Introduction

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Calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM) are related members of the calcitonin family of regulatory peptides, which also includes amylin and calcitonin (Muff et al., 1995). We recently showed that a seven- transmembrane receptor, the calcitonin receptor-like receptor (CRLR), can function as either a CGRP receptor or an ADM receptor, depending upon the coexpression of a family of three, single transmembrane receptor activity modifying proteins or RAMPs. RAMP1 presents CRLR at the plasma membrane as a terminally glycosylated, mature glycoprotein and a CGRP receptor, whereas RAMPs 2 and 3 present CRLR as an immature, core glycosylated ADM receptor (McLatchie et al., 1998).

We have considered a number of possible mechanisms of RAMP activity: First, RAMPs may be directly involved in the binding of CGRP and ADM to CRLR. This hypothesis is supported by the observation that ¹²⁵I-CGRP cross-links to a protein that could be RAMP1 and to CRLR following coexpression in human embryonic kidney (HEK) 293T cells (Stangl et al., 1991; McLatchie et al., 1998). Second, carbohydrate residues linked to CRLR during trafficking and glycosylation might determine the binding of CGRP and ADM. Finally, coexpression of RAMPs might alter the conformation of CRLR and thereby alter its binding characteristics.

In this study we carried out a number of experiments to examine the structural basis of RAMP activity. Detailed pharmacological characterization showed that, in spite of their disparate amino acid sequences, RAMPs 2 and 3 form an identical ADM receptor following coexpression with CRLR in HEK293T cells. Furthermore, experiments using RAMP chimeras showed that both the glycosylation state of CRLR and its ligand specificity are determined by the amino terminus of the coexpressed RAMP protein.

	Experimental Procedures

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Oocytes. Adult female Xenopus laevis (Blades Biologicals) were anesthetized using 0.2% tricaine (3-aminobenzoic acid ethyl ester; Sigma Chemical Co., St. Louis, MO), sacrificed, and the ovaries rapidly removed. Oocytes were defolliculated by collagenase digestion (1.5 mg ml¹; Sigma type I) in divalent cation-free OR2 solution (82.5 mM NaCl, 2.5 mM KCl, 1.2 mM NaH₂PO₄, and 5 mM HEPES, pH 7.5 at 25°C). Single stage V and VI oocytes were transferred to ND96 solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, and 5 mM HEPES, pH 7.5 at 25°C), which contained 50 µg ml¹ gentamycin and stored at 18°C.

Molecular Biology. CRLR was modified to provide a consensus Kozak sequence as described (Aiyar et al., 1996). The Haemaglutinin (HA) epitope tag was fused in frame to the 5' end of cDNA encoding CRLR. The native signal sequence was removed and replaced with that for T8. HA-CRLR was cloned into pCIN6 (a pcDNA3-based vector that contains an internal ribosomal entry site element for the construction of stable cell lines). HA-CRLR behaved in the same way as "native" CRLR in the assays described (data not shown).

Cell Culture and cDNA Transfection. HEK293T cells were maintained in Dulbecco's minimal essential medium (DMEM) supplemented with 10% fetal calf serum and 2 mM glutamine and maintained at 37°C and 95% humidity. NG108-15 cells were cultured in DMEM/Ham's F12 (1:1) supplemented with hypoxantinine aminoptevine (1×) and 10% FCS. Five micrograms of linearized DNA per plasmid was transfected for each T75-cm² flask containing 80% confluent cells. Cells were left 24 h before plating into 96-well plates and were cultured for a further day. For cAMP determinations, the cells were washed with PBS and preincubated in DMEM containing 300 µM 3-isobutyl-1-methylxanthine (Sigma) for 30 min at 37°C. CGRP or ADM was added for a further 30 mins and the cells washed with ice-cold PBS. cAMP levels were determined using scintillation proximity-based assays (Amersham, Arlington Heights, IL).

Radioligand Binding, SDS-Polyacrylamide Gel Electrophoresis (PAGE), and Western Blotting. HEK293T cells were harvested into PBS 48 h following transfection. The cells were pelleted by centrifugation and homogenized in 50 mM HEPES-KOH, pH 7.6 (containing 15 µg/ml aprotinin, 0.25 µg/ml antipain, 0.25 µg/ml leupeptin, 0.1 mg/ml bezamidine, and 0.1 mg/ml bacitracin). After centrifugation at 500g for 20 min at 4°C, the supernatant was removed and centrifuged at 48,000g for 30 min at 4°C. The final pellet was resuspended in homogenization buffer and the protein content measured. SK-N-MC cells were harvested into PBS and subjected to the same membrane preparation in 50 mM HEPES-KOH, 1 mM EDTA, 100 µM leupeptin, and 25 µg/ml bacitracin, pH 7.6. Immediately before the first homogenization, 1 mM phenylmethylsulfonyl fluoride and 2 µM pepstatin A were added. The final pellet was resuspended without phenylmethylsulfonyl fluoride or pepstatin A.

Fluorescence-Activated Cell-Sorting Analysis (FACS) Analysis. HEK293T cells were transiently transfected with cDNA as described above, were harvested 2 days later, and washed three times in PBS. The cells were resuspended in DMEM and incubated with the primary antibody, 9E10 (anti-C-myc) diluted 1:30 for 15 min. Following three further washes, the secondary antibody (sheep anti-mouse Fab₂ coupled with FITC) diluted 1:30 was incubated for 30 min in the dark. For permeabilized cells the Fix and Perm kit (Caltag Labs., South San Francisco, CA) was used. FACS sorting was performed on an EPICS Elite (Coulter Corp., Hialeah, FL); 10,000 cells were sorted in each experiment. Sham-transfected cells did not show any significant fluorescence (data not shown).

	Results

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We have previously reported that RAMPs 2 and 3 interact with CRLR to produce an ADM receptor when coexpressed in Xenopus oocytes (McLatchie et al., 1998). In the present study, detailed pharmacological characterization of the RAMP2/CRLR and RAMP3/CRLR receptors was carried out following transient expression in HEK293T cells. FACS analysis of cells coexpressing RAMPs 1, 2, or 3 with epitope-tagged CRLR demonstrated that the three RAMP proteins transport CRLR to the cell surface with a similar efficiency (Fig. 1). Thus, in cells expressing myc-CRLR but no RAMP, myc epitope appeared at the cell surface in only 2% of cells, whereas in cells coexpressing RAMP1, 2, or 3, approximately 25% of the cells expressed myc-CRLR at the cell surface (transfection efficiency for myc-CRLR was about 70%, as judged by FITC fluorescence of permeabilized cells).

View larger version (24K): [in this window] [in a new window]   Fig. 1.   RAMPs 1, 2, and 3 transport epitope-tagged CRLR to the cell surface with similar efficiencies. FACS analysis of HEK293T cells transiently expressing myc epitope- tagged CRLR with or without RAMPs 1, 2, or 3. Number of cells sorted as showing cell surface myc-CRLR increased from approximately 3 to 25% irrespective of which RAMP is coexpressed.

Analyses of radioligand binding and cAMP accumulation were carried following transient transfection of RAMPs 2 or 3 with CRLR in HEK293T cells.

In nontransfected cells, or cells expressing CRLR alone, no ¹²⁵I-rat ADM binding was detected. However, expression of RAMPs 2 or 3 with CRLR conferred specific, high-affinity ¹²⁵I-rat ADM binding.

Displacement curves were constructed for this binding using a range of ADM and CGRP peptides. In each case, the IC₅₀ values obtained were similar for the receptors produced by RAMP2/CRLR or RAMP3/CRLR, with a rank order of binding affinity of ADM_1-52 > ADM_13-52 > ADM_22-52 > CGRP_8-37 > CGRP2 > CGRP1 (Table 1). Similar values were obtained using membranes from the rat/mouse hybrid NG108-15 cell line, which has been used extensively as a native source of ADM receptors (Zimmermann et al., 1996; Table 1), suggesting that the RAMPs 2- or 3-trafficked CRLR forms an ADM receptor.

Intracellular cAMP accumulation was measured in the same batch of cells using a range of CGRP and ADM receptor agonists. In cells expressing RAMP2/CRLR or RAMP3/CRLR (but not cells expressing CRLR alone), a dose-dependent increase in intracellular cAMP was recorded following incubation with ADM_1-52, ADM_13-52, CGRP1, or CGRP2. Once again, there was no significant difference in the responses of cells transfected with RAMP2/CRLR (Fig. 2, top) and those transfected with RAMP3/CRLR (Fig. 2, middle), with a rank order of agonist potency of ADM_1-52 = ADM_13-52 > CGRP2 > CGRP1 (ED₅₀ values for these experiments shown in Table 2). These data contrast with the rank order of potency of CGRP1 = CGRP2 > ADM_1-52 > ADM_13-52 (Fig. 2, lower panel, and Table 2) recorded in cells coexpressing RAMP1 and CRLR, which show a CGRP receptor pharmacology. Interestingly, in cells expressing RAMP2/CRLR and RAMP3/CRLR, CGRPs 1 and 2 produced a greater increase in cAMP than the ADM peptides.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Functional responses of cells transiently transfected with CRLR and RAMPs 1, 2, or 3 to CGRP and ADM peptides. HEK2993T cells were cotransfected with CRLR and RAMPs 1, 2, or 3. Forty-eight hours later cells were incubated with CGRP1 (), CGRP2 (), ADM1-52 (), and ADM13-52 () was added for a further 30 mins and intracellular cAMP measured and represented relative to cAMP response to forskolin. RAMP2/CRLR and RAMP3/CRLR transfected cells respond to ADM peptides more than CGRP. These cells discriminate between CGRP2 and CGRP1. RAMP1/CRLR receptor responds to CGRP peptides more than ADM and does not discriminate between CGRP1 and CGRP2. Maximum response to CGRP was greater than that to ADM (p < .01 for RAMP2/CRLR and RAMP3/CRLR). Figures are from a single experiment typical of three others. Values represent means ± S.E.M. of quadruplicate determinations. ED50 values (means ± S.E.M.) for intracellular cAMP responses to these peptides over three experiments are given in Table 2.

Despite the relatively low level of homology between RAMPs 2 and 3, both proteins produce an ADM receptor when coexpressed with CRLR in Xenopus oocytes or in HEK293T cells. To further examine the RAMPs 2 and 3 processed CRLR, the glycosylation status of HA-epitope tagged CRLR was assessed by pretreatment with endoglycosidase F (EndoF) or H (EndoH), followed by SDS-PAGE and immunoblotting (McLatchie et al.,1998). As shown in Fig. 3, RAMP 2 and 3-trafficked CRLR showed an identical profile of endoglycosidase sensitivity, with both EndoF and EndoH reducing CRLR in size from a single immunoreactive band at 56 kDa to a band at 48 kDa. These data suggest that the glycosylation status of CRLR may be a key determinant of ligand specificity, with core glycosylation of CRLR (RAMPs 2 and 3) forming an ADM receptor, and terminal glycosylation (RAMP1) switching the ligand specificity to that of a CGRP receptor. To examine the regions of the RAMP protein involved in the interaction and glycosylation of CRLR, two chimeras were constructed between RAMPs 1 and 2. The amino terminal domain of RAMP1 was replaced with that of RAMP2 to produce a RAMP2/1 chimera, and conversely the RAMP1/2 chimera contained the amino terminus of RAMP1 and the transmembrane domain and carboxy-terminus of RAMP2. The junction of the chimera was a point of common sequence at the "extracellular" face of the transmembrane domain corresponding to an aspartic acid residue and two prolines (DPP in single-letter amino acid notation).

View larger version (27K): [in this window] [in a new window]   Fig. 3.   The 56-kDa CRLR species (produced without RAMPs or with RAMPs 2 or 3) is a glycoprotein that reduced with either EndoF or EndoH to a 48-kDa band. The 66-kDa species produced after RAMP1 coexpression reduced to a 48-kDa band after EndoF treatment but it was resistant to EndoH, suggesting that it is a mature glycoprotein. Deglycosylation was carried out exactly according to the supplier's protocols (Boehringer Mannheim) before SDS-PAGE/immunoblotting. Similar results were obtained whether CRLR was tagged with the HA epitope (shown here) or that for myc.

Initial characterization of the chimeric RAMP proteins was carried out following expression in Xenopus oocytes. We have previously reported that RAMP1 will potentiate the endogenous oocyte "CRLR equivalent" to form a functional, type I CGRP receptor, which will respond to CGRP but not to ADM (McLatchie et al., 1998). In contrast, expression of RAMP2 in oocytes does not induce a response to either ADM or CGRP. It may be that RAMP2 is unable to interact with the Xenopus homolog of CRLR. Alternatively, the Xenopus receptor coexpressed with RAMP2 may not recognize human ADM. Whatever the mechanism, this lack of activity is shared by the RAMP2/1 chimera (amino terminus of RAMP2), which had no significant activity with the endogenous oocyte CGRP receptor (Fig. 4). In contrast, expression of the RAMP1/2 chimera induced a large CGRP response in oocytes, which was approximately 40% of the response seen with RAMP1 (Fig. 4).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   CGRP responses by Xenopus oocytes injected with control cRNA and cRNA encoding RAMPs 2 and 1 and chimeric proteins RAMP2/1 and RAMP1/2. CGRP (100 nM) was used in each experiment and was maintained until a maximum response was reached. At least 15 individual oocytes from three different animals were used per determination. Results are presented normalized against the CGRP responses in RAMP1- expressing oocytes and as means ± S.E.M.

These data suggest that the amino terminus of RAMP1 may be critical in determining the activity and ligand specificity of CRLR. To confirm this hypothesis, several experiments were carried out coexpressing RAMP chimeras with CRLR. Expression of RAMP1/2 with CRLR produced responses that were similar to those seen with RAMP1 alone or with RAMP1/2 alone: large responses to CGRP1 but no responses to ADM (Fig. 5). Conversely, coexpression of RAMP2/1 with CRLR produced responses that were similar to those seen with RAMP2 alone (McLatchie et al., 1998): responses to both ADM and, to a lesser extent, CGRP1 (although the magnitude of these responses was only about 10% of those observed with RAMP2 alone).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   CGRP and ADM responses by Xenopus oocytes injected with cRNA encoding chimeric proteins RAMP2/1 and RAMP1/2. CGRP1 (100 nM) and 10 nM ADM1-52 were administered until a maximum response was recorded. At least 15 individual oocytes from three different animals were used per determination. Results are presented as size of elicited current to avoid obscuring different efficiencies of two chimeras in this system and as means ± S.E.M.

The two chimeric RAMP proteins were also examined in a mammalian background following coexpression with HA-CRLR in HEK293T cells. In agreement with the oocyte data, RAMP activity followed the amino terminus of the chimera, with RAMP1/2 and RAMP2/1 generating high-affinity ¹²⁵I-CGRP1 and ¹²⁵I-ADM specific binding, respectively. However, two inconsistencies were observed between the oocyte and mammalian data: 1) RAMP1 and RAMP1/2-trafficked CRLR showed ¹²⁵I-CGRP1 and ¹²⁵I-ADM binding sites (Fig. 6) even though the expression of the same constructs in oocytes produces no ADM responses. 2) RAMP2 and RAMP2/1-trafficked CRLR showed ¹²⁵I ADM but not ¹²⁵I-CGRP1 binding sites (Fig. 6), whereas oocytes expressing these constructs respond to CGRP and ADM.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   125I-rat ADM- and 125I-CGRP1-specific binding to HEK2993T cells cotransfected with cDNA encoding CRLR alone and in combination with RAMPs 1 or 2 or the chimeric proteins RAMP2/1 and RAMP1/2.125I-rat ADM (100 pM) or 30 pM 125I-CGRP1 were used in radioligand binding studies. Kd values for ability of human ADM and CGRP receptors to bind their corresponding radiolabeled peptides have been reported as 200 pM and 0.05 pM, respectively (Sone et al. 1997; Stangl et al. 1991). Therefore results tend to underestimate CGRP binding relative to that for ADM. Plasma membrane from same cells were subject to SDS-PAGE and Western blot with anti-HA antiserum with or without treatment with EndoF (not shown) or EndoH. When expressed alone, with RAMP2 or RAMP2/1, HA-CRLR migrated as a 58-kDa protein (not shown) that was digested to 48-kDa by EndoF (not shown) or EndoH. When expressed with RAMP1, HA-CRLR migrated predominantly as a 66-kDa protein that was sensitive to EndoF (not shown) but not EndoH. When coexpressed with RAMP1/2, some HA-CRLR was sensitive to EndoH treatment. Results are from a single experiment performed in triplicate and are typical of two other experiments.

HEK293T cell membranes from the above experiments were pretreated with EndoF or EndoH, subjected to SDS-PAGE, and immunoblotted to assess the glycosylation status of CRLR. HA-epitope tagged CRLR expressed alone, with RAMP2 or with RAMP2/1 ran as a 56-kDa protein that was digested to a 48-kDa form by EndoF (not shown) and EndoH (Fig. 6, bottom). HA-CRLR expressed with RAMP1 or with RAMP1/2 ran predominantly as a 66-kDa protein that was digested to a 48k-Da form by EndoF (not shown) but not by EndoH (Fig. 6, bottom).

An interesting observation from the HEK293T cell binding experiments was that RAMP1/2-trafficked CRLR showed an increased level of ¹²⁵I-ADM binding relative to RAMP1. To further examine the anomalous pharmacology of the RAMP1/2-trafficked CRLR, a dose-response experiment was carried out titrating increasing amounts of transfected RAMP1/2 cDNA against a fixed amount of cDNA encoding CRLR. At each concentration of RAMP1/2 cDNA, we measured ¹²⁵I-ADM and ¹²⁵I-CGRP1-specific binding as well as analyzing the molecular form of CRLR after EndoH treatment (Fig. 7). Even at low RAMP1/2 cDNA concentrations, the ratio of ADM-specific binding to CGRP-specific binding was greater than that observed with RAMP1, suggesting that differences in pharmacology did not result from the saturation of intracellular glycosylation pathways at high RAMP1/2 concentrations. Use of a gradient gel system provided better resolution of the molecular forms of CRLR following EndoH treatment. As observed previously (McLatchie et al. 1998 and Figs. 3 and 6 in this study), CRLR appeared to lose immunoreactivity following cotransfection with RAMP1, with only small amount of immunoreactive material visible at about 66 kDa. In contrast, the RAMP1/2 chimera generated a different profile, with a strong immunoreactive band at 48 kDa, and three higher molecular mass bands at around 66kDa. Although transfection of increasing amounts of RAMP1/2 led to an increase in the amount of immunoreactive material at 66 kDa, the gel profile never resembled that seen in RAMP1.

View larger version (40K): [in this window] [in a new window]   Fig. 7.   HEK293T cells were transiently transfected with 1.5 µg of HA-CRLR and cotransfected with either vector control, RAMP1, or increasing amounts of RAMP1/2. The cells were analyzed for their expression of 125I-rat ADM- and 125I-CGRP1-specific binding and for their ability to generate EndoH-resistent CRLR as described in Fig. 6. Ratio of CGRP to ADM binding was approximately 4:1 when RAMP1 was coexpressed with CRLR, whereas ratio was no more than 2:1 with RAMP1/2 chimera irrespective of amount of cDNA transfected.

	Discussion

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We recently identified a family of three RAMP proteins (RAMPs 1, 2, and 3) that are required to traffic CRLR to the cell surface as a functional CGRP or ADM receptor. In this study, for the first time, we examined the expression and activity of RAMP3 in a mammalian cell line. In our experiments, RAMPs 2 and 3 appear to be functionally identical, with both proteins trafficking CRLR to the cell surface as an immature, core glycosylated protein. Pharmacological evaluation, using radioligand binding and cAMP assay, revealed that RAMP3-trafficked CRLR forms an ADM receptor that is indistinguishable from that seen following coexpression of RAMP2 and CRLR.

The pharmacology of the ADM receptor created by the coexpression of RAMPs 2 or 3 with CRLR is interesting in itself. CGRPs 1 and 2 differ by only 3 amino acids out of 37 and have previously been regarded as functionally identical (Muff et al., 1995). However, in our experiments, CGRP2 had a higher affinity (lower IC₅₀) in displacement binding assays and gave a lower ED₅₀ in cAMP assays, suggesting that CGRP2 is more potent than CGRP1 at the RAMP2/CRLR and RAMP3/CRLR receptors. We have also observed that CGRPs 1 and 2 are more effective (in terms of maximal response) at increasing intracellular cAMP than the ADM peptides. This may relate to the kinetics of the responses to the two peptides, to their relative stability, or to their absolute efficacy at the RAMP/CRLR receptor.

In this study, we also examined the structural determinants of RAMP activity. RAMPs are type I membrane proteins with a relatively long extracellular amino terminus, a single membrane-spanning domain, and a short intracellular C-terminus. Several lines of evidence suggest that the extracellular N-terminus of RAMP1 plays an important role in determining ligand specificity. First, cross-linking data using ¹²⁵I-CGRP1 suggests that RAMP1 and CRLR are closely associated at the cell membrane (McLatchie et al.,1998). Second, the RAMP1/2 chimera (which contains the amino terminus of RAMP1) traffics CRLR as a CGRP receptor following expression in HEK293T cells. Finally, no ¹²⁵I-CGRP binding was detected in cells coexpressing the reverse chimera, RAMP2/1, with CRLR suggesting that the transmembrane domain and C-terminus of RAMP1 does not contribute to CGRP binding. Taken together these data suggest that RAMP1 could associate with the amino terminus of CRLR to form a binding pocket for CGRP. It is notable that other members of the family B class of G protein-coupled receptors, such as the glucagon and parathyroid hormone receptors, are thought to bind ligand predominantly via their amino termini (Unson et al., 1995; Zhou et al., 1997). Alternatively, the N-terminus of RAMP1 may affect ligand binding indirectly through a change in the glycosylation or conformational state of CRLR.

RAMPs 2 and 3 traffic CRLR to the cell membrane with a similar efficiency to RAMP1, raising the possibility that they can contribute to the ligand binding of CRLR in a similar manner. However, such a hypothesis is difficult to reconcile with the fact that the N-termini of RAMPs 2 and 3 have only 27 out of 110 amino acids in common, 15 of which are shared by RAMP1. It is possible that the secondary and tertiary structure of RAMPs 2 and 3 are more similar than their primary amino acid sequence suggests, or alternatively, that a relatively small portion of RAMP2/RAMP3 may define their ability to generate an ADM receptor when coexpressed with CRLR.

The ligand specificity of CRLR could also be determined by differences in the level of glycosylation because RAMP1, but not RAMPs 2 or 3, traffics CRLR as a terminally glycosylated receptor. Thus, the ability of CRLR to bind and respond to CGRP could be conferred by terminal glycosylation. For several receptors, glycosylation state appears to have no effect upon function: the calcitonin (Quiza et al., 1997), leuteinizing hormone (Davis et al., 1997), parathyroid hormone (Bisello et al., 1996), and vasopressin V2 (Sadeghi et al., 1997) receptors are examples. However, some other receptors such as the calcium (Fan et al., 1997; Ray et al., 1997) and nicotinic acetylcholine receptors (Buller and White, 1990) are not expressed if glycosylation is inhibited. These data demonstrate that transit through the Golgi apparatus is essential for correct receptor processing; whether glycosylation contributes directly to the pharmacology of the receptors is less certain (White et al., 1998). The importance of N-linked glycosylation to the functioning of CRLR as a CGRP or ADM receptor will be tested experimentally by the sequential mutation of its three putative N-linked glycosylation sites.

Despite our data demonstrating the biological significance of the amino terminus of RAMPs, other regions of the RAMP protein may also be involved in determining ligand specificity. This is suggested by experiments with the RAMP1/2 chimera (amino terminus of RAMP1) in which a significantly higher level of ¹²⁵I-ADM binding was seen than with the native RAMP1. Further structure-function studies will serve to elucidate the contribution of the RAMP protein to ligand binding.

These studies have demonstrated that the pharmacology of CRLR is the same whether it is expressed with RAMP2 or RAMP3. They have also suggested the importance of the amino terminal portions of RAMPs in determining their biological activity and provided the basis for a more complete molecular analysis of their mode of action.