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• W2083720922 abstract "Although the involvement of the nonvisual arrestins in the agonist-induced internalization of the human lutropin receptor (hLHR) has been documented previously with the use of dominant-negative mutants, a physical association of the nonvisual arrestins with the hLHR in intact cells has not been established. In the studies presented herein, we used a cross-linking/coimmunoprecipitation/immunoblotting approach as well as confocal microscopy to document the association of the hLHR with the nonvisual arrestins in co-transfected 293 cells. We also used this approach to examine the relative importance of receptor activation and receptor phosphorylation in the formation of this complex. Using hLHR mutants that impair phosphorylation, activation, or both, we show that the formation of the hLHR-nonvisual arrestin complex depends mostly on the agonist-induced activation of the hLHR rather than on the phosphorylation of the hLHR. These results stand in contrast to those obtained with several other G protein-coupled receptors (i.e. the β2-adrenergic receptor, the m2 muscarinic receptor, rhodopsin, and the type 1A angiotensin receptor) where arrestin binding depends mostly on receptor phosphorylation rather than on receptor activation. We have also examined the association of the nonvisual arrestins with naturally occurring gain-of-function mutations of the hLHR found in boys with Leydig cell hyperplasia or Leydig cell adenomas. Our results show that these mutants associate with the nonvisual arrestins in an agonist-independent fashion. Although the involvement of the nonvisual arrestins in the agonist-induced internalization of the human lutropin receptor (hLHR) has been documented previously with the use of dominant-negative mutants, a physical association of the nonvisual arrestins with the hLHR in intact cells has not been established. In the studies presented herein, we used a cross-linking/coimmunoprecipitation/immunoblotting approach as well as confocal microscopy to document the association of the hLHR with the nonvisual arrestins in co-transfected 293 cells. We also used this approach to examine the relative importance of receptor activation and receptor phosphorylation in the formation of this complex. Using hLHR mutants that impair phosphorylation, activation, or both, we show that the formation of the hLHR-nonvisual arrestin complex depends mostly on the agonist-induced activation of the hLHR rather than on the phosphorylation of the hLHR. These results stand in contrast to those obtained with several other G protein-coupled receptors (i.e. the β2-adrenergic receptor, the m2 muscarinic receptor, rhodopsin, and the type 1A angiotensin receptor) where arrestin binding depends mostly on receptor phosphorylation rather than on receptor activation. We have also examined the association of the nonvisual arrestins with naturally occurring gain-of-function mutations of the hLHR found in boys with Leydig cell hyperplasia or Leydig cell adenomas. Our results show that these mutants associate with the nonvisual arrestins in an agonist-independent fashion. G protein-coupled receptor G protein-coupled receptor kinase β2-adrenergic receptor hemagglutinin wild-type dithiobis(succinimidylpropionate) hemagglutinin green fluorescent protein lutropin receptor Internalization of G protein-coupled receptors (GPCRs)1 is a ubiquitous response that follows agonist activation (reviewed in Refs. 1Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar, 2Carman C.V. Benovic J.L. Curr. Opin. Neurobiol. 1998; 8: 335-344Crossref PubMed Scopus (230) Google Scholar, 3Bünemann M. Hosey M.M. J. Physiol. (Lond.). 1999; 517: 5-23Crossref Scopus (162) Google Scholar, 4Ferguson S.S.G. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar). Although GPCRs can be internalized by several distinct pathways (3Bünemann M. Hosey M.M. J. Physiol. (Lond.). 1999; 517: 5-23Crossref Scopus (162) Google Scholar), the most common and best understood pathway is facilitated by the G protein-coupled receptor kinase (GRK)-catalyzed phosphorylation of GPCRs and the subsequent formation of a complex between the agonist-activated and phosphorylated GPCRs and a family of proteins known as the nonvisual arrestins or βarrestins. The nonvisual arrestins (arrestin-2 also known as β-arrestin-1 and arrestin-3, also known as β-arrestin-2) bind with high affinity to clathrin and to adaptor protein-2 (5Goodman J.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 (1140) Google Scholar, 6Laporte 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 (297) Google Scholar) and thus target the activated and phosphorylated GPCRs to clathrin coated pits. Once localized to clathrin-coated pits, the GPCRs are internalized by a process that requires the participation of dynamin, a GTPase involved in the fission of clathrin-coated pits (7Warnock D. Schmid S. BioEssays. 1996; 18: 885-893Crossref PubMed Scopus (137) Google Scholar). Studies from this and other laboratories have shown that the binding of agonist to the rat, mouse, porcine, or human lutropin receptor (LHR) triggers the internalization of the agonist-receptor complex via clathrin-coated pits by a pathway that can be inhibited with dominant-negative mutants of the nonvisual arrestins and a dominant-negative mutant of dynamin (8Ascoli M. J. Cell Biol. 1984; 99: 1242-1250Crossref PubMed Scopus (72) Google Scholar, 9Ghinea N. Vuhai M.T. Groyer-Picard M.-T. Houllier A. Schoëvaërt D. Milgrom E. J. Cell Biol. 1992; 118: 1347-1358Crossref PubMed Scopus (86) Google Scholar, 10Lazari M.F.M. Bertrand J.E. Nakamura K. Liu X. Krupnick J.G. Benovic J.L. Ascoli M. J. Biol. Chem. 1998; 273: 18316-18324Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 11Nakamura K. Liu X. Ascoli M. J. Biol. Chem. 2000; 275: 241-247Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 12Min L. Ascoli M. Mol. Endocrinol. 2000; 14: 1797-1810Crossref PubMed Scopus (59) Google Scholar). Although the large number of studies on the β2-adrenergic receptor emphasize the importance of GPCR phosphorylation on the process of internalization (1Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar, 2Carman C.V. Benovic J.L. Curr. Opin. Neurobiol. 1998; 8: 335-344Crossref PubMed Scopus (230) Google Scholar, 3Bünemann M. Hosey M.M. J. Physiol. (Lond.). 1999; 517: 5-23Crossref Scopus (162) Google Scholar, 4Ferguson S.S.G. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar), our recent mutagenesis studies with the human (h) LHR suggest that the agonist-induced internalization of this receptor is much more dependent on receptor activation than on receptor phosphorylation (12Min L. Ascoli M. Mol. Endocrinol. 2000; 14: 1797-1810Crossref PubMed Scopus (59) Google Scholar,13Li S. Liu X. Min L. Ascoli M. J. Biol. Chem. 2001; 276: 7968-7973Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). This conclusion was based on the following findings. First, an activation-competent but phosphorylation-impaired mutant of the hLHR (constructed by mutation of the phosphorylation sites) lengthens thet½ of internalization of the hLHR less than 2-fold (12Min L. Ascoli M. Mol. Endocrinol. 2000; 14: 1797-1810Crossref PubMed Scopus (59) Google Scholar). Second, two mutations of the second extracellular loop (S512A and V519A) that cause a slight impairment in receptor activation (i.e. a 2–3-fold rightward shift in the EC50for cAMP accumulation) lengthen the t½ of internalization 5–7-fold without affecting receptor phosphorylation (13Li S. Liu X. Min L. Ascoli M. J. Biol. Chem. 2001; 276: 7968-7973Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Third, two other mutants of the hLHR (D405N and Y546F in transmembrane helices 2 and 5, respectively) that cause a more severe impairment in receptor activation (i.e. a 10–50-fold rightward shift in the EC50 for cAMP accumulation) become resistant to agonist-induced phosphorylation but still lengthen thet½ of internalization 5–7-fold (12Min L. Ascoli M. Mol. Endocrinol. 2000; 14: 1797-1810Crossref PubMed Scopus (59) Google Scholar). Interestingly, the phosphorylation of the D405N and Y546F mutants and their long t½ of internalization can be rescued by overexpression of GRK2 but only if the phosphorylation sites are intact. Because dominant-negative mutants of the nonvisual arrestins inhibit the agonist-induced internalization of the hLHR, we hypothesized that the different manipulations described above affected internalization by affecting the association of the hLHR with the nonvisual arrestins. The studies presented here describe a method that can be used to measure the formation of the nonvisual arrestin-hLHR complex and use this method to formally test the hypothesis proposed above. The preparation and properties of a hLHR-wt expression vector modified with the Myc epitope at the N terminus has been described (12Min L. Ascoli M. Mol. Endocrinol. 2000; 14: 1797-1810Crossref PubMed Scopus (59) Google Scholar). All hLHR mutants used here were prepared using this vector as template. The preparation, signaling properties, and internalization properties of all these mutants have also been described (12Min L. Ascoli M. Mol. Endocrinol. 2000; 14: 1797-1810Crossref PubMed Scopus (59) Google Scholar, 13Li S. Liu X. Min L. Ascoli M. J. Biol. Chem. 2001; 276: 7968-7973Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Expression vectors for arrestin-2, arrestin-3, a β2-adrenergic receptor tagged with the HA epitope at the N terminus (in pcDNA3.1), and arrestin-3 tagged with GFP at the C terminus (in pEGFP-N1) were kindly provided by Dr. Jeff Benovic (Thomas Jefferson University, Philadelphia, PA). The nonvisual arrestins were modified (using PCR strategies) with the FLAG epitope at the N terminus and subcloned into pcDNA3.1 for expression. Co-transfection experiments revealed that the FLAG-tagged nonvisual arrestin constructs were as effective as their wild-type counterparts in enhancing the internalization of the hLHR-wt. A full-length clone encoding for bovine GRK2 (14Benovic J.L. DeBlasi A. Stone C.W. Caron M.G. Lefkowitz R.J. Science. 1989; 246: 235-240Crossref PubMed Scopus (325) Google Scholar) was also kindly provided by Dr. Jeff Benovic (Thomas Jefferson University, Philadelphia, PA), and it was subcloned into pcDNA3.1 for expression. HA-tagged dynamin K44A (15Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1031) Google Scholar) was kindly provided by Dr. Sandra Schmid (Scripps Research Institute, La Jolla, CA). An expression vector (pcDNA1.1) encoding for a prenylated, kinase-deficient, mutant of GRK2, designated C20-GRK2-K220M, (16Ferguson S.S.G. Menard L. Barak L.S. Koch W.J. Colapietro A.-M. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), was generously provided by Dr. Marc Caron (Duke University, Durham, NC). 293T cells were maintained in Dulbecco's modified Eagle's medium containing 10 mm Hepes, 10% newborn calf serum, and 50 μg/ml gentamycin, pH 7.4. Transient transfections were performed using the calcium phosphate method of Chen and Okayama (17Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4799) Google Scholar). Cells were plated in gelatin-coated plasticware and transfected when 70–80% confluent with the amounts of plasmid DNA indicated in the figure legends. After an overnight incubation, the cells were washed and used 24 h later. Cells were plated and transfected in 100-mm dishes (in 10 ml of medium) that had been coated with gelatin. At the end of the transfection, the cells were washed, trypsinized, plated in gelatin-coated 100-mm dishes and in gelatin-coated 35-mm wells, and used 24 h later. The cells plated in 35-mm wells were used to assess125I-hCG binding and internalization. The binding of125I-hCG was measured by incubating the transfected cells with a saturating concentration (∼25 nm) of125I-hCG for 1 h at room temperature. These results were used to ensure that the cells were expressing equivalent densities of cell surface receptors and to equalize the amount of receptor to be immunoprecipitated as described below. The internalization of125I-hCG was measured during a 50-min incubation of the cells with a concentration of hCG equivalent to theKd (∼ 2 nm) as described elsewhere (11Nakamura K. Liu X. Ascoli M. J. Biol. Chem. 2000; 275: 241-247Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 18Nakamura K. Liu X. Ascoli M. J. Biol. Chem. 1999; 274: 25426-25432Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 19Nakamura K. Ascoli M. Mol. Pharmacol. 1999; 56: 728-736PubMed Google Scholar). Rates of internalization can be calculated by plotting the internalization index (defined as the ratio of surface to internalized hormone) as a function of time (11Nakamura K. Liu X. Ascoli M. J. Biol. Chem. 2000; 275: 241-247Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 18Nakamura K. Liu X. Ascoli M. J. Biol. Chem. 1999; 274: 25426-25432Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 19Nakamura K. Ascoli M. Mol. Pharmacol. 1999; 56: 728-736PubMed Google Scholar). In all arrestin-3 binding experiments reported herein (using cells co-transfected with dynamin-K44A), the internalization indices for hCG in cells co-transfected with the hLHR-wt or mutants thereof were 0.05–0.1 when measured during a 50-min incubation. This low internalization index measured at this long incubation time is consistent with half-times of internalization in the 150–200-min range (11Nakamura K. Liu X. Ascoli M. J. Biol. Chem. 2000; 275: 241-247Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 18Nakamura K. Liu X. Ascoli M. J. Biol. Chem. 1999; 274: 25426-25432Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 19Nakamura K. Ascoli M. Mol. Pharmacol. 1999; 56: 728-736PubMed Google Scholar). These results were simply used to confirm that all cells co-transfected with dynamin-K44A were internalizing125I-hCG at a slow and comparable rate regardless of the receptor expressed or the amount of arrestin-3 transfected. The cells plated in the 100-mm dishes were used to measure arrestin-3 binding as follows. The monolayers were washed three times with warm 0.15 m NaCl, 20 mm Hepes, pH 7.4 (Buffer A). After addition of 3 ml of the same buffer, each dish received a 60-μl aliquot of vehicle or hCG to give a final concentration of ∼26 nm (the Kd for hCG binding is ∼2 nm; see Ref. 11Nakamura K. Liu X. Ascoli M. J. Biol. Chem. 2000; 275: 241-247Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) and the cells were incubated at 37 °C for the times indicated. At the end of this incubation, each dish received 150 μl of a freshly prepared 25 mm solution of dithiobis(succinimidylpropionate) (DSP) in Me2SO. The cross-linking reaction was allowed to proceed for 30 min at room temperature while the dishes were rocked (20Freedman N. Ament A. Oppermann M. Stoffel R. Exum S. Lefkowitz R. J. Biol. Chem. 1997; 272: 17734-17743Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). After cross-linking the cells were placed on ice, the buffer was aspirated, and the monolayers were washed once with ice-cold Dulbecco's-modified Eagle's medium containing 10 mm Hepes, 10% newborn calf serum, pH 7.4, and then incubated in 5 ml of the same medium for 5 min on ice. Finally, the monolayers were washed two more times with cold 0.15m NaCl, 20 mm Hepes, pH 7.4, and the cells were lysed during a 30-min incubation on ice with 1 ml of lysis buffer (1% Nonidet P-40, 4 mg/ml dodecyl-β-d-maltoside, 0.8 mg/ml cholesteryl hemisuccinate in buffer A) supplemented with 40 μg/ml Complete®, EDTA-free protease inhibitor mixture (Roche Molecular Biochemicals). The lysate was clarified by centrifugation, and aliquots (∼500 μl) containing the same amount of receptor (calculated from the binding experiments done in parallel as described above) were immunoprecipitated with a monoclonal antibody to the Myc epitope (9E10) that had been preabsorbed to agarose-conjugated protein G for 90–120 min at 4 °C as described previously (12Min L. Ascoli M. Mol. Endocrinol. 2000; 14: 1797-1810Crossref PubMed Scopus (59) Google Scholar). After extensive washing the cross-linked complexes were reduced and eluted by vigorous mixing of the beads in SDS sample buffer with reducing agents for 15 min at room temperature. The eluted material was then resolved on SDS gels and electrophoretically transferred to polyvinylidene difluoride membranes as described elsewhere (21Quintana J. Hipkin R.W. Ascoli M. Endocrinology. 1993; 133: 2098-2104Crossref PubMed Scopus (39) Google Scholar). The co-immunoprecipitated FLAG-tagged nonvisual arrestins were visualized in the blots during a 1-h incubation with an anti-FLAG M2 monoclonal antibody covalently coupled to horseradish peroxidase used at a final dilution of 1:500. The presence of equal amounts of receptor in the immunoprecipitates was confirmed by developing the blots with an anti-Myc (9E10) monoclonal antibody covalently coupled to horseradish peroxidase used at a final dilution of 1:1000 (this is documented only in Fig. 3). In experiments that utilized GRK2 co-transfections, the transfected GRK2 was visualized with a commercially available primary antibody (C-15 from Santa Cruz Biotechnology) as described previously (22Lazari M.F.M. Liu X. Nakamura K. Benovic J.L. Ascoli M. Mol. Endocrinol. 1999; 13: 866-878Crossref PubMed Scopus (93) Google Scholar). In some experiments (cf. Fig. 2), the endogenous and/or transfected arrestin-3 were visualized using a 1:2000 dilution of a rabbit polyclonal antibody raised against a glutathioneS-transferase fusion protein containing residues 350–409 of bovine arrestin-3 (23Orsini M.J. Benovic J.L. J. Biol. Chem. 1998; 273: 34616-34622Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). In all of these cases, the appropriate secondary antibodies covalently coupled to horseradish peroxidase were used at a 1:5000 dilution. All immune complexes were ultimately visualized and quantitated using the Super Signal West Femto Maximum Sensitivity system of detection from Pierce and a Kodak digital imaging system. This image capture system is set up to alert us when image saturation occurs and to prevent us from measuring the intensity of such images.Figure 2Agonist-dependent association of the Myc-hLHR-wt with FLAG-arrestin-3 in intact cells expressing increasing amounts of FLAG-arrestin-3.Panels A andB, 293T cells (plated in 100-mm dishes) were transiently co-transfected with a constant amount of Myc-hLHR-wt (10 μg/dish) and dynamin-K44A (2.5 μg/dish) plus the indicated amounts of FLAG-arrestin-3. The transfected cells were washed and incubated for 30 min at 37 °C without or with 26 nm hCG as indicated prior to cross-linking and immunoprecipitation. The results presented show the relevant areas of the blots of a representative experiment in which aliquots of the different lysates (∼18 μl of lysate forpanel A and ∼ 500 μl of lysate for panel B) containing equivalent amounts of receptor (measured by125I-hCG binding as described under “Materials and Methods”) were used to measure the total amount of FLAG-arrestin-3 expressed (panel A) or the amount of FLAG-arrestin-3 immunoprecipitated with the 9E10 monoclonal antibody (panel B) as described in the legend to Fig. 1 and under “Materials and Methods.” The arrows show the amount of FLAG-arrestin-3 used for transfections in the experiments summarized in Figs. 5, 6, 8, and 9. Panel C, a polyclonal antibody to arrestin-3 (23Orsini M.J. Benovic J.L. J. Biol. Chem. 1998; 273: 34616-34622Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) followed by a secondary antibody covalently labeled with horseradish peroxidase were used to reveal the amount of arrestin-3 present in serial dilutions of the lysates of untransfected 293T cells and of the cells shown in panels A and B that were transfected with 0.03 μg of FLAG-arrestin-3.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 293T cells were plated in 100-mm dishes and cotransfected as described above with 10 μg of HA-tagged β2AR, 2.5 μg of dynamin-K44A, 0.25 μg of FLAG-arrestin-3, and 5 μg of empty vector or 5 μg of C20-GRK2-K220M. The transiently transfected cells were washed with the buffers described above and incubated with or without 10 μm isoproterenol for 5 min at 37 °C. The cells were then cross-linked and lysed as described above, except that radioimmune precipitation buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mm NaCl, 5 mm EDTA, 50 mm Tris-Cl, pH 8.0) with protease inhibitors was used. The lysates were clarified by centrifugation, and aliquots (∼500 μl) containing the same amount of protein were immunoprecipitated with a monoclonal antibody to the HA epitope (3F10, 1.5 μg/sample) that had been preabsorbed to agarose-conjugated protein G for 90–120 min at 4 °C as described above. The rest of this procedure was done exactly as described above for the hLHR. The co-immunoprecipitated FLAG-arrestin-3 and the HA-tagged β2AR were visualized in the blots during a 1-h incubation with a horseradish peroxidase-conjugated anti-FLAG M2 monoclonal antibody (final dilution = 1:500) or a horseradish peroxidase-conjugated anti-HA (3F10) monoclonal antibody (final concentration = 25 milliunits/ml). Expression of the transfected C20-GRK2-K220M was monitored using a monoclonal antibody to GRK2 (3A10 from Jeff Benovic), followed by a secondary antibody coupled to horseradish peroxidase. All immune complexes were ultimately visualized and quantitated using the Super Signal West Femto Maximum Sensitivity system of detection from Pierce and a Kodak digital imaging system. This image capture system is set up to alert us when image saturation occurs and to prevent us from measuring the intensity of such images. Cells were plated in eight-chamber coverslip culture vessels coated with polylysine (BioCoat from Becton-Dickinson). They were co-transfected (in a total volume of 400 μl) with 400 ng of Myc-hLHR, 100 ng of dynamin-K44A, and 8 ng of arrestin-3-GFP using the methods described above. Two days after transfection, the cells were incubated with or without hCG for 30 min at 37 °C as described above for the arrestin-3 binding assays. The medium was removed, and the cells were washed twice with phosphate-buffered saline (137 mm NaCl, 2.7 mmKCl, 1.4 mm NaH2PO4, 4.3 mm Na2HPO4, pH 7.4) and fixed during a 30-min incubation at room temperature with 4% paraformaldehyde (dissolved in phosphate-buffered saline). The fixed cells were washed twice again and then incubated for 1 h at room temperature with phosphate-buffered saline containing 50 mg/ml bovine serum albumin. This solution was removed, and the cells were incubated for another h at room temperature with a 1:100 dilution of the anti-Myc monoclonal antibody (9E10) dissolved in phosphate-buffered saline containing 5 mg/ml bovine serum albumin. After washing three times with phosphate-buffered saline, the cells were incubated for another 1 h at room temperature with a 1:2000 dilution of rhodamine-labeled anti-mouse IgG. Finally they were washed three or four times with phosphate-buffered saline, dried, and mounted in Vectashield mounting medium (Vector Laboratories). The rhodamine-labeled hLHR and the arrestin-3-GFP were visualized with a Bio-Rad confocal microscope at the Central Microscopy Facility of the University of Iowa. Human kidney 293T cells are a derivative of 293 cells that express the SV40T antigen (24Margolskee R. McHenry-Rinde B. Horn R. BioTechniques. 1993; 15: 906-911PubMed Google Scholar) and were provided to us by Dr. Marlene Hosey (Northwestern University, Chicago, IL). The 9E10 hybridoma cell line was obtained from the American Type Culture Collection. Purified hCG (CR-127, ∼13,000 IU/mg) was kindly provided by Dr. A. Parlow and the National Hormone and Pituitary Agency (NIDDK, National Institutes of Health, Bethesda, MD) and purified recombinant hCG 2Both preparations were used in this study and were found to be indistinguishable. was provided by Ares Serono (Randolph, MA). 125I-hCG was prepared as described elsewhere (25Ascoli M. Puett D. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 99-102Crossref PubMed Scopus (123) Google Scholar). Partially purified hCG (∼3,000 IU/mg) was purchased from Sigma, and it was used only for the determination of nonspecific binding (see above). 125I-cAMP and cell culture medium were obtained from the Iodination Core and the Media and Cell Production Core, respectively, of the Diabetes and Endocrinology Research Center of the University of Iowa. Concentrated supernatant from the 9E10 cells was prepared by the Hybridoma Facility of the Cancer Center of the University of Iowa. The 9E10 and anti-Flag M2 monoclonal antibodies coupled to horseradish peroxidase were purchased from Roche Molecular Biochemicals and Sigma, respectively. The 3F10 monoclonal antibody to the HA epitope (native or coupled to horseradish peroxidase) were from Roche. Secondary antibodies coupled to horseradish peroxidase or rhodamine were from Bio-Rad and Sigma, respectively. Other cell culture supplies and reagents were obtained from Corning and Invitrogen, respectively. All other chemicals were obtained from commonly used suppliers. The association of several GPCR binding partners such as GRKs and nonvisual arrestins in intact cells has been difficult to assess using standard immunoprecipitation/immunoblotting approaches but can be readily determined if the complexes are cross-linked prior to immunoprecipitation (20Freedman N. Ament A. Oppermann M. Stoffel R. Exum S. Lefkowitz R. J. Biol. Chem. 1997; 272: 17734-17743Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 26Dicker 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, 27Luttrell L.M. Ferguson S.S.G. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Lin F.-T. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Science. 1999; 282: 655-661Crossref Scopus (1234) Google Scholar, 28McDonald P.H. Chow C.-W. Miller W.E. Laporte S.A. Field M.E. Lin F.-T. Davis R.J. Lefkowitz R.J. Science. 2000; 290: 1574-1577Crossref PubMed Google Scholar). We encountered the same difficulties in experiments attempting to ascertain the association of the hLHR with the nonvisual arrestins in co-transfected 293T cells, and the only way to reliably detect such complexes was to treat the cells with DSP, a cell-permeable, homobifunctional, cleavable cross-linking agent, prior to immunoprecipitation. Thus, the basic method used below to detect the association of the hLHR with the nonvisual arrestins consisted of co-transfecting 293T cells with a Myc-tagged hLHR (12Min L. Ascoli M. Mol. Endocrinol. 2000; 14: 1797-1810Crossref PubMed Scopus (59) Google Scholar) and one of the FLAG-tagged nonvisual arrestins. Following incubation with or without agonist, the cells were further incubated with DSP (20Freedman N. Ament A. Oppermann M. Stoffel R. Exum S. Lefkowitz R. J. Biol. Chem. 1997; 272: 17734-17743Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar) prior to solubilization. The cell lysates were then immunoprecipitated with a monoclonal antibody to the Myc epitope (9E10), and the cross-linked complexes were dissociated by incubation of the immunoprecipitates with SDS sample buffer containing reducing agents. The reduced complexes were then resolved on SDS gels, blotted, and probed with a horseradish peroxidase-conjugated monoclonal antibody to the FLAG epitope (M2) to detect the FLAG-tagged nonvisual arrestins co-immunoprecipitated with the hLHR. Fig. 1 shows that the association of the Myc-hLHR-wt with FLAG-arrestin-3 is minimally detectable in unstimulated cells. When exposed to hCG, however, there is a quick and robust association of these two components. A steady state level of association was attained during a 20–30-min incubation with a saturating concentration of hCG. When the same experiment was done in cells co-transfected with dynamin-K44A (a condition that blocks the agonist-induced internalization of the hLHR; see Refs. 11Nakamura K. Liu X. Ascoli M. J. Biol. Chem. 2000; 275: 241-247Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 12Min L. Ascoli M. Mol. Endocrinol. 2000; 14: 1797-1810Crossref PubMed Scopus (59) Google Scholar, 13Li S. Liu X. Min L. Ascoli M. J. Biol. Chem. 2001; 276: 7968-7973Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), the time course of formation of the complex was somewhat slower, but, at steady state, the magnitude of the agonist-induced increase in the amount of complex formed was higher. In several experiments similar to that shown in Fig. 1, the amount of FLAG-arrestin-3·Myc-hLHR-wt complex formed (quantitated as described under “Materials and Methods”) during 30 min with hCG increased 4–10-fold and 20–50-fold in cells co-transfected without or with dynamin-K44A, respectively. Because the aim of the experiments presented here was to compare the association of the nonvisual arrestins with mutants of the hLHR that are internalized at different rates, we wanted to ensure that changes in the formation of the receptor-arrestin-3 complexes we" @default.
• W2083720922 created "2016-06-24" @default.
• W2083720922 creator A5043968508 @default.
• W2083720922 creator A5086369520 @default.
• W2083720922 creator A5089145622 @default.
• W2083720922 date "2002-01-01" @default.
• W2083720922 modified "2023-10-09" @default.
• W2083720922 title "The Association of Arrestin-3 with the Human Lutropin/Choriogonadotropin Receptor Depends Mostly on Receptor Activation Rather than on Receptor Phosphorylation" @default.
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