FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2080232509> ?p ?o ?g. }

• W2080232509 endingPage "29609" @default.
• W2080232509 startingPage "29602" @default.
• W2080232509 abstract "Expression of the calcitonin receptor-like receptor (CRLR) and its receptor activity modifying proteins (RAMPs) can produce calcitonin gene-related peptide (CGRP) receptors (CRLR/RAMP1) and adrenomedullin (AM) receptors (CRLR/RAMP2 or -3). A chimera of the CRLR and green fluorescent protein (CRLR-GFP) was used to study receptor localization and trafficking in stably transduced HEK 293 cells, with or without co-transfection of RAMPs. CRLR-GFP failed to generate responses to CGRP or AM without RAMPs. Furthermore, CRLR-GFP was not found in the plasma membrane and its localization was unchanged after agonist exposure. When stably coexpressed with RAMPs, CRLR-GFP appeared on the cell surface and was fully active in intracellular cAMP production and calcium mobilization. Agonist-mediated internalization of CRLR-GFP was observed in RAMP1/CGRP or AM, RAMP2/AM, and RAMP3/AM, which occurred with similar kinetics, indicating the existence of ligand-specific regulation of CRLR internalization by RAMPs. This internalization was strongly inhibited by hypertonic medium (0.45m sucrose) and paralleled localization of rhodamine-labeled transferrin, suggesting that CRLR endocytosis occurred predominantly through a clathrin-dependent pathway. A significant proportion of CRLR was targeted to lysosomes upon binding of the ligands, and recycling of the internalized CRLR was not efficient. In HEK 293 cells stably expressing CRLR-GFP and Myc-RAMPs, these rhodamine-labeled RAMPs were co-localized with CRLR-GFP in the presence and absence of the ligands. Thus, the CRLR is endocytosed together with RAMPs via clathrin-coated vesicles, and both the internalized molecules are targeted to the degradative pathway. Expression of the calcitonin receptor-like receptor (CRLR) and its receptor activity modifying proteins (RAMPs) can produce calcitonin gene-related peptide (CGRP) receptors (CRLR/RAMP1) and adrenomedullin (AM) receptors (CRLR/RAMP2 or -3). A chimera of the CRLR and green fluorescent protein (CRLR-GFP) was used to study receptor localization and trafficking in stably transduced HEK 293 cells, with or without co-transfection of RAMPs. CRLR-GFP failed to generate responses to CGRP or AM without RAMPs. Furthermore, CRLR-GFP was not found in the plasma membrane and its localization was unchanged after agonist exposure. When stably coexpressed with RAMPs, CRLR-GFP appeared on the cell surface and was fully active in intracellular cAMP production and calcium mobilization. Agonist-mediated internalization of CRLR-GFP was observed in RAMP1/CGRP or AM, RAMP2/AM, and RAMP3/AM, which occurred with similar kinetics, indicating the existence of ligand-specific regulation of CRLR internalization by RAMPs. This internalization was strongly inhibited by hypertonic medium (0.45m sucrose) and paralleled localization of rhodamine-labeled transferrin, suggesting that CRLR endocytosis occurred predominantly through a clathrin-dependent pathway. A significant proportion of CRLR was targeted to lysosomes upon binding of the ligands, and recycling of the internalized CRLR was not efficient. In HEK 293 cells stably expressing CRLR-GFP and Myc-RAMPs, these rhodamine-labeled RAMPs were co-localized with CRLR-GFP in the presence and absence of the ligands. Thus, the CRLR is endocytosed together with RAMPs via clathrin-coated vesicles, and both the internalized molecules are targeted to the degradative pathway. calcitonin gene-related peptide receptor activity-modifying protein calcitonin receptor-like receptor adrenomedullin green fluorescent protein G protein-coupled receptor tetramethylrhodamine isothiocyanate concanavalin A human embryonic kidney cells Dulbecco's modified Eagle's medium phosphate-buffered saline bovine serum albumin β2-andrenergic receptor Calcitonin gene-related peptide (CGRP)1 and adrenomedullin (AM) belong to the calcitonin family of regulatory peptides, both of which are potent vasodilators (1Brain S.D. Williams T.J. Tippins J.R. Morris H.R. MacIntyre I. Nature. 1985; 313: 54-56Crossref PubMed Scopus (1820) Google Scholar, 2Kitamura K. Kangawa K. Kawamoto M. Ichiki Y. Nakamura S. Matsuo H. Eto T. Biochem. Biophys. Res. Commun. 1993; 192: 553-560Crossref PubMed Scopus (2086) Google Scholar). CGRP is a 38-amino acid neuropeptide produced by tissue-specific alternative splicing of the primary transcript of the calcitonin gene (3Amara S.G. Jonas V. Rosenfeld M.G. Nature. 1982; 298: 240-244Crossref PubMed Scopus (1754) Google Scholar). CGRP immunoreactivity is present throughout the central and peripheral nervous system, and release of CGRP from nervous tissue has been demonstrated (4Kruger L. Mantyh P.W. Sternini C. Brecha N.C. Mantyh C.R. Brain. Res. 1988; 463: 223-244Crossref PubMed Scopus (191) Google Scholar, 5Poyner D.R. Pharmacol. Ther. 1992; 56: 23-51Crossref PubMed Scopus (201) Google Scholar, 6Goodman E.C. Iversen L.L. Life Sci. 1986; 38: 2169-2178Crossref PubMed Scopus (203) Google Scholar). Human AM, which consists of 52 amino acids, exhibits diverse biological activities consistent with its wide tissue distribution (2Kitamura K. Kangawa K. Kawamoto M. Ichiki Y. Nakamura S. Matsuo H. Eto T. Biochem. Biophys. Res. Commun. 1993; 192: 553-560Crossref PubMed Scopus (2086) Google Scholar, 7Kitamura K. Eto T. Curr. Opin. Nephrol. Hypertension. 1997; 6: 80-87Crossref PubMed Scopus (46) Google Scholar, 8Samson W.K. Front. Neuroendocrinol. 1998; 19: 100-127Crossref PubMed Scopus (60) Google Scholar). It mainly exerts powerful cardiovascular effects as an autocrine/paracrine regulator. Both peptides have been reported to activate adenylyl cyclase and phospholipase C, and to increase the intracellular calcium concentration in various cell types (9Shimekake Y. Nagata K. Ohta S. Kambayashi Y. Teraoka H. Kitamura K. Eto T. Kangawa K. Matsuo H. J. Biol. Chem. 1995; 270: 4412-4417Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 10Drissi H. Lasmoles F. Mellay V.L. Marie P.J. Lieberherr M. J. Biol. Chem. 1998; 273: 20168-20174Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). This suggests that their receptors activate different G proteins, Gs and Gq. Specific or common binding sites for these peptides have been suggested to be present in both central and peripheral tissues; however, their formal cell receptors have not been identified. Recently, human receptor activity-modifying protein 1 (RAMP1) was identified as an accessory protein that enhances the activity of endogenous CGRP receptors in Xenopus oocytes (11McLatchie L.M. Fraser N.J. Main M.J. Wise A. Brown J. Thompson N. Solari R. Lee M.G. Foord S.M. Nature. 1998; 393: 333-339Crossref PubMed Scopus (1868) Google Scholar). This protein consisted of 148 amino acids including an N-terminal signal sequence and putative single transmembrane region, and was shown to induce trafficking of the seven-transmembrane domain calcitonin receptor-like receptor (CRLR) to the cell surface by fluorescence-activated cell sorting analysis. RAMP2 and -3 cDNAs (175 and 148 amino acids, respectively) were then cloned by expressed sequence tag analysis (11McLatchie L.M. Fraser N.J. Main M.J. Wise A. Brown J. Thompson N. Solari R. Lee M.G. Foord S.M. Nature. 1998; 393: 333-339Crossref PubMed Scopus (1868) Google Scholar). When co-transfected with RAMP2 or -3 in mammalian cells the CRLR became a functional AM receptor (11McLatchie L.M. Fraser N.J. Main M.J. Wise A. Brown J. Thompson N. Solari R. Lee M.G. Foord S.M. Nature. 1998; 393: 333-339Crossref PubMed Scopus (1868) Google Scholar, 12Muff R. Leuthauser K. Buhlmann N. Foord S.M. Fischer J.A. Born W. FEBS Lett. 1998; 441: 366-368Crossref PubMed Scopus (74) Google Scholar, 13Fraser N.J. Wise A. Brown J. McLatchie L.M. Main M.J. Foord S.M. Mol. Pharmacol. 1999; 55: 1054-1059Crossref PubMed Scopus (172) Google Scholar). Although RAMP2 and -3 share only 30% homology and they show different tissue distributions, the CRLR/RAMP2 and CRLR/RAMP3 receptors in HEK 293T cells have been reported to be indistinguishable by radioligand binding, functional assay, and biochemical analysis (13Fraser N.J. Wise A. Brown J. McLatchie L.M. Main M.J. Foord S.M. Mol. Pharmacol. 1999; 55: 1054-1059Crossref PubMed Scopus (172) Google Scholar). Exposure of cells to agonists often leads to a rapid internalization of cell surface G protein-coupled receptors (GPCRs). This agonist-promoted phenomenon is common to a large number of GPCRs (14Koenig J.A. Edwardson J.M. Trends. Pharmacol. Sci. 1997; 18: 276-287Abstract Full Text PDF PubMed Scopus (299) Google Scholar, 15Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (858) Google Scholar). Internalization is believed to involve clathrin-coated vesicles and/or caveolin-rich vesicles. The internalized GPCRs may be recycled back to the plasma membrane rather than being trafficked to lysosomes where they are degraded (14Koenig J.A. Edwardson J.M. Trends. Pharmacol. Sci. 1997; 18: 276-287Abstract Full Text PDF PubMed Scopus (299) Google Scholar, 15Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (858) Google Scholar). These processes have been best studied and characterized for the β2-adrenergic receptor (14Koenig J.A. Edwardson J.M. Trends. Pharmacol. Sci. 1997; 18: 276-287Abstract Full Text PDF PubMed Scopus (299) Google Scholar, 15Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (858) Google Scholar). On the other hand, there have been no reports concerning the trafficking of RAMP molecules after agonist exposure. The recent development of GPCR conjugated with green fluorescent protein (GFP) has provided the opportunity for a more extensive optical analysis of receptor trafficking events in individual cells (16Tarasova N.I. Stauber R.H. Choi J.K. Hudson E.A. Czerwinski G. Miller J.L. Pavlakis G.N. Michejda C.J. Wank S.A. J. Biol. Chem. 1997; 272: 14817-14824Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 17Kallal L. Gagnon A.W. Penn R.B. Benovic J.L. J. Biol. Chem. 1998; 273: 322-328Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 18Tarasova N.I. Stauber R.H. Michejda C.J. J. Biol. Chem. 1998; 273: 15883-15886Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 19Carter R.E. Sorkin A. J. Biol. Chem. 1998; 273: 35000-35007Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). In this study, we characterized the biochemical properties of the functional CRLR chimera with GFP fused to the C terminus of the receptor, CRLR-GFP, with or without co-transfection of RAMPs. Moreover, direct visualization of the localization of CRLR-GFP and rhodamine-labeled RAMPs provided evidence that, when stably coexpressed, agonist-induced endocytosis of both molecules occurred together through the same pathways and this was irreversible. A plasmid containing human CRLR cDNA (20Fluhmann B. Muff R. Hunziker W. Fisher J.A. Born W. Biochem. Biophys. Res. Comun. 1995; 206: 341-347Crossref PubMed Scopus (127) Google Scholar) was a kind gift from Dr. Kenji Kangawa (National Cardiovascular Research Institute, Osaka, Japan). 125I-Labeled human CGRP (specific activity 2177 Ci/mmol) and 125I-labeled human adrenomedullin (specific activity 1260 Ci/mmol) were from Peninsula Laboratories. Human CGRP and adrenomedullin were from Peptide Institute (Osaka, Japan). Fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate (TRITC) were from Dako. Tetramethylrhodamine-concanavalin A (ConA), tetramethylrhodamine-transferrin, and LysoTracker Red were purchased from Molecular Probes Inc. All other reagents were of analytical grade and were obtained from various suppliers. A bright green mutant of GFP, enhanced GFP (CLONTECH), was attached to the C terminus of human CRLR by standard recombinant techniques. Briefly, the full open reading frame of the human CRLR, modified to provide a consensus Kozak sequence (21Aiyar N. Rand K. Elshourbagy N.A. Zeng Z. Adamou J.E. Bergsma D.J. Li Y. J. Biol. Chem. 1996; 271: 11325-11329Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar), was amplified using appropriate primers containingSalI and XbaI restriction sites at the 5′ and 3′ ends, respectively. The 5′ and 3′ ends of CRLR were ligated into theSalI and XbaI sites of modified pEGFP-N1 (CLONTECH), SmaI → XbaI at its multicloning site. The fused product was then cloned into pCAGGS/Neo, termed pCAGGS-CRLR-GFP. RAMP cDNAs, modified to provide a consensus Kozak sequence, were obtained from a human embryonic cDNA library (CLONTECH). Each RAMP was cloned into pIRES1/Hyg (CLONTECH). The Myc epitope tag (EQKLISEEDL) was used in-frame to the 5′ end of cDNAs encoding CRLR and RAMPs. The native signal sequences were removed and replaced with MKTILALSTYIFCLVFA (22Guon X.-M. Kobilka T.S. Kobilka B.K. J. Biol. Chem. 1992; 267: 21995-21998Abstract Full Text PDF PubMed Google Scholar). Myc-CRLR and Myc-RAMPs were cloned into pCAGGS/Neo and pIRES1/Hyg, respectively. CRLR-GFP, Myc-CRLR, RAMPs, and Myc-RAMPs cDNAs were all sequenced using an Applied Biosystems 310 Genetic Analyzer. Human embryonic kidney cells (HEK 293) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 μg/ml penicillin G, 100 units/ml streptomycin, and 500 μg/ml amphotericin B at 37 °C in a humidified atmosphere of 95% air, 5% CO2. Cells grown to 70–80% confluence were transfected with linearized pCAGGS-CRLR-GFP using the LipofectAMINE method (Life Technologies, Inc.). Two days after transfection, cells were plated in selective medium containing 1 mg/ml G418 (Life Technologies, Inc.). A pool of G-418-resistant colonies was trypsinized followed by limited dilution and single cell plating into 96-well plates. Stable transfectants were isolated approximately 3 weeks after transfection and screened for expression of GFP by fluorescence microscopy and flow cytometry. Thereafter, six clones were selected for stable co-transfection. Each clone was then transfected with linearized pIRES-RAMPs or pIRES-Myc-RAMPs using LipofectAMINE. After 2 weeks of hygromicin B (Wako, Osaka, Japan, 0.4–0.2 mg/ml) selection, individual clonal lines of HEK 293 expressing CRLR-GFP/RAMPs or CRLR-GFP/Myc-RAMPs were established by monitoring with both flow cytometry and sequestration studies under fluorescence microscopy. Stable transfectants with stronger fluorescence tended to be poorly internalized (data not shown). These transfectant cell lines were maintained in the same DMEM buffer containing 0.25 mg/ml G418 and 0.1 mg/ml hygromycin B. Myc-CRLR/RAMPs double stable HEK293 cell lines were established by the above-mentioned methods. Flow cytometry was performed to assess expression of constructs of CRLR-GFP in the absence and presence of RAMPs. Cells were dissociated, washed twice with fluorescence-activated cell sorting buffer (1% FBS, 0.02% sodium azide in phosphate-buffered saline (PBS)), and adjusted to 2 × 105 per tube in the same buffer with 5 μg/ml propidium iodide. Samples were subjected to flow cytometry on an EPICS XL flow cytometer (Beckman Coulter) and analyzed using the EXPO 2 software (Beckman Coulter). Fluorophores were excited at 488 nm, and emission was monitored at 530 nm for GFP and 575 nm for phycoerythrin. Viability was assessed by exclusion of propidium iodide. For assay of cAMP accumulation, cells were seeded in 6- or 24-well plates 72 h prior to determination. Cells were replenished with Hanks' buffer containing 20 mm HEPES and 0.1% bovine serum albumin (BSA), and then treated with the indicated agonists in the presence of 0.5 mm3-isobutyl-1-methylxanthine (Sigma) for 15 min at 37 °C. The reactions were terminated by addition of lysis buffer (Amersham Pharmacia Biotech) followed by centrifugation at 2000 rpm for 10 min at 4 °C. Aliquots of the supernatants were removed and cAMP content determined using a commercial enzyme immunoassay kit, according to the manufacturer's instructions for the non-acetylation protocol (Amersham Pharmacia Biotech). Stably transfected HEK 293 cells (1 × 106/ml) were loaded with 2 μm fura-2/AM (Molecular Probes Inc.) in Hanks' buffered salt solution containing 20 mm HEPES and 0.1% BSA for 30 min at 37 °C in the dark. After two washes, cells were resuspended at 1 × 106 cells/ml. Cells (2 ml) were prewarmed to 37 °C and stimulated by 100 nm agonists in a quartz cuvette with a continuously stirring magnetic bar using a Perkin-Elmer LS 50B spectrofluorimeter. Data were recorded as the relative ratio of fluorescence emitted at 510 nm after excitation at 340 and 380 nm (y axis) over time (x axis). HEK 293 cells stably expressing CRLR-GFP, CRLR-GFP/RAMPs, CRLR-GFP/Myc-RAMPs, or Myc-CRLR/RAMPs were plated onto 35-mm dishes containing a centered 22-mm well formed from a glass coverslip seated in a hole in the plastic. After rinsing with PBS, human CGRP or adrenomedullin was added to the dishes at a concentration of 100 nm in prewarmed serum-free DMEM containing 20 mm HEPES and 0.1% BSA for the indicated times at 37 °C. For receptor recycling studies, following 30-min agonist treatment, cells were washed three times with prewarmed PBS and medium was replaced with fresh DMEM for 1 h at 37 °C. Internalization and recycling were stopped with ice-cold PBS. Cells were then fixed with 3.7% formaldehyde/PBS for 15 min at room temperature and washed three times with PBS. The coverslips were mounted using Slow-Fade mounting medium (Molecular Probes Inc.). To examine whether Myc-RAMPs localize with CRLR-GFP during internalization and recycling, after the indicated incubation period, cells were fixed as described above followed by permeabilization with 0.1% Triton X-100/PBS for 5 min at room temperature. Nonspecific binding was blocked with 1% BSA/PBS for 30 min at room temperature. The first antibody (monoclonal c-Myc, Invitrogen) was added at 1:250 dilution for 1 h at room temperature followed by three successive washes with PBS. Incubation with rabbit anti-mouse fluorescein TRITC-conjugated secondary antibody (Dako) diluted 1:30 in 1% BSA/PBS was carried out for 1 h at room temperature. After three washes with PBS, coverslips were mounted using Slow-Fade. To simultaneously observe endocytosis of CRLR-GFP and transferrin, cells were incubated with 100 nm human CGRP or adrenomedullin and 100 μm tetramethylrhodamine-transferrin for 15 min at 37 °C, fixed, washed, and mounted. Events following agonist treatment were observed by confocal microscopy. Whole cell radioligand binding assays were performed as follows. Cells were seeded in 24-well culture plates (3 × 105 cells/well). After 3 days, cells were pretreated with or without the inhibitor (0.45 m sucrose, 15 min) and the incubated with serum-free DMEM containing 20 mm HEPES and 0.1% BSA for the indicated periods (up to 3 h) at 37 °C in the presence or absence of 100 nmagonists. Following agonist exposure, cells were washed twice with ice-cold PBS and then incubated with either 125I-labeled human CGRP (10 pm) or 125I-labeled human adrenomedullin (20 pm) in modified Krebs-Ringers-HEPES medium (20 mm HEPES, pH 7.4, 110 mm NaCl, 5 mm KCl, 1 mm MgCl2, 1.8 mm CaCl2, 25 mmd-glucose, 1% BSA) for 3 h at 4 °C. Cells were harvested with 0.5 m NaOH, and associated radioactivity was counted in a γ-counter. For receptor recycling studies, following 30-min ligand exposure, cells were washed three times with prewarmed PBS and then incubated with fresh DMEM for 60 min at 37 °C. After incubation, the medium was immediately replaced with ice-cold Krebs-Ringers-HEPES medium. Following the same radioligand treatment for 3 h at 4 °C, cells were harvested and then counted in a γ-counter. Nonspecific binding was measured in the presence of 1 μm unlabeled CGRP or AM. Cells were observed under a Fluoview laser scanning confocal microscope (OLYMPUS). GFP was excited using a 488-nm argon/krypton laser, and emitted fluorescence was detected with a 510-nm band pass filter. For tetramethylrhodamine and LysoTracker Red, a 568-nm helium/neon laser was used for excitation, and fluorescence was detected with a 585-nm band pass filter. For microscopic visualization of CRLR molecules, we constructed a chimera consisting of the human CRLR fused to mutant GFP (EGFP) at its C terminus. To examine the functionality of the CRLR-GFP fusion protein, intracellular cAMP concentrations were measured. The intact HEK 293 cells lacked functional CGRP and AM receptors, because they showed little cAMP response to agonist stimulation (Fig.1 A, left). In contrast to other RAMPs, when stably expressed alone in HEK 293 cells, RAMP1 induced cAMP responses to CGRP (maximal cAMP reached approximately 8-fold basal), but not to AM (data not shown), suggesting that RAMP1 and endogenous calcitonin receptor may function as CGRP receptors (23Christopoulos G. Perry K.J. Morfis M. Tiakaratine N. Gao Y. Fraser N.J. Main M.J. Foord S.M. Sexton P.M. Mol. Pharmacol. 1999; 56: 235-242Crossref PubMed Scopus (421) Google Scholar). In HEK 293 cells stably expressing CRLR-GFP alone, AM, but not CGRP, slightly increased cAMP contents; maximal cAMP level reached approximately 2.5-fold higher than the basal level (Fig. 1 A, right). This was consistent with the finding that HEK 293 cells express endogenous RAMP2 (data not shown). After stable co-transfection of individual RAMPs, expression of constructs of the CRLR-GFP was assessed using flow cytometry. The fluorescence of the CRLR-GFP in the cells expressing RAMP1, -2, or -3 together yielded at least 100-fold greater intensity than HEK 293 control cells, with individual single peaks, indicating that the three types of cells expressed CRLR-GFP at almost 100% (Fig. 1 B). Furthermore, RAMPs increased the fluorescence intensity of CRLR-GFP; 11.5% for RAMP1, 6.8% for RAMP2, and 6.7% for RAMP3 (Fig.1 C). When expressed with RAMPs, CRLR-GFP had significant ability to produce cAMP responses to CGRP or AM that were concentration-dependent (Fig.2 A). Expression of CRLR-GFP and RAMP1 (CRLR/RAMP1) induced cAMP responses to CGRP or AM (EC50 of 0.34 or 1.1 nm, respectively), which were 100-fold more potent than that in CRLR-GFP-expressing cells. In contrast, CRLR/RAMP2 or -3 specifically responded to AM, with EC50 of 0.39 or 0.86 nm, respectively. Such ligand specificity of CRLR by RAMPs was also observed in intracellular calcium mobilization (Fig. 2 B). These [Ca2+]i increases by CGRP for CRLR/RAMP1 receptor and by AM for CRLR/RAMP2 or -3 receptor were receptor-mediated, because subsequent rechallenge with the same ligand failed to evoke significant responses, probably due to receptor desensitization. In CRLR/RAMP1 stable transfectants, following exposure to AM or CGRP elicited a small [Ca2+]i increase similar to that in HEK 293 cells stably expressing RAMP1 alone (data not shown), suggesting the existence of another CGRP receptor, calcitonin receptor/RAMP1, as mentioned above. These observations suggested that the fusion of the 238-amino acid GFP protein to the C terminus of the CRLR shows the full functional properties of the receptor in cells coexpressing RAMPs.Figure 2Functional characterization of the cells stably expressing CRLR-GFP and RAMPs. A, cyclic AMP responses. Cells were treated with various concentrations of CGRP or AM for 15 min at 37 °C. Results represent the mean ± S.E. of three experiments. B, calcium mobilization. Cells were exposed to 100 nm CGRP or AM, and then rechallenged with either agonist (100 nm) to examine receptor desensitization. Representative results from three observations are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine the distribution of the fluorescent CRLR-GFP without co-transfection of RAMPs, the cell surface was labeled by brief exposure to tetramethylrhodamine-ConA (50 μg/ml in PBS). The green fluorescence corresponding to CRLR showed little co-localization with the red fluorescence of ConA, and was distributed throughout the cytoplasm. The staining pattern suggested that CRLR-GFP was probably in the endoplasmic reticulum, representing the pool of newly synthesized molecules not yet transported to the plasma membrane (Fig.3 A). After 30-min exposure to 100 nm CGRP or AM, the distribution pattern of the CRLR-GFP was almost unchanged (Fig. 3, B and C). A large proportion of the green fluorescence corresponding to CRLR-GFP was observed on the cell membrane in cells stably co-transfected with RAMPs, whereas a small number of receptors was diffusely distributed throughout the cytoplasm (Fig.4). Measurement of radioligand binding revealed that exposure of cells coexpressing RAMPs to 100 nm CGRP or AM for up to 120 min produced rapid internalization of 50–70% of the cell surface CRLR-GFP, with similar kinetics (Fig.5 A). In all three stably transfected cell lines, addition of ligands caused the green fluorescence of the CRLR to disappear almost completely from the cell surface, depending on the ligand specificity; RAMP1/CGRP or AM, RAMP2/AM, and RAMP3/AM (Fig. 5 B). Pretreating cells with a hypertonic sucrose solution has been demonstrated to be effective in blocking receptor endocytosis via clathrin-coated pits (24Perkins J.P. Hausdroff W.P. Lefkowitz R.J. Perkins J.P. The β-Adrenergic Receptor. Humana Press, Clifton, NJ1991: 125-180Google Scholar, 25Daukas G. Zigmond S.H. J. Cell Biol. 1985; 101: 1673-1679Crossref PubMed Scopus (172) Google Scholar, 26Heuser J.E. Anderson R.G. J. Cell Biol. 1989; 108: 389-400Crossref PubMed Scopus (773) Google Scholar). Therefore, we preincubated our transfected cells with 0.45m sucrose for 15 min, followed by exposure to 100 nm ligands for various periods. Even after 120 min of agonist incubation, almost no receptor sequestration was observed (Fig.6, A and B). Sucrose pretreatment did not affect intracellular cAMP production by ligands (data not shown). The CRLR-GFP fluorescence moved into intracellular vesicles identified by the tetramethylthodamine derivative of transferrin, used as a marker of the endosomal compartment (27Trowbridge I.S. Collawn J.F. Hopkins C.R. Annu. Rev. Cell Biol. 1993; 9: 129-161Crossref PubMed Scopus (704) Google Scholar) (Fig. 7). Then, the majority of receptor molecules showed significant co-localization with the lysosomal marker, LysoTracker Red (28Palmiter R.D. Cole T.B. Findley S.D. EMBO J. 1996; 15: 1784-1791Crossref PubMed Scopus (397) Google Scholar) (Fig.8).Figure 6Inhibition of ligand-induced CRLR-GFP internalization by hypertonic medium. A, cells were pretreated with 0.45 m sucrose for 15 min, and then exposed to 100 nm ligand for different periods. Next, binding to125I-CGRP for CRLR/RAMP1 receptor and to125I-AM for CRLR/RAMP2 or -3 receptor was assessed. Binding at various time points was compared with the unstimulated cells. Data are the mean ± S.E. of three experiments. B, CRLR-GFP/RAMPs double transfectants were pretreated with 0.45m sucrose for 15 min, and then exposed to 100 nm ligand for 30 min. CGRP for CRLR/RAMP1 (top); AM for CRLR/RAMP2 (middle); AM for CRLR/RAMP3 (bottom).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Colocalization of CRLR-GFP (green) with the endosomal marker tetramethylrhodamine transferrin (red) after ligand exposure. Cells stably expressing CRLR-GFP and RAMPs were exposed to the marker and 100 nm ligand for 15 min. A, CGRP for CRLR/RAMP1;B and C, AM for CRLR/RAMP2 and -3, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 8Colocalization of CRLR-GFP with the lysosomal marker LysoTracker Red after addition of the ligand.CRLR-GFP/RAMPs stable transfectants were treated with the marker and 100 nm ligand for 30 min. A, CGRP for CRLR/RAMP1; B and C, AM for CRLR/RAMP2 and -3, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cells expressing CRLR-GFP and RAMPs were pretreated with cycloheximide (25 μg/ml) to inhibit de novo synthesis of receptor molecules and incubated with the ligands (100 nm CGRP or AM) for 30 min at 37 °C. Cycloheximide did not affect ligand-induced internalization of CRLR-GFP (data not shown). The cells were rinsed with medium containing cycloheximide, and incubated for various periods. After removal of the ligands, cell surface CRLR-GFP increased less than 10% in all three stably transfected cell lines (Fig.9, A and B). To investigate whether fusing the C terminus of GFP affects ligand-induced trafficking of CRLR, we have further constructed a chimeric molecule consisting of the CRLR fused to the Myc epitope tag at its N terminus (Myc-CRLR). Examination of numerous HEK293 cells stably co-transfected with RAMPs suggests that the rhodamine-labeled Myc-CRLR was diffusely distributed on the cell surface before agonist treatment (Fig.10). Radioligand binding experiments in three stably transfected cell lines indicate that exposure of 100 nm CGRP or AM caused rapid internalization of the cell surface Myc-CRLR, with similar kinetics (Fig.11 A). In all three transfectants, addition of ligands caused the rhodamine fluorescence of the CRLR to disappear almost completely from the cell surface, depending on the ligand specificity; RAMP1/CGRP or AM, RAMP2/AM, and RAMP3/AM (Fig. 1" @default.
• W2080232509 created "2016-06-24" @default.
• W2080232509 creator A5008677045 @default.
• W2080232509 creator A5025162838 @default.
• W2080232509 creator A5025701712 @default.
• W2080232509 creator A5043069986 @default.
• W2080232509 creator A5044967502 @default.
• W2080232509 creator A5048075263 @default.
• W2080232509 creator A5055974210 @default.
• W2080232509 creator A5071339903 @default.
• W2080232509 creator A5076623813 @default.
• W2080232509 date "2000-09-01" @default.
• W2080232509 modified "2023-09-30" @default.
• W2080232509 title "Visualization of the Calcitonin Receptor-like Receptor and Its Receptor Activity-modifying Proteins during Internalization and Recycling" @default.
• W2080232509 cites W1483189363 @default.
• W2080232509 cites W1507084693 @default.
• W2080232509 cites W1529813475 @default.
• W2080232509 cites W1545475398 @default.
• W2080232509 cites W1552886287 @default.
• W2080232509 cites W1605393199 @default.
• W2080232509 cites W1689798268 @default.
• W2080232509 cites W1964831127 @default.
• W2080232509 cites W1973955512 @default.
• W2080232509 cites W1981578013 @default.
• W2080232509 cites W1984123894 @default.
• W2080232509 cites W1991637403 @default.
• W2080232509 cites W1994392737 @default.
• W2080232509 cites W1996161888 @default.
• W2080232509 cites W1996991086 @default.
• W2080232509 cites W1999334921 @default.
• W2080232509 cites W2017201573 @default.
• W2080232509 cites W2032002058 @default.
• W2080232509 cites W2032504743 @default.
• W2080232509 cites W2039885825 @default.
• W2080232509 cites W2052640354 @default.
• W2080232509 cites W2053277833 @default.
• W2080232509 cites W2073457809 @default.
• W2080232509 cites W2074295150 @default.
• W2080232509 cites W2078373009 @default.
• W2080232509 cites W2079067713 @default.
• W2080232509 cites W2082614214 @default.
• W2080232509 cites W2087956146 @default.
• W2080232509 cites W2090664128 @default.
• W2080232509 cites W2092419989 @default.
• W2080232509 cites W2092448118 @default.
• W2080232509 cites W2109390568 @default.
• W2080232509 cites W2111112233 @default.
• W2080232509 cites W2117309485 @default.
• W2080232509 cites W2125468282 @default.
• W2080232509 cites W2152848856 @default.
• W2080232509 cites W2154810167 @default.
• W2080232509 cites W2168053641 @default.
• W2080232509 cites W2178812731 @default.
• W2080232509 cites W4232524111 @default.
• W2080232509 doi "https://doi.org/10.1074/jbc.m004534200" @default.
• W2080232509 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10882736" @default.
• W2080232509 hasPublicationYear "2000" @default.
• W2080232509 type Work @default.
• W2080232509 sameAs 2080232509 @default.
• W2080232509 citedByCount "152" @default.
• W2080232509 countsByYear W20802325092012 @default.
• W2080232509 countsByYear W20802325092013 @default.
• W2080232509 countsByYear W20802325092014 @default.
• W2080232509 countsByYear W20802325092015 @default.
• W2080232509 countsByYear W20802325092016 @default.
• W2080232509 countsByYear W20802325092017 @default.
• W2080232509 countsByYear W20802325092018 @default.
• W2080232509 countsByYear W20802325092019 @default.
• W2080232509 countsByYear W20802325092020 @default.
• W2080232509 countsByYear W20802325092021 @default.
• W2080232509 countsByYear W20802325092022 @default.
• W2080232509 countsByYear W20802325092023 @default.
• W2080232509 crossrefType "journal-article" @default.
• W2080232509 hasAuthorship W2080232509A5008677045 @default.
• W2080232509 hasAuthorship W2080232509A5025162838 @default.
• W2080232509 hasAuthorship W2080232509A5025701712 @default.
• W2080232509 hasAuthorship W2080232509A5043069986 @default.
• W2080232509 hasAuthorship W2080232509A5044967502 @default.
• W2080232509 hasAuthorship W2080232509A5048075263 @default.
• W2080232509 hasAuthorship W2080232509A5055974210 @default.
• W2080232509 hasAuthorship W2080232509A5071339903 @default.
• W2080232509 hasAuthorship W2080232509A5076623813 @default.
• W2080232509 hasBestOaLocation W20802325091 @default.
• W2080232509 hasConcept C118303440 @default.
• W2080232509 hasConcept C134018914 @default.
• W2080232509 hasConcept C139770010 @default.
• W2080232509 hasConcept C163170386 @default.
• W2080232509 hasConcept C170493617 @default.
• W2080232509 hasConcept C17971392 @default.
• W2080232509 hasConcept C185592680 @default.
• W2080232509 hasConcept C2779627488 @default.
• W2080232509 hasConcept C55493867 @default.
• W2080232509 hasConcept C75184817 @default.
• W2080232509 hasConcept C86803240 @default.
• W2080232509 hasConcept C92316933 @default.
• W2080232509 hasConcept C95444343 @default.
• W2080232509 hasConceptScore W2080232509C118303440 @default.
• W2080232509 hasConceptScore W2080232509C134018914 @default.

• next