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• W2091036574 abstract "Numerous alternatively spliced transcripts are generated from the gene for the G protein-coupled calcitonin receptor, and some of the splice variants show differences in receptor-mediated signaling events. This study showed that the Δe13 splice variant of the rabbit calcitonin receptor is expressed together with the more common C1a in osteoclast-like cells. Since other G protein-coupled receptors form homo- or heterodimers, we examined whether heterodimerization of the calcitonin receptor splice variants occurs and, if so, whether it affects the function of the receptor. Homodimers of both isoforms and Δe13/C1a heterodimers were detected by co-immunoprecipitation and fluorescence resonance energy transfer analysis. In contrast to the C1a isoform, the Δe13 isoform was not efficiently transported to the cell surface. When co-expressed with the C1a splice variant, the Δe13 isoform colocalized with the C1a isoform within the cell but not at the cell surface. Furthermore, the overexpression of the Δe13 variant led to a significant reduction of the C1a surface expression and consequently a reduction of the cAMP response and Erk phosphorylation after ligand stimulation. We therefore suggest that the Δe13 variant of the rabbit calcitonin receptor acts to regulate the surface expression of the C1a isoform. Numerous alternatively spliced transcripts are generated from the gene for the G protein-coupled calcitonin receptor, and some of the splice variants show differences in receptor-mediated signaling events. This study showed that the Δe13 splice variant of the rabbit calcitonin receptor is expressed together with the more common C1a in osteoclast-like cells. Since other G protein-coupled receptors form homo- or heterodimers, we examined whether heterodimerization of the calcitonin receptor splice variants occurs and, if so, whether it affects the function of the receptor. Homodimers of both isoforms and Δe13/C1a heterodimers were detected by co-immunoprecipitation and fluorescence resonance energy transfer analysis. In contrast to the C1a isoform, the Δe13 isoform was not efficiently transported to the cell surface. When co-expressed with the C1a splice variant, the Δe13 isoform colocalized with the C1a isoform within the cell but not at the cell surface. Furthermore, the overexpression of the Δe13 variant led to a significant reduction of the C1a surface expression and consequently a reduction of the cAMP response and Erk phosphorylation after ligand stimulation. We therefore suggest that the Δe13 variant of the rabbit calcitonin receptor acts to regulate the surface expression of the C1a isoform. Calcitonin (CT) 1The abbreviations used are: CT, calcitonin; CTR, calcitonin receptor; Erk, extracellular signal-regulated kinase; FACS, fluorescenceactivated cell sorter; FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; GST, glutathione S-transferase; HEK, human embryonic kidney; MEM, minimal essential medium; mRIPA, modified radioimmune precipitation assay; PBS, phosphate-buffered saline; PE, phycoerythrin; RFP, red fluorescent protein; sCT, salmon calcitonin; OCL, osteoclast-like cell; HA, hemagglutinin. is a 32-amino acid polypeptide that was originally identified as a hypocalcemic factor present in bovine parathyroids (1Copp D.H. Annu. Rev. Pharmacol. 1969; 9: 327-344Crossref PubMed Scopus (25) Google Scholar). CT acts on bone and kidney to maintain calcium homeostasis and is also present in the central nervous system, where it has anorexic and analgesic effects (2Azria M. The Calcitonins: Physiology and Pharmacology. Karger, Basel, Switzerland1989: 43-58Google Scholar). Of the cells present in bone, osteoclasts are the main target of CT. It inhibits motility and induces marked cellular retraction of isolated osteoclasts, two effects that are thought to be essential to the CT-induced inhibition of bone resorption (3Zaidi M. Moonga B.S. Bevis P.J. Bascal Z.A. Breimer L.H. Crit. Rev. Clin. Lab. Sci. 1990; 28: 109-174Crossref PubMed Scopus (85) Google Scholar, 4Su Y. Chakraborty M. Nathanson M.H. Baron R. Endocrinology. 1992; 131: 1497-1502Crossref PubMed Scopus (67) Google Scholar). The calcitonin receptor (CTR) belongs to the class B of G protein-coupled receptors (GPCRs) and was first cloned in 1991 (5Lin H.Y. Harris T.L. Flannery M.S. Aruffo A. Kaji E.H. Gorn A. Kolakowski L.F.J. Yamin M. Lodish H.F. Goldring S.R. Trans. Assoc. Am. Physicians. 1991; 104: 265-272PubMed Google Scholar). It couples to multiple heterotrimeric G proteins, resulting in activation of the effector proteins adenylyl cyclase and phospholipase Cβ (6Chakraborty M. Chatterjee D. Kellokumpu S. Rasmussen H. Baron R. Science. 1991; 251: 1078-1082Crossref PubMed Scopus (131) Google Scholar, 7Chabre O. Conklin B.R. Lin H.Y. Lodish H.F. Wilson E. Ives H.E. Catanzariti L. Hemmings B.A. Bourne H.R. Mol. Endocrinol. 1992; 6: 551-556Crossref PubMed Google Scholar, 8Shyu J.F. Inoue D. Baron R. Horne W.C. J. Biol. Chem. 1996; 271: 31127-31134Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 9Shyu J.F. Zhang Z. Hernandez-Lagunas L. Camerino C. Chen Y. Inoue D. Baron R. Horne W.C. Eur. J. Biochem. 1999; 262: 95-101Crossref PubMed Scopus (23) Google Scholar). We have reported that the rabbit CTR also stimulates Shc tyrosine phosphorylation and Erk1/2 activation, primarily via Gi- and Gq-dependent signaling pathways (10Chen Y. Shyu J.F. Santhanagopal A. Inoue D. David J.P. Dixon S.J. Horne W.C. Baron R. J. Biol. Chem. 1998; 273: 19809-19816Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Recent data indicate that in the presence of “receptor activitymodifying proteins,” the CTR functions as a receptor for amylin, and therefore the complexes of the CTR with receptor activity-modifying protein 1, 2, or 3 are called AMY1-, AMY2-, and AMY3-receptor, respectively (11Spedding M. Bonner T.I. Watson S.P. Pharmacol. Rev. 2002; 54: 231-232Crossref PubMed Scopus (5) Google Scholar). Numerous alternatively spliced transcripts of the CTR have been described in different species. The most common isoform in all species corresponds to the sequence originally cloned from porcine cells (5Lin H.Y. Harris T.L. Flannery M.S. Aruffo A. Kaji E.H. Gorn A. Kolakowski L.F.J. Yamin M. Lodish H.F. Goldring S.R. Trans. Assoc. Am. Physicians. 1991; 104: 265-272PubMed Google Scholar) and the rodent C1a isoform (12Albrandt K. Mull E. Brady E.M. Herich J. Moore C.X. Beaumont K. FEBS Lett. 1993; 325: 225-232Crossref PubMed Scopus (103) Google Scholar). In humans, the less abundant insert-positive form shows a loss of Gq-mediated responses and attenuation of Gs-mediated signaling and also decreased internalization (13Moore E.E. Kuestner R.E. Stroop S.D. Grant F.J. Matthewes S.L. Brady C.L. Sexton P.M. Findlay D.M. Mol. Endocrinol. 1995; 9: 959-968Crossref PubMed Google Scholar). In rodents, the C1b splice variant has an insertion of 37 amino acids in the first intracellular loop and shows only a weak interaction with human CT (12Albrandt K. Mull E. Brady E.M. Herich J. Moore C.X. Beaumont K. FEBS Lett. 1993; 325: 225-232Crossref PubMed Scopus (103) Google Scholar, 14Sexton P.M. Houssami S. Hilton J.M. O'Keeffe L.M. Center R.J. Gillespie M.T. Darcy P. Findlay D.M. Mol. Endocrinol. 1993; 7: 815-821Crossref PubMed Google Scholar, 15Houssami S. Findlay D.M. Brady C.L. Martin T.J. Epand R.M. Moore E.E. Murayama E. Tamura T. Orlowski R.C. Sexton P.M. Mol. Pharmacol. 1995; 47: 798-809PubMed Google Scholar, 16Yamin M. Gorn A.H. Flannery M.R. Jenkins N.A. Gilbert D.J. Copeland N.G. Tapp D.R. Krane S.M. Goldring S.R. Endocrinology. 1994; 135: 2635-2643Crossref PubMed Scopus (60) Google Scholar). Cloning of the rabbit CTR by our group revealed both the C1a transcript and a splice variant with a deletion of exon 13, designated CTRΔe13, which encodes much of the seventh transmembrane domain (8Shyu J.F. Inoue D. Baron R. Horne W.C. J. Biol. Chem. 1996; 271: 31127-31134Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The Δe13 variant failed to induce the production of inositol phosphates or to mobilize intracellular calcium and also showed a decreased cAMP response to salmon and human CT stimulation (8Shyu J.F. Inoue D. Baron R. Horne W.C. J. Biol. Chem. 1996; 271: 31127-31134Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 17Santhanagopal A. Chidiac P. Horne W.C. Baron R. Dixon S.J. Endocrinology. 2001; 142: 4401-4413Crossref PubMed Scopus (18) Google Scholar). Interestingly, the exon deleted in the CTRΔe13 isoform is highly conserved in the class B GPCRs. Receptor-spliced variants resulting in deletion of the homologous 14 amino acids as in the CTRΔe13 variant have been described for two other class B GPCRs, the CRH-R1 (18Grammatopoulos D.K. Dai Y. Randeva H.S. Levine M.A. Karteris E. Easton A.J. Hillhouse E.W. Mol. Endocrinol. 1999; 13: 2189-2202Crossref PubMed Scopus (116) Google Scholar) and the parathyroid hormone/parathyroid hormone-related protein receptors (19Ding C. Racusen L. Wilson P. Burrow C. Levine M.A. J. Bone Miner. Res. 1995; 10: S484Google Scholar). Recent reports suggest that many GPCRs form homodimers and/or heterodimers (20Bouvier M. Nat. Rev. Neurosci. 2001; 2: 274-286Crossref PubMed Scopus (584) Google Scholar, 21George S.R. O'Dowd B.F. Lee S.P. Nat. Rev. Drug Discov. 2002; 1: 808-820Crossref PubMed Scopus (546) Google Scholar, 22Rios C.D. Jordan B.A. Gomes I. Devi L.A. Pharmacol. Ther. 2001; 92: 71-87Crossref PubMed Scopus (291) Google Scholar). The functional relevance of GPCR dimerization was strikingly shown for the GABAB receptor. Several groups simultaneously reported that co-expression of the GBR1 and GBR2 isoforms is a prerequisite for the formation of functional GABA receptors at the cell surface (23White J.H. Wise A. Main M.J. Green A. Fraser N.J. Disney G.H. Barnes A.A. Emson P. Foord S.M. Marshall F.H. Nature. 1998; 396: 679-682Crossref PubMed Scopus (1019) Google Scholar, 24Jones K.A. Borowsky B. Tamm J.A. Craig D.A. Durkin M.M. Dai M. Yao W.J. Johnson M. Gunwaldsen C. Huang L.Y. Tang C. Shen Q. Salon J.A. Morse K. Laz T. Smith K.E. Nagarathnam D. Noble S.A. Branchek T.A. Gerald C. Nature. 1998; 396: 674-679Crossref PubMed Scopus (929) Google Scholar, 25Kaupmann K. Malitschek B. Schuler V. Heid J. Froestl W. Beck P. Mosbacher J. Bischoff S. Kulik A. Shigemoto R. Karschin A. Bettler B. Nature. 1998; 396: 683-687Crossref PubMed Scopus (1018) Google Scholar). Subsequent studies demonstrated that the GBR1 isoform bears an endoplasmic reticulum retention signal that is blocked by dimerization with the GBR2 isoform, allowing endoplasmic reticulum export and plasma membrane targeting (26Margeta-Mitrovic M. Jan Y.N. Jan L.Y. Neuron. 2000; 27: 97-106Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar). Others reported that effects of GPCR dimerization include changing ligand selectivity and altered signal transduction (27Brady A.E. Limbird L.E. Cell Signal. 2002; 14: 297-309Crossref PubMed Scopus (221) Google Scholar). Since there are numerous alternatively spliced transcripts of the CTR gene product, and some of the splice variants show considerable differences in their trafficking or receptor function, dimerization of these splice variants could result in profound changes in the biology of the CTR. We show here that the Δe13 variant accumulates within the cell and is only minimally expressed on the cell surface, whereas the C1a variant of the rabbit CTR is highly expressed on the cell surface. We also show that when both splice variants are expressed in the same cell, they form both homodimers and heterodimers, via interaction of the C-terminal tail but also via at least one other domain of the receptor. Co-expression of the Δe13 and C1a isoforms results in a marked decrease in the surface expression of the C1a isoform. Moreover, the reduced surface expression of the C1a isoform was accompanied by diminished cAMP production and Erk phosphorylation after ligand stimulation, suggesting that the dimerization of the Δe13 isoform with the C1a isoform serves to down-regulate CT-induced signaling by retaining the C1a splice variant in an intracellular compartment. Reagents and Antibodies—Salmon calcitonin (sCT) was purchased from Peninsula Laboratories, Inc. (Belmont, CA). The monoclonal anti-HA antibody (F-7) and the monoclonal anti-Myc antibody (9E10) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); the monoclonal anti-GFP antibody and the monoclonal anti-RFP antibody were from Clontech (Palo Alto, CA); the antibodies against Erk2 and phosphorylated Erk1/2 were from New England Biolabs, Inc. (Beverly, MA); and the monoclonal anti-FLAG antibody (M2) was from Sigma. Enhanced chemiluminescence solutions and nitrocellulose membranes were from Amersham Biosciences and Schleicher & Schuell, respectively. Cell Culture and Transient Transfections—Dulbecco's modified Eagle's medium and fetal bovine serum were purchased from Invitrogen. Minimal essential medium, α modification (α-MEM) was from Sigma. All media were supplemented with 100 μg/ml streptomycin and 100 units/ml penicillin. HEK 293 cells were cultured as described before (28Zhang Z. Hernandez-Lagunas L. Horne W.C. Baron R. J. Biol. Chem. 1999; 274: 25093-25098Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). For transient transfections, cells were grown to 60–70% confluence and then transfected with Fugene 6 (Roche Molecular Biochemicals) according to the protocol of the manufacturer. When not otherwise described (i.e. Fig. 2A), experiments that involved transfecting the CTR isoforms alone or in combination were performed with constant amounts of each cDNA and adding empty vector DNA when only one CTR isoform was expressed to keep the total amount of DNA constant. Production of Rabbit Osteoclast-like Cells—Rabbit osteoclast-like cells (OCLs) were produced as described before (29David J.P. Neff L. Chen Y. Rincon M. Horne W.C. Baron R. J. Bone Miner. Res. 1998; 13: 1730-1738Crossref PubMed Scopus (48) Google Scholar). Briefly, long bones and scapulae from 4-day-old rabbits were isolated and dissected from soft tissue. The bones were minced, the bone particles were allowed to settle, and the cells left in suspension were recovered by centrifugation. Cells were plated in culture dishes with α-MEM and 10% fetal calf serum. After overnight incubation, the nonadherent cells and the adherent stromal cells that could be removed with trypsin/EDTA were pooled and plated in 10-cm dishes in the presence of dihydroxyvitamin D (10–8m). After 7–9 days, the layer of stromal cells was removed from the differentiated OCLs by repeated pipetting of medium across the cell layer. The OCLs were detached by incubation in 50 mm EDTA in PBS at 37 °C for 5 min, washed in α-MEM, and resuspended in α-MEM containing 5% fetal calf serum. Single Osteoclast-like Cell Reverse Transcriptase-PCR—Single cells from a rabbit OCL suspension in α-MEM containing 5% fetal calf serum were sorted in individual wells of a 96-well PCR plate (Costar Thermowell, Cambridge, MA) with a FACS device (Becton Dickinson, Franklin Lakes, NJ), selecting cells in the top 30% region of the cell size scatter. The wells of the PCR plate contained 10 μl of cold reverse transcription buffer (Sensiscript reverse transcription kit, Qiagen, Hilden, Germany) with 0.5 units/μl RNase inhibitor (Roche Applied Science). After sorting, plates were immediately placed on dry ice and stored at –80 °C. After thawing, which lysed the cells, 10 μl of 2× master mix of the Sensiscript reverse transcription kit (Qiagen) containing oligo(dT) primer (final concentration 1 μm) was added, and the reaction was incubated for 60 min at 37 °C. 10 μl of this reaction was used to amplify the CTR-specific cDNA by PCR, using primers on the 5′ and 3′ sides of exon 13, resulting in different length PCR products amplified from the C1a (356-bp) and Δe13 (308-bp) cDNAs. The forward primer (5′-GATGGCAGCTCTGGTGGTCAAT) corresponded to nucleotides 1190–1211 of the rabbit C1a cDNA, and the reverse primer (5′-GGTGGGCGGGGCGTCTT) corresponded to nucleotides 1529–1545 of the rabbit C1a cDNA (8Shyu J.F. Inoue D. Baron R. Horne W.C. J. Biol. Chem. 1996; 271: 31127-31134Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The PCR was done in the presence of 1.5 mm MgCl2 and 0.3 μm each primer by performing 40 cycles consisting of 1 min at 94 °C, 1 min at 55 °C, and 1 min at 72 °C. DNA Constructs—cDNAs encoding the rabbit C1a and Δe13 variants of the CTR were generated by PCR and cloned in the KpnI/HindIII sites of the p3XFLAG-CMV-13 vector (Sigma) to obtain CTRs with a C-terminal 3-fold FLAG tag. The same PCR products were cloned in the KpnI/HindIII site of pEGFP-N1 (Clontech) to obtain C-terminal GFP-tagged CTR splice variants. The PCR fragments were also cloned in the KpnI/HindIII site of pDsRed2-N1 (Clontech) to obtain C-terminal RFP-tagged CTR splice variants. The GFP constructs were published before and compared with the wild type CTR in respect to ligand binding, phosphorylation after ligand stimulation, and cAMP generation and were found to be indistinguishable (30Seck T. Baron R. Horne W.C. J. Biol. Chem. 2003; 278: 10408-10416Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). For this study, we also compared the cellular localization of the GFP- and RFP-tagged CTR constructs with the FLAG-tagged constructs by immunocytochemistry. They were found to be indistinguishable (data not shown). A fragment spanning the complete receptor including the seventh transmembrane domain but lacking the intracellular C-terminal tail of the receptor (amino acids 1–397) was cloned in the KpnI/HindIII site of the pEGFP-N1 vector and designated C1aΔ397-GFP. We also cloned these fragments in the KpnI/HindIII site of the p3XFLAG-CMV-13 vector. Constructs expressing each of the two splice variants with three tandem Myc epitope tags at the C terminus were generated as described before (9Shyu J.F. Zhang Z. Hernandez-Lagunas L. Camerino C. Chen Y. Inoue D. Baron R. Horne W.C. Eur. J. Biochem. 1999; 262: 95-101Crossref PubMed Scopus (23) Google Scholar). All constructs contained an HA tag in the extracellular N terminus of the receptor (after amino acid 29 of the original sequence). We also generated a C-terminally GFP-tagged Δe13 construct containing a Myc tag instead of an HA tag in the extracellular N terminus by mutagenesis using the QuikChange mutagenesis kit (Stratagene, Cedar Creek, TX) according to the instructions of the manufacturer. The ligand binding and cAMP production of all full-length CTR constructs were measured and were found to be indistinguishable from the wild type CTR (data not shown). A fragment spanning the C-terminal tail of the CTR (amino acids 397–474) was cloned between the HindIII and KpnI sites of the p3XFLAG-CMV-13 vector after generation by PCR. A PCR-derived fragment spanning the same region was cloned in the EcoRI/SalI sites of the pGEX-4T-2 vector (Amersham Biosciences) to generate a GST-C-terminal tail fusion protein called GST-C-tail. The C-terminally RFP-tagged V1a vasopressin receptor was a generous gift from Dr. M. Nathanson (Yale University). All PCR-derived constructs were sequenced by the Yale Keck Sequencing Facility. Co-immunoprecipitation and Western Blotting—Cells were lysed in mRIPA buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.1% IGEPAL, 1% sodium deoxycholate, 10 mm NaF, 1 μg/ml pepstatin, and 1 mm phenylmethylsulfonyl fluoride) and incubated at 4 °C for 30 min. Lysates were then centrifuged for 30 min at 4 °C, 16,000 × g, the protein concentrations were measured with the BCA protein assay kit (Pierce), and equal amounts of protein were used for immunoprecipitation. 30 μl of protein G-agarose slurry and typically 5 μg of antibody were suspended in 500 μl of PBS and incubated for 1 h at 4 °C. The beads were washed three times in mRIPA buffer, and then 500 μg of protein lysate and bovine serum albumin (0.2% w/v) were added, and the mix was incubated for 2 h at 4 °C. The immune complexes on the beads were washed four times with washing buffer containing 300 mm NaCl and 0.1% Triton X-100 and once with PBS. Beads were boiled in 2× SDS-PAGE buffer, and samples were electrophoresed on precast 10% SDS-PAGE gels (Invitrogen). Proteins were transferred to nitrocellulose membranes, and the transfer was verified by staining with 0.2% Ponceau S in 3% trichloroacetic acid. Nonspecific binding was blocked by incubating the membranes in 5% nonfat milk in TBST buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.1% Tween 20) for 1 h. Membranes were incubated in the primary antibody for 2 h, washed three times for 15 min in TBST, and incubated for 1 h in 1:10,000 diluted horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG antibody (Promega). Blots were developed using the enhanced chemiluminescence system from Amersham Biosciences. Biotinylation of Surface Proteins—Cells were washed two times with ice-cold PBS, cooled on ice, and incubated in the presence of 0.5 mg/ml sulfo-N-hydroxysuccinimide-biotin (Pierce) at 4 °C for 30 min to biotinylate proteins on the cell surface. Excess biotin was quenched by incubating in 50 mm Tris-HCl for 10 min at 4 °C. Cells were washed twice and harvested in mRIPA buffer. For isolating the cell surface-expressed CTR, biotinylated proteins were bound to 25 μl of resin of immobilized streptavidin (Pierce) by incubating for2hat4 °C in the presence of 0.1% bovine serum albumin (w/v). After the incubation, the resin was pelleted by centrifugation and washed four times with immunoprecipitation-washing buffer containing 0.1% Triton X-100 and 500 mm NaCl. The bound proteins were eluted by using 100 μl of 1% SDS, 25 mm Tris-HCl, pH 7.4, which was diluted with 900 μl of PBS prior to purifying the CTR by immunoprecipitation. For analysis of receptor surface expression after biotinylation, cells were washed twice with ice-cold PBS and harvested in mRIPA. The FLAG-tagged CTR was immunoprecipitated using the M2 monoclonal antibody (Sigma). The immune complexes were resolved on a 10% SDS-PAGE and transferred onto nitrocellulose. The biotinylated receptor was detected using horseradish peroxidase-conjugated avidin D (Vector Laboratories, Burlingame, CA). Fluorescence Resonance Energy Transfer (FRET) Experiments— FRET analysis was performed using the C-terminal GFP- and RFP-tagged receptor constructs as described in Ref. 31Cornea A. Janovick J.A. Maya-Nunez G. Conn P.M. J. Biol. Chem. 2001; 276: 2153-2158Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar. HEK 293 cells were seeded in 35-mm glass bottom dishes (MatTek Corp., Ashland, OR) and 24 h later transfected with the GFP- and RFP-tagged CTR constructs as described above. Images were taken by placing the dish in a heated stage (DH-35; Warner Instruments, Hamden, CT) in a Zeiss LSM 510 confocal microscope (Zeiss, Jena, Germany) to maintain a temperature of 37 °C. The culture medium was replaced by 37 °C warm HEPES buffer containing glucose. A 63 × 1.25 numerical aperture water immersion objective was used with the pinhole set to 2.5 Airy disc units. GFP was excited at 488 nm by an argon laser, and RFP was excited at 568 nm using a krypton laser. Emission was measured in the green channel from 505 to 530 nm and in the red channel from 610 to 670 nm. FRET was measured by imaging GFP before and after photobleaching the RFP with the 568-nm krypton laser. An increase of the donor fluorescence (GFP) was interpreted as evidence of FRET from GFP to RFP. An unbleached area of membrane in the same cell served as control. All experiments analyzed at least 10 cells, the mean change of GFP fluorescence was calculated, and statistical analysis of the results was performed by using Student's t test. For time lapse studies, cells were prepared as described above. For the imaging, the excitation energy was lowered to minimize photobleaching and cell damage caused by absorption of light energy. The region of interest was set to membrane areas, and the GFP and RFP fluorescence was measured over the time course, using the same channels as described above. Overexpression and Purification of GST Fusion Proteins—Escherichia coli, strain BL-21, harboring the empty GST vector or GST-C-tail were grown overnight in LB ampicillin medium. The next morning, cells were diluted 1:10 and grown to an A600 of 0.5 and then induced with 0.1 mm isopropyl-1-thio-β-d-galactopyranoside to express the fusion proteins. After 3 h, cells were harvested by pelleting at 4,000 × g at 4 °C, resuspended in PBS containing protease inhibitors (10 mm NaF, 1 μg/ml pepstatin, and 1 mm phenylmethylsulfonyl fluoride), and sonicated for a total time of 60 s. The bacterial lysate was solubilized by the addition of Triton X-100 to a final concentration of 1% and centrifuged at 14,000 × g at 4 °C to remove insoluble material. Glutathione-agarose beads (Amersham Biosciences) were added to the supernatant and incubated for 1 h at 4 °C. The beads were washed four times in PBS containing 0.1% Triton X-100 and 300 mm NaCl. The integrity of the of the GST fusion protein bound to the beads was analyzed by resolving the proteins on SDS-PAGE followed by Coomassie Blue staining. In Vitro Characterization of C-terminal Domain Interactions—5 μgof GST or GST-C-tail bound to glutathione-agarose beads were incubated with 300 μg of total cell lysate of HEK 293 cells transfected with the C1a-GFP or the C1aΔ397-GFP construct. After a 2-h incubation at 4 °C with continuous rocking, beads were washed three times with washing buffer containing 300 mm NaCl and 0.1% Triton X-100 and once with PBS. Bound proteins were solubilized in 2× SDS-PAGE buffer and analyzed on SDS-PAGE. Measurement of Receptor Cell Surface Expression by FACS Analysis—Cells in six-well plates were incubated with trypsin-EDTA solution at room temperature. All further steps were performed at 4 °C. The trypsin activity was neutralized by the addition of growth medium containing 10% dialyzed fetal bovine serum. Cells were collected by centrifugation at 800 × g for 3 min. The cell pellet was resuspended in 100 μl of ice-cold PBS. Usually about 3 × 105 cells were used for each experiment. Normal goat IgG was added to a final concentration of 200 μg/ml. After 10 min, the antibody against the N-terminal tag (HA except where otherwise noted) was added to a final concentration of 10 μg/ml. A 30-min incubation step was followed by resuspending in 100 μl of PBS containing 50 μg/ml phycoerythrin (PE)-conjugated goat anti-mouse IgG antibody (Molecular Probes, Inc., Eugene, OR). After a final washing step, cells were resuspended in PBS containing 2% formaldehyde to fix the sample. Bound antibody was analyzed by fluorescence flow cytometry (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ). Win MDI software version 2.8 was used for data analysis. cAMP Measurement—cAMP was measured as described before (8Shyu J.F. Inoue D. Baron R. Horne W.C. J. Biol. Chem. 1996; 271: 31127-31134Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Cells transfected with the CTR were plated in a 96-well plate (8,000 cells/well). Cells were preincubated with 1 mm 3-isobutyl-1-methylxanthine (Sigma) for 10 min before stimulation with sCT for 10 min at 37 °C. The reaction was stopped with 95% ethanol containing 3 mm HCl. cAMP was measured by a scintillation proximity assay (Amersham Biosciences) following the manufacturer's instructions. The Cell Surface Expression Levels of the C1a and the Δe13 Isoform of the Rabbit CTR Differ—The decreased amplitude of the cAMP response previously reported for the Δe13 isoform of the CTR could reflect, at least in part, different levels of cell surface expression of the two isoforms. To examine the subcellular localization of the two isoforms, we first generated C-terminal GFP constructs for the C1a and the Δe13 isoforms and expressed both in HEK 293 cells. Both isoforms showed marked accumulation within the cell (Fig. 1A). Similar intracellular accumulations were found when the CTR isoforms were tagged with the FLAG epitope (data not shown). We previously reported that these accumulations are a recycling compartment for the C1a isoform (30Seck T. Baron R. Horne W.C. J. Biol. Chem. 2003; 278: 10408-10416Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Moreover, we found that much less of the Δe13-GFP chimera (Fig. 1A, right panel) was expressed on the cell surface relative to the C1a-GFP chimera (Fig. 1A, left panel). To better quantify the difference in the cell surface expression, we used a flow cytometric method to measure the cell surface expression of the two constructs. An HA tag near the N terminus of the C1a- and Δe13-GFP constructs was used to label the receptors on the cell surface with fluorescent phycoerythrin (PE). FACS analysis showed that whereas the expressions of the two GFP-tagged CTR isoforms were similar (Fig. 1B, upper panels), there was much less PE labeling of the cells expressing the GFP-Δe13 chimera (Fig. 1B, lower panel), indicating that the C1a isoform is much more highly expressed on the cell surface of HEK 293 cells than the Δe13 isoform. The difference was statistically significant (p < 0.001). The C1a and the Δe13 Isoform of the Rabbit CTR Form Dimers—To address the question of whether or not the two isoforms of the CTR form homo- or heterodimers, we coexpressed FLAG- and/or GFP-tagged C1a and Δe13 receptors in HEK 293 cells as described in Fig. 2. After immunoprecipitation of the FLAG-tagged CTR, we blotted the immune complexes with the anti-GFP antibody. In all cases, we found that the immune complexes contained both the CTR-FLAG chimera and the CTR-GFP chimera, suggesting that the CTR forms dimers or oligomers (Fig. 2A, upper panel). We found C1a/Δe13 heterodim" @default.
• W2091036574 created "2016-06-24" @default.
• W2091036574 creator A5028618728 @default.
• W2091036574 creator A5053072500 @default.
• W2091036574 creator A5059497956 @default.
• W2091036574 date "2003-06-01" @default.
• W2091036574 modified "2023-10-16" @default.
• W2091036574 title "The Alternatively Spliced Δe13 Transcript of the Rabbit Calcitonin Receptor Dimerizes with the C1a Isoform and Inhibits Its Surface Expression" @default.
• W2091036574 cites W1571586310 @default.
• W2091036574 cites W1595449036 @default.
• W2091036574 cites W1654156243 @default.
• W2091036574 cites W1852077611 @default.
• W2091036574 cites W1945680072 @default.
• W2091036574 cites W1966815718 @default.
• W2091036574 cites W1980236160 @default.
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• W2091036574 cites W2009411531 @default.
• W2091036574 cites W2014825849 @default.
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• W2091036574 cites W2019086342 @default.
• W2091036574 cites W2026444780 @default.
• W2091036574 cites W2040067193 @default.
• W2091036574 cites W2067466593 @default.
• W2091036574 cites W2070411747 @default.
• W2091036574 cites W2070805067 @default.
• W2091036574 cites W2071274951 @default.
• W2091036574 cites W2093628054 @default.
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• W2091036574 cites W2157907012 @default.
• W2091036574 cites W2165133988 @default.
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