1 is quickly followed by the internalization of the agonist-receptor complex into endocytic vesicles. The model developed from studies of the β2-adrenergic receptor views internalization as a process facilitated by binding of β-arrestin proteins to agonist-activated receptors after phosphorylation of the receptors by G protein-coupled receptor kinases (GRKs) (1Gurevich V.V. Dion S.B. Onorato J.J. Ptasienski J. Kim C.M. Sterne-Marr R. Hosey M.M. Benovic J.L. J. Biol. Chem. 1995; 270: 720-731Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 2Pippig S. Andexinger S. Lohse M.J. Mol. Pharmacol. 1995; 47: 666-676PubMed Google Scholar). Phosphorylation of GPCRs by GRKs is a prerequisite for the mobilization of cytosolic β-arrestins. Binding of β-arrestins to GRK-phosphorylated receptors results in the physical uncoupling of receptors from their cognate G proteins and terminates agonist-mediated signaling (3Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (857) Google Scholar, 4Pippig S. Andexinger S. Daniel K. Puzicha M. Caron M.G. Lefkowitz R.J. Lohse M.J. J. Biol. Chem. 1993; 268: 3201-3208Abstract Full Text PDF PubMed Google Scholar). It was shown recently that β-arrestins bind clathrin, a major component of the clathrin-based endocytic machinery, with high affinity and serve as an adaptor that targets activated and phosphorylated receptors to clathrin-coated pits (5Krupnick J.G. Goodman Jr., O.B. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 15011-15016Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 6Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1156) Google Scholar, 7Ferguson S.S.G. Downey III, W.E. Colapietro A.-M. Barak L.S. Ménard L. Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (841) Google Scholar). In the case of the β2-adrenergic receptor, receptor internalization is the consequence of the formation of a complex between β-arrestin2, the clathrin adaptor protein AP2, clathrin, and the activated receptor (8Laporte S.A. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S.G. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Crossref PubMed Scopus (520) Google Scholar). Internalization has at least two outcomes: directing the receptor to a compartment where the phosphates are removed, allowing resensitization, and movement of the receptor to lysosomes for degradation (2Pippig S. Andexinger S. Lohse M.J. Mol. Pharmacol. 1995; 47: 666-676PubMed Google Scholar, 9Laporte S.A. Oakley R.H. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 2000; 275: 23120-23126Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar,10Ferguson S.S.G. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar). Little is known about the structural determinants of GPCRs involved in receptor internalization and/or in arrestin interaction. The current model for the understanding of the arrestin-receptor interaction mechanism is based on various studies of visual arrestin interaction with rhodopsin (11Scheicher A. Kuhn H. Hofmann K.P. Biochemistry. 1989; 28: 1770-1775Crossref PubMed Scopus (166) Google Scholar, 12Granzin J. Wilden U. Choe H.W. Labahn J. Krafft B. Büldt G. Nature. 1998; 391: 918-921Crossref PubMed Scopus (208) Google Scholar, 13Hirsch J.A. Schubert C. Gurevich V.V. Sigler P.B. Cell. 1999; 97: 257-269Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar, 14Vishnivetskiy S.A. Paz C.L. Schubert C. Hirsch J.A. Sigler P.B. Gurevich V.V. J. Biol. Chem. 1999; 274: 11451-11454Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 15Gurevich V.V. J. Biol. Chem. 1998; 273: 15501-15506Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 16Vishnivetskiy S.A. Schubert C. Climaco G.C. Gurevich Y.V. Velez M.-G. Gurevich V.V. J. Biol. Chem. 1999; 275: 41049-41057Abstract Full Text Full Text PDF Scopus (153) Google Scholar). Visual arrestin binding with rhodopsin requires GRK-1 phosphorylated residues, and the contact of phosphorylated residues with a cationic region of arrestin switches it into an active conformation, enabling interaction with an exposed binding site on activated rhodopsin. The receptor for parathyroid hormone (PTH) and PTH-related protein (PTHrP) is involved in the regulation of calcium homeostasis and in bone remodeling (17Gardella T.J. Jüppner H. Rev. Endocrinol. Metab. 2000; 1: 317-329Crossref PubMed Scopus (46) Google Scholar). Agonist occupancy of the PTH/PTHrP receptor (PTHR) leads to activation of adenylyl cyclase (via Gs), and phosphatidylinositol-specific phospholipase Cβ (via Gq). PTH-induced activation of the PTHR results in the internalization of the PTH-receptor complex via the clathrin-coated pit pathway and involves β-arrestin2 (18Ferrari S.L. Behar V. Chorev M. Rosenblatt M. Bisello A. J. Biol. Chem. 1999; 274: 29968-29975Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 19Ferrari S.L. Bisello A. Mol. Endocrinol. 2001; 15: 149-163Crossref PubMed Scopus (53) Google Scholar). Recently, we have demonstrated that neither the internalization nor the mobilization of β-arrestins (i.e. β-arrestin1 and β-arrestin2) to PTHR required the receptor activation isoform that is necessary for activation of both Gs and Gq proteins (i.e. relative displacement of helix 3 and helix 6 of the receptor; Ref. 20Vilardaga J.-P. Frank M. Krasel C. Dees C. Nissenson R.A. Lohse M.J. J. Biol. Chem. 2001; 276: 33435-33443Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). This indicates that G protein activation is not an absolute prerequisite for receptor internalization. In the present study, we investigated the molecular basis of PTHR internalization by examining the interaction of β-arrestins with a series of internalization-deficient receptors. PTH-(1–34) was obtained from Bachem. β-Arrestin2 and β-arrestin1 were fused to the GFP as described (20Vilardaga J.-P. Frank M. Krasel C. Dees C. Nissenson R.A. Lohse M.J. J. Biol. Chem. 2001; 276: 33435-33443Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar,21Groarke D.A. Wilson S. Krasel C. Milligan G. J. Biol. Chem. 1999; 274: 23263-23269Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The preparation of β-arrestin1 antibody has been described previously (22Söhlemann P. Hekmann M. Puzicka M. Buchen C. Lohse M.J. Eur. J. Biochem. 1995; 232: 464-472Crossref PubMed Scopus (41) Google Scholar). PTHR and GFP antibodies were obtained from BAbCO (Richmond, CA) and CLONTECH (Palo Alto, CA), respectively. Expression vectors pCDNA3 and pCEP4 were from Invitrogen (San Diego, CA). Site-directed mutagenesis was performed on the opossum PTH/PTHrP receptor cDNA (23Jüppner H. Abou-Samra A.B. Freeman M. Kong X.F. Schipani E. Richards J. Kolakowski Jr., L.F. Hock J. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Science. 1991; 254: 1024-1026Crossref PubMed Scopus (1140) Google Scholar). Constructions of PTHR-PD (phosphorylation-deficient receptor, mutation of serine residues at positions 483, 485, 486, 488, 495, and 498 to alanine residues), PTHR-TR (carboxyl terminus-truncated receptor after the residue 474) and K382A (mutation of Lys-382 to Ala, in the third intracellular loop near helix 5) have been described previously (24Huang Z. Chen Y. Pratt S. Chen T.-S. Bambino T. Nissenson R.A. Shoback D.M. J. Biol. Chem. 1996; 271: 33382-33389Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 25Malecz N. Bambino T. Bencsik M. Nissenson R.A. Mol. Endocrinol. 1998; 12: 1846-1856Crossref PubMed Google Scholar, 26Huang Z. Cheng Y. Pratt S. Chen T.-H. Bambino T. Shoback D.M. Nissenson R.A. Mol. Endocrinol. 1995; 9: 1240-1249PubMed Google Scholar). Construction of N289A (mutation of Asn-289 to Ala, in helix 3) was performed using the transformer site-directed mutagenesis kit (CLONTECH, Palo Alto, CA) based on the method described by Deng and Nickoloff (27Deng W.P. Nickoloff J.A. Anal. Biochem. 1992; 200: 81-88Crossref PubMed Scopus (1079) Google Scholar). Mutations were verified by sequencing. Receptor cDNAs were cloned into pCDNA3 and pCEP4 for transient and stable expression, respectively. Arrestin cDNAs (β-arrestin1, β-arrestin2, β-arrestin1-GFP, and β-arrestin2-GFP) were cloned into pCDNA3. HEK-293 cells (ATCC, CRL 1573) served as the expression system for the wild type and mutant receptors. Cells were maintained in culture at 37 °C under a humidified atmosphere with 7% CO2 in Dulbecco's modified Eagle's medium with 10% fetal calf serum (Invitrogen). Cells were transfected using a protocol based on the calcium-phosphate method as described previously (28Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1997Google Scholar). Selection of stably transfected cells was initiated 2 days after transfection by the addition of hygromycin (200 μg/ml). Selection was generally complete after 3–4 weeks of hygromycin treatment. Stock cell lines were cultured in the continuous presence of hygromycin, except when subcultured for experiments in which case hygromycin was omitted. To measure phosphorylation of the PTH receptors in intact cells, transiently transfected HEK-293 cells were labeled in six-well plates with 100 μCi/well [32P]orthophosphate in phosphate-free Dulbecco's modified Eagle's medium for 2 h. Labeled cells were stimulated as indicated, solubilized in 0.8 ml of radioimmune precipitation buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mm Tris, pH 7.4, 100 mm NaCl, 2 mmEDTA, 50 mm NaF) for 30 min on ice, and the receptors were immunoprecipitated with 10 μg of mouse monoclonal anti-PTHR antibody. Immunoprecipitated proteins were analyzed by SDS-polyacrylamide gel electrophoresis and PhosphorImager analysis. Transfected cells were grown for 3 days in six-well dishes. After incubation for 1 h at 37 °C with 1 ml of DHB (serum-free Dulbecco's modified Eagle's medium containing 20 mm HEPES and 0.1% bovine serum albumin), cells were incubated with 0.5 ml of DHB containing 125I-PTHrP-(1–34) (100,000 cpm) at 37 °C for the indicated times. Previous studies have demonstrated that PTH-(1–34) and PTHrP-(1–34) bind equipotently the PTH/PTHrP receptor (23Jüppner H. Abou-Samra A.B. Freeman M. Kong X.F. Schipani E. Richards J. Kolakowski Jr., L.F. Hock J. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Science. 1991; 254: 1024-1026Crossref PubMed Scopus (1140) Google Scholar). To stop the incubation, the cells were placed on ice and rapidly washed with 1 ml of ice-cold phosphate-buffered saline (PBS). The cells were incubated for 2 × 5 min in 0.5 ml of acid wash solution (150 mm glycine/50 mm acetic acid, pH = 3) to remove the surface-bound radioligand. The supernatants containing the acid-released radioactivity were collected, and the cells were treated with 1 ml of 0.8 n NaOH to solubilize the acid-resistant radioactivity. The nonspecific binding was measured in parallel experiments using 10−6m PTH-(1–34). The radioactivity was quantified in a γ-counter. The percentage of internalization was calculated after deduction of the respective nonspecific value: % internalization = [(cpm acid-resistant)/(cpm acid-resistant + cpm acid-released)] × 100. The data were fitted to single-phase exponential curves. The endocytic rate constantk e was determined as reported previously (20Vilardaga J.-P. Frank M. Krasel C. Dees C. Nissenson R.A. Lohse M.J. J. Biol. Chem. 2001; 276: 33435-33443Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Ligand binding and cAMP assays were carried out following our previously published procedures (29Vilardaga J.-P. Lin I. Nissenson R.A. Mol. Endocrinol. 2001; 15: 1186-1199Crossref PubMed Scopus (21) Google Scholar). In brief, for binding studies, transfected cells were incubated in DHB buffer for 2 h at 0 °C, followed by a 2-h incubation with the same buffer containing 100,000 cpm 125I-PTH-(1–34) with or without unlabeled PTH-(1–34). Cells were washed twice with iced phosphate-buffered saline and extracted with 0.8 n NaOH, and cell-associated 125I-PTH-(1–34) was counted. Competition curves were fitted to a one-site competitive binding curve. For cAMP assays, cells were washed twice with HEPES buffer and incubated with isobutylmethylxanthine (0.5 mm) in the same buffer for 1 h at room temperature. Cells were stimulated with different PTH-(1–34) concentrations at 37 °C for 10 min. Cellular cAMP was extracted and measured by radioimmunoassay (Immunotech). β-Arrestin translocation was monitored by two assays: confocal scanning microscopy visualization in intact cells and immunoblot analysis. β-Arrestin1 or β-arrestin2 translocation assays were performed in transiently transfected HEK-293 cells. The cells were grown on glass coverslips coated with poly-l-lysine and cotransfected with cDNAs of β-arrestin2 fused to GFP (β-arrestin2-GFP) or β-arrestin1-GFP and PTHR. 48 h after transfection, cells were incubated with 100 nm PTH-(1–34) for different times, and fixed for 20 min in 4% paraformaldehyde. Fixed cells were observed with an oil immersion objective (×40) using a Leica (TCS) confocal laser microscope. Colocalization of β-arrestin2-GFP with rhodamine-labeled wild type or mutant receptors was performed on HEK-293 cells stably expressing the wild type or the mutant PTHR. Cells were grown on glass coverslips and transiently transfected with β-arrestin2-GFP cDNA. After 48 h of transfection, the cells were incubated with 1 μm PTH for different times at 37 °C. Then, the cells were fixed for 20 min in 4% paraformaldehyde and permeabilized using 0.2% Triton. The cells were incubated with mouse OK-1 PTHR antibody at 4 °C overnight. After washing with PBS, the cells were incubated with donkey anti-mouse IgG-rhodamine conjugate secondary antibody at room temperature for 1 h. The cells were mounted on glass slides and examined with a Leica TCS NT/SP confocal microscope. For immunodetection, transiently transfected HEK-293 cells were detached with a rubber policeman and pelleted by low speed centrifugation; the supernatant was discarded and the cells were lysed in PBS containing protease inhibitors (Calbiochem) by sonication. The lysate was centrifuged at 800–1000 × g for 10 min to remove unbroken cells, and the supernatant was further centrifuged at 20,000 × g for 30 min. The resulting supernatant represents the cytosolic fraction and the pellet the membrane fraction. Membrane proteins solubilized in RIPA buffer (150 mm NaCl, 50 mm Tris-HCl, pH 7.5, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS) supplemented with a protease inhibitor mixture (Calbiochem) and soluble protein fractions of transfected cells coexpressing PTHR and β-arrestin1-GFP or β-arrestin2-GFP were separated on 7.5% SDS-PAGE. Proteins were transferred onto an Immobilon P transfer membrane (Millipore, Bedford, MA). Membranes were reacted with anti-β-arrestin1 antibody (1:3000) or anti-GFP (1:5000), and anti-rabbit IgG (1:20,000) coupled to peroxidase, and immunoreactive bands were visualized using chemiluminescence (Pierce). The data were calibrated for similar levels of surface receptors. Concentration/response, competition and internalization kinetics were analyzed by computer-assisted nonlinear regression (GraphPad, San Diego, CA). If not otherwise indicated, all experiments were replicated at least three times in independent experiments, and the results are expressed as mean ± S.E. As shown in Fig.1 A, the PTHR rapidly internalizes in response to agonist when expressed transiently in HEK-293 cells. As monitored by radioligand binding, the maximal extent of internalization was observed after 20 min and represents 55–60% of the cell-specific associated [125I-PTHrP]. The presence of 0.45 m sucrose (an agent that destabilizes clathrin-coated pits; Ref. 30Hansen S.H. Sandvig K. van Deurs B. J. Cell Biol. 1993; 121: 61-72Crossref PubMed Scopus (297) Google Scholar) reduced the maximal extent of PTHR internalization by 80%. This supports the involvement of clathrin-coated pits in PTH-mediated PTHR internalization (Fig.1 A). Overexpression of β-arrestin2 enhanced the maximal extent of PTHR internalization by 15–20%, whereas overexpression of β-arrestin1 did not significantly increase the magnitude of PTHR internalization (Fig. 1 A). Similar effects were observed after overexpression of functional β-arrestins fused to the green fluorescent protein (i.e. β-arrestin1-GFP or β-arrestin2-GFP, data not shown). Overexpression of β-arrestins did not modify the endocytic rate constant of the PTHR (k e ≈ 0.10 min−1 for both β-arrestin isoforms versus k e = 0.12 ± 0.03 min−1 for PTHR). Confocal scanning microscopy was used to visualize the translocation of β-arrestin1-GFP and β-arrestin2-GFP to the PTHR in response to PTH in transiently transfected HEK-293 cells. Fig. 1 B shows that β-arrestin1-GFP and β-arrestin2-GFP were homogeneously localized in the cytosol of cells expressing the PTHR at the resting state. Within minutes, upon PTH stimulation (100 nm), β-arrestin1-GFP and β-arrestin2-GFP translocated equally well from the cytosol to the cell membrane (Fig. 1 B, 1 min). After longer PTH exposure, β-arrestin1-GFP and β-arrestin2-GFP clusters were present inside the cell (Fig. 1 B, 20 min). The punctated pattern of β-arrestin-GFP fluorescence presumably reflected internalization of PTHR with β-arrestins into endocytic vesicles. These data demonstrated that both β-arrestin isoforms translocate equally well to the activated receptor and show, as do recent studies for β-arrestin2 (18Ferrari S.L. Behar V. Chorev M. Rosenblatt M. Bisello A. J. Biol. Chem. 1999; 274: 29968-29975Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 19Ferrari S.L. Bisello A. Mol. Endocrinol. 2001; 15: 149-163Crossref PubMed Scopus (53) Google Scholar), the involvement of β-arrestins in PTH-mediated PTHR internalization. We found a series of PTHR mutants with a reduced ability to internalize [125I-PTHrP] when transiently expressed in HEK-293 cells (Fig. 2, A and B). Truncation of the cytoplasmic tail (PTHR-TR) resulted in a severe loss of internalization (50% relative to the wild type PTHR). Mutation of the cytoplasmic tail to remove the sites of PTH-stimulated phosphorylation (PTHR-PD) produced a 30% reduction (relative to the wild type receptor) in PTHR internalization. Two additional mutations in the body of the PTHR, N289A (in the third transmembrane helix) and mutation K382A (in the third cytoplasmic loop), were found to reduce receptor internalization by 60%. Similar internalization deficiency was also observed in HEK-293 cells stably expressing PTHR-TR, N289A, or K382A (data not shown). In contrast, however, PTHR-PD stably expressed in HEK-293 cells internalizes to levels similar to that observed for PTHR in response to agonist (reported previously in Ref. 25Malecz N. Bambino T. Bencsik M. Nissenson R.A. Mol. Endocrinol. 1998; 12: 1846-1856Crossref PubMed Google Scholar). The pharmacological properties of PTHR, PTHR-PD, PTHR-TR, and K382A have been described in our previous studies (24Huang Z. Chen Y. Pratt S. Chen T.-S. Bambino T. Nissenson R.A. Shoback D.M. J. Biol. Chem. 1996; 271: 33382-33389Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 25Malecz N. Bambino T. Bencsik M. Nissenson R.A. Mol. Endocrinol. 1998; 12: 1846-1856Crossref PubMed Google Scholar, 26Huang Z. Cheng Y. Pratt S. Chen T.-H. Bambino T. Shoback D.M. Nissenson R.A. Mol. Endocrinol. 1995; 9: 1240-1249PubMed Google Scholar). The wild type as well as mutant receptors displayed comparable cell surface receptor expression and similar high PTH binding affinity (TableI). N289A displayed a 4-fold decrease in binding affinity and a 2-fold higher EC50 value (Table I). K382A displayed a strong reduction of the cAMP (Table I) as well as inositol phosphate responses (data not shown), confirming our previous results (24Huang Z. Chen Y. Pratt S. Chen T.-S. Bambino T. Nissenson R.A. Shoback D.M. J. Biol. Chem. 1996; 271: 33382-33389Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar).Table IBinding and signaling characteristics of wild type and mutants of PTHRReceptors/cellK iPTH-mediated cAMP% of maximumEC50× 106nmnmPTHR0.80 ± 0.022.3 ± 0.11000.25 ± 0.05N289A0.90 ± 0.089.4 ± 1.1100 ± 170.50 ± 0.05K382A0.86 ± 0.052.9 ± 0.55 ± 0.80.30 ± 0.03PTHR-TR0.69 ± 0.031.4 ± 0.2120 ± 50.31 ± 0.16PTHR-PD0.61 ± 0.051.8 ± 0.3100 ± 150.17 ± 0.08HEK-293 cells transiently expressing the receptors were examined for125I-PTHrP binding competition and PTH-(1–34)-mediated cAMP production. Basal and maximal PTH-(1–34)-mediated cAMP production were 2–3 and 70–80 pmol/30-mm well (n = 3), respectively. Open table in a new tab HEK-293 cells transiently expressing the receptors were examined for125I-PTHrP binding competition and PTH-(1–34)-mediated cAMP production. Basal and maximal PTH-(1–34)-mediated cAMP production were 2–3 and 70–80 pmol/30-mm well (n = 3), respectively. Because agonist-mediated receptor phosphorylation might facilitate PTH-mediated receptor internalization, we examined the relationship between PTH-dependent internalization and phosphorylation of the receptors. As demonstrated previously (25Malecz N. Bambino T. Bencsik M. Nissenson R.A. Mol. Endocrinol. 1998; 12: 1846-1856Crossref PubMed Google Scholar, 31Dicker F. Quitterer U. Winstel R. Honold K. Lohse M.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5476-5481Crossref PubMed Scopus (115) Google Scholar), PTHR undergoes a PTH-dependent ≈3-fold increase in phosphorylation, and then runs as a broad band at approximately 85 kDa on an autoradiograph (data not shown). Truncation of the tail (PTHR-TR) or mutation of certain Ser residues in the tail (as in PTHR-PD) abolished PTH-promoted receptor phosphorylation. In contrast, the mutant N289A and the signaling-impaired receptor K382A maintained intact phosphorylation characteristics (Fig. 3). These results show that the absence of phosphorylation in PTHR-TR and PTHR-PD may be responsible for the internalization defect. However, our data for N289A and K382A indicate that the decrease of PTH-mediated internalization can occur even when there is no reduction in receptor phosphorylation. Thus, additional mechanisms must regulate PTHR internalization. Given that β-arrestins are involved in the process of PTHR internalization, we next assessed if the diverse mutations reduce PTHR internalization through a β-arrestin-dependent mechanism. We reasoned that overexpression of β-arrestins with the internalization-deficient receptors might compensate for a possible decrease in receptor affinity for β-arrestins. We coexpressed β-arrestin2 or β-arrestin1 with the different mutants in transiently transfected HEK-293 cells and tested by a radioligand assay the specific agonist-mediated internalization of the wild type and mutant receptors. Fig.4 shows that overexpression of β-arrestin2 restored the internalization defect of the phosphorylation-deficient and the truncated PTHRs to 80–85% of that seen with the wild type PTHR in presence of overexpressed β-arrestin2. As observed for β-arrestin2, overexpression of β-arrestin1 ameliorated the internalization extent of PTHR-TR (to 75% of the wild type PTHR+β-arrestin1) but was less able to increase the internalization defect of PTHR-PD. In contrast to the cytoplasmic tail mutants, β-arrestin1 and β-arrestin2 were much less effective in promoting internalization of N289A and K382A mutant receptors; β-arrestin2 or β-arrestin1 only weakly rescued the internalization of these receptors (40–45% of wild type PTHR+β-arrestins). Kinetic analysis of receptor internalization was performed in the absence or presence of exogenous β-arrestin2. Fig.5 shows that, in the absence of β-arrestin2, the internalization-deficient receptors internalized at a lower rate than wild-type PTHR (k e ≈ 0.06–0.09 min−1 for the mutants versus 0.12 min−1 for the wild-type receptor). Coexpression of β-arrestin2 had no effect on the internalization rate of N289A and K382A (k e = 0.060 ± 0.004 min−1 and 0.080 ± 0.004 min−1, respectively), but resulted in a significant increase in the internalization rate constant of PTHR-TR and PTHR-PD (k e in min−1 for PTHR-TR = 0.17 ± 0.01 and for PTHR-PD = 0.12 ± 0.03). These results demonstrated the existence of two classes of receptor mutants. Class 1 mutants (the cytoplasmic tail mutants: PTHR-TR and PTHR-PD) are those where overexpression of β-arrestins can partially rescue agonist-mediated internalization; class 2 mutants (N289A and K382A) are those where overexpression of β-arrestins was not or poorly effective in the amelioration of PTHR internalization. The absence of phosphorylation in the class 1 mutants could reduce the affinity of the PTHR for β-arrestins, and overexpression of β-arrestins might therefore partially compensate for the decrease in receptor affinity for arrestin. The reduced ability of exogenous β-arrestins to restore internalization of class 2 mutants might be the consequence of a weak (or absent) PTH-induced β-arrestin association. Alternatively, the absence of sensitivity to overexpressed β-arrestins might reflect a defect in the internalization process of N289A and K382A that is independent of β-arrestin. To examine more directly the association of β-arrestin2, wild type, and mutant receptors, we visualized by confocal scanning microscopy the trafficking of β-arrestin2-GFP in response to PTH in HEK-293 cells stably expressing the receptors. For these experiments, receptors were labeled with the rhodamine-conjugated anti-PTHR mouse monoclonal antibody and the colocalization between the receptor and the transiently expressed β-arrestin2-GFP was visualized by merging the green and red fluorescence, resulting in a yellow signal. Fig. 6 A shows that in the absence of PTH, the PTHR was present at the
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