From the First Department of Internal Medicine, Miyazaki Medical College, Miyazaki 889-1692, Japan
Received for publication, March 13, 2003 , and in revised form, April 7, 2003.
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ABSTRACT |
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INTRODUCTION |
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RAMPs are a recently identified group of single transmembrane domain accessory proteins that serve to transport CRLR to the cell surface where they form functional CGRP and AM receptors (6). The three RAMP isoforms (RAMP1, RAMP2, and RAMP3), which share only 30% sequence identity and differ in their tissue distributions, are all comprised of 160 amino acids that make up a large extracellular N-terminal domain, a single membrane-spanning domain, and a very short cytoplasmic domain (6, 7). Co-expression of CRLR with RAMP1 leads to both proteins being presented at the plasma membrane as a heterodimeric CGRP receptor, whereas co-expression of CRLR with RAMP2 or RAMP3 enables the resultant heterodimer to function as a AM receptor (6, 8, 9). Upon binding their respective agonist, both receptors mediate a rise in intracellular cAMP levels as well as Ca2+ mobilization and undergo internalization and intracellular trafficking with similar kinetics (9). One recent study (10) shows that the CRLR/RAMP1 heterodimer functions fully as both an AM and a CGRP receptor, which may explain why many actions of AM are potently antagonized by the CGRP receptor antagonist CGRP-(837).
An analysis of various RAMP1/2 chimeras showed the extracellular N-terminal domain to be crucial for defining CGRP and AM selectivity (11), which is consistent with earlier radioligand binding and functional assays (8). A more detailed analysis using various deletion mutants showed that a seven-residue segment situated between the residues (Trp-Cys and Tyr) conserved in human, rat, and mouse RAMP2 and RAMP3 is essential for high affinity agonist binding to AM receptors and that RAMP mutants in which these segments were deleted act as negative regulators of AM receptor function (12, 13). By contrast, there has been no detailed analysis of the extracellular domain(s) of RAMP1 that confer agonist specificity, although sequential truncation mutation of hRAMP1 showed the transmembrane domain to be critical for the functional expression of a CGRP receptor (14). To address this question, we analyzed the effects of co-expression in HEK-293 cells of CRLR with human (h) RAMP1 containing various deletion mutations in its extracellular domain. Our results indicate that residues 91103 of hRAMP1 are key determinants of high affinity agonist binding to the CRLR/RAMP1 heterodimer and that their deletion results in the formation of a dominant negative mutant capable of inhibiting endogenous hRAMP1 function.
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EXPERIMENTAL PROCEDURES |
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Expression ConstructsHuman CRLR and RAMP1 were modified to provide a consensus Kozak sequence as described previously (15). A HA epitope tag (YPYDVPDYA) was ligated in-frame to the 5' end of the hRAMP1 cDNA, and the native signal sequences were removed and replaced with MKTILALSTYIFCLVFA, yielding HA-hRAMP1 (16). Human CRLR and HA-hRAMP1 were cloned into the mammalian expression vector pCAGGS/Neo (9) using the 5'-XhoI and 3'-NotI sites. The sequences of the resultant constructs were all verified using an Applied Biosystems 310 genetic analyzer. HA-hRAMP1 was compared with the native sequence in the assays and was found to behave identically (data not shown).
Deletion mutations and single amino acid substitutions were carried out using a QuikChange kit (Stratagene, San Diego, CA) according to the manufacturer's instructions with pIRES-HA-hRAMP1, which was constructed by subcloning the coding sequence of HA-hRAMP1 into pIRES1/Neo (Clontech, Palo Alto, CA). For each mutation, two complementary 30-mer oligonucleotides (sense and antisense) were designed to contain the desired mutation in their middle. To enable rapid screening of mutated clones, the primers carried an additional silent mutation introducing (or removing) a restriction site. The presence of each mutation of interest and the absence of undesired ones were confirmed by DNA sequencing. Individual HA-hRAMP1 mutants were then cloned into pCAGGS/Neo.
Cell Culture and DNA TransfectionHEK-293 and HEK-293T 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 7080% confluence, were transiently co-transfected with hCRLR plus HA-hRAMP1, one of the HA-tagged hRAMP1 mutants, or empty vector using LipofectAMINE transfection reagents (Invitrogen) 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 reagent, and 2 µl/well LipofectAMINE. As a control, some cells were transfected with empty vector (pCAGGS/Neo) (Mock). All of the experiments were performed 48 h after transfection.
FACS AnalysisFlow cytometry was carried out to assess the levels of whole cell and cell surface expression of HA-hRAMP1 or the HA-hRAMP1 mutant 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 (9), and then incubated with anti-HA-FITC antibody (1:50 dilution) for 60 min at 4 °C in the dark. 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-HA-FITC antibody (1:50 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).
Radioligand BindingTo assess whole-cell radioligand binding, transfected HEK-293 cells in 24-well plates were washed twice with prewarmed PBS and incubated for 20 min at 37 °C with 0.1% bovine serum albumin/PBS to reduce endogenous AM binding, after which the remaining adherent cells were washed with ice-cold PBS. The cells were then incubated for 4 h at 4 °C with [125I]hCGRP (100 pM) in the presence (for nonspecific binding) or absence (for total binding) of 1 µM unlabeled h
CGRP in modified Krebs-Ringer-HEPES medium (9). They were then washed twice with ice-cold PBS and harvested with 0.5 M NaOH, and the associated cellular radioactivity was measured in a
-counter.
cAMP AssayThe analyses of intracellular cAMP accumulation were carried out following transfection of the indicated cDNAs into HEK-293 or HEK-293T 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 or hAM for 15 min at 37 °C in the presence of 0.5 mM 3-isobutyl-1-methylxanthine (Sigma). 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 content were determined using a commercial enzyme immuno-assay kit according to the manufacturer's (Amersham Biosciences) instructions for a nonacetylation protocol.
mRNA Expression Measured by Real-time Quantitative PCRTotal RNA was extracted from transfected HEK-293 or HEK-293T cells using Total RNA isolation reagent (Invitrogen) and then reverse-transcribed using SuperScript reverse transcriptase (Invitrogen), yielding the respective cDNAs. The expression of mRNAs encoding hCRLR, the three hRAMP isoforms, and hAM was assessed using real-time quantitative PCR (Prism 7700 Sequence Detector, Applied Biosystems, Foster City, CA) as described previously (17, 18). Oligonucleotide primers and fluorescently labeled probes were prepared as described previously (10, 18). The levels of hCRLR, hRAMP, and hAM mRNA were normalized to those of glyceraldehyde-3-phosphate dehydrogenase mRNA, which served as an internal control. DNA sequence analysis confirmed that the amplified products were identical to those of hCRLR and the three hRAMP isoforms (6, 19, 20).
Statistical AnalysisResults are expressed as means ± S.E. of at least three independent experiments. Differences between two groups were evaluated with Student's t test. The differences among multiple groups were evaluated with one-way analysis of variance followed by Scheffe's tests. Values of p < 0.05 were considered significant.
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RESULTS |
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We initially analyzed the whole-cell expression of epitope-tagged hCRLR mutants in permeabilized cells using FACS (Fig. 2A). Immunoreactivity was detected in only 1.9% cells expressing the empty vector (Mock), which is well within the 2% limit of resolution characteristic of FACS analysis. When expressed alone or with hCRLR, HA-hRAMP1 was detected in 60.3 and 58.8% of cells, respectively. Similarly, co-expression of hCRLR with one of the hRAMP1 deletion mutants led to their full expression in 3752% cells. Thus, the transfection efficacy for the mutant receptor was comparable with that for hCRLR/HA-hRAMP1. Moreover, the staining pattern suggested that these proteins were probably in the endoplasmic reticulum, representing a pool of newly synthesized molecules not yet transported to the cell surface (data not shown).
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We next analyzed the cell surface expression of the hRAMP1 mutants in nonpermeabilized cells (Fig. 2B). Empty vector and HA-hRAMP1 appeared at the surface of 1.7 ± 0.10% and 4.2 ± 0.28% of cells, respectively, which is consistent with earlier studies showing that most HA-hRAMP1 molecules are not transported to the cell surface when expressed alone (6, 23). When hCRLR was co-expressed with HA-hRAMP1, cell surface immunoreactivity was detected in 23.7 ± 2.3% cells. Among the 17 deletion mutants, D2833, D3439, D7476, D7880, D105107, D109112, and D113118 were detected at the surface of 1430% cells when co-transfected with hCRLR, which is comparable with the frequency seen with hCRLR/HA-hRAMP1. Cell surface immunoreactivity was detected in fewer cells co-transfected with hCRLR and each of the remaining 10 mutants (713%), but it still tended to be higher than was seen with transfection of HA-hRAMP1 alone.
The functionality of the hRAMP1 mutants was assessed by measuring agonist-induced intracellular cAMP accumulation (Fig. 3). Neither 10 nM hCGRP nor hAM elicited significant increases in cAMP in nontransfected HEK-293 cells or cells transfected with HA-hRAMP1 or hCRLR alone. On the other hand, the coexpression of hCRLR with HA-hRAMP1 enabled CGRP and AM to each elicit significant increases in cAMP, which is consistent with previous reports that CGRP and AM bind to the CRLR/RAMP1 heterodimer with similar affinities (8, 9, 12, 24, 25). Similar CGRP- and AM-evoked increases in cAMP levels were seen when hCRLR was co-expressed with D7476, D105107, or D109112. By contrast, cells expressing hCRLR with D2833, D3439, D4650, D5155, D7880, D8386, D8890, or D113118 responded more selectively to CGRP. In particular, CGRP-induced cAMP production mediated by hCRLR/D7880 or D8890 was nearly 10-fold greater than that induced by AM, despite the fact that [125I]CGRP binding to the mutant receptors was much diminished (Fig. 4).
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Six of the mutants (D4145, D5965, D6771, D9194, D96100, and D101103) did not support either [125I]CGRP binding (Fig. 4) or agonist-evoked cAMP accumulation (Fig. 3), even though their cell surface expression was similar to that of hCRLR/D7880 and D8890, which did support receptor function. To better understand what accounts for this difference, we carried out a detailed FACS analysis in which the behavior of D4145, D5965, D6771, D9194, D96100, and D101103 was compared with that of D4650, D5155, and D8890. When whole-cell expression of the epitope-tagged hRAMP1 mutants was analyzed, we found that all were expressed in 4253% cells and these levels changed little when the mutants were co-transfected with hCRLR (Fig. 5A). An analysis of their cell surface expression showed that when the mutants were expressed alone, cell surface immunoreactivity was detected in 3.34.7% cells. This is comparable with the levels seen with transfection of HA-hRAMP1 alone (compare Figs. 2B and 5B) and suggests that similar to HA-hRAMP1, these mutants interact with the small amounts of endogenous CRLR or calcitonin receptor expressed in HEK-293 cells (22, 26). This interaction is not sufficient to produce any detectable [125I]CGRP binding or mediate CGRP-evoked cAMP production (Figs. 3 and 4).
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Cell surface expression of all of the mutants was significantly increased when they were co-transfected with hCRLR. In particular, cell surface immunoreactivity was detected in 9.4 ± 0.12% cells co-expressing D5155 and hCRLR. This is 2.7-fold higher than that observed in the absence of hCRLR and led to significant increases in both [125I]CGRP binding and CGRP-induced cAMP accumulation (Figs. 3 and 4). Similar levels of cell surface immunoreactivity (8.89.3%) were detected in cells expressing hCRLR and D9194, D96100, or D101103. However, although their cell surface expression was 2.0- to 2.4-fold greater than in cells not expressing hCRLR, there was a complete absence of [125I]CGRP binding and signaling.
Single Amino Acid Substitutions of Residues 91103 of hRAMP1The aforementioned findings suggest that CGRP selectivity is determined within a region of hRAMP1 spanning amino acids 91103. To assess the extent to which any single amino acid residue within that segment affects the capacity of CGRP to induce cAMP production, residues 91103 were substituted one at a time with alanine. We initially analyzed the cell surface expression of epitope-tagged mutants using FACS (Fig. 6). Cell surface immunoreactivity was detected in 24.0 ± 1.0% cells co-transfected with HA-hRAMP1 and hCRLR. Co-transfection of hCRLR with F93A, Y100A, or F101A led to their expression at the cell surface in only 811% cells, although the proteins were observed to be diffusely distributed throughout the cytoplasm of most cells (data not shown). The other eight mutants (R91A, F92A, V96A, H97A, G98A, R99A, F102A, and S103A) appeared at the surface of 1824% cells, a level comparable to hCRLR/HA-hRAMP1. Apparently, the cell surface expression of D9194, D96100, and D101103 is specifically inhibited by the substitution of Phe93, Tyr100, and Phe101, respectively. Surprisingly, cell surface expression of hCRLR/L94A was 1.6-fold higher than that of hCRLR/HA-hRAMP1.
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As a negative control, we also constructed a C104A mutant because this cysteine residue is conserved among all three RAMPs and is essential for the cell surface expression of the receptor (21). As expected, hCRLR/C104A was detected at the surface of only 4% cells.
To confirm their ligand selectivity, we examined the binding of [125I]CGRP to receptor heterodimers comprised of hCRLR complexed with the indicated point mutant (Fig. 7). The specific CGRP binding to cells co-expressing hCRLR and HA-hRAMP1 was 7300 cpm/well, and the nonspecific/total binding ratio was 0.26. Similar levels of specific binding (53007400 cpm/well) were seen in cells co-expressing hCRLR with R91A, V96A, R99A, R102A, S103A, D7678, or D7982, whereas levels of specific binding in cells co-expressing hCRLR with F92A, F93A, H97A, or Y100A were somewhat lower (17002400 cpm/well). Consistent with their level of presentation at the cell surface (Fig. 6), CGRP binding to cells expressing hCRLR with L94A or G98A was 42 and 27%, respectively, higher than in cells expressing hCRLR/HA-hRAMP1. Conversely, CGRP binding to cells co-expressing hCRLR with F101A was 94% lower than in cells expressing hCRLR/HA-hRAMP1.
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The functionality of the receptors comprised of hCRLR and each of the point mutants was then evaluated by measuring CGRP-evoked cAMP production. Consistent with the binding assays (Fig. 7), the C104A substitution markedly impaired CGRP-evoked cAMP production. In all of the other cases, however, the EC50 values obtained with the mutant receptors were not significantly different from that obtained with hCRLR/HA-hRAMP1 (Table I). Cells expressing hCRLR/L94A exhibited the strongest responses, which were 2.5-fold greater than in cells expressing hCRLR/HA-hRAMP1, whereas cells expressing hCRLR/F101A exhibited the smallest response, which was 42% smaller than that seen with hCRLR/HA-hRAMP1. Taken together, these results indicate that among residues 91103 of hRAMP1, no single amino acid contributes significantly to the ligand selectivity of the CRLR/RAMP1 heterodimer.
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Characterization of hRAMP1 Double MutantsTo further examine the effects of the L94A point mutation on the cell surface expression of D9193, D96100, and D101103, we constructed three double mutants, L94A/D9193, L94A/D96100, and L94A/D101103, and analyzed their expression using FACS (Fig. 8A). Whole-cell expression of the double mutants was similar to that seen with the various deletion mutants (compare Figs. 2A and 8A). An analysis of the cell surface expression showed that when expressed alone, L94A/D9193, L94A/D96100, and L94A/D101103 appeared at the surface of 4.0 ± 0.44, 3.8 ± 0.56, and 6.8 ± 0.68% cells, respectively, which was similar to HA-hRAMP1 alone (compare Figs. 2B and 8B). Their cell surface expression increased significantly to 10.6 ± 1.1, 9.3 ± 0.68, and 18.9 ± 1.4% cells when co-expressed with hCRLR. Notably, cell surface expression of hCRLR/L94A/D101103 was comparable to that of hCRLR/HA-hRAMP1.
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We then examined the effects of L94A/D101103 on CGRP-evoked cAMP production via hCRLR/hRAMP1. As mentioned above, HEK-293 cells lack functional CGRP or AM receptors, even when transfected with hCRLR, which was consistent with our finding that they endogenously express no hRAMP1 and only low levels of hRAMP2 (Fig. 9). On the other hand, HEK-293T cells abundantly express endogenous hRAMP1 as well as a smaller amount of hRAMP2, which is consistent with earlier findings (6). Both CGRP and AM had a small effect on the cAMP content of HEK-293T cells expressing empty vector (Mock). Maximal cAMP levels reached 6-fold over base line (EC50 = 0.82 or 52 nM, respectively) (Figs. 10, A and B). In cells transfected with hCRLR alone, CGRP and AM elicited cAMP accumulation that were 6- and 10-fold, respectively, greater than that in mock-transfected cells (EC50 = 0.41 or 32 nM, respectively). Co-transfection of hCRLR with L94A/D101103 reduced CGRP-evoked cAMP accumulation by 3851% (Fig. 10A) and AM-evoked accumulation by 2342% (Fig. 10B). Thus, L94A/D101103 attenuated the functional effect of both endogenous hRAMP1 and hRAMP2, presumably because of competition between the mutant and the endogenous proteins.
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DISCUSSION |
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Co-transfection of hCRLR and hRAMP1 deletion mutants D4145, D5965, D6771, D9194, D96100, or D101103 eliminated CGRP and AM binding as well as evoked cAMP accumulation. When expressed alone, the six deletion mutants were detected at the cell surface of only 4%, which is similar to HA-hRAMP1. That cell surface expression of the deletion mutants was significantly increased by co-transfection of hCRLR is consistent with recent FACS and immunohistochemical findings that the appearance of epitope-tagged RAMP molecules at the cell surface is significantly increased by CRLR (6, 13, 21) or other Class II G protein-coupled receptors (28). In addition, Flahaut et al. (23) recently showed that in a Xenopus oocyte expression system, RAMP1 is neither N-glycosylated nor transported to the plasma membrane when expressed alone but that introduction of N-glycosylation sites into its sequence (D58N/G60S, Y71N, and K103N/P105S) allowed its cell surface expression at levels similar to those seen when wild-type RAMP1 is co-expressed with CRLR. Still, CRLR-induced increases in the cell surface expression of the deletion mutants were 2.5-fold lower than was seen with HA-hRAMP1. In the context of earlier studies (22, 26), this finding suggests that these mutants probably interact more potently with calcitonin receptor than with CRLR, both of which are endogenously expressed in HEK-293 cells.
The CRLR-induced increases in the cell surface expression of D9194, D96100, and D101103 were very similar to that seen with D5155. Our finding that hCRLR/D5155 mediated full CGRP binding and signaling, whereas the others did not, suggests that residues 91103 of hRAMP1 are especially important for high affinity agonist binding and signaling. Residues 5965 of hRAMP1 correspond to residues 8692 of hRAMP2 and 5965 of hRAMP3, which as mentioned above we found to be crucial for high affinity agonist binding to hAM receptors and to be situated between the conserved residues (Trp-Cys and Tyr) common to humans, rats, and mice (7). Notably, hRAMP1 residues 91103 are also situated between the conserved residues (Pro-Asn and Cys).
When alanines were substituted one at a time for residues 91103 of hRAMP1, co-expression of F101A with hCRLR reduced the magnitude maximal responses by 42% as compared with those observed with hCRLR/HA-hRAMP1 and [125I]CGRP binding to the mutant receptor was also markedly diminished. In addition, this amino acid residue is conserved among all three RAMPs, although its precise role in determining agonist specificity is still not completely clear. The remaining amino acid residues show little sequence identity among RAMPs, suggesting that the three segments (residues 9194, 96100, and 101103) do not directly interact with the binding agonist but confer selectivity by contributing to the structure of the ligand binding pocket or through allosteric modulation of the conformation of CRLR.
The expression of L94A up-regulated surface expression of the receptor heterodimer more potently than did wild-type hRAMP1, leading to increased CGRP binding and signaling. To our knowledge, there has been only one other study showing such up-regulation. In the C terminus of the 2-adrenergic receptor, L339A substitution caused a 1.8-fold increase in the number of binding sites as compared with the wild-type receptor with no changes in ligand affinity (29). The mechanism by which the substitution of the hydrophilic leucine residue with hydrophobic alanine increased the number of binding sites is not clear. However, we did find that whereas L94A substitution significantly increased cell surface expression of hCRLR/D101103, there were no differences in the cell surface expression of heterodimers comprised of CRLR and the L94A/D9193, L94A/D96100, D9194, or D96100. This finding suggests that residues 9193 and 96100 are necessary for the increase in cell surface expression of L94A.
CGRP and AM bind to CRLR/RAMP1 with similar affinities (810, 12, 24, 25), and in the present study, both elicited accumulation of cAMP in cells co-expressing hCRLR with D7476, D105107, or D109112 that was comparable to cells expressing hCRLR/HA-hRAMP1. By contrast, cells expressing hCRLR and D2833, D3439, D4650, D5155, D7880, D8386, D8890, or D113118 responded more selectively to CGRP. The selectivity seen with hCRLR/D7880 or D8890 was particularly high. Because the ring structure (Cys16Cys21) and amidation of the C-terminal residue of the CGRP and AM molecules are both essential for agonist binding and receptor activation (3), deleted segments that strongly inhibited both CGRP- and AM-evoked cAMP production may be involved in the binding of those structures. Deletions that selectively reduced only AM-evoked responses may contribute to the interaction with agonist residues other than those making up the ring and amidated C terminus.
We also tested the hypothesis that the L94A/D101103 double mutant can act as a negative regulator of CGRP or AM receptor function. To address that question, we used HEK-293T cells transfected with hCRLR, which form functional CGRP and AM receptors with endogenous hRAMP1 and hRAMP2. Consistent with our hypothesis, co-expression of L94A/D101103 with hCRLR significantly diminished CGRP- and AM-evoked cAMP accumulation. Previous studies have shown that the dominant negative activity of truncated V2 vasopressin receptors resulted from the reduction of cell surface expression of the full-length receptor protein and the formation of heterodimeric complexes involving the truncated and full-length forms (30). Although several studies (3134) have investigated the relative affinities of various RAMP forms for CRLR, it remains unclear whether the inhibition of receptor function by dominant negative RAMP mutants is attributed to competitive inhibition, formation of heterodimeric complexes, or both. A detailed analysis of dominant negative effects of L94A/D101103 is currently ongoing.
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FOOTNOTES |
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To whom correspondence should be addressed: First Department of Internal Medicine, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan. Tel.: 81-985-85-0872; Fax: 81-985-85-6596; E-mail: kuwasako{at}fc.miyazaki-med.ac.jp.
1 The abbreviations used are: CGRP, calcitonin gene-related peptide; AM, adrenomedullin; RAMP, receptor activity-modifying protein; CRLR, calcitonin receptor-like receptor; h, human; FITC, fluorescein isothiocyanate; HA, human hemagglutinin; HEK, human embryonic kidney; FACS, fluorescence-activated cell-sorting; PBS, phosphate-buffered saline; D, deletion.
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REFERENCES |
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