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Originally published In Press as doi:10.1074/jbc.M511147200 on January 11, 2006

J. Biol. Chem., Vol. 281, Issue 11, 7205-7213, March 17, 2006
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Functions of the Cytoplasmic Tails of the Human Receptor Activity-modifying Protein Components of Calcitonin Gene-related Peptide and Adrenomedullin Receptors*Formula

Kenji Kuwasako{ddagger}1, Yuan-Ning Cao{ddagger}, Chun-Ping Chu§, Shuji Iwatsubo{ddagger}, Tanenao Eto{ddagger}, and Kazuo Kitamura{ddagger}

From the {ddagger}First and §Third Departments of Internal Medicine, Miyazaki Medical College, University of Miyazaki, Miyazaki 889-1692, Japan

Received for publication, October 13, 2005 , and in revised form, January 6, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Receptor activity-modifying proteins (RAMPs) enable calcitonin receptor-like receptor (CRLR) to function as a calcitonin gene-related peptide receptor (CRLR/RAMP1) or an adrenomedullin (AM) receptor (CRLR/RAMP2 or -3). Here we investigated the functions of the cytoplasmic C-terminal tails (C-tails) of human RAMP1, -2, and -3 (hRAMP1, -2, and -3) by cotransfecting their C-terminal deletion or progressive truncation mutants into HEK-293 cells stably expressing hCRLR. Deletion of the C-tail from hRAMP1 had little effect on the surface expression, function, or intracellular trafficking of the mutant heterodimers. By contrast, deletion of the C-tail from hRAMP2 disrupted transport of hCRLR to the cell surface, resulting in significant reductions in 125I-hAM binding and evoked cAMP accumulation. The transfection efficiency for the hRAMP2 mutant was comparable with that for wild-type hRAMP2; moreover, immunocytochemical analysis showed that the mutant hRAMP2 remained within the endoplasmic reticulum. FACS analysis revealed that deleting the C-tail from hRAMP3 markedly enhances AM-evoked internalization of the mutant heterodimers, although there was no change in agonist affinity. Truncating the C-tails by removing the six C-terminal amino acids of hRAMP2 and -3 or exchanging their C-tails with one another had no effect on surface expression, agonist affinity, or internalization of hCRLR, which suggests that the highly conserved Ser-Lys sequence within hRAMP C-tails is involved in cellular trafficking of the two AM receptors. Notably, deleting the respective C-tails from hRAMPs had no effect on lysosomal sorting of hCRLR. Thus, the respective C-tails of hRAMP2 and -3 differentially affect hCRLR surface delivery and internalization.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
CGRP2 and AM belong to the calcitonin family of regulatory molecules and exert a wide variety of biological effects, including potent vasorelaxation (1–3). The receptors that mediate these effects are heterodimers composed of CRLR and RAMP, a novel accessory protein (4). The three RAMP isoforms (RAMP1, RAMP2, and RAMP3) are each composed of ~160 amino acids, and all exhibit a common structure that includes a large extracellular N-terminal domain, a single membrane-spanning domain, and a very short C-tail, but they share less than 30% sequence identity and differ in their tissue distributions (4, 5). When acting as a chaperone, each RAMP forms a 1:1 heterodimer with CRLR, probably in the ER (4, 6). They then mediate the transport of CRLR to the cell surface, where the heterodimers form functional CGRP or AM receptors: CRLR/RAMP1 forms the CGRP1 receptor (4), which can also be activated by high concentrations of AM (7, 8); CRLR/RAMP2 forms an AM-specific receptor that is sensitive to the AM receptor antagonist AM-(22–52) (AM1 receptor) (7, 9); and CRLR/RAMP3 forms an AM receptor that is sensitive to both the CGRP1 receptor antagonist CGRP-(8–37) and AM-(22–52) (AM2 receptor) (7, 9). It is the RAMP extracellular domain that mediates agonist binding to CRLR/RAMP heterodimers (11–13), which in turn mediate intracellular cAMP production and Ca2+ mobilization (4, 10).

Exposing cells that express GPCRs to their respective agonists frequently leads to a rapid internalization of the receptor in a process believed to involve clathrin-coated vesicles, caveolin-rich vesicles, or both (14, 15). The internalized GPCRs may be recycled back to the plasma membrane in order to promote functional restoration of signal transduction, or they may be trafficked to lysosomes, where they are degraded (14, 15). Similarly, upon binding their respective agonist, hCRLR/RAMP heterodimers stably expressed in HEK-293 cells are rapidly internalized without dissociation via clathrin-coated vesicles (6, 10) in a process that is blocked by dominant negative mutants of dynamin and beta-arrestin 2 (6). In that regard, it is well known that G protein-coupled receptor kinases phosphorylate serine/threonine sites located in many GPCR C-tails, enabling beta-arrestins to bind there (16). After internalization, both CRLR and RAMP are targeted to lysosomes (10), where they are degraded (6).

Although short, the RAMP C-tails do contain potential sites of interaction with other proteins (5, 17). For instance, the hRAMP3 C-tail possesses a classical type I PDZ (PSD-95/Disc-large/ZO-1) binding motif (TLL) (5, 17), and the binding of NSF to the PDZ motif of hRAMP3 was found to promote slow recycling of internalized hCRLR/hRAMP3 heterodimers in HEK-293 cells (18). In addition, a five-residue motif (QSKRT) in the hRAMP1 C-tail can act as an ER retention signal (19). The C-tails of RAMPs, like that of CRLR, also contain potential phosphorylation and ubiquitination sites (5, 17). Ubiquitination is the post-translational attachment of ubiquitin lysine residues in the substrate proteins (20, 21); it is not crucial for receptor internalization but is essential for proper trafficking to lysosomes for degradation (22, 23). Whether the phosphorylation and ubiquitination sites are also involved in intracellular trafficking of CRLR/RAMP heterodimers remains unknown. To address that issue, we examined the effects of expressing various hRAMP C-tail deletion and progressive truncation mutants and chimeras in which the C-tails were exchanged among the three hRAMPs in HEK-293 cells stably expressing hCRLR.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—125I-[Tyr0]h{alpha}CGRP (specific activity 2 µCi/pmol) (24), which contains an extra N-terminal tyrosine residue (Tyr0), and 125I-hAM (specific activity 2 µCi/pmol) (1) were both produced in our laboratory. Human {alpha}CGRP was purchased from Peptide Institute (Osaka, Japan). [Tyr0]h{alpha}CGRP was from Phoenix Pharmaceuticals, Inc. Human AM was kindly donated by Shionogi & Co. (Osaka, Japan). Mouse anti-hNSF antibody was from Calbiochem. Mouse anti-V5 antibody and FITC-conjugated mouse anti-V5 monoclonal antibody (anti-V5-FITC antibody) were from Invitrogen. Rabbit anti-calnexin antibody was from Stressgen Biotechnologies Corp. (Victoria, Canada), and Alexa Fluor® 594 (biotin- and fluorescent dye-labeled goat anti-rabbit IgG antibody) were from Molecular Probes, Inc. (Eugene, OR). PE-conjugated rabbit anti-mouse secondary antibody was from Exalpha Biologicals, Inc. All other reagents were of analytical grade and were obtained from various commercial suppliers.

Expression Constructs—hNSF (GenBankTM accession number BC030613 [GenBank] ) was cloned from cDNA obtained from human heart (Clontech) using PCR with the appropriate primers and then modified to provide a consensus Kozak sequence as previously described (25). hRAMP1, -2, and -3 (4) were also modified to provide the same Kozak sequence. A double V5 epitope tag (GKPIPNPLLGLDST) was ligated, in frame, to the 5'-end of the cDNAs encoding each intact hRAMP, and the native signal sequences were removed and replaced with MKTILALSTYIFCLVFA (26), yielding V5-hRAMP1, -2, and -3. The deletion and progressive truncation mutations in the V5-hRAMP C-tails were created by using 3'-primers that introduced a translational stop codon at the desired positions (Fig. 1); with RAMP3, for instance, {Delta}139 represents a mutant in which a stop codon was introduced after residue 139. In addition, various V5-hRAMP chimeras were constructed by exchanging the 9 C-terminal amino acid residues among the three hRAMPs. The hNSF, V5-hRAMPs, V5-hRAMP deletion and truncation mutants, and V5-hRAMP chimeras were then respectively cloned into the mammalian expression vector pCAGGS/Neo (10) using the 5'-XhoI and 3'-NotI sites, and the sequences of the resultant constructs were all verified using an Applied Biosystems 310 Genetic Analyzer. The individual V5-hRAMPs were compared with the native sequence in the assays and were found to behave identically (data not shown).

Cell Culture and DNA Transfection—HEK-293 cells stably expressing a hCRLR-GFP chimera (10) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B, and 0.25 mg/ml G 418 at 37 °C under a humidified atmosphere of 95% air, 5% CO2. For experimentation, cells were seeded into 6- or 24-well plates and, upon reaching 70–80% confluence, were transiently cotransfected with the indicated cDNAs using Lipofectamine transfection reagents (Invitrogen) according to the manufacturer's instructions. Briefly, the cells were incubated for 4 h in Opti-MEM I medium containing plasmid DNAs, Plus reagent, and Lipofectamine (see Ref. 27 for 6-well and Ref. 11 for 24-well plates). As a control, some cells were transfected with empty vector (mock). All experiments were carried out 48 h after transfection.

FACS Analysis—Flow cytometry was carried out to assess the levels of cell surface expression of V5-hRAMPs, V5-hRAMP truncation mutants, or V5-hRAMP chimeras in HEK-293 cells. To evaluate cell surface expression, cells were harvested following transient transfection, washed twice with PBS, resuspended in ice-cold FACS buffer (27), and then incubated for 60 min at 4 °C in the dark with anti-V5 monoclonal antibody (1:1000 dilution). Following two additional washes with FACS buffer, the cells were incubated for 60 min at 4 °C in the dark with PE-conjugated rabbit anti-mouse secondary antibody (1:400 dilution) in ice-cold FACS buffer. For evaluation of whole cell expression, cells were first permeabilized using IntraPrepTM reagents (Beckman Coulter, Fullerton, CA) according to the manufacturer's instructions and then incubated with anti-V5-FITC antibody (1:500 dilution) for 15 min at room temperature in the dark. Following two successive washes with FACS buffer, both groups of cells were subjected to flow cytometry in an EPICS XL flow cytometer (Beckman Coulter) and analyzed using EXPO 2 software (Beckman Coulter). Fluorophores were excited at 488 nm, and the emission was monitored at 530 nm for GFP and 575 nm for PE. Viability was assessed by exclusion of propidium iodide.


Figure 1
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  		FIGURE 1.
Amino acid sequence alignment of the cytoplasmic tails of hRAMP1, -2, and -3. The sequences are aligned for maximum homology; alignment of the entire sequences was presented by MacLatchie et al. (4). The numbers indicate the amino acid positions in accordance with Sexton et al. (5). Black circles indicate conserved amino acids; arrows indicate potential phosphorylation sites; and the PDZ binding motif is boxed. The progressive hRAMP C-tail truncation mutants were created using 3'-primers that introduced a translational stop codon at the indicated positions; for instance, {Delta}139 represents a truncation mutant that introduced a stop codon after residue 139.

 
Immunofluorescence Microscopy—HEK-293 cells stably expressing hCRLR-GFP were plated onto 35-mm glass-bottomed dishes (Iwaki, Tokyo, Japan). As determined by the experimental protocol, some cells were then transiently transfected with V5-hRAMP2, {Delta}166-hRAMP2, or {Delta}167-hRAMP2. The cells were then fixed with 3.7% formaldehyde in PBS for 20 min at room temperature, washed twice with PBS, and permeabilized with 0.25% Triton X-100 in PBS for 10 min. Thereafter, the cells were incubated at room temperature for 30 min in blocking buffer (PBS containing 1% bovine serum albumin), followed by incubation for 60 min first with rabbit anti-calnexin (1:200 dilution) and mouse anti-V5-FITC antibody (1:500 dilution) and then, after washing four times with PBS, with the Alexa Fluor® 594 diluted 1:100 in blocking buffer. After another three washes with PBS, the cells were mounted using Slow-Fade mounting medium (Molecular Probes, Inc.), and a 22-mm glass coverslip was seated in the center of each dish. Double labeling was viewed using a TCS-SP2 AOBS confocal laser-scanning microscope (Leica) equipped with a x 63/1.32 numerical aperture immersion lens (Leica).

Whole-cell Radioligand Binding Assays—Transfected HEK-293 cells in 24-well plates were washed twice with prewarmed PBS and then incubated for 5 h at 4 °C with 125I-[Tyr0]h{alpha}CGRP (100 pM) or 125I-hAM (20 pM) in the presence (for nonspecific binding) or absence (for total binding) of 1 µM unlabeled h{alpha}CGRP or hAM in modified Krebs-Ringer-HEPES medium (10), after which they were washed twice more with ice-cold PBS and harvested with 0.5 M NaOH. The associated cellular radioactivity was measured in a {gamma}-counter. Specific binding was defined as the difference between total binding and nonspecific binding.

cAMP Measurements—Transfectants in 24-well plates were incubated for 15 min at 37 °C in Hanks' buffer containing 20 mM HEPES, 0.2% bovine serum albumin, 0.5 mM 3-isobutyl-1-methylxanthine (Sigma), and the indicated concentrations of h{alpha}CGRP or hAM. The reaction mixture was then replaced with 20 mM HCl and 1 M acetic acid to extract the intracellular cAMP, after which the resultant extracts were lyophilized and stored at -30 °C until assayed. The cAMP concentrations were measured using our specific radioimmunoassay (1).

FACS Analysis of Receptor Internalization and Recycling—Following cotransfection of the indicated cDNAs into HEK-293 cells stably expressing hCRLR-GFP in 6-well plates, the cells were exposed to selected concentrations of h{alpha}CGRP or hAM in prewarmed serum-free Dulbecco's modified Eagle's medium containing 20 mM HEPES and 0.2% bovine serum albumin for the indicated periods (up to 2 h) at 37 °C. For receptor recycling studies, the cells were incubating for 60 min with the agonist plus 10 µg/ml cycloheximide and 10 µg/ml brefeldin A and then washed three times with prewarmed PBS. The medium was then replaced with prewarmed Dulbecco's modified Eagle's medium containing 20 mM HEPES, 10% fetal bovine serum, 10 µg/ml cycloheximide, and 10 µg/ml brefeldin A for the indicated periods (up to 4 h) at 37 °C. Internalization and recycling were stopped by adding ice-cold PBS, after which the cells were harvested, resuspended in ice-cold FACS buffer, and labeled with anti-V5 monoclonal antibody and fluorescein PE-conjugated rabbit anti-mouse secondary antibody. The cells were then subjected to flow cytometry and analyzed as described above.

mRNA Expression Measured by Real Time Quantitative PCR—Total RNAs were extracted from HEK-293 cells either untransfected or transfected as indicated using total RNA isolation reagent (Invitrogen). Thereafter, the target cDNAs were synthesized from the respective mRNAs by reverse transcription using SuperScript reverse transcriptase (Invitrogen). The expression of mRNAs encoding hNSF was assessed using real time quantitative PCR (Prism 7700 Sequence Detector, Applied Biosystems, Foster City, CA) with original oligonucleotide primers (sense, 5'-AGAACAGTGACCGCACACCAT-3'; antisense, 5'-TCCACAACCACACAACTGAGC-3') and a fluorescently labeled probe (5'-AGCGTGCTTCTGGAAGGCCCTCCTCACAGT-3'). The size of the amplified DNA was 223 bp. The levels of hNSF mRNA were normalized to those of glyceraldehyde-3-phosphate dehydrogenase mRNA, which served as an internal control.

Western Analysis—Following transient transfection of hNSF into cells plated in 6-well plates, the transfectants were washed twice with ice-cold PBS, harvested in 1 ml of sample buffer (28), and boiled for 10 min. Equal aliquots of protein (20 µg) were then subjected to 10% SDS gel electrophoresis and transferred to a Hybond-P membrane (Amersham Biosciences). The membrane was then blocked with 5% block reagent (Amersham Biosciences), washed, and incubated first for 1 h at room temperature with rabbit anti-hNSF antibody (1:1,000 dilution) and then with secondary antibody (1:10,000 dilution). hNSF proteins were detected using an ECL Plus chemiluminescence kit (Amersham Biosciences), after which they were quantitated by densitometry using Image Gauge (LAS-1000; Fujifilm).

Statistical Analysis—Results are expressed as means ± S.E. of at least three independent experiments. Differences between two groups were evaluated using Student's t tests; differences among multiple groups were evaluated with a one-way analysis of variance followed by Scheffe's tests. Values of p < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Deletion of hRAMP C-tails—We previously established HEK-293 cells stably expressing hCRLR-GFP alone or together with Myc-hRAMPs and used them to visualize the cellular localization and trafficking of hCRLR (10). Following AM exposure, hCRLR is rapidly internalized together with its associated hRAMP, and then both are trafficked to lysosomes; fusion of GFP to the C terminus of hCRLR had no apparent effect on the trafficking of the heterodimeric receptor. In the present study, therefore, we used HEK-293 cells stably expressing hCRLR-GFP to examine the functions of the C-tails of the three hRAMPs within the respective CRLR/RAMP heterodimers.

We initially tested the effect of completely deleting the C-tails of the three V5-epitope tagged hRAMPs (Fig. 2). When coexpressed with hCRLR, V5-RAMP1, -2, and -3 were detected at the surfaces of 45.9, 21.9, and 38.9% of cells, respectively. On the other hand, the V5-RAMP deletion mutants {Delta}139-RAMP1, {Delta}166-RAMP2, and {Delta}139-RAMP3 appeared at the surface of 43.7, 5.0, and 38.8% of cells, respectively. Thus, deletion of the C-tail significantly reduced surface delivery of only RAMP2. Surface immunoreactivity was detected in only 0.55% of cells expressing the empty vector (Mock), which is well within the 2% limit of resolution characteristic of FACS analysis.

We next evaluated the binding profiles of 125I-[Tyr0]h{alpha}CGRP and 125I-hAM to cells expressing each of the wild-type and mutant receptors (Fig. 3, A and B). When CRLR-GFP was coexpressed with empty vector (Mock), the cells showed only very low levels of specific binding of 125I-[Tyr0]{alpha}CGRP and 125I-AM. Co-transfection of RAMP1 led to markedly higher specific 125I-[Tyr0]{alpha}CGRP binding than was seen with {Delta}139-RAMP1 (Fig. 3A), although there was no difference in the surface expression of either heterodimeric receptor (Fig. 2). Likewise, cotransfection of RAMP2 significantly increased the specific binding of 125I-AM to CRLR-GFP-expressing cells, whereas cotransfection of {Delta}166-RAMP2 did not. In this case, the reduced binding could be due to reduced surface delivery of the mutant receptors (Fig. 2). Finally, deletion of the RAMP3 C-tail had no effect on specific 125I-AM binding.


Figure 2
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  		FIGURE 2.
FACS analysis of HEK-293 cells coexpressing hCRLR with intact hRAMP or a C-terminal deletion mutant. HEK-293 cells stably expressing CRLR-GFP were transiently transfected with empty vector (Mock) or the indicated V5-RAMP or deletion mutant. The cells were incubated first with an anti-V5 monoclonal antibody and then with a fluorescein PE-conjugated rabbit anti-mouse secondary antibody. Samples incubated with only secondary antibody served as the control. Cell surface expression of each construct was estimated by flow cytometry. The bars represent means ± S.E. of six independent experiments. *, p < 0.001 versus control (Mock); #, p < 0.02 versus corresponding wild-type V5-RAMP.

 


Figure 3
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  		FIGURE 3.
Effects of hRAMP C-terminal deletion on agonist binding and evoked cAMP production in HEK-293 cells stably expressing hCRLR. A, specific binding of 125I-h{alpha}CGRP and 125I-hAM. HEK-293 cells expressing CRLR-GFP were transiently transfected with empty vector (Mock) or the indicated V5-RAMP or deletion mutant, after which the cells were incubated for 5 h at 4 °C with 125I-{alpha}CGRP (100 pM)or 125I-AM (20 pM) in the presence or absence of 1 µM unlabeled {alpha}CGRP or AM. Bars represent means ± S.E. of three experiments. *, p < 0.002 versus Mock; #, p < 0.001 versus corresponding wild-type V5-RAMP. B, displacement of radioligand. Cells were transfected and radiolabeled as in A either alone or with the indicated concentration of unlabeled ligand ({alpha}CGRP or AM). Bars represent means ± S.E. of three experiments. C and D, evoked cAMP production. After transient transfection of V5-RAMPs and the corresponding deletion mutants, cells were exposed to the indicated concentrations of {alpha}CGRP, [Tyr0]{alpha}CGRP, or AM for 15 min at 37 °C and then lysed. The resultant lysates were analyzed for cAMP content. Bars represent means ± S.E. of three experiments.

 
Fig. 3B shows a set of 125I-[Tyr0]{alpha}CGRP and 125I-AM competition curves for the wild-type and mutant receptors. The IC50 values derived from the curves obtained with cotransfection of {Delta}139-RAMP1 or {Delta}166-RAMP2 were both >1000 nM, which is much higher than those for RAMP1 and -2 (43.0 and 12.2 nM, respectively). By contrast, the IC50 values obtained with expression of RAMP3 or {Delta}139-RAMP3 were within the same order of magnitude (560 and 257 nM, respectively).

We then further characterized the mutant receptors by measuring agonist-induced intracellular cAMP accumulation (Fig. 3, C and D). {alpha}CGRP and AM elicited little or no cAMP production in HEK-293 cells expressing CRLR-GFP alone, indicating that the stable transfectants used in this study express no functional RAMP proteins. In cells coexpressing RAMP1 and CRLR-GFP, by contrast, {alpha}CGRP (EC50 = 0.18 nM) elicited marked increases in cAMP (Fig. 3C). In cells expressing {Delta}139-RAMP1 with CRLR-GFP, the EC50 for {alpha}CGRP was increased only a little, as compared with RAMP1 (to 0.49 nM), and the maximal responses were also similar to those seen with RAMP1 (Fig. 3C). Interestingly, the responses to [Tyr0]{alpha}CGRP by cells expressing CRLR-GFP/{Delta}139-RAMP1 (EC50 = 8.5 nM) were significantly smaller than those seen in cells expressing CRLR-GFP/RAMP1 (EC50 = 0.79 nM). In cells transfected with RAMP2, AM elicited significant increases in cAMP (EC50 = 0.38 nM), but these responses were diminished by 62.1% in cells transfected with {Delta}166-RAMP2, although there was no significant change in EC50 (0.16 nM) (Fig. 3D). AM-evoked cAMP production did not significantly differ in cells expressing RAMP3 or {Delta}139-RAMP3 (EC50 = 0.41 and 0.20 nM, respectively) (Fig. 3D). That the cAMP production elicited via the respective receptors largely paralleled the profile of radioligand binding (Fig. 3, A and B) suggests that the C-tails of RAMP1 and -3 have little or no involvement with agonist binding and signaling.

We previously quantified the internalization and recycling of AM receptors (CRLR/RAMP heterodimers) using radioligand binding assays; however, interpretation of those experiments was complicated by the high degree of nonspecific AM binding, which reflected the highly hydrophobic and basic nature of the native peptide (1, 10, 11). In the present study, therefore, we used FACS to evaluate agonist-mediated internalization and recycling of wild-type and mutant CRLR-GFP/RAMP heterodimers. Fig. 4A shows the receptor internalization induced by 1 µM {alpha}CGRP- or AM. Exposure to the appropriate agonist elicited rapid declines in cell surface expression of wild-type CRLR-GFP/RAMP1 and -3 that led to 40–60% reductions in signal strength within 2 h and to 90% reduction in cell surface CRLR-GFP/RAMP2 within 30 min. Heterodimers composed of CRLR-GFP plus {Delta}139-RAMP1 or {Delta}166-RAMP2 tended to be internalized somewhat less efficiently, whereas internalization of the CRLR-GFP/{Delta}139-RAMP3 heterodimer was markedly enhanced. Notably, these phenomena occurred with no changes in cell surface CRLR-GFP expression or AM binding and signaling (Figs. 2 and 3).

The dose dependence of the agonist-evoked receptor internalization is illustrated in Fig. 4B. {alpha}CGRP elicited equivalent dose-dependent internalization of CRLR-GFP/RAMP1 and CRLR-GFP/{Delta}139-RAMP1. AM dose-dependently induced RAMP2-mediated internalization of CRLR-GFP, which was more efficient than RAMP1-mediated internalization. And {Delta}166-RAMP2 mediated internalization of CRLR-GFP even more efficiently than did wild-type RAMP2, although the basal surface expression of CRLR-GFP/{Delta}166-RAMP2 was much lower (Fig. 2A). {Delta}139-RAMP3-mediated internalization of CRLR-GFP only differed from that mediated by wild-type RAMP3 at high AM concentrations (100 nM and 1 µM), at which time internalization of the mutant was more efficient.

Progressive Truncation of the C-Tails of hRAMP2 and -3—The results presented so far show that for hRAMP1, the C-tail is not necessary for cell surface delivery and internalization of CRLR (Figs. 2 and 4). For hRAMP2 and -3, by contrast, the respective C-tails do appear to be involved in determining the surface expression and internalization kinetics of CRLR.

To determine more precisely which sites on the C-tails of hRAMP2 and -3 regulate the cellular trafficking of CRLR/RAMP heterodimers, we constructed a group of progressive C-tail truncation mutants (Fig. 1) and then transfected each RAMP construct into HEK-293 cells stably expressing CRLR-GFP. When {Delta}166- or {Delta}167-RAMP2 was individually transfected into HEK-293 cells, its transfection efficiency (~15%) was comparable with that for wild-type RAMP2 (Fig. 5). Moreover, immunocytochemical analysis showed that almost all of both mutants remained in the ER, representing a pool of newly synthesized molecules not yet transported to the cell surface (Fig. 6). Although they were transfected into CRLR-GFP-expressing cells, however, {Delta}166- and {Delta}167-RAMP2 largely failed to transport CRLR-GFP to the cell surface, resulting in significant reductions in specific 125I-AM binding and AM-evoked cAMP production (Table 1). This suggests that the 168th and 169th amino acids (SK sequence) of RAMP2 are important for cell surface delivery of CRLR. The reason for the discrepancy between the IC50 and EC50 remains unclear, however. By contrast, {Delta}168- and {Delta}169-RAMP mediated substantially greater levels of cell surface CRLR-GFP expression, so that AM binding and signaling were equivalent to that seen with wild-type RAMP2 (Table 1). All of the RAMP3 truncation mutants appeared together with CRLR-GFP at the cell surface (Fig. 6A), and the resultant receptors showed AM binding and signaling that was comparable with that seen with wild-type RAMP3 (Table 1).


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  		TABLE 1
Characterization of HEK-293 cells expressing CRLR-GFP and progressive V5-RAMP2 and -3 truncation mutants The indicated RAMP constructs were transiently transfected into CRLR-GFP-expressing HEK-293 cells. Surface expression of each construct was estimated by flow cytometry. The results represent the means ± S.E. of three independent experiments.

 


Figure 4
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  		FIGURE 4.
FACS analysis of the time course of agonist-induced internalization of hCRLR with hRAMPs or their C-terminal deletion mutants. A, time-dependent loss of surface CRLR/RAMP heterodimers. HEK-293 cells stably expressing CRLR-GFP were transiently transfected with V5-RAMPs or their C-terminal deletion mutants and then treated with 1 µM {alpha}CGRP or AM for the indicated times. Cell surface expression of each construct was estimated by flow cytometry. The symbols represent means ± S.E. of three independent experiments; *, p < 0.006 versus control. B, agonist-evoked internalization of hCRLR with hRAMPs or their deletion mutants. Each transfectant was incubated for 60 min with the indicated concentrations of {alpha}CGRP or AM. Cell surface expression of each construct was estimated by flow cytometry. Symbols represent means ± S.E. of three independent experiments; *, p < 0.03 versus control.

 


Figure 5
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  		FIGURE 5.
FACS analysis of whole cell expression of hRAMP2 and its truncation mutants. The indicated RAMP constructs were transiently transfected into HEK-293 cells. Forty-eight hours after transfection, cells were permeabilized with IntraPrepTM reagent and then incubated for 15 min at room temperature with anti-V5-FITC antibody; mock incubation with the antibody served as the control. Whole cell expression of each construct was estimated by flow cytometry. The symbols represent means ± S.E. of three independent experiments; *, p < 0.001 versus control.

 
Among the RAMP2 mutants, {Delta}166 significantly reduced internalization of CRLR-GFP (Fig. 7). Conversely, the {Delta}139- and {Delta}140-RAMP3 mutants mediated significantly greater CRLR-GFP internalization than wild-type RAMP3 (Fig. 7). Such increases were not seen with {Delta}141-RAMP3, and CRLR-GFP internalization mediated by {Delta}142, {Delta}143, and {Delta}144 was equal to that seen with wild-type RAMP3. Thus, the presence of amino acids 141 and 142 (SK sequence) of RAMP3 leads to significant decreases in CRLR internalization.

Characteristics of hRAMP C-tail Chimeras—The SK sequence is highly conserved in the C-tails of all three hRAMPs (Fig. 1) as well as in RAMP isoforms from other species (17). We therefore tested whether exchanging C-tails would affect the cellular trafficking of CRLR-GFP. Given that hRAMP2 promoted CRLR internalization more effectively than other hRAMP isoforms did and that there were no differences in CRLR surface delivery and internalization by hRAMP1 and -3, we constructed four RAMP chimeras (RAMP1/2, -2/1, -2/3, and -3/2) by taking advantage of unique restriction sites that enabled us to generate four hybrid genes. These RAMP chimeras were then transiently transfected into HEK293 cells stably expressing CRLR-GFP and characterized by FACS analysis. As shown in Table 2, cell surface expression and evoked CRLR-GFP internalization of all four chimeras was comparable with that seen with the respective wild-type RAMPs. Thus, exchanging C-tails did not affect RAMP-mediated surface delivery or internalization of CRLR.


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  		TABLE 2
FACS analysis of cell surface delivery and internalization of CRLR-GFP and V5-RAMP chimeras in which the C-tails were exchanged among the three RAMPs The indicated V5-RAMPs or their chimeras were transiently transfected into CRLR-GFP-expressing HEK-293 cells. Surface expression of each construct was estimated by flow cytometry before and after exposing cells to 1 µM {alpha}CGRP or AM for 60 min. The results represent the means ± S.E. of three independent experiments.

 


Figure 6
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  		FIGURE 6.
Intracellular localization of V5-hRAMP2 and its truncation mutants. The indicated RAMP2 constructs were transiently transfected into HEK-293 cells, after which their subcellular distribution was assessed by confocal microscopy in permeabilized cells using anti-V5-FITC antibody. Calnexin (for the ER) was visualized using the appropriate secondary antibody (Alexa Fluor® 594). Co-localization with the ER marker was determined by overlay of the images (lower panels). HEK-293 cells stably expressing CRLR-GFP served as the control for co-localization of CRLR-GFP and the ER (left panels). Bar, ~10 µm.

 
Effect of hRAMP C-tail Deletion on Receptor Recycling—For recycling studies, cells were pretreated with 10 µg/ml cycloheximide and 10 µg/ml brefeldin A to respectively inhibit de novo protein synthesis and cause disassembly of the Golgi apparatus (29); neither of these reagents had any effect of their own on CGRP- or AM-induced internalization of CRLR-GFP/RAMPs (data not shown). Subsequent treatment of cells with 1 µM {alpha}CGRP or AM for 60 min elicited a loss of cell surface receptors that persisted for at least 2 h after washing out the ligands (Fig. 8A). Very similar results were seen with {Delta}139-RAMP1, {Delta}166-RAMP2, and {Delta}139-RAMP3, suggesting the RAMP C-tails are not involved in lysosomal sorting of CRLR or the binding of protein(s) that determine the fate of internalized receptors.

Internalization and Recycling of AM2 Receptors in Cells Cotransfected with hNSF—It was recently shown that, unlike NHERF, NSF contains no recognizable PDZ domains (30) but nonetheless interacts with the PDZ motif of hRAMP3, enabling internalized AM2 receptors to undergo slow recycling (18). Conversely, NSF enhances beta-arrestin 1-mediated beta2-AR internalization (31). Notably, although rat and mouse RAMP3 C-tails contain a RLL sequence instead of a PDZ motif, NSF also promotes recycling of these CRLR/RAMP3 heterodimers (18). With the aim of better understanding the role of NSF in AM2 receptor trafficking, in the present study, we tested whether the reported effects of hNSF on CRLR/RAMP3 trafficking are reproduced in HEK-293 cells endogenously expressing hNSF and in hNSF transfectants.

We first confirmed that NSF was indeed endogenously expressed in HEK-293 cells by identifying both NSF mRNA and protein in the cells (Fig. 8, B and C). Thereafter, we determined that the levels of the transcript were unaffected by transfection of CRLR-GFP alone or together with V5-RAMP3 (Fig. 8D). When NSF was transfected into otherwise untransfected HEK-293 cells and into CRLR-GFP transfectants, levels of its transcript were markedly higher than their endogenous levels in both (Fig. 8B). In those cases, Western analysis of NSF protein yielded a single, strong 76-kDa band that was identical to the band obtained when endogenous NSF was probed (Fig. 8C), which confirmed that our NSF transfection system worked appropriately. Exposure to 10 nM or 1 µM AM for 60 min induced a rapid decline in cell surface CRLR-GFP/V5-RAMP3 that persisted for at least 4 h after washing out the AM (Fig. 8D). Cotransfection of NSF had no effect on these internalization kinetics, and recycling of CRLR/RAMP3 heterodimers, if it occurred, was highly inefficient, even in the presence of abundant NSF (Fig. 8D).


Figure 7
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  		FIGURE 7.
FACS analysis of internalization of hCRLR with progressive hRAMP2 and -3 truncation mutants. The indicated RAMP constructs were transiently transfected into CRLR-GFP-expressing HEK-293 cells. Surface expression of each construct was estimated by flow cytometry before and after exposing cells to 1 µM AM for 60 min. The results represent the means ± S.E. of three independent experiments; *, p < 0.003 versus corresponding control (V5-RAMP2 or -3).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Since their discovery, there have been only a few reports addressing the functions of the respective C-tails of the three RAMP isoforms (17–19, 32, 33); more extensively studied have been their extracellular domains (11–13, 17, 32, 34, 35). Consequently, whereas nearly all highly conserved residues are known to play key roles in the function and trafficking of cell surface receptors, nothing is known about the functions of the highly conserved Ser and Lys resides within RAMP C-tails. In the present study, we showed that deleting the C-tail from RAMP3 ({Delta}139-RAMP3) significantly enhanced AM-induced CRLR-GFP internalization but did not affect the targeting of CRLR-GFP to the cell surface or AM binding and signaling. It is therefore unlikely that the truncation of RAMP3 promoted AM-induced conformational changes in the heterodimeric receptors that would alter the binding of AM and/or the interaction of the receptor with G proteins. On the other hand, like wild-type RAMP3, all of the tested RAMP3 truncation mutants that contained the SK sequence ({Delta}142-, {Delta}143-, and {Delta}144-RAMP3) mediated CRLR-GFP internalization less efficiently than those that did not ({Delta}139-, {Delta}140-, or {Delta}141-RAMP3). We also found that substituting the RAMP3 C-tail with the RAMP2 C-tail, which also contains a SK sequence, had no effect on the AM-induced CRLR-GFP internalization. Taken together, these results suggest that the SK sequence participates in the negative regulation of CRLR/RAMP3 internalization.

The Ser/Thr residues present in the C-tails of the three RAMP have been thought to be potential phosphorylation sites, but Hilairet et al. (6) showed that in HEK-293 cells overexpressing CRLR/RAMP heterodimers, agonists rapidly promote phosphorylation of CRLR but not RAMP. They also demonstrated that internalization of the heterodimeric receptors was dependent on beta-arrestins (6). In the present study, complete removal of the respective RAMP C-tails did not diminish the maximal extent of internalization. It therefore seems unlikely that beta-arrestins interact with the RAMP C-tails.

Similar in function to the SK sequence, a dileucine (LL) motif, which is conserved among GPCRs (36), is also present in the C-tail of RAMP3. This motif negatively regulates lutropin/choriogonadotropin receptor (LHR) internalization, since leucine-to-alanine mutations increased the agonist-stimulated internalization of the receptor (37). It is thought that the LL motif participates in protein sorting through direct interaction with two clathrin adaptor protein (AP) complexes, AP-1 and AP-2 (38–40), and the point mutations disrupted the interaction of the LHR with AP-2 at the plasma membrane (39). On the other hand, these mutations are believed to enhance the binding of beta-arrestins to the LHR, thereby promoting bridge formation between beta-arrestins and clathrin (37). We suggest that, instead of beta-arrestins, the RAMP3 C-tail may interact with other intracellular proteins similar to LRP6, another GPCR accessory protein that interacts with axin and catinin (41).

We believe it is noteworthy that the hRAMP3 C-tail contains not only a LL sequence but also a type I PDZ binding sequence (see Fig. 1). Recently, NHERF-1 was found to interact with the PDZ motif of hRAMP3, resulting in complete inhibition of CRLR/RAMP3 internalization (33). In that case, NHERF-1 is thought to act by tethering surface AM2 receptors to the actin cytoskeleton in a manner also seen with epidermal growth factor receptors (42). By contrast, NHERF-1 promotes the agonist-mediated recycling of beta2-ARs, which also have a PDZ motif (SLL) (30). The mechanism by which NHERF-1 exerts these differing effects on different GPCRs remains unknown. In the present study, three RAMP3 truncation mutants ({Delta}142-, {Delta}143-, and {Delta}144-RAMP3) and the RAMP3/2 chimera, all of which lack both the LL and PDZ sequences, failed to enhance the AM-induced CRLR-GFP internalization. However, this does not preclude the possibility that the level of endogenous NHERF-1 expression in HEK-293 cells used was insufficient to modulate the behavior of the overexpressed RAMP3.

We also showed that deleting the C-tail of RAMP2 impaired the targeting of the CRLR-GFP to the cell surface, thereby markedly reducing AM binding and signaling. Most of the newly synthesized {Delta}166-RAMP2 remained in the ER along with CRLR-GFP. By contrast, removing the C-tail from RAMP1 or -3 did not diminish surface delivery of the respective receptors. All of the RAMP2 mutants containing an SK sequence ({Delta}168-, {Delta}169-, and {Delta}172-RAMP2) showed better surface CRLR-GFP expression than was seen with {Delta}166-RAMP2, which lacked the SK sequence. Apparently, the SK sequence in the RAMP2 C-tail is involved in the proper membrane localization of the CRLR/RAMP2. To our knowledge, there have been no studies on the relation between the SK sequence and surface delivery of other GPCRs, but the LL sequence in the C-tail of the V2 vasopressin receptor was found to be crucial for ER-to-Golgi transfer of that receptor, presumably by helping establish a correct and transport-competent folding state (36). Similarly, RAMPs appear to mediate transport of the CRLR from the ER to the Golgi, since CRLR was restricted to the ER in the absence of RAMPs (6). It therefore seems likely that the SK sequence in the RAMP2 C-tail, but not that in the RAMP3 C-tail, is essential for the escape of CRLR from the ER. The mechanism underlying the differential effect of the SK sequence on the cellular trafficking of these two AM receptors remains to be determined.


Figure 8
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  		FIGURE 8.
The fates of internalized hCRLR and hRAMPs and their C-terminal deletion mutants. A, FACS analysis of recycling of the internalized heterodimeric receptors. HEK-293 cells stably expressing CRLR-GFP were transiently transfected with each RAMP construct (symbols are the same as in Fig. 3B), after which they were incubated for 60 min with 1 µM {alpha}CGRP or AM plus 10 µg/ml cycloheximide and 10 µg/ml brefeldin A. The cells were then washed with prewarmed PBS and labeled with the indicated antibodies. Cell surface expression of each construct was estimated by flow cytometry. Bars represent means ± S.E. of three independent experiments. B, endogenous and transiently transfected hNSF mRNA levels. 1, intact HEK-293 cells; 2, HEK-293 cells stably expressing CRLR-GFP; 3, NSF-transfected HEK-293 cells; 4, NSF-transfected HEK-293 cells stably expressing CRLR-GFP. Expression of NSF mRNA was assessed using real time quantitative PCR with the appropriate primers and probes. Levels of NSF mRNA were normalized to that of GAPDH mRNA, which served as an internal control. Bars represent the average of two experiments. C, Western analysis of endogenous NSF and overexpressed NSF in HEK-293 cells. Equal aliquots of protein (20 µg) were subjected to 10% SDS gel electrophoresis. Lane 1, cells expressing empty vector (Mock); lane 2, cells stably expressing CRLR-GFP; lane 3, cells transiently expressing NSF; lane 4, cells expressing both CRLR-GFP and NSF. The blots shown are representative of three independent experiments. D, FACS analysis of the recycling of CRLR and RAMP3 in the presence and absence of NSF. CRLR-GFP-expressing cells were transiently transfected with V5-RAMP3 plus empty vector or NSF, after which they were incubated for 60 min with 10 nM or 1 µM AM plus 10 µg/ml cycloheximide and 10 µg/ml brefeldin A. The cells were then treated as described in A. Cell surface expression of each construct was estimated by flow cytometry. The symbols represent means ± S.E. of three independent experiments.

 
There have been two studies on the effects of CGRP in HEK-293 cells coexpressing hCRLR with a hRAMP1 C-tail deletion mutant (19, 32). One found that the C-tail deletion resulted in a 1-order of magnitude reduction in CGRP-stimulated cAMP formation, but no data on CGRP binding were shown (19). In that case, the 140th residue Gln, but not the SK sequence, most likely determined the affinity of CGRP for the receptor. The other study found no significant changes in CGRP binding or signaling (32). Similarly, we found that deleting the C-tail of RAMP1 had little effect on receptor signaling, as reflected by CGRP-evoked cAMP production, although the binding profile we obtained differed from that obtained in the earlier study (32). This absence of a significant change in the potency of CGRP may indicate that there was little change in the internalization of CRLR-GFP. In contrast to the {alpha}CGRP responses, 125I-[Tyr0]{alpha}CGRP binding to CRLR-GFP/{Delta}139-RAMP1 was much diminished. This discrepancy could be due in part to interference by the extra N-terminal tyrosine residue (Tyr0). In any event, the contribution of the RAMP1 C-tail to CGRP potency is much smaller than that made by its extracellular domain (13, 17, 32, 34, 35).

Several earlier studies showed that internalized CRLR/RAMP heterodimers are trafficked to lysosomes for degradation (6, 10). It is now recognized that receptor ubiquitination is essential for proper trafficking to lysosomes (43). Indeed, our CRLR-GFP-transfected HEK-293 cells abundantly expressed endogenous ubiquitin, which attaches to lysine residues within the substrate proteins (data not shown). Nevertheless, truncation of RAMP C-tails that removed the Lys residues failed to promote recycling of CRLR-GFP. This raises the possibility that expression of intracellular proteins involved in mediating appropriate receptor recycling was inadequate in the HEK-293 cells used. NSF, like NHERF, is believed to enhance recycling of internalized receptors (44), and it was recently shown that NSF interacts with the PDZ motif of hRAMP3, enabling internalized AM2 receptors to undergo slow recycling (18). Notably, although rat and mouse RAMP3 tails contain an RLL sequence instead of a PDZ motif, NSF also promoted recycling of CRLR/RAMP3 heterodimers (18). In the present study, however, the cells abundantly expressed endogenous NSF, making it unlikely that poor expression of NSF underlies the trafficking of CRLR-GFP/RAMP3 to lysosomes without recycling. Even overexpression of NSF in these cells did not alter the AM-mediated trafficking of AM2 receptors.

The rapid recycling pathway has been best studied and characterized for the beta2-AR, which has a PDZ motif in its C-tail (14, 15, 30) and which requires both NHERF and NSF for its recycling (30, 44). Indeed, among 59 representative seven-transmembrane segment GPCRs tested, NSF bound most strongly to the beta2-AR tail (44). However, a point mutation within the PDZ motif that disrupted the binding of NSF, but not that of NHERF, had no effect on beta2-AR recycling (30). In addition, the beta1-AR and cystic fibrosis transmembrane regulator each contain a C-terminal PDZ binding sequence (SKV and TRL, respectively) and undergo rapid recycling, despite their failure to bind NSF (30). Thus, NSF binding to GPCRs and RAMP3 is not required for recycling of proteins containing PDZ binding sequences.

In summary, our results indicate that the C-tails of hRAMP2 and -3 are involved in hCRLR surface delivery and internalization, respectively, and that the highly conserved SK sequence within their C-tails is a key determinant of the cellular behavior of the AM1 and AM2 receptors.


   FOOTNOTES
 
* This work was supported in part by grants-in-aid for scientific research on priority areas and for the 21st Century Centers of Excellence Program (Life Science) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains one supplemental figure. Back

1 To whom correspondence should be addressed. Tel.: 81-985-85-0872; Fax: 81-985-85-6596; E-mail: kuwasako{at}fc.miyazaki-med.ac.jp.

2 The abbreviations used are: CGRP, calcitonin gene-related peptide; h{alpha}CGRP, human {alpha}CGRP; AM, adrenomedullin; hAM, human AM; CRLR, calcitonin receptor-like receptor; hCRLR, human CRLR; RAMP, receptor activity-modifying protein; C-tail, cytoplasmic C-terminal tail; ER, endoplasmic reticulum; GPCRs, G protein-coupled receptors; NSF, N-ethylmaleimide-sensitive factor; hNSF, human NSF; PDZ, PSD-95/Disc-large/ZO-1; FITC, fluorescein isothionate; PE, phycoerythrin; HEK, human embryonic kidney; GFP, green fluorescent protein; FACS, fluorescence-activated cell sorting; PBS, phosphate-buffered saline; NHERF, Na+/H+ exchanger regulatory factor; beta2-AR, beta2-adrenergic receptor. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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