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Research ArticleArticle

Multiple Amylin Receptors Arise from Receptor Activity-Modifying Protein Interaction with the Calcitonin Receptor Gene Product

George Christopoulos, Katie J. Perry, Maria Morfis, Nanda Tilakaratne, Yongyi Gao, Neil J. Fraser, Martin J. Main, Steven M. Foord and Patrick M. Sexton
Molecular Pharmacology July 1999, 56 (1) 235-242; DOI: https://doi.org/10.1124/mol.56.1.235

Abstract

Receptor activity-modifying proteins (RAMPs) are single-transmembrane proteins that transport the calcitonin receptor-like receptor (CRLR) to the cell surface. RAMP 1-transported CRLR is a calcitonin gene-related peptide (CGRP) receptor. RAMP 2- or RAMP 3-transported CRLR is an adrenomedullin receptor. The role of RAMPs beyond their interaction with CRLR, a class II G protein-coupled receptor, is unclear. In this study, we have examined the role of RAMPs in generating amylin receptor phenotypes from the calcitonin (CT) receptor gene product. Cotransfection of RAMP 1 or RAMP 3 with the human CT receptor lacking the 16-amino acid insert in intracellular domain 1 (hCTRI1−) into COS-7 cells induced specific125I-labeled rat amylin binding. RAMP 2 or vector cotransfection did not cause significant increases in specific amylin binding. Competition-binding characterization of the RAMP-induced amylin receptors revealed two distinct phenotypes. The RAMP 1-derived amylin receptor demonstrated the highest affinity for salmon CT (IC50, 3.01 ± 1.44 × 10−10 M), a high to moderate affinity for rat amylin (IC50, 7.86 ± 4.49 × 10−9 M) and human CGRPα (IC50, 2.09 ± 1.63 × 10−8 M), and a low affinity for human CT (IC50, 4.47 ± 0.78 × 10−7 M). In contrast, whereas affinities for amylin and the CTs were similar for the RAMP 3-derived receptor, the efficacy of human CGRPα was markedly reduced (IC50, 1.12 ± 0.45 × 10−7 M; P < .05 versus RAMP 1). Functional cyclic AMP responses in COS-7 cells cotransfected with individual RAMPs and hCTRI1− were reflective of the phenotypes seen in competition for amylin binding. Confocal microscopic localization of c-myc-tagged RAMP 1 indicated that, when transfected alone, RAMP 1 almost exclusively was located intracellularly. Cotransfection with calcitonin receptor (CTR)I1− induced cell surface expression of RAMP 1. The results of experiments cross-linking 125I-labeled amylin to RAMP 1/hCTR-transfected cells with bis succidimidyl suberate were suggestive of a cell-surface association of RAMP 1 and the receptors. Our data suggest that in the CT family of receptors, and potentially in other class II G protein-coupled receptors, the cellular phenotype is likely to be dynamic in regard to the level and combination of both the receptor and the RAMP proteins.

Amylin is a 37-amino acid pancreatic hormone that shares amino acid homology with the calcitonin gene-related peptide (CGRP), calcitonin (CT), and the adrenomedullin family of peptides. It has the highest identity (∼45%) with the CGRPs, an ∼22% identity with the 38 C-terminal amino acids of adrenomedullin, and an 18 and 33% identity (with a gapped alignment) with rat/human and avian/teleost CTs, respectively. The physiology of these peptides has been reviewed in detail (Muff et al., 1995; Wimalawansa, 1997). Circulating levels of amylin are raised in response to meal ingestion, and the peptide acts to potently inhibit gastric emptying, postprandial glucagon secretion, and food intake. Amylin also opposes the metabolic actions of insulin in skeletal muscle (Sexton and Perry, 1996; Young, 1997). Transgenic mice lacking the amylin gene show abnormal weight gain, an observation that also suggests an important metabolic role for amylin (Devine and Young, 1998; Gebre-Medhin et al., 1998). An independent gene encoding the amylin receptor has not been identified.

McLatchie et al. (1998) recently identified and cloned a family of accessory proteins termed receptor activity-modifying proteins (RAMPs), which was comprised of three members designated RAMP 1, RAMP 2, and RAMP 3. These single-transmembrane domain proteins induced trafficking of the calcitonin receptor-like receptor (CRLR) to the cell surface, where it exhibited either a CGRP receptor phenotype (RAMP 1) or adrenomedullin receptor phenotypes (RAMP 2 or RAMP 3). RAMPs therefore provided a novel mechanism for engendering novel receptor phenotypes. Amylin shows more sequence homology to CGRP than adrenomedullin, but it does not activate or bind to combinations of CRLR and RAMPs (McLatchie et al., 1998). Amylin has even less sequence identity with the CTs, but there is evidence that links amylin receptors with those for CT. The receptors tend to be colocalized (Sexton and Perry, 1996), and both receptors are recognized by antibodies raised against the hypervariable C terminus of the CT receptor (Perry et al., 1997). Furthermore, transfection of human CT receptors into human embryonic kidney (HEK)-293 cells, but not into Ti ni insect cells, induces low levels of amylin receptors in addition to high levels of CT receptors (Chen et al., 1997). Moreover, transfection of the porcine CT receptor into different cellular backgrounds gives rise to different receptor phenotypes with transfection into Chinese hamster ovary (CHO)-K1 cells, yielding a receptor similar to the rat C1a CT receptor (Christmanson et al., 1994), whereas transfection into COS-7 or HEK-293 cells yields a receptor with moderate to high affinity for amylin and poor responsiveness to human CT (Christmanson et al., 1994; Sexton et al., 1994a).

These observations prompted an investigation of whether RAMP coexpression may also underlie the expression of amylin receptors from the CT receptor gene product. Our data indicate that at least two independent amylin receptor phenotypes may be engendered by specific RAMP interaction.

Experimental Procedures

Materials.

Salmon CT, human CT, human adrenomedullin, human CGRPα, and rat amylin were obtained from Bachem (Torrance, CA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), HEPES, G418, and Lipofectamine were obtained from GIBCO-BRL Life Technologies (Grand Island, NY). BSA was obtained from Commonwealth Serum Laboratories (Parkville, Australia), anti-c-myc antibody was obtained from Invitrogen (Carlsbad, CA), and Alexa 488-conjugated goat anti-mouse sera and TOTO-3 were obtained from Molecular Probes (Eugene, OR). Isobutylmethylxanthine was obtained from Sigma Chemical Co. (St. Louis, MO), tissue culture plates and flasks were obtained from Nunc (Roskilde, Denmark), and anti-cyclic AMP (cAMP) antibody was a gift from Dr. Philip Marley (Department of Pharmacology, University of Melbourne, Melbourne, Australia). Endo F was obtained from Boehringer Mannheim (Mannheim, Germany), and bissuccidimidyl suberate (BS3) was obtained from Pierce Chemical Co. (Rockford, IL). Na125I and125I-labeled rat amylin (specific activity, 2000 Ci/mmol) were obtained from Amersham (Buckinghamshire, UK).125I-labeled salmon CT (specific activity, ∼700 Ci/mmol) was prepared as described previously (Nicholson et al., 1986). All other chemicals were of reagent grade or better.

Cell Culture and cDNA Transfection.

Green monkey kidney COS-7 cells were maintained in 175-cm2 flasks at 37°C in a humidified atmosphere with 95% O2:5% CO2, in complete DMEM supplemented with 10% FBS, 80 mg/l gentamycin, 1 mg/l minocycline, and 15 mM HEPES. Human embryonic kidney (HEK)-293 cells stably expressing the rat C1a CT receptor [clones F12 (∼60,000 receptors/cell) and D11 (∼600,000 receptors/cell); Houssami et al., 1994] were maintained in antibiotic- and HEPES-supplemented DMEM containing 5% FBS and 200 μg/ml G418. CHO-K1 cells (a gift from Dr. Steve Rees, GlaxoWellcome Medicines Research Center, Stevenage, UK) were maintained in DMEM/Ham’s F12 media (50:50) supplemented with 10% FBS, 2 mM glutamine, and 0.5 mg/ml hygromycin B.

Coexpression of CT Receptor and RAMP cDNA.

Cells in 24- or 6-well plates were grown to 70 to 80% confluency and transfected with 0.1 μg (unless otherwise specified) of plasmid DNA encoding the most commonly expressed CT receptor isoforms from human [hCTRIl−; a gift from Dr. Emma Moore, Zymogenetics Inc., Seattle, WA (Kuestner et al., 1994)] or rat (C1a;Albrandt et al., 1993; Sexton et al., 1993) and RAMP cDNA (McLatchie et al., 1998) per 2 cm2 by using Lipofectamine, according to the manufacturer’s instructions. The CT receptor isoforms from rat and human are equivalent in their exon splicing and have been denoted hCTRI1− and rCTRI1− for this study. In some experiments, increasing concentrations of RAMP DNA were transfected. Radioligand binding and cAMP assays were performed 48 h after transfection.

Radioligand Binding.

Receptor-expressing cells in 24-well plates, at 90 to 100% confluency, were incubated in binding buffer [DMEM containing 0.1% (w/v) BSA] with ∼80 pM125I-labeled salmon CT or ∼70 pM125I-labeled rat amylin (Sexton et al., 1993), in the absence (total binding) or presence of increasing concentrations of unlabeled ligands. Nonspecific binding was defined as binding in the presence of 1 μM homologous unlabeled peptide. After incubation for 60 min at 37°C, cells were washed with ice-cold PBS (140 mM NaCl, 2 mM KCl, 1 mM KH2PO4, and 8 mM Na2HPO4, pH 7.3) and solubilized with 0.5 M NaOH. Competition binding curves were analyzed with the Equilibrium Binding Data Analysis/Ligand software package (Biosoft, Cambridge, UK).

cAMP Assay.

Transfected cells in 24-well plates, at 90 to 100% confluency, were preincubated in cyclase buffer [DMEM containing 0.1% (w/v) BSA and 1 mM isobutylmethylxanthine] for 20 min at 37°C. Cells subsequently were incubated for 25 min in the absence (basal) or presence of increasing concentrations of ligand. After incubation, cells were washed with ice-cold PBS, and cAMP was extracted with 0.5 ml of absolute ethanol. cAMP levels were assayed by radioimmunoassay as described previously (Sexton et al., 1994a).

Covalent Cross-Linking Analysis.

Transfected cells in six-well plates were incubated for 60 min in binding buffer with an ∼4 nM concentration of the specified radioligand in the absence (total binding), or presence of 1 μM homologous unlabeled peptide (nonspecific binding). After incubation, cells were washed with PBS and cross-linked on ice for 35 min with 1 mM BS3. Cells were collected and solubilized in sample buffer [50 mM Tris HCl (pH 6.8) containing 2% (w/v) SDS, 0.1% bromophenol blue, 10% (v/v) glycerol, and 100 mM dithiothreitol] and centrifuged at 12,000g for 30 min at 4°C, and the supernatants were analyzed by 10% (w/v) SDS-polyacrylamide gel electrophoresis (Quiza et al., 1997). Gels were stained with Coomassie blue R-250, destained, dried, and exposed to phosphor screens (Molecular Dynamics, Sunnyvale, CA). Deglycosylation was performed as described previously (Quiza et al., 1997). The M r of labeled bands was determined from a standard curve generated from the electrophoretic mobility of molecular weight markers that were coelectrophoresed with the samples.

Confocal Microscopic Localization of c-myc-Tagged RAMP 1.

RAMP 1 epitope tagged with the c-myc epitope at the N terminus (McLatchie et al., 1998) was transfected transiently into COS-7 cells seeded onto 22-mm glass coverslips in six-well plates, either alone or together with the I1− isoform of the rat or human CT receptor. Then, 48 h after transfection, cells were fixed with 3.2% paraformaldehyde for 30 min at 22°C, the reaction was stopped with 150 mM glycine in PBS, and the cells were washed three times for 5 min in either PBS or PBS containing 0.3% Triton X-100. All subsequent treatments and washes were performed in either PBS for cell surface labeling or in PBS-Triton to allow intracellular identification of epitope-tagged protein. Cells were preblocked with 10% lamb serum for 30 min at 22°C, washed once with PBS or PBS-Triton, and then incubated with anti-c-myc antisera at a dilution of 1:500 for 1 h at 22°C. After incubation with primary antisera, cells were washed once and then incubated in the dark with Alexa 488-conjugated goat anti-mouse sera (1:100) for 1 h at 22°C. Cells were further incubated in the dark with PBS-Triton containing TOTO-3 (1:1000) and RNase (1:250) for 1 h, washed twice with PBS-Triton for 5 min, dipped in distilled water, and mounted onto glass slides with fluorescent mounting media. Cells were visualized with a Bio-Rad confocal microscope (Bio-Rad Laboratories, Hercules, CA).

Results

Cotransfection of hCTRI1− with increasing concentrations of RAMP 1 or RAMP 3 into COS-7 cells induced specific and high-affinity 125I-labeled amylin binding (Fig. 1, a and e). RAMP 2 had no significant effect (Fig. 1, a and e). Similar results were seen with the rCTRI1− receptor isoform (Fig. 1b). In contrast, in HEK-293 cells stably expressing the rCTRI1−, only RAMP 1 induced alteration in the level of specific 125I-labeled amylin binding (Fig. 1c). No significant change was seen in the level of125I-labeled salmon CT binding for any of the CT receptors studied (Fig. 1, d and f). Transfection of increasing levels of RAMP into CHO-K1 cells, which endogenously express CT receptors, demonstrated induction of 125I-labeled amylin binding with RAMPs 1 and 3 but not with RAMP 2 or a vector control (Fig. 2). The expression of RAMP alone into COS-7 cells did not enable binding of either125I-labeled amylin or125I-labeled salmon CT (not shown).

Figure 1
Figure 1

Effect of cotransfection of RAMP 1, RAMP 2, RAMP 3, or vector DNA on the expression of 125I-labeled amylin (a, b, c, e) or 125I-labeled salmon CT (d, f) binding in COS-7 cells transiently transfected with 100 ng of CT receptor (a, b, e, f) or HEK-293 cells stably expressing the rCTRI1− (c, d). a,125I-labeled amylin binding in cells transfected with 100 ng of hCTRI1−. b, 125I-labeled amylin binding in cells transfected with 100 ng of rCTRI1−. c,125I-labeled amylin binding in cells stably expressing the rat CTRI1−. d, 125I-labeled salmon CT binding in cells stably expressing the rCTRI1−. Representative experiments (n ≥ 3) with triplicate repeats. e and f, pooled data for specific 125I-labeled amylin binding (e) or 125I-labeled salmon CT binding (f) after cotransfection with 100 ng of RAMP and 100 ng of hCTRI1−. ∗,P < .05, ANOVA, multiple comparisons versus control (Tukey’s Test; n = 7). ●, RAMP 1. ○, RAMP 2. ▾, RAMP 3. ▿, vector.

Figure 2
Figure 2

Effect of transfection of RAMP 1 (●), RAMP 2 (○), RAMP 3 (▾), or vector (▿) DNA on the expression of125I-labeled amylin binding in CHO-K1 cells that endogenously express a native CT receptor. RAMP 1 and RAMP 3, but not RAMP 2 or vector DNA, increased specific 125I-labeled amylin binding. Data are from a representative experiment with triplicate repeats (n = three separate experiments).

The amylin receptors generated by the coexpression of the hCTRI1− isoform with RAMP 1 and with RAMP 3 were analyzed in competition binding studies. K dvalues for amylin and salmon CT were 4.48 × 10−9 m and 9.43 × 10−10 m, respectively, for CTR/RAMP 1 and 5.38 × 10−9 M (amylin) and 5.8 × 10−9 M (salmon CT) for CTR/RAMP 3. The hCTRI1−/RAMP 1 combination generated an amylin receptor equivalent to that identified previously in α-thyroid-stimulating hormone thyrotroph cells (α-TSH cells; Perry et al., 1997). It had the highest affinity for salmon CT, a high to moderate affinity for rat amylin and human CGRPα, and a low affinity for human CT (Fig. 3a; Table1). Human adrenomedullin had little interaction with this receptor, being at least 10-fold less potent than human CT (not shown). In contrast, the binding of125I-labeled amylin to the hCTRI1−/RAMP 3 combination was competed for by salmon CT, amylin, and human CT in a manner similar to that seen with hCTRI1−/RAMP 1, but human CGRPα was ∼30-fold less effective (Fig. 3b; Table 1). As for the RAMP 1-induced phenotype, human adrenomedullin had the lowest affinity for this receptor and essentially did not compete for binding except at micromolar concentrations (not shown). Little change was seen in the level and specificity of 125I-labeled salmon CT binding to COS-7 cells after cotransfection with any of the RAMPs (Table2). Similar results were seen with the rCTRI1− isoform (not shown).

Figure 3
Figure 3

RAMP 1 and RAMP 3 generate two distinct amylin receptors. a, competition of 125I-labeled rat amylin binding to COS-7 cells cotransfected with 100 ng of hCTRI1− and 100 ng of RAMP 1 DNA. b, competition of125I-labeled amylin binding to cells cotransfected with hCTRI1− and RAMP 3 DNA. Salmon CT (sCT, ●), rat amylin (AMY, ♦), human CGRPα (CGRP, ▴), human CT (hCT, ▪). Data are from a single representative experiment with triplicate repeats (n = 3). IC50 values for pooled data are shown in Table 1.

View this table:
Table 1

IC50 values (nM) for peptides in competition for125I-labeled rat amylin binding to COS-7 cells cotransfected with 100 ng of hCTRI1− and 100 ng of RAMP (n = 3)

View this table:
Table 2

IC50 values (M) for peptides in competition for125I-labeled salmon CT binding to COS-7 cells cotransfected with 100 ng of hCTRI1− and 100 ng of RAMP (n = 3)

The functional cAMP responses in COS-7 cells cotransfected with individual RAMPs and hCTRI1− were consistent with the pharmacology of the amylin binding they induced. Cells cotransfected with receptor and vector control showed responses typical of a CT receptor (Kuestner et al., 1994; Albrandt et al., 1995; Gorn et al., 1995), with salmon and human CT displaying similar efficacy and amylin and CGRP only weakly stimulating cAMP accumulation. RAMP 1 and RAMP 3 increased amylin potency (Fig.4a), whereas only RAMP 1 increased CGRP potency (Fig. 4b). RAMP cotransfection caused a decrease in the efficacy of human CT (Fig. 4c), whereas the efficacy of salmon CT essentially was unaltered by RAMP treatment (Fig. 4d). Consistent with its limited effect on specific amylin binding, RAMP 2 had little effect on peptide specificity and potency (Fig. 4).

Figure 4
Figure 4

Effect of RAMPs on ligand-induced cAMP responses in COS-7 cells cotransfected with 100 ng of hCTRI1− and 100 ng of vector control (▵), RAMP 1 (●), RAMP 2 (○), or RAMP 3 (▴) DNA. a, amylin responses; b, CGRP responses; c, human CT responses; d, salmon CT responses. Data are from a single representative experiment (n = 3). Arrows indicate the direction of potency shift for each of the peptides in response to RAMP cotransfection. Basal cAMP levels were ∼350, 600, 720, and 400 pmol/ml/106 cells for vector-, RAMP 1-, RAMP 2-, and RAMP 3-cotransfected cells, respectively. Maximal responses were ∼2150, 2700, 3800, and 2200 pmol/ml/106 cells for vector-, RAMP 1-, RAMP 2-, and RAMP 3-cotransfected cells, respectively.

BS3 cross-linking of125I-labeled amylin to RAMP 1/hCTRI1−-transfected cells revealed a broad receptor-binding protein with a M r of ∼80,000, whereas cells transfected with receptor plus vector control exhibited essentially no specific amylin binding (Fig.5). 125I-labeled salmon CT labeled a band with a M r of ∼80,000 in both RAMP 1- and vector control-transfected cells. Endo F deglycosylation reduced the size of the125I-labeled salmon CT-binding protein to aM r of ∼54,000, consistent with the predicted size of the core receptor protein (Quiza et al., 1997). In contrast, deglycosylation of the 125I-labeled amylin-binding protein with Endo F generated two distinct bands, a lower band with a M r of ∼54,000 and an upper band with a M r of ∼68,000 (Fig. 5). Similar results were seen in HEK-293 cells stably transfected with rCTRI1− receptor homolog and transfected with RAMP 1 (not shown).

Figure 5
Figure 5

Transfection of RAMP 1 with hCTRI1− into COS-7 cells enables BS3 to specifically cross-link125I-labeled rat amylin to a broad glycosylated protein with a M r of ∼80,000. Binding of125I-labeled sCT essentially was unaltered by cotransfection of RAMP 1. However, after Endo F deglycosylation of cross-linked RAMP 1 transfected cells, two bands with aM r of ∼68,000 and ∼54,000 were seen with125I-labeled amylin. In cells transfected with hCTRI1− alone, only the lowerM r band was seen after deglycosylation of125I-labeled salmon CT cross-linked receptor. The data are from an individual representative experiment (n = 3).

Confocal microscopic localization of RAMP 1 incorporating a c-myc epitope tag in the N terminus revealed that, when transfected alone, little of the protein was expressed on the cell surface (Fig.6, a and b). However, when cotransfected with rCTRI1−, significant cell-surface expression of the protein was observed (Fig. 6c).

Figure 6
Figure 6

Confocal microscopic localization of c-myc RAMP 1 transfected alone (a, b) or with the rCTRI1− (c). a and c, cell surface labeling in nonpermeabilized cells. b, labeling in cells permeabilized with 0.3% Triton X-100. The data are from an individual representative experiment (n = 6). The data indicate the strong induction of cell-surface expression of RAMP 1 with receptor coexpression.

Discussion

The discovery of RAMPs and the elucidation of their role in the trafficking of CRLR and its expressed cell-surface phenotype provided a novel potential mechanism for the diversification and the regulation of receptor function. However, the role of RAMPs beyond their interaction with CRLR is unclear. CRLR shares ∼55% amino acid sequence identity with the CT receptor and is almost 80% identical in the transmembrane regions. This homology suggested that the CT receptor protein might also be a target for RAMP interaction. In this study, we demonstrate that RAMPs do indeed interact with the CT receptor gene product to induce novel receptor phenotypes. RAMP 1 cotransfection with CT receptors generated an amylin receptor equivalent to that identified in mouse α-TSH cells (Hanna et al., 1995; Perry et al., 1997). The profile of peptide interaction was also similar to amylin receptors characterized in the nucleus accumbens (Beaumont et al., 1993), the kidney (Wookey et al., 1996), and skeletal muscle (Pittner et al., 1996). Although the affinity of peptides interacting with the nucleus accumbens appears higher (Beaumont et al., 1993), this is likely attributable to, at least in part, the difference in assay format, with live cells being used in the current study and membranes being used for the measurement of nucleus accumbens binding. Indeed, analysis of binding competition in brain slices also yields lower affinity for competing peptides (Sexton et al., 1994b).

Comparison of the RAMP 1- and RAMP 3-induced receptor phenotypes indicates that there are different forms of amylin receptor with differential sensitivity to CGRP, and there is evidence for this in tissue preparations. Differential sensitivity of amylin binding to competition by CGRP within rat brain nuclei has been suggested by the results of autoradiographic studies (van Rossum et al., 1994). The disparity in affinity is modest, ∼10-fold at most, but it is consistent with the difference between RAMP 1- and RAMP 3-induced receptor profiles. Amylin binding to regions such as the dorsomedial and arcuate hypothalmic nuclei (low CGRP affinity) is consistent predominantly with the RAMP 3-induced phenotype. Amylin receptors in the nucleus accumbens core and the amygdala (high CGRP affinity) resemble the RAMP 1-induced phenotype. Elsewhere, the affinity of CGRP is intermediate, which may imply varying levels of mixed-receptor phenotypes. Both RAMP 1 and RAMP 3 are expressed significantly in brain (McLatchie et al., 1998). For cells in which an amylin receptor phenotype is induced, it is unclear why the relative potency of ligands in competition for 125I-labeled salmon CT binding is not significantly altered. However, it is likely that cells cotransfected with CT receptor and RAMP 1 or RAMP 3 express mixed amylin-CT receptor phenotypes. Furthermore, we have speculated that specificity of peptides in competition125I-labeled salmon CT binding is more reflective of affinity for inactive state receptor (Houssami et al., 1995).

These data show that in cells expressing CTRI1−, RAMP expression determines the extent to which they respond to the CT family of peptides. For the majority of experiments, the expression of CTRI1−, through cotransfection with RAMP, occurs together with RAMP. However, we also have demonstrated that RAMP expression gives rise to novel amylin receptors in CHO-K1 cells endogenously expressing CT receptors and in cells stably expressing the rCTRI1−. In the latter cell line, unlike COS-7 cells, only RAMP 1 was capable of inducing amylin-receptor binding, which suggests that cellular background, including native RAMP levels and, potentially, other components such as G protein levels, plays a significant role in the derived receptor phenotype.

As observed previously for CGRP and adrenomedullin (McLatchie et al., 1998), the expression of RAMP alone did not enable binding of either amylin or salmon CT, indicating that RAMPs are not receptors by themselves. Confocal microscopic analysis of RAMP 1 distribution indicated that little cell-surface expression of the protein occurred when transfected alone, although significant intracellular protein expression was observed. Cotransfection of CT receptor with RAMP 1 induced the appearance of the RAMP at the cell surface, as has been observed previously with cotransfection of CRLR and RAMP 1 (McLatchie et al., 1998). However, unlike CRLR, which does not traffic to the cell surface in the absence of RAMP, CT receptor alone is strongly expressed at the cell surface, yielding a classical CT receptor phenotype. Thus, for the CT receptor gene product, RAMP appears to be acting principally as a phenotypic modulator and not as a trafficking protein. However, the possibility that RAMP may affect the processing and trafficking of newly formed CT receptor protein in the expression of novel receptor phenotypes cannot be excluded.

Although inconclusive, the results of deglycosylation studies with125I-labeled amylin cross-linked to cell surface-expressed amylin receptor were suggestive of an association between the “CT receptor” protein and a protein the size of which was equivalent to RAMP 1, with the appearance of a band with aM r of ∼64,000, in addition to the core protein band with a M r of ∼54,000. Although it is possible that the higher-molecular-weight band reflects partial deglycosylation of the receptor, we believe that this is unlikely, because the vast majority of the salmon CT-binding protein runs as core protein under equivalent conditions. Thus, the data may indicate a close cell surface association of RAMP 1 (BS3 cross-links primary amino groups within ∼20 Å) and the CT receptor gene product in the expression of the amylin receptor phenotypes. Although differences in the pattern of glycosylation between the major amylin- and salmon CT-binding proteins occur in α-TSH cells (Perry et al., 1997), no apparent differences were seen in the current study, suggesting that alteration in the level of glycosylation is not required for the expression of amylin receptor phenotype. For the α-TSH amylin receptor, the additional carbohydrate occurred at a site susceptible to deglycosylation under nondenaturing conditions (Perry et al., 1997). We have shown previously that glycosylation at a similar site occurs in CT receptors expressed in CT-receptor-naı̈ve cells in the absence of the overt expression of an amylin receptor phenotype (Quiza et al., 1997), and that this carbohydrate may be removed without affecting ligand binding. It remains possible, however, that small changes in either the degree or the site of glycosylation, beyond the resolution of the current experiments, do occur. At the CRLR, terminal modification of the carbohydrate is induced by RAMP 1 in generating the high affinity CGRP receptor (McLatchie et al., 1998), although it is unclear whether this modification contributes directly to CGRP binding. Nonetheless, the induction of a novel receptor phenotype by RAMP does not necessarily involve alterations to receptor glycosylation as RAMP 2- or RAMP 3-induced adrenomedullin-like receptors occur without carbohydrate modification (McLatchie et al., 1998).

We have demonstrated for the first time that receptors for amylin can be created by the coexpression of CT receptors and RAMPs. The novel amylin receptors have pharmacologies consistent with those observed in tissue preparations. RAMPs have now been shown to determine the pharmacology of both CT receptor and CRLR. These receptors are homologous, particularly in their transmembrane domains. The physiological significance of these regulatory mechanisms is uncertain, but they suggest a way in which cells could change their sensitivity to peptides in the CT/CGRP family and possibly other class II G protein-coupled receptors.

Footnotes

    • Received April 14, 1999.
    • Accepted May 18, 1999.

  • Send reprint requests to: Dr. Patrick M. Sexton, Department of Pharmacology, University of Melbourne, Parkville 3052, Victoria, Australia. E-mail:p.sexton{at}pharmacology.unimelb.edu.au

  • This work was funded in part by the National Health and Medical Research Council of Australia and by GlaxoWellcome, Australia. P.M.S. is a Research Fellow of the National Health and Medical Research Council of Australia.

Abbreviations

CGRP
calcitonin gene-related peptide
CT
calcitonin
RAMP
receptor activity-modifying protein
CRLR
calcitonin receptor-like receptor
α-TSH cells
α-thyroid-stimulating hormone thyrotroph cells
BS3
bissuccidimidyl suberate
cAMP
cyclic AMP
CHO
Chinese hamster ovary
CTR
calcitonin receptor
DMEM
Dulbecco’s modified Eagle’s medium
FBS
fetal bovine serum
hCTRI1−
human CT receptor lacking the 16 amino acid insert in intracellular domain 1
HEK
human embryonic kidney
rCTRI1−
rat insert negative CT receptor isoform equivalent to rat C1a CT receptor

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Multiple Amylin Receptors Arise from Receptor Activity-Modifying Protein Interaction with the Calcitonin Receptor Gene Product

George Christopoulos, Katie J. Perry, Maria Morfis, Nanda Tilakaratne, Yongyi Gao, Neil J. Fraser, Martin J. Main, Steven M. Foord and Patrick M. Sexton
Molecular Pharmacology July 1, 1999, 56 (1) 235-242; DOI: https://doi.org/10.1124/mol.56.1.235
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