The Extracellular Domain of Receptor Activity-modifying Protein 1 Is Sufficient for Calcitonin Receptor-like Receptor Function
2003; Elsevier BV; Volume: 278; Issue: 16 Linguagem: Inglês
10.1074/jbc.m211946200
ISSN1083-351X
AutoresTimothy J. Fitzsimmons, Xilin Zhao, Stephen A. Wank,
Tópico(s)Signaling Pathways in Disease
ResumoA functional calcitonin gene-related peptide (CGRP) receptor requires dimerization of calcitonin receptor-like receptor (CRLR) with receptor activity-modifying protein 1 (RAMP 1). To determine the function of the three domains (extracellular, ECD; transmembrane, TM; and tail domains) of human RAMP 1, three mutants were constructed: RAMP 1 without the cytoplasmic tail, a chimera consisting of the ECD of RAMP 1 and the TM and tail of the platelet-derived growth factor receptor, and the ECD of RAMP 1 alone. These RAMP 1 mutants were examined for their ability to associate with CRLR to effect CGRP-stimulated cAMP accumulation, CGRP binding, CRLR trafficking, and cell surface expression. All RAMP 1 mutants were able to associate with CRLR with full efficacy for CGRP-stimulated cAMP accumulation. However, the RAMP 1/platelet-derived growth factor receptor chimera demonstrated a 10-fold decrease in potency for CGRP signaling and binding, and the RAMP 1-ECD mutant had a 4000-fold decrease in potency. In conclusion, the ECD of RAMP 1 is sufficient for normal CRLR association and efficacy. The presence of a TM domain and the specific sequence of the RAMP 1 TM domain contribute to CGRP affinity and potency. The C-terminal tail of RAMP 1 is unnecessary for CRLR function. A functional calcitonin gene-related peptide (CGRP) receptor requires dimerization of calcitonin receptor-like receptor (CRLR) with receptor activity-modifying protein 1 (RAMP 1). To determine the function of the three domains (extracellular, ECD; transmembrane, TM; and tail domains) of human RAMP 1, three mutants were constructed: RAMP 1 without the cytoplasmic tail, a chimera consisting of the ECD of RAMP 1 and the TM and tail of the platelet-derived growth factor receptor, and the ECD of RAMP 1 alone. These RAMP 1 mutants were examined for their ability to associate with CRLR to effect CGRP-stimulated cAMP accumulation, CGRP binding, CRLR trafficking, and cell surface expression. All RAMP 1 mutants were able to associate with CRLR with full efficacy for CGRP-stimulated cAMP accumulation. However, the RAMP 1/platelet-derived growth factor receptor chimera demonstrated a 10-fold decrease in potency for CGRP signaling and binding, and the RAMP 1-ECD mutant had a 4000-fold decrease in potency. In conclusion, the ECD of RAMP 1 is sufficient for normal CRLR association and efficacy. The presence of a TM domain and the specific sequence of the RAMP 1 TM domain contribute to CGRP affinity and potency. The C-terminal tail of RAMP 1 is unnecessary for CRLR function. Calcitonin gene-related peptide (CGRP) 1The abbreviations used are: CGRPcalcitonin gene-related peptideRAMPreceptor activity-modifying proteinECDextracellular domainTMtransmembraneCRLRcalcitonin receptor-like receptorPDGFplatelet-derived growth factorPDGFRPDGF receptorGFPgreen fluorescent proteinHAhemagglutininGPCRG protein-coupled receptorPTHparathyroid hormoneDMEMDulbecco's modified Eagle's mediumPBSphosphate-buffered salineBSAbovine serum albuminMOPS4-morpholinepropanesulfonic acidPBSTPBS containing 0.1% Tween 20CIconfidence interval is a neuropeptide found in the central and peripheral nervous systems. CGRP mediates sensory neurotransmission, decreases vascular tone, gastrointestinal motility and secretion, and inhibits the action of insulin on carbohydrate metabolism (1Wimalawansa S.J. Crit. Rev. Neurobiol. 1997; 11: 167-239Crossref PubMed Scopus (393) Google Scholar). CGRP is a member of a family of neurotransmitters and hormones including calcitonin, adrenomedullin, and amylin that have 24–46% sequence homology. calcitonin gene-related peptide receptor activity-modifying protein extracellular domain transmembrane calcitonin receptor-like receptor platelet-derived growth factor PDGF receptor green fluorescent protein hemagglutinin G protein-coupled receptor parathyroid hormone Dulbecco's modified Eagle's medium phosphate-buffered saline bovine serum albumin 4-morpholinepropanesulfonic acid PBS containing 0.1% Tween 20 confidence interval The receptors for CGRP, adrenomedullin, calcitonin, and amylin also belong to a subfamily of seven transmembrane G protein-coupled receptors (GPCR) that also includes the receptors for secretin, vasoactive intestinal peptide and parathyroid hormone (PTH), among others in the “B family” of GPCRs. The receptor for CGRP is one of very few GPCR that requires an accessory protein other than a G protein for its function (2McLatchie 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). The receptor for CGRP, the calcitonin receptor-like receptor (CRLR) requires interaction with the accessory protein, receptor activity-modifying protein 1 (RAMP 1), to form a functional CGRP receptor. RAMP 1 belongs to a three-member family of integral membrane proteins that share ∼30% sequence homology. The RAMPs are 150–177 amino acids in size with a cleavable signal peptide, relatively large N-terminal extracellular domain, one transmembrane-spanning domain, and a nine-amino acid intracellular C-terminal domain (see Fig. 1). CRLR associated with RAMP 1 has high affinity for CGRP, whereas association with RAMP 2 and RAMP 3 results in higher affinity for adrenomedullin (2McLatchie 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). The association of CRLR and RAMP 1 occurs early after translation. The first report of the discovery of the RAMPs (2McLatchie 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) and subsequent studies show that RAMP 1 influences the glycosylation and trafficking of CRLR to the cell surface (3Hilairet S. Foord S.M. Marshall F.H. Bouvier M. J. Biol. Chem. 2001; 276: 29575-29581Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Glycosylation of RAMP 2 and 3 is required for their cell surface expression in the absence of CRLR (4Flahaut M. Rossier B.C. Firsov D. J. Biol. Chem. 2002; 277: 14731-14737Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). RAMP 1, unlike RAMP 2 and 3, is not glycosylated and therefore requires association with CRLR for cell surface expression. This functional interaction continues at the cell surface where RAMP 1 enables CGRP binding and signaling, and the heterodimer can be cross-linked and immunoprecipitated with CGRP (5Aldecoa A. Gujer R. Fischer J.A. Born W. FEBS Lett. 2000; 471: 156-160Crossref PubMed Scopus (54) Google Scholar). The extracellular domain (ECD) of RAMP is largely responsible for determining CRLR ligand specificity for either CGRP or adrenomedullin although domain swapping between the ECD and TM/tail of RAMPs 1 and 2 suggests that the TM and/or tail may play a minor role (6Fraser 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). Similar to the RAMPs, the extracellular N terminus of several members of the B class of receptors also plays an important role in receptor-ligand association as demonstrated by cross-linking (7Dong M. Wang Y. Pinon D.I. Hadac E.M. Miller L.J. J. Biol. Chem. 1999; 274: 903-909Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), chimera (8Turner P.R. Bambino T. Nissenson R.A. J. Biol. Chem. 1996; 271: 9205-9208Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), and site-directed mutagenesis studies (9Carter P.H. Shimizu M. Luck M.D. Gardella T.J. J. Biol. Chem. 1999; 274: 31955-31960Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). In fact, the exogenously expressed N-terminal domain of the PTH receptor is able to associate with PTH (10Grauschopf U. Lilie H. Honold K. Wozny M. Reusch D. Esswein A. Schafer W. Rucknagel K.P. Rudolph R. Biochemistry. 2000; 39: 8878-8887Crossref PubMed Scopus (106) Google Scholar). Another accessory protein known to assist a GPCR in its function is LRP6. LRP6 is also a single transmembrane-spanning protein that associates with the GPCR, frizzled, and promotes its activity. The ECD of LRP6 can associate with frizzled, and an LRP6 mutant lacking its cytosolic tail is a dominant negative for frizzled function (11Tamai K. Semenov M. Kato Y. Spokony R. Liu C. Katsuyama Y. Hess F. Saint-Jeannet J.P. He X. Nature. 2000; 407: 530-535Crossref PubMed Scopus (1103) Google Scholar). To determine the function of each of the three domains (ECD, TM, and tail) of human RAMP 1, we made the following three mutants: the ECD and TM of RAMP 1 without the tail, a chimera of the ECD of RAMP 1 with the TM and tail from the platelet-derived growth factor (PDGF) receptor, and the ECD of RAMP 1 alone. These RAMP 1 mutants were examined for their ability to functionally couple with CRLR to effect CGRP-stimulated intracellular cAMP accumulation, CGRP binding, CRLR trafficking, and cell surface expression. All RAMP 1 mutants were able to associate with CRLR with full efficacy for CGRP-stimulated cAMP accumulation. However, the RAMP 1-ECD/PDGFR TM/tail mutant demonstrated a moderate decrease in potency for CGRP signaling and binding, whereas the RAMP-ECD mutant was more severely affected. CRLR cell surface expression was retained with all RAMP 1 mutants, although the RAMP 1 mutant consisting of the ECD alone functioned less efficiently and was secreted into the medium. The ECD of RAMP 1 was also shown to interact specifically with the N terminus of CRLR. The full-length human RAMP 1 was PCR-amplified from human brain cDNA (Clontech, Palo Alto, CA) and cloned into the TOPO TA cloning vector (Invitrogen). An oligonucleotide primer encoding a 5′ HindIII restriction site, followed by theHaemophilus influenza hemagglutinin cleavable signal peptide sequence (MKTILALSTYIFCLVFA) (12Guan X.-M. Kobilka T.S. Kobilka B.K. J. Biol. Chem. 1992; 267: 21995-21998Abstract Full Text PDF PubMed Google Scholar) and the c-myc antigen epitope sequence (EQKLISEEDL) was used to replace the native signal peptide in-frame with the remaining RAMP 1 sequence using PCR. The RAMP 1 C-terminal tail truncation was made by PCR amplification of wild type RAMP 1 cDNA using an antisense primer that inserted a stop codon after Trp-139, the last predicted amino acid of the transmembrane domain (see Fig. 1). The RAMP 1 chimera consisting of the RAMP 1-ECD and the PDGF receptor TM and tail domains was constructed by PCR amplification of wild type RAMP 1 cDNA using a long antisense primer coding for the TM and first nine amino acids of the C-terminal tail of the human PDGFR (see Fig. 1). The RAMP 1-ECD was made by PCR amplification of wild type RAMP 1 cDNA using an antisense primer that replaced the codon for the first amino acid in the transmembrane domain with a stop codon (see Fig. 1). The full-length human CRLR was PCR-amplified from human brain cDNA (Clontech, Palo Alto, CA) and cloned into the TOPO TA cloning vector (Invitrogen). An oligonucleotide primer encoding a 5′ HindIII restriction site, followed by the H. influenza hemagglutinin cleavable signal peptide sequence (MKTILALSTYIFCLVFA) (12Guan X.-M. Kobilka T.S. Kobilka B.K. J. Biol. Chem. 1992; 267: 21995-21998Abstract Full Text PDF PubMed Google Scholar) and the influenza virus hemagglutinin antigen epitope (HA1) sequence (YPYDVPVYA), was used to replace the native signal peptide in-frame with the remaining CRLR sequence using PCR. This construct of CRLR and a similar construct containing an enhanced GFP (Clontech, Palo Alto, CA) C-terminal fusion (CRLR-GFP) were cloned into PEAK 12 vector (Edge Biosystems, Gaithersburg, MD) at the HindIII and NotI restriction sites. All studies were performed using human embryonic kidney 293 tsA 201 cells (tsA 201) (a kind gift from Ronald Li, Institute of Molecular Cardiobiology, The Johns Hopkins University School of Medicine, Baltimore, MD). tsA 201 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (BioFluids Inc., Rockville, MD) and 1% penicillin/streptomycin and incubated in humidified air supplemented with 5% CO2 at 37 °C. Transfections of tsA 201 cells were performed in either six-well culture plates (5 × 105 cells per well) or 10-cm round culture plates (5 × 106 cells) using Polyfect (Qiagen, Santa Clarita, CA) according to the manufacturer's protocol. For the cAMP and binding studies, cells were split 8 h after transfection and cultured in the above medium until assayed at 24 h post-transfection. Eight h after transfection, cells were trypsinized and reseeded in 24-well plates at a density of 100,000 cells per well. The cells were washed once with cold phosphate-buffered saline (PBS), pH 7.4, containing 0.1% bovine serum albumin (BSA) and incubated in DMEM containing 0.1% BSA for 60 min at 37 °C, 50 pm [125I]CGRP (human 8–37) (2200 Ci/mmol) (PerkinElmer Life Sciences) for 30 min with and without increasing concentrations of unlabelled human CGRP. The cells were washed one time with PBS containing 1% BSA and were collected with 0.5 ml of 0.1 n NaOH added to each well. Radioactivity was detected in a γ-counter (Packard Instruments, Downers Grove, IL). Nonspecific binding (determined in the presence of 1 μm CGRP) was always less than 10% of total binding. Binding assays were performed in triplicate in at least three separate experiments. IC50 values were determined using a nonlinear regression curve-fitting computer program within PRISM, version 2.0 (Graph-Pad Software, Inc., San Diego, CA). Intracellular cAMP levels were assayed using a modification of the procedure described by Salomon et al.(13Salomon Y. Londos C. Rodbell M. Anal. Biochem. 1974; 58: 541-548Crossref PubMed Scopus (3374) Google Scholar). Briefly, 8 h after transfection, tsA 201 cells were trypsinized and reseeded in 24-well plates at a density of 100,000 cells per well in DMEM containing 10% calf serum and [3H]adenine (2 mCi/ml). The cells were washed with DMEM alone for 10 min and incubated in DMEM with or without the indicated concentrations of human CGRP, BSA (1%), and isobutylmethylxanthine for 30 min. Following the aspiration of the incubation medium, 100 μl of 5% SDS/1 mm cAMP solution was used to lyse the cells. Intracellular [3H]cAMP released into the lysate was measured by successive column chromatography using a Dowex AG-50W-X4 resin (Bio-Rad) followed by aluminum oxide (Sigma). Eluates were collected in scintillation vials, and radioactivity was counted using a liquid scintillation counter (Beckman). cAMP assays were performed in triplicate in at least six separate experiments. Each assay included an experimental positive control group of cells expressing native human HA-CRLR-GFP and native RAMP 1. Positive control cells stimulated 20-fold over basal, whereas mock (vector alone) and untransfected cells were unresponsive to CGRP. Results were expressed as a percent of the maximal response observed for the positive control group of cells, and EC50 values were determined using a nonlinear regression curve-fitting computer program within PRISM, version 2.0 (Graph-Pad Software, Inc., San Diego, CA). tsA 201 cells were grown in six-well culture plates (5 × 105 cells/well) and processed 24 h post-transfection. The cells were solubilized after washing away the growth medium and incubating with 1 ml of PBS containing 1% Triton X-100, 0.2% SDS, and a protease inhibitor mixture (Roche Molecular Biochemicals) for 1 h at 4 °C. Following centrifugation (20,000 × g at 4 °C for 15 min), a fraction of the supernatant was mixed with sample buffer (final concentration: 1% SDS, 4 m urea, and 50 mmdithiothreitol), separated on a 4–12% NuPAGE gel (Invitrogen) run in MOPS buffer, and elector-transferred onto nitrocellulose membranes. Blots were blocked in 5% non-fat milk in PBS containing 0.1% Tween 20 (PBST) for 1 h at room temperature, incubated with primary antibody, either monoclonal HA.11 (anti-HA ascites; 1:3000) or 9E10 (anti-c-myc; 1:3000) (BabCo, Berkeley, CA), in PBST/1% milk for 1 h at room temperature. The blots were then washed in three changes of PBST for 15 min, incubated with secondary antibody, goat anti-mouse IgG, horseradish peroxidase-conjugated (Kirkegaard and Perry Laboratories, Gaithersburg, MD), in PBST/1% milk for 1 h at room temperature, and washed in three changes of PBST for 15 min at room temperature. Bands were visualized using enhanced chemiluminescence (ECL plus kit; Amersham Biosciences). tsA 201 cells were grown in six-well culture plates (5 × 105 cells/well) and processed 24 h post-transfection. The cells were solubilized after washing away the growth medium and incubating with 1 ml of PBS containing 1% Triton X-100, 0.2% SDS, and a protease inhibitor mixture (Roche Molecular Biochemicals) for 1 h at 4 °C. Following centrifugation (20,000 × g at 4 °C for 15 min), the supernatant was mixed on a rocker with either HA.11 (1:200) or 9E10 (1:200) antibody overnight at 4 °C. The immune complexes were bound to protein G-Sepharose beads (Amersham Biosciences) after rocking for 1 h at 4 °C. The beads were washed with PBS/0.1% Triton X-100 and eluted with sample buffer (final concentration: 1% SDS, 4 m urea, and 50 mm dithiothreitol). Samples were analyzed by gel electrophoresis and Western blotting as described above. tsA 201 cells, grown in six-well culture plates (5 × 105 cells), were incubated 24 h post-transfection by washing three times with PBS and incubated with blocking buffer (PBS/0.2% bovine serum albumin) for 1 h on ice (4Flahaut M. Rossier B.C. Firsov D. J. Biol. Chem. 2002; 277: 14731-14737Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). HA.11 anti-HA antibody was added (1:200) for 2 h on ice. The cells were washed once with 4 °C blocking buffer and twice with 4 °C PBS and then lysed, centrifuged, bound to protein G beads, and Western blotted as described above. tsA 201 cells transiently expressing either wild type or one of the mutant RAMP 1 constructs and N-terminal HA epitope-tagged human CRLR-GFP (HA-CRLR-GFP) were harvested 24 h post-transfection using Versene (Invitrogen). The cells were washed with DMEM, 10% fetal bovine serum at 4 °C and resuspended at 1 × 106 cells/ml in the same medium containing a 1:100 dilution of anti-HA antibody (HA.11) (BabCo, Berkeley, CA) for 60 min at 4 °C. The cells were subsequently washed twice with DMEM/10% fetal bovine serum at 4 °C and resuspended at 1 × 106 cells/ml in the same medium containing a 1:100 dilution of phycoerythrin-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, OR) for 60 min at 4 °C. The cells were washed twice with DMEM/10% fetal bovine serum and resuspended in the same media at 1 × 106 cells/ml at 4 °C. Flow cytometric analyses were performed on 1 × 104 cells using an EPICS ELITE ESP flow cytometer (Coulter, Hialeah, FL). Argon laser excitation and either a 525 or 575 nm bandpass filtering was used to detect GFP or phycoerythrin. Cells that fluoresced at least two standard deviations above the mean autofluorescence of unmanipulated tsA 201 cells were defined as positive. Differences between mean values were analyzed by Student's t test. Differences were considered statistically significant for p values <0.05. EC50 and IC50 values and their 95% confidence intervals (CI), were determined using a nonlinear regression curve-fitting computer program within PRISM, version 2.0 (Graph-Pad Software, Inc., San Diego, CA). To determine the role of the ECD, TM, and tail domains of human RAMP 1, three mutants were constructed. The role of the cytoplasmic tail was evaluated using a mutant consisting of the ECD and TM (ECD/TM). The importance of the specific sequence of the TM and tail was determined using a chimera composed of the ECD of RAMP 1 and the TM and tail of the PDGF receptor (R1-ECD/PDGF-TM/tail). A mutant consisting of the ECD alone (ECD) was used to further evaluate the role of the TM domain, as well as the role of the ECD (see “Experimental Procedures” and Fig. 1). tsA 201 cells were transiently transfected with human HA-CRLR-GFP and either wild type human RAMP 1 or one of the RAMP 1 mutants. Twenty-four h post-transfection, dose response assays for CGRP stimulation of intracellular cAMP accumulation were performed. CGRP stimulated cAMP accumulation with an EC50 of 36 pm (95% CI, 24–55 pm) (see Fig. 2and Table I). Truncation of the nine-amino acid intracellular C-tail of RAMP 1 to form the ECD/TM mutant resulted in an ∼4-fold increase in EC50(EC50 = 125 pm, 95% CI, 52–299 pm) that was not significantly different from wild type RAMP 1 (Table I). Maximal stimulation (efficacy) was unaffected by the tail truncation, and therefore the ECD/TM mutant was statistically indistinguishable from wild type RAMP 1 in its biological function as measured by cAMP stimulation.Table IEC50 and IC50 values for CGRP-stimulated increases in intracellular cAMP accumulation and [125I]CGRP binding for wild type RAMP 1 and each of the RAMP 1 mutants derived from Figs. 2and 6, respectivelyHEK 293 tsA 201 cell transfection, HA-CRLR-GFPcAMP accumulation EC50 (95% CI)[125I]CGRP Binding IC50 (95% CI)nm+ RAMP 1 WT0.036 (0.024–0.055)2.8 (2.1–3.7)+ RAMP 1-ECD/TM0.125 (0.052–0.299)4.5 (3.5–5.9)+ RAMP 1-ECD/PDGFR-TM/tail0.470 (0.201–1.099)23 (7.3–74)+ RAMP 1-ECD145 (78–272)ND Open table in a new tab The importance of the TM domain of RAMP 1 was examined by replacing the TM and tail with the TM and first nine amino acids of the C-terminal tail of the PDGF receptor (Fig. 1). This chimera allows the exploration of the contribution of the specific sequence of the RAMP 1 TM domainversus a nonspecific membrane-tethering role it may serve for the ECD. The EC50 for the CGRP dose response curve for cAMP stimulation in cells expressing the RAMP 1-ECD/PDGFR-TM/tail chimera and HA-CRLR-GFP was increased ∼12-fold compared with wild type RAMP 1 (470 pm, 95% CI, 201–1099 pm) (see Fig. 2 and Table I), and the maximal response (efficacy) was unaffected. These data indicate that the specific sequence of the TM of RAMP 1 contributes to CGRP sensitivity, although the remote possibility that the PDGFR tail interferes with HA-CRLR-GFP action cannot be ruled out. This RAMP 1/PDGFR chimera shows that although the TM domain is important for CGRP sensitivity, it is still unclear whether the TM domain is necessary for full efficacy or whether the ECD of RAMP 1 alone is sufficient for full biologic function. Therefore, the ECD mutant of RAMP 1 was transiently expressed with HA-CRLR-GFP in tsA 201 cells, and the cAMP response to increasing doses of CGRP was determined. Although the EC50 for the ECD of RAMP 1 was increased ∼4000-fold (145 nm, 95% CI, 78–272 nm) compared with wild type RAMP 1, surprisingly, the maximal response was increased nearly 2-fold. CGRP stimulation of HA-CRLR-GFP expressed alone was the same as non-transfected tsA 201 cells (data not shown). These data indicate that the ECD alone is sufficient for association with HA-CRLR-GFP and full biological efficacy as determined by the cAMP response to CGRP and that the specific amino acid sequence and tethering function of the TM domain are required for full receptor sensitivity to CGRP. Previous studies have shown that RAMP 1 associates early with CRLR to form a heterodimer and functions to promote their trafficking through the Golgi to the cell surface. As a result of passing through the Golgi, CRLR increases in size as it becomes terminally glycosylated. This increase in the size of CRLR can be used to assess the functional association and trafficking of CRLR and RAMP 1. To determine whether the decrease in CGRP potency for stimulation of cAMP observed for the various mutants of RAMP 1 was because of impaired association and trafficking with CRLR, we examined the effect of each mutant of RAMP 1 on HA-CRLR-GFP glycosylation. Total lysates of tsA 201 cells expressing HA-CRLR-GFP alone or with RAMP 1 were analyzed by SDS-PAGE (4–12%) and immunoblotting with mouse monoclonal anti-HA antibody. In the absence of RAMP 1, HA-CRLR-GFP runs as two bands of ∼70 and 82 kDa (Fig.3A) that have been shown previously to represent intracellular core-glycosylated and cell surface terminally glycosylated forms, respectively (4Flahaut M. Rossier B.C. Firsov D. J. Biol. Chem. 2002; 277: 14731-14737Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). HA-CRLR-GFP co-expressed with wild type RAMP 1 ran at an intermediate size of 75 kDa. Similar to the wild type RAMP 1, all three RAMP 1 mutants, the ECD/TM mutant lacking the nine-amino acid intracellular tail and the RAMP 1-ECD/PDGFR-TM/tail chimera (Fig. 3A), as well as the RAMP 1-ECD alone (Fig. 3B), were able to completely convert the 70- and 82-kDa bands to the 75-kDa form of HA-CRLR-GFP. The significance of the differences in the intensity of the bands of HA-CRLR-GFP co-expressed with the various mutants is difficult to interpret because of the variability in transfection efficiency and mutant expression. Nonetheless, this size shift functional assay demonstrates that only the ECD of RAMP 1 is necessary for the early association and trafficking with HA-CRLR-GFP. Therefore the decreased potency for CGRP stimulation of cAMP observed for the RAMP 1 mutants is not because of impaired early association and trafficking with HA-CRLR-GFP. Several expression studies in a variety of heterologous cells have shown that RAMP 1 association with CRLR is necessary for full expression of CRLR at the cell surface (2McLatchie 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). Although one study has questioned this requirement for RAMP 1, the data still suggest that RAMP 1 is important for surface expression of the functional CRLR/RAMP 1 heterodimer (5Aldecoa A. Gujer R. Fischer J.A. Born W. FEBS Lett. 2000; 471: 156-160Crossref PubMed Scopus (54) Google Scholar). Therefore, to determine whether the absence or mutation of one domain within RAMP 1 that resulted in the decreased potency for cAMP production reported above could be because of impaired surface expression of CRLR, each of the RAMP 1 mutants was compared with wild type RAMP 1 for its ability to promote HA-CRLR-GFP cell surface expression. Cell surface expression of HA-CRLR-GFP was detected with anti-HA monoclonal antibody and phycoerythrin-labeled, goat anti-mouse secondary antibody. The GFP fused to the C terminus of CRLR-GFP was used to measure the level of whole cell expression of HA-CRLR-GFP. Fluorescent flow cytometric analysis was applied for the simultaneous measurement of both the phycoerythrin and whole cell GFP (HA-CRLR-GFP) fluorescent signals. Fluorescent measurements for 10,000 cells are depicted on scatter plots (Fig.4A). HA-CRLR-GFP surface expression could be then be normalized for the level of total cell receptor expression of GFP and expressed as a ratio that could be meaningfully compared among cells co-expressing wild typeversus mutant or absent domains of RAMP 1 (Fig.4B). Among all the mutant RAMPs, both the RAMP 1-ECD/PDGFR-TM-tail chimera and the RAMP 1-ECD mutant lacking the TM and tail domains were impaired with statistical significance (70.3 ± 11.2%) and (56 ± 1.5%, respectively) compared with wild type RAMP 1 in their ability to promote HA-CRLR-GFP surface expression. Although the impaired function of the mutant that resulted in a 50% reduction in HA-CRLR-GFP surface expression may contribute to the observed 4000-fold decrease in CGRP potency for cAMP stimulation, it cannot account for all of the reduction. We demonstrated that the ECD of RAMP 1 could associate and traffic with HA-CRLR-GFP similar to wild type RAMP 1 using an N-linked glycosylation maturation assay (see “Experimental Procedures” and Fig. 3B). To determine whether the heterodimer consisting of the ECD of RAMP 1 and HA-CRLR-GFP was unstable, perhaps because of the absence of the TM and tail of RAMP 1, the media of cells either expressing myc-ECD of RAMP 1 alone or co-expressed with HA-CRLR-GFP was examined for the presence of the ECD of RAMP 1. Myc-ECD of RAMP 1 was immunoprecipitated from the medium using mouse monoclonal anti-myc antibody and analyzed by PAGE and immunoblotting with the same anti-myc antibody. The presence of RAMP 1-myc-ECD in the media of cells expressing RAMP 1-Myc-ECD alone was nearly undetectable (Fig5, lane 1) whereas there was a dramatic increase of the 11-kDa RAMP 1-Myc-ECD in the media of cells co-expressing HA-CRLR-GFP (Fig. 5, lane 2). These data confirm that the ECD of RAMP 1 alone is capable of associating with HA-CRLR-GFP and trafficking through the Golgi to the cell surface as shown above. In addition, the presence of the ECD of RAMP 1 in the media indicates that the ECD has dissociated from HA-CRLR-GFP presumably as a result of a decrease in the stability of its interaction with HA-CRLR-GFP at the surface. Although it is difficult to quantitate the degree of instability between the ECD and HA-CRLR-GFP, because there are so many unknown variables, the interaction is stable enough to allow full efficacy of CGRP stimulation of intracellular cAMP accumulation. RAMP 1 association with CRLR at the cell surface is necessary for CGRP high affinity binding and activation of signal transduction. Having demonstrated that the RAMP 1-ECD is sufficient for association, trafficking, and cell surface expression of HA-CRLR-GFP, the effect of the mutations of each of the RAMP 1 domains on CGRP affinity was examined as a possible basis for the observed decrease in potency for stimulating cAMP accumulation. Radioligand binding dose inhibition studies were performed in tsA 201 cells co-expressing HA-CRLR-GFP and each of the RAMP 1 mutants using 50 pm [125I]CGRP and inc
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