The Extracellular Domain of Receptor Activity-modifying Protein 1 Is Sufficient for Calcitonin Receptor-like Receptor Function*

Timothy J. Fitzsimmons, Xilin Zhao, and Stephen A. WankDagger

From the Digestive Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-1804

Received for publication, November 22, 2002, and in revised form, February 5, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Calcitonin gene-related peptide (CGRP)1 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 (1). CGRP is a member of a family of neurotransmitters and hormones including calcitonin, adrenomedullin, and amylin that have 24-46% sequence homology.

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 (2). 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 (2).

The association of CRLR and RAMP 1 occurs early after translation. The first report of the discovery of the RAMPs (2) and subsequent studies show that RAMP 1 influences the glycosylation and trafficking of CRLR to the cell surface (3). Glycosylation of RAMP 2 and 3 is required for their cell surface expression in the absence of CRLR (4). 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 (5).

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 (6).

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 (7), chimera (8), and site-directed mutagenesis studies (9). In fact, the exogenously expressed N-terminal domain of the PTH receptor is able to associate with PTH (10).

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 (11).

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Construction of Wild Type and Mutant RAMP 1-- 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 the Haemophilus influenza hemagglutinin cleavable signal peptide sequence (MKTILALSTYIFCLVFA) (12) 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).

Construction of HA-CRLR-GFP-- 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) (12) 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.

Cell Culture and cDNA Transfection-- 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.

Radioligand Binding to Transfected tsA 201 Cells-- 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 gamma -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).

cAMP Assays-- Intracellular cAMP levels were assayed using a modification of the procedure described by Salomon et al. (13). 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).

Immunoblotting-- 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 mM dithiothreitol), 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).

Immunoprecipitation-- 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.

Surface Immunoprecipitation-- 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 (4). 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.

Flow Cytometry-- 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.

Statistical Analysis-- 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).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Role of RAMP 1 Domains in CGRP-stimulated Signal Transduction-- 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. 2 and 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.


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Fig. 1.   A model illustrating the sequence modifications of human RAMP 1 used to construct myc-RAMP 1 and the truncation and chimeric mutants. In all constructs the native signal peptide was replaced with the hemagglutinin signal peptide and the myc epitope. RAMP 1 was truncated after Trp-139 to form the ECD/TM mutant and after Ile-118 to form the ECD mutant. The RAMP 1-ECD/PDGFR-TM/tail chimera was constructed by replacing the TM and tail of RAMP 1 with the TM and tail of PDGFR at the position denoted by the line separating the extracellular and transmembrane domains.


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Fig. 2.   CGRP-stimulated intracellular cAMP accumulation in tsA 201 cells expressing HA-CRLR-GFP with each RAMP 1 mutant. Eight h after transfection with human HA-CRLR-GFP and RAMP 1, or one of the RAMP 1 mutants, tsA 201 cells were trypsinized and reseeded in 24-well plates at a density of 100,000 cells per well. Twenty-four h post transfection, the cells were stimulated with the indicated concentrations of human CGRP for 30 min at 37 °C and subsequently assayed for intracellular cAMP accumulation. Results are expressed as a percent of the maximal stimulation (mean, 95% CI; n > 5) observed for positive control cells expressing wild type RAMP 1 and HA-CRLR-GFP.


                              
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Table I
EC50 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. 2 and 6, respectively
Values were determined using a nonlinear regression curve-fitting computer program within PRISM, version 2.0. ND, not detected.

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 domain versus 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.

Role of RAMP 1 Domains in CRLR Trafficking-- 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 (4). HA-CRLR-GFP co-expressed with wild type RAMP 1 ran at an intermediate size of 75 kDa.


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Fig. 3.   Immunoblot analysis of HA-CRLR-GFP alone and with RAMP 1 and RAMP 1 mutants. Twenty-four h after transient transfection of tsA 201 cells, total lysates from solubilized cells were analyzed by SDS-PAGE (4-12%) and immunoblotted with mouse monoclonal anti-HA antibody. Comparison of the size of HA-CRLR-GFP expressed either alone or with wild type (WT), tail truncation (ECD/TM), chimeric (R1-ECD/PDGFR-TM/Tail) (A) or ECD truncation (B) mutants of RAMP 1 is shown. Arrows indicate the molecular mass of the receptors extrapolated from linear regression analysis of four protein standards (ranging from 39 to 97 kDa). Data were derived from at least three separate experiments.

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.

Role of RAMP 1 Domains in CRLR Surface Expression-- 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 (2). 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 (5). 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 type versus 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.


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Fig. 4.   Fluorescent flow cytometric analysis of the effect of RAMP 1 mutants on the cell surface expression of HA-CRLR-GFP. A, tsA 201 cells transiently co-transfected with wild type or mutant RAMP 1 and HA-CRLR-GFP were incubated with monoclonal anti-HA antibody for 60 min at 4 °C, washed, incubated with phycoerythrin-conjugated goat anti-mouse IgG for 60 min at 4 °C, and washed again prior to analysis. Flow cytometric analyses were performed on 1 × 104 cells per experimental group of transfected cells. 525- or 527-nm bandpass filtering was used to detect GFP (abscissa) and phycoerythrin (ordinate) fluorescence for each cell, respectively. Cells that fluoresced at least two standard deviations above the mean autofluorescence of unmanipulated tsA 201 cells were defined as positive. B, the relative mean fluorescent intensity at 575 nm (phycoerythrin) relative to 525 nm (GFP) for each group of transfected cells shown in A was plotted as a percent of the control wild type HA-CRLR-GFP/RAMP 1-transfected cells. Results are expressed as the mean ± S.E. from three separate experiments. R1, RAMP 1. Differences between mean values were analyzed by Student's t test and considered statistically significant for p values < 0.05.

Role of Transmembrane Domain of RAMP 1 in RAMP 1 CRLR Heterodimer Stability-- 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 (Fig 5, 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.


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Fig. 5.   Immunoblot analysis of the stability of the interaction between the ECD of RAMP 1 and HA-CRLR-GFP. Forty-eight h post-transfection of tsA 201 cells with Myc-ECD of RAMP 1 alone (lane 1) or with HA-CRLR-GFP (lane 2), the medium was examined for the presence of the ECD of RAMP 1. The Myc-ECD was detected in the medium by immunoprecipitation with mouse monoclonal anti-myc antibody. Immunoprecipitates were analyzed by PAGE (4-12%) and immunoblotting with the same anti-myc antibody. The arrow indicates the position of the Myc-ECD determined to be 11 kDa on the basis of linear regression analysis of three protein standards (ranging from 6-17 kDa). The position of two dark upper bands in both lanes is consistent with nonspecific immunostaining of the heavy and light chains of the immunoprecipitating antibody.

Role of RAMP 1 Domains in CGRP Affinity-- 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 increasing concentrations of unlabeled CGRP. The results of the competitive binding studies were consistent with the results from the cAMP studies. Similar to the results of the cAMP studies, CGRP affinity for HA-CRLR-GFP co-expressed with the RAMP 1-ECD-TM (tail truncation) mutant (IC50 = 4.5 nM, 95% CI, 3.5-5.9 nM) was not significantly different from co-expression with wild type RAMP 1 (IC50 = 2.8 nM, 95% CI, 2.1-3.7 nM) (see Fig. 6 and Table I). Also similar to the cAMP studies, the RAMP 1-ECD/PDGFR-TM-tail chimera had a nearly 10-fold decrease in CGRP affinity compared with wild type RAMP 1 (IC50 = 2.8 nM, 95% CI 2.1-3.7 versus 23 nM, 95% CI 7.3-74 nM, respectively). As might be expected from the 4000-fold increase in EC50 for cAMP stimulation, the IC50 for CGRP binding to the RAMP 1-ECD mutant/HA-CRLR-GFP heterodimer was too great to be determined using radioligand binding dose inhibition studies. The parallel decrease in potency for CGRP stimulation of cAMP with the decrease in CGRP binding affinity suggests that the majority of the loss in biologic function for both the RAMP 1-ECD/PDGFR-TM/tail chimera and the RAMP 1-ECD mutant is attributable to loss in CGRP affinity. Taken together, the data indicate that the stability conferred by the presence of the TM of RAMP 1 is important for high affinity binding of CGRP to the RAMP 1/CRLR heterodimer.


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Fig. 6.   Displacement of [125I]CGRP binding to tsA 201 cells expressing wild type RAMP 1 or RAMP 1 mutants with HA-CRLR-GFP. Eight h after transfection with human HA-CRLR-GFP and RAMP 1 or RAMP 1 mutants, tsA 201 cells were trypsinized and reseeded in 24-well plates at a density of 100,000 cells per well. Twenty-four h post-transfection, the cells were incubated with [125I]CGRP (50 pM) either alone or with the indicated concentrations of unlabeled CGRP for 60 min at 37 °C. Results are expressed as a percent of the saturable binding in the absence of unlabeled ligand. Each value represents the mean and 95% CI (n > 5).

Interaction of the ECD of RAMP 1 with the N Terminus of CRLR-- Having demonstrated the ability of the RAMP 1-ECD to associate with HA-CRLR-GFP early in protein processing and traffic through the Golgi to the cell surface, we sought to identify the region of CRLR that was interacting with the ECD of RAMP 1. The N terminus of several B class GPCRs have been shown to be major determinants for ligand binding (7-10). In addition, the N-terminal ECD of RAMP 1 has been shown to determine CGRP versus adrenomedullin specificity when co-expressed with CRLR (6). Therefore, a chimera was constructed in which the N-terminal extracellular domain of CRLR replaced the N-terminal extracellular domain of the secretin receptor. The secretin receptor was chosen for chimerization, because it does not associate with RAMP 1, and a variety of secretin/PTH receptor chimeras have maintained biologic function (9, 10). Because the N terminus of CRLR contains all of the N-linked glycosylation sites, we were able to use the previously demonstrated RAMP 1 associated change in glycosylation as a measurement of the functional interaction between RAMP 1 and the N terminus of CRLR (Fig. 7A). As expected, RAMP 1 had no effect on the mobility of the HA epitope-tagged secretin receptor as determined by immunoblot (Fig. 7B). However, the HA epitope-tagged CRLR/secretin receptor chimera demonstrated the familiar shift in size when co-expressed with RAMP 1 (Fig. 7C). The total and surface immunoprecipitation of the HA-CRLR/secretin receptor chimera showed a pattern similar to the wild type HA-CRLR-GFP (Fig. 7A), namely two bands when expressed alone and one intermediate band when co-expressed with RAMP 1. The surface immunoprecipitation shows that in the absence of RAMP 1, the lower band does not make it to the cell surface unlike the upper band and the solitary intermediate size band formed in the presence of RAMP 1 (Fig. 7, B and C). The remarkable similarity in the size shift of the CRLR/secretin chimera and the wild type CRLR because of RAMP 1-associated changes in glycosylation suggests that RAMP 1 interacts with the N-terminal extracellular domain of CRLR.


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Fig. 7.   Immunoblot analysis of the interaction of RAMP 1 with HA-CRLR, HA-secretin receptor, and chimeric HA-CRLR-secretin receptor. All receptors were expressed 24 h following transient transfection of tsA 201 cells and analyzed by SDS-PAGE (4-12%) and immunoblotted with mouse monoclonal anti-HA antibody. Immunoprecipitation of HA-CRLR (A), HA-secretin receptor (B), and chimeric HA-CRLR-secretin receptor (C) expressed alone or with RAMP 1 from lysates of whole cells (total IP) or whole cells that were incubated with mouse monoclonal anti-HA antibody and washed prior to lysis (surface IP) is shown. Arrows indicate the molecular mass of the receptors extrapolated from linear regression analysis of four protein standards (ranging from 39 to 97 kDa). Data were derived from at least three separate experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The human RAMP 1 protein can be divided into at least three structural domains, the N-terminal ECD, a transmembrane domain, and a nine-amino acid intracellular C-terminal tail domain. Three mutants of RAMP 1 were constructed to help ascribe a potential biological function to each of these domains: 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 PDGFR, and the ECD of RAMP 1 alone. Each of the mutants was compared with wild type RAMP 1 for their ability to associate and traffic with HA-CRLR-GFP from the endoplasmic reticulum, through Golgi to the cell surface, where they were assessed for their ability to bind CGRP and transduce a signal leading to the measured accumulation of intracellular cAMP. On the basis of these functional criteria, it appears that the C-terminal tail of RAMP 1 is unnecessary. The specific sequence and presence of the TM domain of RAMP 1 appears to contribute to CGRP affinity and thereby also the potency for stimulation of receptor-mediated signal transduction. The ECD of RAMP 1 is sufficient for normal association and trafficking of HA-CRLR-GFP. However, the surface expression of HA-CRLR-GFP, as well as CGRP affinity and potency for signal transduction, were all diminished. This observed decrease in the function of the ECD is possibly a result of the unstable association of the RAMP 1-ECD with HA-CRLR-GFP as evidenced by the secretion of the ECD in the media. Finally, the functional interaction of RAMP 1 with the N-terminal CRLR/secretin receptor chimera indicates that the N-terminal ECD of RAMP 1 interacts with the N terminus of CRLR to form a functional heterodimer capable of trafficking to the cell surface.

The RAMP 1-ECD/TM mutant lacking the nine-amino acid tail domain was functionally indistinguishable from wild type RAMP 1. Loss of the C-terminal tail had no affect on the association and trafficking with HA-CRLR-GFP. Similarly, surface expression and CGRP affinity and potency for signal transduction were unimpaired. During the preparation of this manuscript, a report was published (14) confirming that this same RAMP 1 tail truncation mutant interacted and trafficked with HA-CRLR-GFP to the cell surface normally. However, unlike the results presented here, this report found a 10-fold decrease in potency and a 35% decrease in efficacy for CGRP-stimulated cAMP response in a similar HEK 293 cell line transiently expressing the tail-truncated mutant (14). The reason for this discrepancy is unclear.

Considering the highly conserved (Q/R)(S/T)K(R/D)(S/T) sequence within the nine-amino acid tail of all three members of the RAMP family, it was somewhat unexpected to find that truncation of the tail was without a functional consequence. However, also during the preparation of this manuscript, it was reported that the QSKRT sequence in the tail of human RAMP 1 functions as an intracellular retention motif that can be overcome by the association of RAMP 1 with CRLR (14). Although trafficking of RAMP 1 expressed alone was not addressed in this study, this finding provides a suspected function for the conserved sequence in the C-terminal tail. This reported retention function for the C-terminal tail of RAMP 1 does not exclude other potential functions such as interaction with intracellular proteins similar to LRP6, another accessory protein for the GPCR, frizzled, that interacts with axin and catinin (11).

A chimera replacing the TM and tail of RAMP 1 with the TM and first nine amino acids in the C terminus of the PDGF receptor had no effect on the efficacy of CGRP-stimulated cAMP accumulation; however, both the affinity and potency of CGRP were decreased to a similar extent of 10-12-fold. These results, along with the data from the tail truncation mutant, indicate that the TM of RAMP 1 can be substituted without a loss in maximal function. However, the loss in CGRP affinity and potency suggests that the specific sequence of the TM domain of RAMP 1 is required for normal function. Although it is possible that the PDGFR tail hinders the function of CRLR, the small size, intracellular location, and irrelevance of the native C-tail suggest that this is unlikely. The importance of the TM domain in ligand sensitivity found here is not consistent with a previous study of RAMP 1 and RAMP 2 chimeras (6). In this study, the RAMP 1-ECD/RAMP 2-TM-tail chimera function, as measured by the maximal CGRP-induced membrane channel current in Xenopus laevis oocytes, was 40% of RAMP 1. In addition, this chimera exhibited increased affinity for adrenomedullin compared with RAMP 1 when expressed in HEK 293 cells. Although the ECD is the major determinant for ligand affinity and specificity, these results indicate that the TM domain of RAMP 1 contributes to ligand affinity, as well.

Co-expression of just the ECD of RAMP 1 with HA-CRLR-GFP resulted in full HA-CRLR-GFP glycosylation and a maximal response to CGRP greater than observed for full-length RAMP 1; however, surface expression and affinity for CGRP were impaired. These data indicate that the ECD alone is sufficient for association with HA-CRLR-GFP and full biological efficacy and that the tethering function of the TM domain is only required for full receptor sensitivity to CGRP. A recent study (14) reported that removal of the C-terminal tail plus 2, 7, or 20 amino acids from the 22-amino acid TM domain of RAMP 1 resulted in the complete loss of CGRP-stimulated increase in cAMP in HEK 293 cells transiently co-expressing CRLR despite normal CRLR glycosylation and RAMP 1 truncation mutant cell surface expression for all but the smallest mutant. From these non-functioning truncation mutants, they conclude that the TM domain is essential for CGRP receptor function. Our data using an ECD mutant of RAMP 1 that was truncated one amino acid shorter than their shortest truncation mutant demonstrated impairment only in affinity and potency. Not only was the TM domain of RAMP 1 not essential, but the efficacy for CGRP-stimulated cAMP accumulation in the absence of the TM domain was actually greater than with the TM domain present. The discrepancy between their negative and our positive functional data is presently unknown and may be on the basis of single amino acid difference in length between their construct and ours.

The doubling of the biological efficacy of the RAMP 1-ECD mutant was unexpected. The ability to recover the ECD from the culture medium by immunoprecipitation indicates the importance of the TM domain for anchoring RAMP 1. In the absence of the TM domain, the interaction of the ECD of RAMP 1 with the N terminus of HA-CRLR-GFP is of low enough affinity to allow it to dissociate into the medium. The dissociation of the ECD of RAMP 1 should result in a diminution in functional cell surface receptors and hence the paradox presented by the observed increase in efficacy. Possible explanations for the paradox could include loss of inhibition of signaling by the TM domain or increased receptor turnover leading to more active receptors on the surface. Although these hypotheses are not the focus of this work, they could be interesting for future studies.

The ability of RAMP 1-ECD to functionally interact with HA-CRLR-GFP suggests that the region of interaction is in the extracellular N terminus and/or loops of CRLR. The fact that RAMP 1 was able to interact with the N terminus of CRLR fused to the secretin receptor to recapitulate the glycosylation pattern characteristic of the holo-CRLR, combined with the fact that RAMP 1-ECD can change the glycosylation pattern of HA-CRLR-GFP, indicates that the main interaction is between the ECD of RAMP 1 and the N terminus of CRLR. Because the CRLR-secretin receptor chimera doesn't have signaling capability with CGRP or secretin (data not shown), it is premature to conclude more about what portion of CRLR is sufficient for the effect of RAMP 1 on CRLR specificity and functional signaling.

In conclusion, we find that the RAMP 1-ECD interacts with the N-terminal domain of CRLR and that this interaction is sufficient for trafficking from the endoplasmic reticulum through the Golgi to the cell surface. Furthermore, the interaction of the ECD of RAMP 1 with CRLR is sufficient for full biological efficacy. Although the ECD is sufficient for full efficacy, the TM domain of RAMP 1 is important for achieving full CGRP affinity and potency, as well as surface expression of CRLR, perhaps as a result of tethering the ECD to the membrane and specifically interacting with the TM domain of CRLR. In view of these findings, future structural studies of the interaction of the ECD of RAMP 1 with the N terminus of CRLR may be feasible.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 301-402-3704; Fax: 301-402-0600; E-mail: stevew@bdg10.niddk.nih.gov.

Published, JBC Papers in Press, February 6, 2003, DOI 10.1074/jbc.M211946200

    ABBREVIATIONS

The abbreviations used are: CGRP, calcitonin gene-related peptide; RAMP, receptor activity-modifying protein; ECD, extracellular domain; TM, transmembrane; CRLR, calcitonin receptor-like receptor; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; GFP, green fluorescent protein; HA, hemagglutinin; GPCR, G protein-coupled receptor; PTH, parathyroid hormone; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; BSA, bovine serum albumin; MOPS, 4-morpholinepropanesulfonic acid; PBST, PBS containing 0.1% Tween 20; CI, confidence interval.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Wimalawansa, S. J. (1997) Crit. Rev. Neurobiol. 11, 167-239[Medline] [Order article via Infotrieve]
2. McLatchie, L. M., Fraser, N. J., Main, M. J., Wise, A., Brown, J., Thompson, N., Solari, R., Lee, M. G., and Foord, S. M. (1998) Nature 393, 333-339[CrossRef][Medline] [Order article via Infotrieve]
3. Hilairet, S., Foord, S. M., Marshall, F. H., and Bouvier, M. (2001) J. Biol. Chem. 276, 29575-29581[Abstract/Free Full Text]
4. Flahaut, M., Rossier, B. C., and Firsov, D. (2002) J. Biol. Chem. 277, 14731-14737[Abstract/Free Full Text]
5. Aldecoa, A., Gujer, R., Fischer, J. A., and Born, W. (2000) FEBS Lett. 471, 156-160[CrossRef][Medline] [Order article via Infotrieve]
6. Fraser, N. J., Wise, A., Brown, J., McLatchie, L. M., Main, M. J., and Foord, S. M. (1999) Mol. Pharmacol. 55, 1054-1059[Abstract/Free Full Text]
7. Dong, M., Wang, Y., Pinon, D. I., Hadac, E. M., and Miller, L. J. (1999) J. Biol. Chem. 274, 903-909[Abstract/Free Full Text]
8. Turner, P. R., Bambino, T., and Nissenson, R. A. (1996) J. Biol. Chem. 271, 9205-9208[Abstract/Free Full Text]
9. Carter, P. H., Shimizu, M., Luck, M. D., and Gardella, T. J. (1999) J. Biol. Chem. 274, 31955-31960[Abstract/Free Full Text]
10. Grauschopf, U., Lilie, H., Honold, K., Wozny, M., Reusch, D., Esswein, A., Schafer, W., Rucknagel, K. P., and Rudolph, R. (2000) Biochemistry 39, 8878-8887[CrossRef][Medline] [Order article via Infotrieve]
11. Tamai, K., Semenov, M., Kato, Y., Spokony, R., Liu, C., Katsuyama, Y., Hess, F., Saint-Jeannet, J. P., and He, X. (2000) Nature 407, 530-535[CrossRef][Medline] [Order article via Infotrieve]
12. Guan, X.-M., Kobilka, T. S., and Kobilka, B. K. (1992) J. Biol. Chem. 267, 21995-21998[Abstract/Free Full Text]
13. Salomon, Y., Londos, C., and Rodbell, M. (1974) Anal. Biochem. 58, 541-548[Medline] [Order article via Infotrieve]
14. Steiner, S., Muff, R., Gujer, R., Fischer, J. A., and Born, W. (2002) Biochemistry 41, 11398-11404[CrossRef][Medline] [Order article via Infotrieve]


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