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• W2095664544 abstract "Previous studies from our laboratory using co-immunoprecipitation techniques suggested that the human lutropin receptor (hLHR) constitutively self-associates into dimers/oligomers and that agonist treatment of cells either increased hLHR dimerization/oligomerization and/or stabilized hLHR dimers/oligomers to detergent solubilization (Tao, Y. X., Johnson, N. B., and Segaloff, D. L. (2004) J. Biol. Chem. 279, 5904–5914). In this study, bioluminescence resonance energy transfer (BRET2) analyses confirmed that the hLHR constitutively self-associates in living cells. After subcellular fractionation, hLHR dimers/oligomers were detected in both the plasma membrane and endoplasmic reticulum (ER). Further evidence supporting the constitutive formation of hLHR dimer/oligomers in the ER is provided by data showing homodimerization of misfolded hLHR mutants that are retained in the ER. These mutants, when co-expressed with wild-type receptor, are shown by BRET2 to heterodimerize, accounting for their dominant-negative effects on cell surface receptor expression. Hormone desorption assays using intact cells demonstrate allosterism between hLHR protomers, indicating functional cell surface hLHR dimers. However, quantitative BRET2 analyses in intact cells indicate a lack of effect of agonist on the propensity of the hLHR to dimerize. Using purified plasma membranes, human chorionic gonadotropin was similarly observed to have no effect on the BRET2 signal. An examination of the propensity for constitutively active and signaling inactive hLHR mutants to dimerize further showed no correlation between dimerization and the activation state of the hLHR. Taken altogether, our data suggest that hLHR dimers/oligomers are formed early in the biosynthetic pathway in the ER, are constitutively expressed on the plasma membrane, and are not affected by the activation state of the hLHR. Previous studies from our laboratory using co-immunoprecipitation techniques suggested that the human lutropin receptor (hLHR) constitutively self-associates into dimers/oligomers and that agonist treatment of cells either increased hLHR dimerization/oligomerization and/or stabilized hLHR dimers/oligomers to detergent solubilization (Tao, Y. X., Johnson, N. B., and Segaloff, D. L. (2004) J. Biol. Chem. 279, 5904–5914). In this study, bioluminescence resonance energy transfer (BRET2) analyses confirmed that the hLHR constitutively self-associates in living cells. After subcellular fractionation, hLHR dimers/oligomers were detected in both the plasma membrane and endoplasmic reticulum (ER). Further evidence supporting the constitutive formation of hLHR dimer/oligomers in the ER is provided by data showing homodimerization of misfolded hLHR mutants that are retained in the ER. These mutants, when co-expressed with wild-type receptor, are shown by BRET2 to heterodimerize, accounting for their dominant-negative effects on cell surface receptor expression. Hormone desorption assays using intact cells demonstrate allosterism between hLHR protomers, indicating functional cell surface hLHR dimers. However, quantitative BRET2 analyses in intact cells indicate a lack of effect of agonist on the propensity of the hLHR to dimerize. Using purified plasma membranes, human chorionic gonadotropin was similarly observed to have no effect on the BRET2 signal. An examination of the propensity for constitutively active and signaling inactive hLHR mutants to dimerize further showed no correlation between dimerization and the activation state of the hLHR. Taken altogether, our data suggest that hLHR dimers/oligomers are formed early in the biosynthetic pathway in the ER, are constitutively expressed on the plasma membrane, and are not affected by the activation state of the hLHR. The human lutropin receptor (hLHR) 3The abbreviations used are: hLHR, human lutropin receptor; LHR, lutropin receptor; LH, lutropin; hCG, human chorionic gonadotropin; GFP, green fluorescent protein; D-PBS, Dulbecco's phosphate-buffered saline; GPCR, protein-coupled receptor; BRET, bioluminescence resonance energy transfer; ER, endoplasmic reticulum; CAM, constitutively active mutant; FRET, fluorescent resonance energy transfer; RET, resonance energy transfer. is a G protein-coupled receptor (GPCR) that plays a central role in reproductive physiology. In women, the LHR is expressed primarily in the ovary, where it mediates the actions of pituitary LH by stimulating the synthesis of androgens (which are mostly converted to estrogens) and ovulation. The hLHR also binds the nearly identical placental glycoprotein hormone hCG. As such, in pregnant women it rescues the corpus luteum, thus maintaining pregnancy. In males the hLHR is expressed predominantly in the testes, stimulating androgen biosynthesis in response to LH. The physiological importance of the LHR to reproductive physiology is underscored by disorders arising from mutations of the hLHR gene (1Latronico A. Segaloff D. Am. J. Hum. Genet. 1999; 65: 949-958Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 2Themmen A.P.N. Huhtaniemi I.T. Endocr. Rev. 2000; 21: 551-583Crossref PubMed Google Scholar). Thus, whereas constitutively active mutants (CAMs) of the hLHR are associated with gonadotropin-independent precocious puberty in males, loss-of-function mutations of the hLHR result in Leydig cell hypoplasia in males and infertility in females. Structurally, the hLHR is composed of two structural domains. There is the canonical seven transmembrane serpentine region common to GPCRs as well as a large extracellular domain that is composed of numerous leucine-rich repeats, which bind LH or hCG with high affinity (3Ascoli M. Fanelli F. Segaloff D.L. Endocr. Rev. 2002; 23: 141-174Crossref PubMed Scopus (500) Google Scholar). The hLHR is most closely related to the other glycoprotein hormone receptors for follitropin and thyrotropin, and is a member of the family A or rhodopsin-like family of GPCRs (4Vassart G. Pardo L. Costagliola S. Trends Biochem. Sci. 2004; 29: 119-126Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Agonist-occupied wild-type hLHR or hLHR CAMs activate the Gs, Gi/o, and Gq/11 families of G proteins (5Hirakawa T. Ascoli M. Endocrinology. 2003; 144: 3872-3878Crossref PubMed Scopus (35) Google Scholar, 6Ascoli M. Mol. Cell. Endocrinol. 2007; 260–262: 244-248Crossref PubMed Scopus (21) Google Scholar), with the primary actions of the hLHR being mediated by Gs. A large body of work published in recent years supports the concept that GPCRs can form self-associated dimers and higher ordered oligomers within cells (for reviews see Refs. 7Bulenger S. Marullo S. Bouvier M. Trends Pharmacol. Sci. 2005; 26: 131-137Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar, 8Milligan G. Biochim. Biophys. Acta. 2007; 1768: 825-835Crossref PubMed Scopus (154) Google Scholar, 9Dalrymple M.B. Pfleger K.D. Eidne K.A. Pharmacol. Ther. 2008; 118: 359-371Crossref PubMed Scopus (86) Google Scholar, 10Hebert T.E. Gales C. Rebois R.V. Cell Biochem. Biophys. 2006; 45: 85-109Crossref PubMed Google Scholar). In addition to GPCRs forming homodimers, distinct GPCRs can in some cases physically associate as heterodimers. The functional ramifications of GPCR dimerization are most clearly evident in heterodimers, where in many cases the pharmacological properties of heterodimers between two different GPCRs are unique as compared with those of each GPCR alone (11Mellado M. Rodriguez-Frade J.M. Vila-Coro A.J. Fernandez S. Martin de Ana A. Jones D.R. Toran J.L. Martinez A.C. EMBO J. 2001; 20: 2497-2507Crossref PubMed Scopus (384) Google Scholar, 12Rocheville M. Lange D.C. Kumar U. Patel S.C. Patel R.C. Patel Y.C. Science. 2000; 288: 154-157Crossref PubMed Scopus (755) Google Scholar, 13AbdAlla S. Lother H. Quitterer U. Nature. 2000; 407: 94-98Crossref PubMed Scopus (441) Google Scholar, 14Breit A. Gagnidze K. Devi L.A. Lagace M. Bouvier M. Mol. Pharmacol. 2006; 70: 686-696Crossref PubMed Scopus (55) Google Scholar, 15El-Asmar L. Springael J.Y. Ballet S. Andrieu E.U. Vassart G. Parmentier M. Mol. Pharmacol. 2005; 67: 460-469Crossref PubMed Scopus (164) Google Scholar, 16Ellis J. Pediani J.D. Canals M. Milasta S. Milligan G. J. Biol. Chem. 2006; 281: 38812-38824Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 17Gomes I. Gupta A. Filipovska J. Szeto H.H. Pintar J.E. Devi L.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5135-5139Crossref PubMed Scopus (352) Google Scholar, 18Gomes I. Jordan B.A. Gupta A. Trapaidze N. Nagy V. Devi L.A. J. Neurosci. 2000; 20: RC110Crossref PubMed Google Scholar, 19Hilairet S. Bouaboula M. Carriere D. Le Fur G. Casellas P. J. Biol. Chem. 2003; 278: 23731-23737Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). In contrast, the role of GPCR homodimerization is less clear. It has been hypothesized that agonist binding may alter the dimerization state of the receptor. Although this question has been addressed in numerous studies, there is little consensus with some studies suggesting that agonist promotes GPCR dimer formation (20Rodriguez-Frade J.M. Vila-Coro A.J. de Ana A.M. Albar J.P. Martinez A.C. Mellado M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3628-3633Crossref PubMed Scopus (204) Google Scholar, 21Vila-Coro A.J. Mellado M. de Ana A.M. Lucas P. del Real G. Martinez A.C. Rodriguez-Frade J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3388-3393Crossref PubMed Scopus (130) Google Scholar, 22Vila-Coro A.J. Rodriguez-Frade J.M. De Ana A.M. Moreno-Ortiz M.C. Martinez A.C. Mellado M. FASEB J. 1999; 13: 1699-1710Crossref PubMed Scopus (443) Google Scholar, 23AbdAlla S. Zaki E. Lother H. Quitterer U. J. Biol. Chem. 1999; 274: 26079-26084Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 24Kroeger K.M. Hanyaloglu A.C. Seeber R.M. Miles L.E. Eidne K.A. J. Biol. Chem. 2001; 276: 12736-12743Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 25Horvat R.D. Roess D.A. Nelson S.E. Barisas B.G. Clay C.M. Mol. Endocrinol. 2001; 15: 695-703Crossref PubMed Scopus (55) Google Scholar), others that agonist causes the dissociation of GPCR dimers (26Latif R. Graves P. Davies T.F. J. Biol. Chem. 2002; 277: 45059-45067Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), and yet others that the GPCR dimers are constitutively expressed and unaffected by agonist (27Issafras H. Angers S. Bulenger S. Blanpain C. Parmentier M. Labbe-Jullie C. Bouvier M. Marullo S. J. Biol. Chem. 2002; 277: 34666-34673Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 28Terrillon S. Durroux T. Mouillac B. Breit A. Ayoub M.A. Taulan M. Jockers R. Barberis C. Bouvier M. Mol. Endocrinol. 2003; 17: 677-691Crossref PubMed Scopus (280) Google Scholar, 29Romano C. Yang W.L. O'Malley K.L. J. Biol. Chem. 1996; 271: 28612-28616Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar, 30Bai M. Trivedi S. Brown E.M. J. Biol. Chem. 1998; 273: 23605-23610Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). As discussed in a recent review, unappreciated limitations to some of the methods used may have contributed to the apparently discrepant results (10Hebert T.E. Gales C. Rebois R.V. Cell Biochem. Biophys. 2006; 45: 85-109Crossref PubMed Google Scholar). Previous studies have suggested that the LHR can self-associate into dimers/oligomers. Evidence pointing toward this goes back to as early as the 1980s when data from equilibrium sedimentation of detergent-solubilized LHR and radiation inactivation of LHRs on gonadal cells suggested that the LHR may be present in a dimeric or oligomeric form (31Kusuda S. Dufau M.L. J. Biol. Chem. 1988; 263: 3046-3049Abstract Full Text PDF PubMed Google Scholar, 32Crine P. Aubry M. Potier M. Ann. N. Y. Acad. Sci. 1984; 438: 224-237Crossref PubMed Scopus (6) Google Scholar). More recently, functional complementation studies further suggested the dimerization of the LHR (33Lee C.-W. Ji I. Ryu K.-S. Song Y.-S. Conn P.M. Ji H. J. Biol. Chem. 2002; 277: 15795-15800Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 34Ji I. Lee C.W. Song Y.S. Conn P.M. Ji T.H. Mol. Endocrinol. 2002; 16: 1299-1308Crossref PubMed Scopus (80) Google Scholar). Direct evidence in support of dimerization of the LHR has been obtained using fluorescent resonance energy transfer (FRET) (35Roess D.A. Horvat R.D. Munnelly H. Barisas B.G. Endocrinology. 2000; 141: 4518-4523Crossref PubMed Scopus (67) Google Scholar, 36Roess D.A. Smith S.M. Biol. Reprod. 2003; 69: 1765-1770Crossref PubMed Scopus (34) Google Scholar, 37Hunzicker-Dunn M. Barisas G. Song J. Roess D.A. J. Biol. Chem. 2003; 278: 42744-42749Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 38Lei Y. Hagen G.M. Smith S.M. Liu J. Barisas G. Roess D.A. Mol. Cell. Endocrinol. 2007; 260: 65-72Crossref PubMed Scopus (22) Google Scholar) as well as our studies demonstrating specific co-immunoprecipitation of differentially tagged forms of the LHR (39Tao Y.X. Johnson N.B. Segaloff D.L. J. Biol. Chem. 2004; 279: 5904-5914Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). In this latter study it was shown that under basal conditions the hLHR physically self-associates into complexes of sizes consistent with dimers and higher ordered oligomers of the receptor. It was also observed that hCG pretreatment of cells prior to detergent solubilization led to an increase in the abundance of hLHR dimers/oligomers relative to monomers as detected by Western blotting. Although these results could be explained by an agonist-dependent induction of hLHR dimerization, it may also reflect a stabilization of preformed receptor dimers/oligomers to detergent solubilization when they are occupied by hCG. To address this question, this work was undertaken utilizing bioluminescence resonance energy transfer (BRET) as an independent means to probe hLHR dimerization and to study the effects of receptor activation on dimerization. BRET is a highly sensitive means to assess protein-protein interactions in living cells, thus avoiding the detergent solubilization required for co-immunoprecipitation experiments (10Hebert T.E. Gales C. Rebois R.V. Cell Biochem. Biophys. 2006; 45: 85-109Crossref PubMed Google Scholar, 40Milligan G. Bouvier M. FEBS J. 2005; 272: 2914-2925Crossref PubMed Scopus (189) Google Scholar, 41Pfleger K.D. Eidne K.A. Nat. Meth. 2006; 3: 165-174Crossref PubMed Scopus (440) Google Scholar). In the BRET paradigm, a given protein is fused to the energy donor Renilla luciferase (Rluc), which is co-expressed with the same or a different protein fused to an energy acceptor, typically a variant of green fluorescent protein (GFP). Addition of substrate for Rluc results in bioluminescence, which can also cause the excitation of GFP via resonance energy transfer only if the donor and acceptor are within 100 Å of each other, a distance consistent with the predicted size of a GPCR dimer (10Hebert T.E. Gales C. Rebois R.V. Cell Biochem. Biophys. 2006; 45: 85-109Crossref PubMed Google Scholar, 40Milligan G. Bouvier M. FEBS J. 2005; 272: 2914-2925Crossref PubMed Scopus (189) Google Scholar, 41Pfleger K.D. Eidne K.A. Nat. Meth. 2006; 3: 165-174Crossref PubMed Scopus (440) Google Scholar). Although identical in principle, BRET2 is a second generation modification of BRET that achieves a greater spectral resolution between donor and acceptor emissions (10Hebert T.E. Gales C. Rebois R.V. Cell Biochem. Biophys. 2006; 45: 85-109Crossref PubMed Google Scholar, 40Milligan G. Bouvier M. FEBS J. 2005; 272: 2914-2925Crossref PubMed Scopus (189) Google Scholar, 41Pfleger K.D. Eidne K.A. Nat. Meth. 2006; 3: 165-174Crossref PubMed Scopus (440) Google Scholar). Both have been widely used in recent years to examine homo- and heterodimerization of a number of different GPCRs (10Hebert T.E. Gales C. Rebois R.V. Cell Biochem. Biophys. 2006; 45: 85-109Crossref PubMed Google Scholar). A limitation of BRET/BRET2 (as well as the related method of FRET) is that a positive signal from a single ratio of energy donor and acceptor is not necessarily reflective of the affinity of GPCR protomers to dimerize nor the extent of GPCR dimerization. This is because RET is dependent not only on the distance between the energy donor and acceptor, but on their relative orientations as well (10Hebert T.E. Gales C. Rebois R.V. Cell Biochem. Biophys. 2006; 45: 85-109Crossref PubMed Google Scholar, 40Milligan G. Bouvier M. FEBS J. 2005; 272: 2914-2925Crossref PubMed Scopus (189) Google Scholar, 41Pfleger K.D. Eidne K.A. Nat. Meth. 2006; 3: 165-174Crossref PubMed Scopus (440) Google Scholar). A method of quantitative BRET analyses was therefore proposed by Mercier et al. (42Mercier J.-F. Salahpour A. Angers S. Breit A. Bouvier M. J. Biol. Chem. 2002; 277: 44925-44931Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar) to permit the determination of the relative affinities of pairs of GPCR protomers to form homo- or heterodimers. Based on theoretical considerations of RET, this approach utilizes saturation curves (analogous to hormone-receptor binding curves) to calculate the half-maximal BRET signal (BRET50), which is related to the relative affinity of the protomers for each other (41Pfleger K.D. Eidne K.A. Nat. Meth. 2006; 3: 165-174Crossref PubMed Scopus (440) Google Scholar, 42Mercier J.-F. Salahpour A. Angers S. Breit A. Bouvier M. J. Biol. Chem. 2002; 277: 44925-44931Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). Using quantitative BRET2 analyses, we demonstrate that hLHR dimers can be detected under basal conditions in living cells, with constitutively expressed hLHR dimers/oligomers observed in both the plasma membrane as well as the endoplasmic reticulum. From a number of different experimental strategies, it is further concluded that dimerization/oligomerization of the hLHR is not affected by the activation state of the hLHR. Plasmids and Hormones-The hLHR cDNA was kindly given to us by Ares Advanced Technology (Ares-Serono Group, Randolph, MA). Mutants of the hLHR were made using standard techniques. The wild-type and mutant forms of the hLHR were all modified to contain a Myc epitope tag at the N terminus. Before use, the coding sequence of each construct was determined by the DNA Core of the University of Iowa. For the BRET2 studies, the cDNAs were subcloned into pRluc or pGFP2 vectors (PerkinElmer Life Sciences), which insert Renilla luciferase (Rluc) or GFP2, respectively, in-frame at the C terminus of the protein. The cDNA encoding KvLQT1 was used as described previously (43Rebois R.V. Robitaille M. Gales C. Dupre D.J. Baragli A. Trieu P. Ethier N. Bouvier M. Hebert T.E. J. Cell Sci. 2006; 119: 2807-2818Crossref PubMed Scopus (119) Google Scholar). Highly purified preparations of recombinant hCG and recombinant hLH were purchased from Dr. A. Parlow and the National Hormone and Pituitary Program, NIDDK, National Institutes of Health. hLH was iodinated as described previously for hCG (44Ascoli M. Puett D. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 99-102Crossref PubMed Scopus (123) Google Scholar). A crude preparation of hCG (used solely for the determination of nonspecific binding of 125I-hLH) was purchased from Sigma. Cells and Transfections-Human embryonic kidney (HEK) 293 and 293T cells were obtained from the American Type Tissue Collection (Manassas, VA). Cells were maintained at 5% CO2 in growth media consisting of high glucose Dulbecco's modified Eagle's medium containing 50 μg/ml gentamicin, 10 mm HEPES, and 10% newborn calf serum. For experiments, cells were plated onto wells that had been precoated for 45 min with 0.1% gelatin in Dulbecco's phosphate-buffered saline, pH 7.1 (D-PBS), that was calcium- and magnesium-free. Cells were transiently transfected as described previously (45Zhang M. Mizrachi D. Fanelli F. Segaloff D.L. J. Biol. Chem. 2005; 280: 26169-26176Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) and used for experiments 24 h after removing the mixture. BRET2 Assay-HEK293T cells in 6-well plates were transiently co-transfected with vectors encoding Rluc fusion or GFP2 fusion proteins. In a given experiment, the total amount of plasmid transfected was made constant by the addition of empty vector. On the day of the experiment, cells were washed two times with calcium- and magnesium-free D-PBS and then detached from the well in 1 ml of D-PBS. Protein concentrations were measured, and then equal protein aliquots were distributed into microcentrifuge tubes and collected by gentle centrifugation. The cell pellets were resuspended in a small volume of D-PBS and transferred to a white-bottomed 96-well microplate (white Optiplate; PerkinElmer Life Sciences) such that all samples were of equal volume and protein concentration. Total fluorescence of the cell suspensions was measured using a POLARstar Optima plate reader (BMG Labtech, Offenburg, Germany), with an excitation filter at 485 nm and an emission filter at 520 nm, and was corrected for the fluorescence measured in cells transfected with empty vector only. The substrate Coelenterazine 400a (Biosynth; Zurich, Switzerland) was then added at a final concentration of 5 μm, and readings at 410/80 nm (reflecting the bioluminescence given off by Rluc) and 515/30 nm (reflecting the resonance energy transfer from Rluc to GFP2) were measured simultaneously. Bioluminescence readings were corrected for those obtained from cells transfected with empty vector only. The BRET2 ratio was calculated as the ratio of the light emitted by the receptor-GFP2 (515/30 nm) over the light emitted by the receptor-Rluc (410/80 nm). The BRET2 ratios reported were corrected by subtracting the ratios obtained when receptor-Rluc was expressed alone. Within a given experiment, each data point was obtained using duplicate wells of cells that were transfected and collected for BRET2 analyses. Data shown are the mean ± range of the duplicate determinations from one representative experiment (of at least three independent experiments). BRET2 Titration Curves-For saturation curves, HEK293T cells in 6-well plates were co-transfected with a fixed concentration of Rluc fusion protein and increasing concentrations of a GFP2 fusion protein. When more than one curve was performed in a given experiment, the concentrations of plasmids encoding the Rluc fusion proteins were adjusted so that, after substrate addition, the bioluminescence values of the Rluc fusion proteins expressed alone were similar. Data were expressed as the net BRET2 ratio, calculated as described above, relative to the ratio of acceptor to donor. The data were plotted using GraphPad Prism (San Diego), and the Bmax and KD determinations were taken as the BRETmax and BRET50, respectively. Subcellular Fractionation-HEK293T cells were co-transfected with hLHR-Rluc and hLHR-GFP2 or with KvLQT1-Rluc and hLHR-GFP2 as a negative control. On the day of the experiment, the cells were placed on ice, washed three times with ice-cold D-PBS, and scraped off the dish in ice-cold hypotonic lysis buffer (20 mm HEPES, pH 7.4, 2 mm EDTA, 2 mm EGTA, and 6 mm magnesium chloride) containing complete protease inhibitor mixture (Roche Diagnostics). Cells were homogenized with 50 strokes in a tight fitting Dounce homogenizer on ice. After centrifugation at 1000 × g for 5 min at 4 °C, the post-nuclear supernatant was collected and assayed for protein concentration. Each cell lysate was adjusted to 1 mg of protein in 463 μl of hypotonic lysis buffer and brought to contain a final sucrose concentration of 2 m and placed in the bottom of a 5-ml ultracentrifuge tube for a Beckman SW55 Ti rotor. A discontinuous step sucrose gradient was then made in each tube as follows. Applied above the sample, 237 μl of lysis buffer containing 1.9 m sucrose was added. Then 700 μl each of hypotonic lysis buffer containing 1.75, 1.5, 1.25, 1.0, 0.75, and 0.5 m sucrose were added. Samples were centrifuged 16 h at 50,000 rpm at 4 °C. Aliquots of 400 μl were taken from the top of each tube. BRET2 ratios were determined from 100-μl samples. Equal volume samples were also taken from each aliquot for Western blots (see below) to detect calnexin (a marker for the endoplasmic reticulum), Na+/K+-ATPase (a marker for the plasma membrane), or the myc-hLHR. Protein concentrations were measured in each aliquot. Western blots for myc-hLHR were also analyzed when the gels were run after applying equal amounts of protein to each well. Western Blotting-Samples were diluted 1:6 into a 6-fold concentrated Laemmli sample buffer containing reducing agents (12% w/v SDS, 40% glycerol, 109 mm EDTA, 1.5 m Tris/HCl, 98 mg/ml dithiothreitol and 6% v/v β-mercaptoethanol), incubated 1 h at room temperature, fractionated by SDS-PAGE on 7.5% gels, and transferred to polyvinylidene difluoride membranes. Membranes were probed with anti-Myc monoclonal antibody 9E10 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), anti-calnexin polyclonal antibody (1:1000; StressGen, Ann Arbor, MI), or anti-Na+/K+-ATPase monoclonal antibody (1:250; Sigma). The membranes were then incubated with either horseradish peroxidase-conjugated sheep anti-mouse antibody (1:25,000; GE Healthcare) or donkey anti-rabbit antibody (1:100,000; GE Healthcare). The immunoreactive bands were visualized using the Amersham Biosciences ECL detection system (GE Healthcare). Hormone Desorption Experiments-HEK293 cells were seeded on 24-well culture plates that had been coated with gelatin as described above. Cells were transfected as described above and used for experiments 24 h after removing the mixture. On the day of the experiment, the cells were washed two times with binding medium consisting of warm Waymouth's MB752/1 containing 50 μg/ml gentamicin and 1 mg/ml bovine serum albumin. The cells were then incubated 1 h at room temperature in binding medium containing a saturating concentration of 125I-hLH (final concentration 1000 ng/ml) with or without an excess of unlabeled crude hCG (final concentration 50 IU/ml) to determine nonspecific binding. The end of the binding assay was defined as t = 0 relative to the subsequent desorption phase of the experiment. One group of cells was used to determine the maximal binding at t = 0. These cells were set on ice, washed three times with ice-cold Hanks' balanced salt solution modified to contain 50 μg/ml gentamicin and 1 mg/ml bovine serum albumin, solubilized in 0.5 n NaOH, transferred to plastic test tubes with cotton swabs, and counted in a gamma counter. Another group of cells was used to determine the time course of desorption of the prebound 125I-hLH. These cells were washed three times with warm binding medium, incubated in binding medium containing hCG (final concentration 500 ng/ml), hLH (1000 ng/ml), or vehicle only and incubated for increasing times at room temperature. At a given time point, the cells were placed on ice, and the 125I-hLH released into the medium was determined by collecting the medium and precipitating intact hormone with trichloroacetic acid. To determine the amount of 125I-hLH remaining bound to the cells, the cells were washed and solubilized, and the bound radioactivity was counted as described for the t = 0 time point. To determine whether hLHR self-association could be detected in living cells, we examined whether specific BRET2 signals would be observed in cells co-transfected hLHR(wt)-Rluc and hLHR(wt)-GFP2. As shown in Fig. 1, titration curves were performed in which cells were transfected with a subsaturating and fixed concentration of the energy donor hLHR(wt)-Rluc and increasing concentrations of the energy acceptor hLHR(wt)-GFP2. As the ratio of energy acceptor to donor was increased, the BRET2 ratio increased, as would be predicted by molecules that were clustered together and not randomly distributed (42Mercier J.-F. Salahpour A. Angers S. Breit A. Bouvier M. J. Biol. Chem. 2002; 277: 44925-44931Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 46Kenworthy A.K. Edidin M. J. Cell Biol. 1998; 142: 69-84Crossref PubMed Scopus (404) Google Scholar). At the higher ratios of hLHR(wt)-Rluc to hLHR(wt)-GFP2, the BRET2 signal reached a plateau, indicative of a saturable interaction between the energy donor and acceptor (42Mercier J.-F. Salahpour A. Angers S. Breit A. Bouvier M. J. Biol. Chem. 2002; 277: 44925-44931Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). As a negative control, cells were co-transfected with the plasma membrane cardiac voltage-gated K+ channel KvLQT1 fused to Rluc and hLHR(wt)-GFP2. Little or no detectable BRET2 ratios were detected under those conditions, suggesting that the BRET2 signals observed between hLHR(wt)-Rluc and hLHR(wt)-GFP2 are because of the physical interaction between the hLHR portions of the fusion proteins. To ensure that the BRET2 observed between hLHR(wt)-Rluc and hLHR(wt)-GFP2 was not because of spurious bystander interactions between the two molecules, we examined the effect of decreasing the protein expression levels of the receptor, while maintaining the ratio of hLHR(wt)-Rluc/hLHR(wt)-GFP2 constant. As shown in Fig. 2, the BRET2 ratio remained detectable at low levels of receptor expression, consistent with what would be predicted by specific clustering of molecules and not random collisions (46Kenworthy A.K. Edidin M. J. Cell Biol. 1998; 142: 69-84Crossref PubMed Scopus (404) Google Scholar).FIGURE 2BRET2 signal resulting from co-expression of hLHR(wt)-Rluc and hLHR(wt)-GFP2 is not a function of random self-association. HEK293T cells were transiently transfected with varying total amounts of a fixed ratio (1:5) of hLHR(wt)-Rluc and hLHR(wt)-GFP2. A, data shown are the net BRET2 ratios as a function of the total amount of plasmid transfected. B, expression of hLHR(wt)-GFP2 (as mea" @default.
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• W2095664544 creator A5080216648 @default.
• W2095664544 date "2009-03-01" @default.
• W2095664544 modified "2023-10-16" @default.
• W2095664544 title "Bioluminescence Resonance Energy Transfer Studies Reveal Constitutive Dimerization of the Human Lutropin Receptor and a Lack of Correlation between Receptor Activation and the Propensity for Dimerization" @default.
• W2095664544 cites W1559798894 @default.
• W2095664544 cites W1568442781 @default.
• W2095664544 cites W1586056486 @default.
• W2095664544 cites W1593172464 @default.
• W2095664544 cites W1922502842 @default.
• W2095664544 cites W1967738476 @default.
• W2095664544 cites W1970375638 @default.
• W2095664544 cites W1970443640 @default.
• W2095664544 cites W1982192731 @default.
• W2095664544 cites W1984429646 @default.
• W2095664544 cites W1985986593 @default.
• W2095664544 cites W1988395319 @default.
• W2095664544 cites W1991990669 @default.
• W2095664544 cites W1992303275 @default.
• W2095664544 cites W1994511844 @default.
• W2095664544 cites W1994975210 @default.
• W2095664544 cites W1997098110 @default.
• W2095664544 cites W2000007478 @default.
• W2095664544 cites W2000092232 @default.
• W2095664544 cites W2002154523 @default.
• W2095664544 cites W2007051067 @default.
• W2095664544 cites W2007149985 @default.
• W2095664544 cites W2013087877 @default.
• W2095664544 cites W2014134434 @default.
• W2095664544 cites W2014348198 @default.
• W2095664544 cites W2017977773 @default.
• W2095664544 cites W2020538114 @default.
• W2095664544 cites W2021035723 @default.
• W2095664544 cites W2021879786 @default.
• W2095664544 cites W2024292538 @default.
• W2095664544 cites W2027591014 @default.
• W2095664544 cites W2035290373 @default.
• W2095664544 cites W2035423945 @default.
• W2095664544 cites W2039079134 @default.
• W2095664544 cites W2041138404 @default.
• W2095664544 cites W2043026342 @default.
• W2095664544 cites W2043944866 @default.
• W2095664544 cites W2047517608 @default.
• W2095664544 cites W2049464937 @default.
• W2095664544 cites W2052441056 @default.
• W2095664544 cites W2053912544 @default.
• W2095664544 cites W2054004217 @default.
• W2095664544 cites W2062333166 @default.
• W2095664544 cites W2066738995 @default.
• W2095664544 cites W2066964651 @default.
• W2095664544 cites W2067466593 @default.
• W2095664544 cites W2071129811 @default.
• W2095664544 cites W2071327910 @default.
• W2095664544 cites W2074381517 @default.
• W2095664544 cites W2074537257 @default.
• W2095664544 cites W2075503178 @default.
• W2095664544 cites W2077591028 @default.
• W2095664544 cites W2079052124 @default.
• W2095664544 cites W2079871914 @default.
• W2095664544 cites W2080985839 @default.
• W2095664544 cites W2081819792 @default.
• W2095664544 cites W2084606789 @default.
• W2095664544 cites W2085309553 @default.
• W2095664544 cites W2092443517 @default.
• W2095664544 cites W2093306312 @default.
• W2095664544 cites W2093691852 @default.
• W2095664544 cites W2095775529 @default.
• W2095664544 cites W2101255369 @default.
• W2095664544 cites W2103897720 @default.
• W2095664544 cites W2105041061 @default.
• W2095664544 cites W2120365849 @default.
• W2095664544 cites W2122336578 @default.
• W2095664544 cites W2133344686 @default.
• W2095664544 cites W2151426675 @default.
• W2095664544 cites W2151711371 @default.
• W2095664544 cites W2151983621 @default.
• W2095664544 cites W2156070415 @default.
• W2095664544 cites W2162670271 @default.
• W2095664544 cites W2167948597 @default.
• W2095664544 cites W2168941494 @default.
• W2095664544 cites W2313881658 @default.
• W2095664544 cites W2767182304 @default.
• W2095664544 cites W4297627420 @default.
• W2095664544 doi "https://doi.org/10.1074/jbc.m809150200" @default.
• W2095664544 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2658044" @default.
• W2095664544 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19147490" @default.
• W2095664544 hasPublicationYear "2009" @default.
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