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First Department of Internal Medicine, Miyazaki Medical College, University of Miyazaki, Miyazaki, Japan
Received July 29, 2003; accepted October 8, 2003.
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Abstract |
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analogs. Fluorescence-activated cell-sorting analysis revealed that their cotransfection with CTR2 induced cell surface expression of all three RAMPs, and the three CTR2/RAMP heterodimers mediated equivalent levels of cAMP production in response to hCGRP that were approximately 50-fold greater than were seen with CTR2 alone. By contrast, [Tyr0]hCGRP binding and signaling were markedly weaker with CTR2/RAMP2 or -3 than with CTR2/RAMP1 or CRLR/RAMP1; likewise, 125I-[His10]hCGRP bound most potently to CTR2/RAMP1. When CTR2 was coexpressed with DN RAMP1 or -2, hCGRP-evoked responses were similar to those seen with CTR2 alone, despite the expression of both CTR2 and DN RAMP at the cell surface. But coexpression of DN RAMP3 with CTR2 significantly diminished hCGRP signaling compared with that seen with CTR2 alone, indicating that DN RAMP3 is able to function as a negative regulator of CTR2 function. Competition experiments showed the relative agonist sensitivity of the four receptors to be hCGRP > [Tyr0]hCGRP > [Cys(Et)2,7]hCGRP > [Cys(ACM)2,7]hCGRP. Of the linear analogs, [Cys(ACM)2,7]hCGRP (ACM, acetylmethoxy) enhanced cAMP formation only via CTR2/RAMP1, whereas [Cys(Et2,7)]hCGRP acted via CRLR/RAMP1 and somewhat less potently via CTR2/RAMP1. Thus, among the three CGRP8–37-insensitive receptors, CTR2/RAMP1 is most sensitive to the two linear analogs, suggesting that it could be classified as a CGRP2 receptor. Moreover, the combined use of iodinated CGRP analogs may be useful for defining the CGRP1 receptor.
form of calcitonin gene-related peptide (CGRP) is a 37-amino acid peptide generated from alternate tissue-specific splicing of the calcitonin gene (Amara et al., 1982
), and, like adrenomedullin and amylin, it belongs to the calcitonin family of regulatory peptides (Wimalawansa, 1997). Notably, the 
form of the peptide (CGRP) is not derived from the calcitonin gene, despite its high sequence homology with CGRP (Amara et al., 1985). CGRP and - each contain a disulfide bridge between cysteine residues at positions 2 and 7 and a C-terminal phenylalanine amide, both of which are required for biological activity (Wimalawansa, 1997). Their binding sites are widely distributed among peripheral tissues and in the central nervous system, enabling CGRP to exert a wide variety of biological effects, including potent vasorelaxation (Van Rossum et al., 1997).
The existence of at least two CGRP receptor subtypes has been proposed from differential antagonist affinities and agonist potencies in a variety of in vivo and in vitro bioassays (Dennis et al., 1989, 1990; Dumont et al., 1997). The CGRP1 receptor subtype was found to be particularly sensitive to the antagonist fragment CGRP8–37 (Chiba et al., 1989; Dennis et al., 1990; Mimeault et al., 1991). By contrast, the CGRP2 receptor was sensitive to the linear analogs [Cys(ACM)2,7]and [Cys(Et)2,7]hCGRP but was insensitive to CGRP8–37 (Dennis et al., 1989, 1990; Dumont et al., 1997). In 1998, the CGRP1 receptor was identified as a heterodimer composed of a novel single transmembrane domain accessory protein, receptor activity-modifying protein 1 (RAMP1), and an orphan receptor, calcitonin receptor-like receptor (CRLR) (McLatchie et al., 1998). RAMP2 and RAMP3 also transport CRLR to the cell surface, where the CRLR/RAMP complex exhibits a 1:1 stoichiometry and forms functional adrenomedullin receptors (McLatchie et al., 1998; Hilairet et al., 2001). The three RAMPs, which share only 30% sequence identity and differ in their tissue distributions, are all composed of 
160 amino acids, which make up a large extracellular N-terminal domain, a single membrane-spanning domain, and a very short cytoplasmic domain (Sexton et al., 2001). Upon binding their respective agonist, these receptors mediate mobilization of intracellular cAMP and Ca2+ (Kuwasako et al., 2000). We recently identified the individual RAMP domains responsible for agonist binding to CRLR/RAMP heterodimers and their deletion yielded a group of dominant-negative (DN) RAMP mutants (Kuwasako et al., 2001, 2002, 2003a).
CRLR shares 55% overall amino acid sequence identity with the calcitonin receptor (CTR), although the transmembrane domains are almost 80% identical (Poyner et al., 2002). The best characterized splice variants of hCTR differ depending on the presence (CTR1) or absence (CTR2) of 16 amino acids in the first intracellular loop (Poyner et al., 2002). In that regard, cotransfection of RAMPs with the most common variant, CTR2, leads to the formation of a 1:1 dimer at the cell surface (Christopoulos et al., 1999) and augmentation of responses evoked by amylin or CGRP (Christopoulos et al., 1999; Leuthauser et al., 2000; Tilakaratne et al., 2000). The CGRP-evoked responses mediated via CTR/RAMPs were blocked by the selective receptor antagonist calcitonin8-32 but not by CGRP8–37 (Kuwasako et al., 2003b; Leuthauser et al., 2000). Although calcitonin8-32 should therefore be useful for defining the CGRP2 receptor subtype, whether the functional properties of this receptor are similar to those mediated by CTR2/RAMP heterodimers remains unclear. In addition, nothing is known about the effects of DN RAMPs on CTR2 function. We therefore cotransfected hCTR2 and individual hRAMPs into human embryonic kidney (HEK) 293 cells, which express no functional CGRP or amylin receptors, and examined their pharmacological features using the aforementioned DN RAMPs and various hCGRP peptide analogs.
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Materials and Methods |
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(specific activity, 2 µCi/pmol), which contains an extra N-terminal tyrosine residue (Tyr0), was produced in our laboratory using a modification of a method described previously (Kitamura et al., 1993; Kuwasako et al., 2003a). 125I-[His10]hCGRP (specific activity, 2 µCi/pmol) was obtained from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK). Human CGRP was purchased from Peptide Institute (Osaka, Japan). [Tyr0]hCGRP, [Cys(ACM)2,7]hCGRP (ACM, acetylmethoxy), [Cys(Et)2,7]hCGRP (Et, ethylamide), and rat amylin were from Phoenix Pharmaceuticals, Inc. (Belmont, CA). Rat FITC-conjugated monoclonal anti-HA antibody was from Roche Applied Science (Indianapolis, IN). All of the other reagents were of analytical grade and were obtained from various commercial suppliers.
Expression Constructs. Human CTR2 (Kuestner et al., 1994) and the three hRAMPs (McLatchie et al., 1998) were modified to provide a consensus Kozak sequence as described previously (Aiyar et al., 1996). An HA epitope tag (YPYDVPDYA) was ligated, inframe, to the 5' end of cDNAs encoding the intact and DN hRAMPs (DN hRAMP1, L94A/D101-103; DN hRAMP2, D86-92; DN hRAMP3, D59-65) used for CRLR/RAMP heterodimers (Kuwasako et al., 2001, 2003a), and the native signal sequences were removed and replaced with MKTILALSTYIFCLVFA (Guan et al., 1992), yielding HA hRAMPs and HA DN hRAMPs. Human CTR2 and individual HA hRAMPs and HA DN hRAMPs were cloned into the mammalian expression vector pCAGGS/Neo (Kuwasako et al., 2000) using the 5'-XhoI and 3'-NotI sites. The sequences of the resultant constructs were all verified using the 310 Genetic Analyzer (Applied Biosystems, Foster City, CA). Each of the HA hRAMPs was compared with the native sequence in the assays and was found to behave identically (data not shown).
Cell Culture and DNA Transfection. HEK 293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B at 37°C under a humidified atmosphere of 95% air/5% CO2. For experimentation, cells were seeded into 24-well plates and, upon reaching 70 to 80% confluence, were transiently cotransfected with hCTR2 plus HA hRAMP, HA DN hRAMP, or empty vector using LipofectAMINE transfection reagents (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The cells were incubated for 4 h in 250 µl of OptiMEM 1 medium containing 200 ng/well plasmid DNA, 2 µl/well Plus regent, and 2 µl/well LipofectAMINE. As a control, some cells were transfected with empty vector (pCAGGS/Neo) (Mock). All experiments were carried out 48 h after transfection.
FACS Analysis. Flow cytometry was carried out to assess the levels of whole-cell and cell-surface expression of each HA hRAMP or HA DN hRAMP mutant in HEK 293 cells. To evaluate cell-surface expression, cells were harvested after transient transfection, washed twice with PBS, resuspended in ice-cold FACS buffer (Kuwasako et al., 2000), and then incubated with anti-HA FITC antibody (1:50 dilution) for 60 min at 4°C in the dark. For the evaluation of whole-cell expression, cells were first permeabilized using IntraPrep reagents (Beckman Coulter, Fullerton, CA) according to the manufacturer's instructions and then incubated with anti-HA FITC antibody (1:50 dilution) for 15 min at room temperature in the dark. After 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).
Radioligand Binding. To assess whole-cell radioligand binding, transfected HEK 293 cells in 24-well plates were washed twice with ice-cold PBS and then incubated with 100 pM 125I-[Tyr0]hCGRP or 125I-[His10]hCGRP in binding buffer (modified Krebs-Ringers-HEPES medium) (Kuwasako et al., 2000) in the absence (total binding) or presence of increasing concentrations of unlabeled hCGRP. The total incubation volume was 300 µl. Nonspecific binding was defined as binding in the presence of 1 µM unlabeled hCGRP. After incubation for 4 h at 4°C, the cells were washed twice with ice-cold PBS and solubilized with 0.5 M NaOH, after which the associated cellular radioactivity was measured in a 
-counter.
cAMP Assay. Analysis of intracellular cAMP accumulation was carried out after transfection of the indicated cDNAs into HEK 293 cells. In Hanks' buffer solution containing 20 mM HEPES and 0.1% bovine serum albumin, the cells were exposed to the indicated concentrations of hCGRP, [Tyr0]hCGRP, [Cys(ACM)2,7]hCGRP, [Cys(Et)2,7]hCGRP, or rat amylin for 15 min at 37°C in the presence of 0.5 mM 3-isobutyl-1-methylxanthine (Sigma Chemical, St. Louis, MO). The reactions were terminated by the addition of lysis buffer (Amersham Biosciences). The resultant lysates were centrifuged at 2000 rpm for 10 min at 4°C, after which aliquots of the supernatants were collected, and the cAMP contents were determined using a commercial enzyme immunoassay kit according to the manufacturer's instructions for a nonacetylation protocol (Amersham Biosciences).
Statistical Analysis. Results are expressed as means ± S.E. of at least three independent experiments. Differences between two groups were evaluated with the use of Student's t tests. The differences among multiple groups were evaluated with one-way analysis of variance followed by Scheffé's tests. Values of P < 0.05 were considered significant.
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binding was not increased (Fig. 1B). Cotransfection of hRAMP1 with increasing concentrations of hCTR2 markedly increased specific 125I-[Tyr0]hCGRP binding, whereas hCTR2/hRAMP2 or -3 showed only small increases in the specific binding. In all cases, 125I-[Tyr0]hCGRP binding was approximately maximal when 100 ng of hCTR2 DNA was transfected (EC50 = 50 ng).
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The four hCGRP receptors generated by coexpression of each of the hRAMPs with hCTR2 or hCRLR were characterized using the two 125I-labeled CGRP analogs 125I-[Tyr0]hCGRP and 125I-[His10]hCGRP (Fig. 2). There was little specific binding of either iodinated analog to HEK 293 cells transfected with empty vector (Mock) or hCTR2 alone. The specific binding of [Tyr0]hCGRP to cells coexpressing hCTR2 with hRAMP1 was 26,000 cpm/well (nonspecific/total binding ratio = 0.11) (Fig. 2A). Similar levels of specific binding were obtained in cells coexpressing hCRLR with hRAMP1 (17,500 cpm/well), whereas the specific binding to cells coexpressing hCTR2 with hRAMP2 or -3 was somewhat lower (2800 and 4500 cpm/well, respectively) (Fig. 2A). On the other hand, hRAMP1 markedly increased levels of specific 125I-[His10]hCGRP binding when coexpressed with hCTR2 (39,800 cpm) but failed to significantly affect binding when coexpressed with hCRLR (13,100 cpm) (Fig. 2B). Much lower levels of specific binding were observed when hCTR2 was coexpressed with hRAMP2 or -3 (9200 and 6000 cpm, respectively) (Fig. 2B).
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The functionality of these receptors was assessed by measuring agonist-induced intracellular cAMP accumulation (Fig. 3). Although they endogenously express functional hCTR2 (Kuwasako et al., 2003b), high concentrations of hCGRP or [Tyr0]hCGRP elicited no significant increases in cAMP in HEK 293 cells expressing empty vector (Mock) (Fig. 3A). This may be caused by the low level of hCTR2 protein expression. In HEK 293 cells transfected with hRAMP1, however, hCGRP increased the cAMP content slightly; maximal cAMP levels (92 pmol/mg protein) reached 8-fold over baseline (data not shown). This suggests that hRAMP1 and endogenous hCTR2 are capable of forming a functional CGRP receptor (Christopoulos et al., 1999). Transfection with hCTR2 enabled hCGRP (EC50 = 3.8 nM) and [Tyr0]hCGRP (EC50 = 19 nM) to elicit significant increases in cAMP (Fig. 3B), and cotransfection of hCTR2 and hRAMP1 markedly increased the potency of hCGRP (EC50 = 0.014 nM) (Fig. 3C). hCGRP acted somewhat less potently in cells expressing hCTR2/hRAMP2 or -3 or hCRLR/hRAMP1 (EC50 = 0.20, 0.21, and 0.18 nM, respectively) (Fig. 3C). In cells expressing hCTR2/hRAMP, [Tyr0]hCGRP was approximately 10 to 20-fold less potent than hCGRP (EC50 = 0.30 nM for hCTR2/hRAMP1 versus 4.8 nM for hCTR2/hRAMP2 versus 2.3 nM for hCTR2/hRAMP3), but the two analogs were equipotent in cells expressing hCRLR/hRAMP1 (EC50 = 0.48 nM for [Tyr0]hCGRP) (Fig. 3D). Notably, the responses to [Tyr0]hCGRP closely paralleled the profile of 125I-[Tyr0]hCGRP binding (Fig. 2A). Rat amylin was highly potent in cells expressing hCTR2/hRAMP1, -2, or -3 (EC50 = 0.02, 0.35, and 0.15 nM, respectively) but not hCRLR/hRAMP1 (EC50 > 180 nM) (data not shown). Thus, coexpressed hCTR2 with hRAMP seems to form high-affinity receptors for both CGRP and amylin.
To explore further the role of hRAMP in hCTR2 function, the effects of expressing three hRAMP deletion mutants (DN hRAMPs) on the whole-cell and cell-surface expression of epitope-tagged hRAMPs were analyzed. After transient transfection, all of the mutants were expressed in HEK 293 cells at levels similar to those seen with intact hRAMPs (Table 1). Moreover, the cell-surface expression of all three mutants was significantly increased by cotransfection with hCTR2, again reaching levels similar to those seen in intact hRAMPs. This may reflect the fact that the mutants encode all of the cysteine residues and N-glycosylation sites required for cell-surface expression of RAMPs (McLatchie et al., 1998; Flahaut et al., 2002; Kuwasako et al., 2003c). Total [Tyr0]hCGRP binding obtained with mutant receptor heterodimers composed of hCTR2 complexed with each of the DN hRAMPs was not significantly different from that obtained with hCTR2 alone (Fig. 4A). The levels of specific binding of [Tyr0]hCGRP to cells coexpressing hCTR2 with hRAMP1, -2, or -3 (250–630 cpm/well) were also similar to those seen with hCTR2 alone (140 cpm/well) (Fig. 4A).
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The functionality of the receptors composed of hCRLR and each of the DN RAMPs was then evaluated by measuring hCGRP-evoked cAMP production (Fig. 4B). The EC50 values obtained in cells coexpressing hCTR2 and DN hRAMP1 or -2 (2.0 and 1.3 nM, respectively) did not differ from that obtained with hCTR2 alone (1.6 nM). Surprisingly, coexpression of hCTR2 with DN hRAMP3 reduced hCGRP-evoked cAMP accumulation by 20 to 88% at concentrations ranging from 10-11 Mto10-7 M (EC50 = 14 nM), suggesting that DN hRAMP3 is able to act as a negative regulator of hCTR2 function.
Using transfectants expressing each of the four CGRP receptors (Table 2), the affinities of various hCGRP analogs were evaluated in 125I-[Tyr0]hCGRP radioreceptor assays. With the exception of rat amylin, the IC50 values for the various analogs were similar with both hCTR2/hRAMP1 and hCRLR/hRAMP1, and the relative sensitivities of the two receptors were the following: hCGRP > [Tyr0]hCGRP > [Cys(Et)2,7]hCGRP > [Cys(ACM)2,7]hCGRP. The profiles of the competition curves obtained with hCTR2/hRAMP2 and hCTR2/hRAMP3 were similar for each of the analogs tested, although the IC50 values were much lower than were seen with hCTR2/hRAMP1 and hCRLR/hRAMP1.
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As shown in Fig. 5, the linear analogs [Cys(ACM)2,7]hCGRP and [Cys(Et)2,7]hCGRP were virtually inactive at micromolar concentrations in cAMP assays, even in cells expressing hCTR2 or hCRLR. Both linear analogs had a moderate effect on the cAMP content of HEK 293 cells expressing hCTR2/hRAMP1 (EC50 = 54 and 4.1 nM, respectively), which was 10-fold greater than was seen in cells expressing hCTR2/hRAMP2 or hCTR2/hRAMP3. The most potent responses were elicited by [Cys(Et)2,7]hCGRP in cells expressing hCRLR/hRAMP1 (EC50 = 0.56 nM). In this case, however, there was not a comparable increase in potency with [Cys(ACM)2,7]hCGRP.
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10-fold greater potency than those mediated by RAMP2 or -3. These results differ somewhat from earlier findings indicating that in monkey COS-7 cells, CGRP-evoked responses were augmented only by RAMP1, whereas amylin-evoked responses were augmented by RAMP1 and -3 (Christopoulos et al., 1999; Leuthauser et al., 2000; Tilakaratne et al., 2000). We suggest that although both COS-7 and HEK 293 cells are derived from kidney, this discrepancy mainly reflects differences in cell background. In contrast to CRLR, a large number of CTR2 molecules appeared at the surface of HEK 293 cells transfected with 100 ng of CTR2 but not those cotransfected with RAMP, and the cell-surface expression levels were unaffected by RAMP transfection (100 ng) (Kuwasako et al., 2003b). Conversely, CTR2, like CRLR, increased the appearance of epitope-tagged RAMP molecules at the cell surface in this and earlier studies (McLatchie et al., 1998; Kuwasako et al., 2002, 2003a). Taken together, these results suggest that although RAMP accessory proteins do not chaperone CTR2 to the cell surface as they do CRLR, they nevertheless interact with CTR2 effectively modifying the binding of both CGRP and amylin.
In addition to the cell-surface RAMP expression, 125I-[Tyr0]hCGRP binding was found to reach approximately maximum levels with transfection of 100 ng of hCTR2 DNA (Fig. 1). Christopoulos et al. (1999) previously investigated concentration-dependent effects of each hRAMP on 125I-labeled amylin binding in HEK 293 cells transiently transfected with 100 ng of hCTR2. In that study, too, specific 125I-amylin binding was almost maximal with cotransfection of 100 ng of hRAMP1 or hRAMP3. Moreover, using covalent cross-linking analysis, those investigators were able to show that hRAMPs form a 1:1 dimer at the cell surface. Collectively, those results, along with the findings presented herein, suggest that the CTR2/RAMP complex exhibits 1:1 stoichiometry, although we have no data directly measuring the molar ratio of individually expressed proteins.
We have iodized the tyrosines of various vasoactive peptides, including adrenomedullin (Kitamura et al., 1993), and in the present study, we iodized a tyrosine residue added to the N terminus of hCGRP. In cells transfected with hCTR2 alone, both hCGRP and [Tyr0]hCGRP elicited moderate increases in cAMP, albeit that [Tyr0]hCGRP responses were 10-fold weaker than those evoked by hCGRP. Nevertheless, there was little specific binding of 125I-[Tyr0]hCGRP to hCTR2. This is probably because the structures added at the N terminus of CGRP interfere with the nearest disulfide bond required for both binding to the receptor and biological activity. Interestingly, cotransfection of hRAMP1, but not hRAMP2 or -3, led to significant increases in the specific binding of 125I-[Tyr0]hCGRP to hCTR2 and in [Tyr0]hCGRP-evoked responses, although [Tyr0]hCGRP exhibited 10 to 20 times less potency for the three hCTR2/hRAMP heterodimers than for hCGRP. In contrast, hCRLR/hRAMP1 responded equally to both analogs, which suggests that [Tyr0]hCGRP could be used to define the non-CGRP1 receptor subtype. Interestingly, 125I-[His10]CGRP bound most potently to cells expressing hCTR2/hRAMP1, and binding to hCRLR/hRAMP1 was much lower than the levels seen with 125I-[Tyr0]CGRP. This suggests that the combined use of the two iodinated CGRP analogs may enable the definition of the CGRP1 receptor.
Recent cross-linking and mutagenesis studies suggest that RAMPs determine ligand specificity by contributing to the structure of the ligand-binding pocket or by allosteric modulation of the conformation of CRLR (Hilairet et al., 2001; Kuwasako et al., 2001, 2003a). The responsible domains were found to be a seven-residue segment situated between three residues (Trp to Cys and Tyr) in human and rat RAMP2 and RAMP3 (Kuwasako et al., 2001, 2002). Moreover, their deletion (D) mutants were able to serve as negative regulators of endogenous adrenomedullin receptor function (Kuwasako et al., 2001, 2002). Very recently, expression of an hRAMP1 L94A/D101-103 double mutant was found to markedly attenuate the activity of endogenous hCGRP receptor (Kuwasako et al., 2003a). Although there have been several studies of the relative affinities of various RAMPs for CRLR (Muff et al., 1998; Buhlmann et al., 1999; Husmann et al., 2000), it remains unclear whether the inhibition of receptor function by DN RAMP mutants was caused by competitive inhibition, formation of heterodimeric complexes, or both. In the present study, hCGRP-evoked responses in cells coexpressing hCTR2 with hRAMP1 L94A/D101-103 or hRAMP2 D86-92 were not enhanced, despite hCTR2-induced cell-surface expression of the RAMP mutants. This raises the possibility that both mutants might also inhibit the function of endogenous hCTR2/hRAMP receptors. It is of particular interest to us that coexpression of hRAMP3 D59-65 with hCTR2 led to a significant reduction in hCGRP signaling via hCTR2 because it suggests that D59-65 is able to function as a negative regulator for hCTR2 function.
It has been reported that the linear CGRP analogs [Cys(ACM)2,7]- and [Cys(Et)2,7]hCGRP are reportedly more potent agonists at CGRP2 than at CGRP1 receptors (Dennis et al., 1989; Dumont et al., 1997; Moreno et al., 2002). Our competition experiments showed the relative agonist sensitivity of the four CGRP receptors tested to be hCGRP > [Cys(Et)2,7]hCGRP > [Cys(ACM)2,7]hCGRP. We also showed that [Cys(ACM)2,7]hCGRP weakly stimulated cAMP formation only via CTR2/RAMP1 (EC50 = 54 nM), whereas [Cys(Et2,7)]hCGRP acted via both hCRLR/hRAMP1 (EC50 = 0.56 nM) and hCTR2/hRAMP1 (EC50 = 4.1 nM). The EC50 values reflecting the affinity of [Cys(ACM)2,7]and [Cys(Et2,7)]hCGRP for hCTR2/hRAMP1 were very similar to those obtained in the rat vas deferens, which is believed to express typical CGRP2 receptors (Dennis et al., 1989; Dumont et al., 1997; Juaneda et al., 2000). Collectively, these results suggest that among the three CGRP8–37–insensitive receptors, CTR2/RAMP1 could be classified as a CGRP2 receptor, although a single use of [Cys(Et2,7)]hCGRP could not draw a sharp distinction between the CGRP1 and CGRP2 receptor subtypes. Because RAMPs, CTR, and CRLR commonly coexist in native tissues and cells (Sexton et al., 2001; Poyner et al., 2002), additional studies will be needed to clarify the precise relationships among the endogenously expressed agonists, receptors, and receptor functions.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: CGRP, calcitonin gene-related peptide; CT, calcitonin; CTR, calcitonin receptor; RAMP, receptor activity-modifying protein; CRLR, calcitonin receptor-like receptor; ACM, acetylmethoxy; Et, ethylamide; h, human; DN, dominant-negative; FITC, fluorescein isothionate; HA, human hemagglutinin; FACS, fluorescence-activated cell-sorting; HEK, human embryonic kidney; PBS, phosphate-buffered saline; D, deletion; Mock, empty vector pCAGGS/Neo.
Address correspondence to: Dr. Kenji Kuwasako, First Department of Internal Medicine, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan. E-mail: kuwasako{at}fc.miyazaki-med.ac.jp
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) and nonspecific (
) binding. Cells were transiently transfected with the indicated plasmids and then incubated for 4 h at 4°C with 100 pM 125I-[Tyr0]hCGRP
, P < 0.01 versus corresponding nonspecific binding).
