High affinity interaction with filamin A protects against calcium-sensing receptor degradation.

Calcium-sensing receptors (CaR) regulate cell proliferation, differentiation, and apoptosis through the MAPK pathway. MAPK pathway activation requires the cytoskeletal scaffold protein filamin A. Here we examine the interactions of CaR with filamin A in HEK-293 and M2 or A7 melanoma cells to determine how interactions with filamin A facilitate signaling. Filamin A interacts with CaR through two predicted beta-strands from residues 962 to 981; interactions between filamin A and CaR are greatly enhanced by exposure to 5 mM Ca2+. Truncations or deletions (from 972 to 997 or 962 to 981) of the CaR carboxyl terminus eliminate high affinity interactions with filamin A, but CaR-mediated MAPK pathway activation still occurs. CaR-mediated ERK phosphorylation can be localized to a predicted alpha-helix proximal to the membrane, which has been shown to be important for G protein-mediated signaling (residues 868-879). In M2 cells (-filamin A), CaR expression levels are very low; cotransfection of CaR with filamin A increases total cellular CaR and increases plasma membrane localization of CaR, facilitating CaR signaling to the MAPK pathway; similar results were obtained in HEK-293 cells. Interaction with filamin A increases cellular CaR by preventing CaR degradation, thereby facilitating CaR signaling. In addition, filamin A facilitates signaling to the MAPK pathway even by CaR truncations or deletion mutants that cannot engage in high affinity interactions with filamin A, suggesting the targeting of critical signaling proteins to CaR.

The calcium-sensing receptor (CaR) 1 is a G protein-coupled receptor expressed in tissues involved in organismal calcium homeostasis (parathyroid, kidney, bone, and intestine) and in many other cell types (nerve, glial, and epithelial cells) where its role(s) is less well defined (reviewed in Refs. 1 and 2). CaR activation acutely regulates cellular functions through heterotrimeric G proteins (G q and/or G i , depending upon cell type) and activation of phospholipase C, resulting in production of inositol 1,4,5-trisphosphate and diacylglycerol, increases in intracellular Ca 2ϩ , activation of phospholipases D and A 2 , alter-ations in lipid metabolism, and regulation of ion channels (2). CaR contributes to long term regulation of cellular processes, including cell proliferation, differentiation, and apoptosis through activation of the mitogen-activated protein kinase (MAPK) cascade (reviewed in Ref. 3). Most interestingly, CaRmediated activation of MAPK signaling requires direct interaction of the CaR carboxyl terminus with the cytoskeletal scaffold protein filamin A (4,5). Yeast two-hybrid analyses have identified strong interactions between filamin A and the CaR carboxyl terminus from residues 907 to 997 (4) and 972 to1031 (5), suggesting that the minimal interaction domain is likely to be between residues 972 and 997.
Interaction of CaR with filamin A is required for activation of MAPK signaling (4,5). CaR expressed in M2 cells, a melanoma cell line that does not express filamin A (4,19), does not activate the MAPK pathway, whereas CaR expressed in A7 cells (M2 cells stably transfected with human filamin A (4,19)) are able to signal the MAPK pathway. CaR and filamin A can also be coimmunoprecipitated with proteins required for RhoAmediated nuclear signaling, including RhoA and RhoGEF (20). In this study, we refine the location of the high affinity binding site for filamin A on the CaR carboxyl terminus, and we determine the consequences on MAPK signaling of selective deletion of the filamin A-binding site or CaR carboxyl-terminal truncations. Paradoxically, CaR-mediated activation of the MAPK pathway is not inhibited by deletions or truncations of the carboxyl terminus which remove the high affinity filamin A interaction domain. MAPK signaling is dependent upon the presence of an ␣-helix (residues 868 -879) shown previously to be involved in G protein-mediated signaling (21)(22)(23). We find that interaction with filamin A stabilizes CaR and attenuates its degradation rate, facilitating MAPK signaling.

EXPERIMENTAL PROCEDURES
Materials-Tissue culture medium and serum were obtained from Invitrogen. The HEK-293 cells were from the American Tissue Culture Collection (Manassas, VA). The human filamin A construct and the M2 and A7 cell lines (19) were provided by Drs. Thomas Stossel and Yasutaka Ohta (Harvard Medical School, Cambridge, MA). Human CaR cDNA was obtained from Klaus Seuwen (Novartis Pharma, AG, Basel, Switzerland). Restriction enzymes were from New England Biolabs and Promega. M 2 -FLAG monoclonal antibody was purchased from Sigma. The rabbit polyclonal antibody specific for the phosphorylated forms of ERK1/2 (p42/44) and antibody against total ERK were obtained from Cell Signaling Technology. The rabbit polyclonal antibody against CaR was generated against the "LRG" epitope defined by Goldsmith et al. (24). EZ-link Sulfo-NHS-Biotin was from Pierce. Chemicals were purchased from Sigma unless otherwise noted.
Cell Culture and Transfections-HEK-293 cells were grown in high glucose Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal calf serum, 50 units/ml penicillin, and 50 g/ml streptomycin (37°C, 5% CO 2 ). M2 and A7 cells were cultured in minimal essential medium containing 8% newborn calf serum and 2% fetal calf serum. A7 medium also contained 500 g/ml G418. HEK-293, M2, or A7 cells were transiently transfected with up to 4 g of each human CaR cDNA construct with 5 l of Novafector (Venn Nova LLC, Pompana, FL) and 100 l of DMEM or minimal essential medium, according to manufacturer's instructions. Cells were grown on collagen-coated plates, and experiments were carried out 48 h after transfection.
Generation of CaR Truncations, Domain Deletions, and Point Mutants-Human CaR and a construct containing an amino-terminal FLAG epitope and a carboxyl-terminal fusion to EGFP (F-CaR-EGFP) were prepared as described previously (25). CaR truncations also contained an amino-terminal FLAG epitope (but no carboxyl-terminal EGFP) and were prepared by generating a PCR product containing an XhoI site and an AflII site, using the original F-CaR-EGFP as the template. One forward primer and a series of reverse primers were used for subsequent PCRs (primer sequences available upon request). After amplification, the products were cut with XhoI-AflII and subcloned in-frame into the original F-CaR-EGFP construct, which had been cut with XhoI/AflII. For deletion of the CaR residues from 972 to 997 or 962 to 981, two separate fragments were produced by PCR, using two pairs of primers that contained restriction sites XhoI/AgeI and AgeI/AflII (sequences available upon request). The PCR products were digested with XhoI/AgeI and Age1/AflII, respectively, and subsequently were subcloned into the F-CaR-EGFP plasmid. Point mutations were generated by a modified inverse PCR mutagenesis method (26) using Pfu-Turbo DNA polymerase (Stratagene, La Jolla, CA). All CaR truncations and point mutants were verified by dideoxy-DNA sequencing (DNA Sequencing Facility, Cornell University, Ithaca, NY).
Assay of Extracellular Signal-regulated Kinase (ERK1/2) Activation-HEK-293 cells were transiently transfected with wtCaR, CaR truncations, or domain deletions and grown in 12-well plates for 48 h. Prior to the experiment, cells were incubated overnight in 0.5 mM Ca 2ϩ , serum-free DMEM containing 0.2% bovine serum albumin. Cells were exposed to varying levels of extracellular Ca 2ϩ (0.5-5 mM) for 10 min (or for times as indicated) at 37°C, and reactions were halted with 1 mM EDTA in PBS on ice. The cells were lysed (25 mM HEPES, pH 7.5, 5 mM MgCl 2 , 5 mM EDTA, 1% Triton X-100, 2 mM phenylmethylsulfonyl fluoride, 1 tablet protease inhibitor mixture (Complete, EDTA-free, Roche Diagnostics)), and Western blotting analysis was performed as described by Hjalm et al. (5). Blot were probed with anti-ERK1/2 or anti-phospho-ERK1/2 (p42/44) polyclonal antibodies (Cell Signaling Technology, Beverly, MA) and detected by enhanced chemiluminescence (Pierce). Densitometry analysis was performed with Kodak Digital Science TM 1D Image Analysis software.
Coimmunoprecipitation-Transfected HEK-293 cells were washed with PBS and lysed in buffer containing 5 mM EDTA, 0.5% Triton X-100, 10 mM iodoacetamide, and protease inhibitor mixture (Roche Diagnostics). After brief sonication (6 ϫ 10 s) on ice, the lysate was agitated at 4°C for 30 min and incubated with 20 l of protein Gagarose (Invitrogen) at 4°C for 1 h to minimize nonspecific binding. Samples were centrifuged at 14,000 rpm for 2 min at 4°C; 2 l of anti-FLAG antibody (Sigma) or anti-filamin A antibody (Novocastra Laboratories Ltd., Newcastle, UK) was added to 600 l of supernatant and incubated for 4 h at 4°C. 20 l of protein G-agarose (Invitrogen) was added to each tube and rotated at 4°C for 18 h. The resin was washed five times with ice-cold lysis buffer and incubated in 25 l of Western blot loading buffer (12 M urea, 4% SDS, 0.01% bromphenol blue, 100 mM ␤-mercaptoethanol in 200 mM Tris) for 30 min at room temperature. Samples were run on 4 -15% SDS-polyacrylamide gels as described previously (27), and blots were probed with anti-filamin A, anti-FLAG, or anti-CaR (LRG) antibody at 1:4000 dilutions.
Cell Surface Biotinylation-Biotinylation of plasma membrane-localized proteins was performed with EZ-link Sulfo-NHS-Biotin (Pierce) according to the manufacturer's instructions. Briefly, cells were washed with ice-cold PBS, pH 8.0. The cell suspension in PBS, pH 8.0, was incubated in 1 mg/ml biotin reagent at room temperature for 30 min. Cells were subsequently washed three times with PBS plus 100 mM glycine, and cell lysates were immunoprecipitated with streptavidinagarose beads (Invitrogen) for Western blot analysis.
Antisense Inhibition of CaR Biosynthesis-Antisense against CaR was used to inhibit CaR biosynthesis. A 1373-bp human CaR cDNA EcoRI fragment was subcloned in antisense orientation into pcDNA3.1, as described previously (41). HEK-293 or M2 melanoma cells were transiently transfected with CaR (2 g/35-mm dish) ϩ filamin A or vector pcDNA3 (5 g/35-mm dish) for 48 h and then transfected with 15 g of antisense CaR cDNA in 100 l of DMEM containing 10 l of NovaFector. The medium was replaced with DMEM containing 10% fetal calf serum after 24 h. After 48 or 72 h, cells were washed twice in PBS, 1 mM EDTA on ice. Cells were collected and lysed in immunoprecipitation buffer. Homogenates were cleared by centrifugation (10,000 rpm, 4°C), and protein concentration was measured by BCA protein assay (Pierce). Equivalent amounts of protein extracts (20 g) were electrophoresed through 4 -15% gradient polyacrylamide gels (Bio-Rad) and blotted onto nitrocellulose membranes. The blot was incubated with a polyclonal anti-CaR antibody (LRG) in 5% serum/TBST buffer at 4°C overnight. The blot was then incubated with horseradish peroxidase-conjugated secondary anti-rabbit antibody for 1 h at room temperature. Bands were visualized by the SuperSignal West Pico chemiluminescent kit (Pierce).

RESULTS
Filamin A Facilitates CaR-mediated MAPK Activation-ERK1/2 phosphorylation (ERK1/2ϳP) was assayed in HEK-293 cells expressing human CaR. As shown in Fig. 1A, 5 mM Ca 2ϩ induces a robust elevation of ERK1/2ϳP in cells stably transfected with CaR; increases in ERK1/2ϳP occur within 5 min and persist for over 2 h. Untransfected HEK-293 cells show a negligible increase in ERK1/2ϳP in response to a 10-min exposure to 5 mM Ca 2ϩ . The total amount of ERK1/2 was not changed significantly during a 2-h treatment with 5 mM Ca 2ϩ . Subsequent experiments utilize a 10-min exposure to 5 mM Ca 2ϩ to assess CaR-mediated MAPK signaling.
The requirement for filamin A in CaR-mediated ERK1/2ϳP is illustrated in Fig. 1B. M2 melanoma cells do not express endogenous filamin A and are an important cell type in which to study filamin A-mediated processes (19). A7 cells are M2 melanoma cells stably transfected with filamin A, restoring filamin A-dependent functions in the M2 cell background. Exposure of M2 cells transiently expressing wtCaR to 5 mM Ca 2ϩ does not induce significant ERK1/2ϳP (Fig. 1B). In contrast, both M2 cells transiently transfected with wtCaR plus filamin A or A7 cells transfected with wtCaR display significant increases in ERK1/2ϳP after 10 min of exposure to 5 mM Ca 2ϩ (Fig. 1B), supporting the requirement for filamin A in CaRmediated MAPK signaling.

Filamin A Stabilizes CaR
Ca 2ϩ for 10 min (37°C). Cell lysates were immunoprecipitated with an anti-filamin A antibody. Western blots were probed with an anti-FLAG antibody to test for the presence of CaR. Fig. 2A illustrates the carboxyl-terminal regions of the receptor constructs studied, from the end of transmembrane helix 7 at residues 860/861 (indicated by black box); the dark gray box indicates the location of the filamin A interaction domain (residues 972-997) defined as the minimal overlap encompassed by the results of two published yeast two-hybrid screens (4,5), and the white box indicates the location of the carboxyl-terminal helix shown to be involved in G protein signaling (21)(22)(23). The anti-filamin A antibody was able to immunoprecipitate wtCaR and the CaR⌬1024 truncation, i.e. only those receptors containing the filamin A interaction domain (Fig. 2B). All other CaR truncations and the deletion CaR972⌬ did not coprecipitate with filamin A. Transfection with filamin A greatly enhanced coprecipitation with CaR as illustrated in Fig. 2B (all lanes except the wt lane to right of the vertical line were derived from cells cotransfected with filamin A). To determine whether high affinity interactions of CaR and filamin A (defined by stability during immunoprecipitation) are dependent upon receptor activation, filamin A was immunoprecipitated from HEK-293 cells (expressing either wtCaR or CaR972⌬) that had been exposed to either 0.5 or 5 mM extracellular Ca 2ϩ for 10 min. Western blots of the immunoprecipitated proteins were probed for the presence of CaR with anti-FLAG antibody. As shown in Fig. 2C, wtCaR was robustly immunoprecipitated with filamin A from cells exposed to 5 mM Ca 2ϩ and weakly immunoprecipitated at 0.5 mM Ca 2ϩ . CaR972⌬ did not coimmunoprecipitate with filamin A at either 0.5 or 5 mM Ca 2ϩ . These results suggest that CaR activation facilitates high affinity interactions with filamin A.
The results of Fig. 2B suggest that CaR carboxyl-terminal residues 972-997 contain all or part of the site for high affinity interactions of CaR with filamin A, i.e. interactions that are stable during immunoprecipitation. Secondary structure analysis of the CaR carboxyl terminus suggests the presence of two ␤-strands from residues 962 to 981, which include the initial portion of the putative filamin A interaction domain (residues 972-997). We tested whether deletion of the two ␤-strands (residues 962-981, termed CaR962⌬) would influence filamin A binding. Fig. 2A illustrates the location of the putative ␤-strands (indicated by white arrows), the second strand (from 972 to 979) overlaps the initial portion of the region identified FIG. 1. Time course of CaR-mediated ERK1/2ϳP. A, HEK-293 cells stably transfected with CaR were exposed to 5 mM Ca 2ϩ for various times as indicated. ERK1/2ϳP was assayed by Western blot using antibodies against phosphorylated ERK1/2 (indicated as ERK1/ 2ϳP, upper panel) and total ERK1/2 (lower panel) as described under "Experimental Procedures." Untransfected HEK-293 cells were used as a control (assayed at 0 and 10 min of exposure to 5 mM Ca 2ϩ ). Data are representative of three independent experiments. B, comparison of wtCaR-mediated ERK1/2ϳP in M2 cells, M2 cells cotransfected with filamin A, or A7 cells. Cells were exposed to 0.5 or 5 mM Ca 2ϩ for 10 min, followed by Western blotting with antibodies against phosphorylated ERK1/2.

Filamin A Stabilizes CaR
from Y2H studies as part of the high affinity filamin A interaction domain (4,5). Cells transiently transfected with CaR962⌬ were treated with 0.5 or 5 mM Ca 2ϩ for 10 min at 37°C, followed by immunoprecipitation with anti-filamin A antibody. Western blots were probed with anti-FLAG antibodies and as illustrated in Fig. 2C; only wtCaR at 5 mM Ca 2ϩ had high affinity interactions with filamin A.
To confirm the specificity of the ␤-strand region (residues 962-981) in high affinity CaR interactions with filamin A, we generated additional deletion constructs of the carboxyl terminus including deletion of the ␣-helix involved in G protein signaling (residues 870 -879, termed 870⌬), and deletion of a predicted disordered region between the proximal ␣-helix and the filamin A binding domain (residues 909 -960, termed 909⌬). The deletion constructs (962⌬, 870⌬, and 909⌬) or wt-CaR were transfected in HEK-293 cells. Cells were exposed to 5 mM Ca 2ϩ for 10 min at 37°C, followed by immunoprecipitation of filamin A; Western blots were probed with anti-FLAG antibody to assess the presence of CaR. As illustrated in Fig.  2D, CaR coprecipitated with filamin A when the CaR deletion mutants contained the ␤-strand region from residues 962 to 981 (wtCaR, 909⌬, and 870⌬), despite the significant changes to the CaR carboxyl terminus, i.e. deletion 909⌬ removed 51 amino acids. In contrast, the CaR deletion that removed the ␤-strand region from residues 962 to 981 did not coprecipitate with filamin A, strongly suggesting that high affinity interactions between CaR and filamin A are specific and mediated by the ␤-strand region.

ERK1/2ϳP Does Not Require High Affinity Interaction of CaR with Filamin A-Filamin
A is required for CaR-mediated activation of ERK1/2ϳP. To determine whether the high affinity interaction between CaR and filamin A is required for activation of the MAPK pathway, we compared the abilities of CaR carboxyl-terminal truncations ( Fig. 2A) and CaR972⌬ to stimulate ERK1/2ϳP. All of the truncations and the deletion constructs were expressed in HEK-293 cells and stimulated with 5 mM Ca 2ϩ for 10 min. As shown in the experiment in Fig.  3A, the CaR truncations and CaR972⌬ exhibit a graded ability to stimulate ERK1/2ϳP. Most surprisingly, truncations that eliminate the high affinity filamin A interaction domain (CaR⌬886 and CaR⌬908) as well as the deletion CaR972⌬ retain the capacity to promote ERK1/2ϳP. More severe truncations of the carboxyl terminus (CaR⌬868 and CaR⌬880) showed a significant attenuation of ERK1/2ϳP, close to that of untransfected HEK-293 cells (Fig. 3A). Averaged data from five individual experiments are illustrated in Fig. 3B and indicate that CaR-mediated ERKϳP does not require the presence of the high affinity filamin A interaction domain. The regions of the CaR carboxyl terminus critical to activation of MAPK pathway signaling lie within the carboxyl terminus proximal to the membrane, from residues 860/861 to 880, a region containing a predicted ␣-helix (including residues 870 -879) having residues critical to G protein activation (21)(22)(23). To confirm this result, we compared the ability of several domain deletion mutants to activate ERK1/2ϳP, including CaR870⌬ (which has a deletion from residues 870 to 879, disrupting the proximal ␣-helix), CaR962⌬ (which has a deletion from residues 962 to 981, eliminating the ␤-strand filamin A interaction domain), and CaR909⌬ (which has a large deletion from residues 909 to 960, eliminating a disordered domain separating the ␣-helix from the ␤-strand domains). Most interestingly, wtCaR and the two mutant CaRs containing deletions within their carboxyl termini, which eliminate high affinity interactions with filamin A, are still able to activate ERK1/2ϳP when exposed to 5 mM Ca 2ϩ (Fig. 3C). In contrast, deletion of part of the ␣-helix (CaR870⌬) inhibits ERK1/2ϳP (Fig. 3C), despite the ability of CaR870⌬ to participate in high affinity interactions with filamin A (Fig.  2D). These results suggest that high affinity interactions between CaR and filamin A are not required for activation of ERK1/2ϳP. Filamin A Is Required to Stabilize Expression and Plasma Membrane Targeting of CaR-High affinity interactions between CaR and filamin A are not required for CaR-mediated activation of the MAPK pathway (Fig. 3), but wtCaR in the absence of filamin A does not signal to the MAPK pathway (Fig.  1B). We sought to reconcile these results by determining whether filamin A interactions contribute to CaR targeting to the plasma membrane. Filamin A has been shown to be involved in plasma membrane targeting and/or recycling of several G protein-coupled receptors (calcitonin (7) and D 2 dopamine (10, 11) receptors), the Kir2.1 potassium channel (30), and the serine endoprotease furin (18). In HEK-293 cells, we measured both total CaR expression (Western blots probed with anti-CaR LRG antibody) and plasma membrane-localized CaR (biotinylation followed by precipitation with streptavidin and Western blot probed with anti-CaR LRG antibody) when CaR (0.5 g cDNA) was transiently expressed in either the absence (5 g of empty vector) or presence (5 g of cDNA) of filamin A. As illustrated in Fig. 4A, both total CaR expression and the plasma membrane-localized CaR increased in the presence of

FIG. 3. CaR-mediated activation of ERK1/2 does not require high affinity CaR-filamin A interactions. A, CaR truncations and
CaR972⌬ were expressed in HEK-293 cells and stimulated with 0.5 or 5 mM Ca 2ϩ for 10 min. HEK indicates untransfected cells (control). Cell lysates were probed with anti-phospho-ERK1/2 antibody by Western blot analysis. Data shown are representative of five independent experiments. B, five independent experiments in which the Ca 2ϩ -stimulated ERK1/2ϳP was determined in response to a 10-min exposure to 5 mM Ca 2ϩ (as in A) were normalized to the response of wtCaR (Normalized ERK1/2ϳP) and averaged (mean Ϯ S.D.). C, HEK-293 cells transiently transfected with the deletion constructs 870⌬, 962⌬, or 909⌬, wtCaR, or empty vector (HEK) were treated with 0.5 or 5 mM Ca 2ϩ for 10 min followed by Western blot analysis using anti-phospho-ERK1/2 antibody. Plot indicates the increase in ERK1/2ϳP induced by 5 mM Ca 2ϩ (band intensity in 5 mM Ca 2ϩ minus the band intensity in 0.5 mM Ca 2ϩ ). Data are representative of three independent experiments.

Filamin A Stabilizes CaR
cotransfected filamin A (both monomer and dimer bands were quantified by scanning and pixel analysis). The bar graph in Fig. 4B illustrates the average of three such experiments, normalized to the band intensities obtained in the presence of filamin A (ϩFLNA); there was a 1.7-fold increase in plasma membrane-localized CaR and a 1.8-fold increase in total CaR. In similar experiments, we transfected M2 cells with either CaR alone or CaR plus filamin A. Fig. 5A illustrates that in the absence of filamin A, surface localization of CaR in M2 cells is low but cotransfection of the same amount of CaR cDNA with filamin A results in a significant increase in plasma membrane-localized CaR as determined by surface biotinylation. Similarly, filamin A significantly increases the total cellular CaR in M2 cells. The bar graph in Fig. 5B summarizes the results from three independent experiments; there was a 1.8fold increase in plasma membrane CaR and a 1.5-fold increase in total CaR upon filamin A cotransfection. Fig. 1B indicates that CaR activation induces increases in ERKϳP only in M2 cells cotransfected with filamin A or A7 cells (which stably express filamin A). When combined with the data in Fig. 5, these results suggest that filamin A is required to increase total cellular CaR, leading to parallel increases in plasma membrane-localized CaR, which is a prerequisite for MAPK pathway activation.
We hypothesized that interaction with filamin A may stabilize CaR against degradation. To test this hypothesis, we used a CaR antisense strategy. We have shown previously that transient transfection with CaR antisense cDNA can inhibit expression of transiently transfected CaR (41). HEK-293 cells were transfected with wtCaR Ϯ filamin A and were allowed to express these proteins for 48 h. Cells were then transfected a second time with a CaR antisense cDNA and were cultured for an additional 0 -72 h. At 0, 48, and 72 h, cells were lysed, and the amount of cellular CaR was analyzed by Western blotting with anti-CaR antibody LRG. Fig. 6A illustrates the results of such an experiment. Cells transfected with CaR without CaR antisense showed a stable level of CaR expression over the period from 0 to 72 h, whereas transfection with CaR antisense at time 0 resulted in a significant decrease in CaR at both 48 and 72 h. Cells initially transfected with both CaR and filamin A exhibited a marked resistance to the effects of CaR antisense, with levels of cellular CaR comparable with those observed without antisense. Fig. 6B summarizes the results of three independent experiments such as in Fig. 6A. CaR antisense caused a significant decrease (Ͼ70%) in total CaR protein over the 72-h experimental period. It is likely that the residual CaR expression represents cells that did not take up the antisense cDNA. In contrast, cells that did not receive CaR antisense exhibited stable levels of CaR protein over the 72-h experimental period. Cotransfection of CaR with filamin A minimized the effects of subsequent CaR antisense transfection and resulted in cellular levels of CaR protein comparable with cells that were not exposed to CaR antisense. Comparable results were observed when M2 cells expressing CaR Ϯ filamin A were exposed to CaR antisense cDNA, i.e. filamin A prevented CaR degradation and, in fact, increased the amount of CaR in cells despite the presence of CaR antisense (Fig. 6C). These results indicate that high affinity interaction between CaR and filamin A stabilizes CaR and attenuates CaR degradation.
Requirement for Filamin A in CaR-mediated MAPK Signaling-Truncations of the CaR carboxyl terminus that eliminate the high affinity filamin A interaction domain are also capable of stimulating ERK1/2ϳP in HEK-293 cells (Fig. 3). We considered two possible explanations. First, elimination of the filamin A interaction domain and/or potential epitopes that target CaR for degradation may stabilize CaR expression and plasma membrane targeting. Indeed, truncations beyond residue 903 have been used to promote plasma membrane targeting of receptor mutants (31,32). Second, filamin A may be required for CaR signaling to the MAPK pathway via low affinity site(s) on CaR (4,5) or associated signaling proteins. To discriminate between these possibilities, we compared the ability of wtCaR, the truncation CaR⌬886, and the deletion CaR972⌬ to stimulate ERK1/2ϳP in M2 versus A7 cells (Fig.  7A). wtCaR, CaR⌬886, and CaR972⌬ were able to stimulate ERK1/2ϳP only in A7 cells, implying that activation of the MAPK pathway requires the presence of filamin A, independent of the ability of CaR to participate in high affinity interactions with filamin A (neither CaR⌬886 nor CaR972⌬ can be immunoprecipitated with filamin A, Fig. 2B). CaR⌬886 and

FIG. 4. Filamin A increases both surface localization and total cellular CaR in HEK-293 cells.
A, HEK-293 cells stably expressing wtCaR were transiently transfected with either empty vector or filamin A (FLNA). Plasma membrane-localized CaR was assayed by biotinylation with subsequent precipitation with streptavidin; total CaR was determined by isolating membranes from disrupted cells. Western blots were probed with anti-CaR LRG antibody. Both monomeric and dimeric CaR immunoreactivity were observed on the blots. B, quantification of pixel intensity of both monomer and dimer bands for three independent experiments as illustrated in A was done by scanning and analysis with Kodak Digital Science TM 1D Image Analysis software and plotted as normalized intensity units (normalized to the signal obtained in the presence of filamin A).

FIG. 5. Filamin A enhances surface localization and total cellular expression of CaR in M2 cells.
A, M2 cells were transiently transfected with wtCaR in the absence or presence of cotransfected filamin A (FLNA). Plasma membrane-localized CaR was determined by biotinylation followed by precipitation with streptavidin; total CaR was determined by isolation of total cell membranes followed by Western blotting. Western blots were probed with anti-CaR LRG antibody. B, quantification of effects of filamin A cotransfection on CaR expression and surface localization in M2 cells averaged from three independent experiments. Quantification was described in Fig. 4B legend.
CaR972⌬ are able to activate ERK1/2ϳP (90 -120% of the activity of wtCaR) in HEK-293 cells (Fig. 3) and in A7 cells (Fig.  7A). The levels of endogenous filamin A expression in M2, A7, and HEK-293 cells were determined by Western blotting with anti-filamin A antibody (Fig. 7B). Although A7 cells express filamin A (Fig. 7, A and B), HEK-293 cells express 2.6-fold more filamin A (Fig. 7B). Thus, robust stimulation of ERK1/2ϳP by CaR truncations or deletions that eliminate high affinity interactions with filamin A requires the presence of filamin A, suggesting a second contribution of filamin A to the CaRmediated MAPK pathway activation, possibly through a second low affinity site proximal to the membrane. DISCUSSION CaR activation of the MAPK pathway requires interaction of CaR with the cytoskeletal scaffold protein filamin A (4,5). In this report, we localize high affinity interactions between CaR and filamin A to two putative ␤-strands from residues 962 to 981, whereas the region of the carboxyl terminus critical to activation of the MAPK pathway is located more proximal to the last transmembrane helix, from residues 860/861 to 886. Intensive mutagenesis suggests that the domain proximal to transmembrane helix 7 within the carboxyl terminus is involved in CaR-mediated activation of G q (21,22,37), protein kinase C-mediated desensitization (33)(34)(35)(36), signaling to phosphatidylinositol phospholipase C (21,22,37), and induction and maintenance of intracellular Ca 2ϩ oscillations (23,36). Furthermore, the residues from 877 to 891 within this critical region are predicted to form an ␣-helical structure (22), which may contribute to regulation and protein interactions in this region of the carboxyl terminus. As defined here, the residues from 880 to 886 are critical for MAPK pathway activation. Between residues 890 and the filamin A interaction domain at residues 962-981, which contains two predicted ␤-strands separated by 4 residues, there is no predicted secondary structure. The filamin A interaction domain is located approximately halfway between the end of transmembrane helix 7 and the carboxyl terminus and is surrounded on both sides by large segments (Ͼ70 residues) predicted to be disordered by both DisEMBL (29) and PONDR (28), two programs that identify disordered regions in proteins. It is tempting to speculate that the predicted ␤-strands of the filamin A interaction domain present a binding surface to filamin A repeats 14 and 15, which are composed of two interlocking ␤-strand domains of 96 amino acid residues each (6). The disordered region from residues 890 to 972 of the CaR carboxyl terminus would thus form a highly flexible "tether" permitting the filamin A interaction domain to find its binding partner on filamin A. It is of interest that activation of CaR by exposure to 5 mM extracellular Ca 2ϩ enhances high affinity interactions between CaR and filamin A. There are two potential explanations for this observation, i.e. either CaR activation induces a conformational change within the CaR carboxyl terminus which promotes interaction with filamin A or filamin A undergoes an activation-induced change that facilitates interaction with CaR. There is precedence for the latter, because activation of calcitonin receptors decreases constitutive calpain-induced cleavage of filamin A, transiently increasing the filamin A concentration and stabilizing calcitonin receptors against degradation (7). It remains to be determined whether interaction with CaR affects filamin A stability.
In this report we demonstrate that high affinity interactions between CaR and filamin A stabilize CaR levels and increase surface membrane localization of CaR in proportion to the overall increase in total CaR in both HEK-293 cells (which express endogenous filamin A) and in M2 cells that are deficient in filamin A. In M2 cells in particular, levels of plasma membrane-localized CaR are very low in the absence of filamin A, offering an explanation for the lack of CaR signaling in M2 cells observed in a previous study (4) and in the current report, i.e. insufficient CaR. In both HEK-293 cells and M2 cells, total CaR and plasma membrane-localized CaR increase in parallel, suggesting that filamin A does not preferentially stabilize CaR at the plasma membrane but rather decreases the CaR degradation rate. A similar effect of filamin A on calcitonin receptor levels in HEK-293 cells has been described, with filamin A slowing calcitonin receptor degradation by 2-fold when rates were compared in M2 versus A7 cells (7). In contrast, filamin A stabilizes dopamine D 2 receptors at the cell surface without an effect on overall D 2 receptor expression (11), and similar effects on cell surface stabilization by filamin A have been described for Kir2.1 channels (30).
Removal of the high affinity filamin A interaction site on the CaR carboxyl terminus by either truncation (CaR⌬886) or deletion of the interaction region (CaR972⌬) does not eliminate signaling to the MAPK pathway in HEK-293 cells. In contrast, neither CaR⌬886 nor CaR972⌬ is capable of stimulating ERK1/ 2ϳP in M2 cells but is functional in A7 cells. These results hint at a second role for filamin A in organizing or facilitating MAPK signaling in addition to stabilizing CaR expression. With a series of deletion constructs, we have mapped high affinity interactions of CaR with filamin A and the ability to activate MAPK signaling to distinct regions of the CaR carboxyl terminus, with MAPK signaling requiring the proximal carboxyl terminus from residues 860/861 to 886. CaR mutants that do not participate in high affinity interactions with filamin A still require the presence of filamin A for MAPK signaling. The initial yeast two-hybrid studies that identified the high affinity filamin A interaction site on CaR demonstrated stronger ␤-galactosidase activity when the CaR construct contained residues from 860 to 1078 (4) suggesting that there may be additional site(s) for CaR interactions with filamin A distinct from the high affinity site at residues 962-981. Alternatively, filamin A targets a number of signaling molecules to the plasma membrane through interactions with carboxyl-terminal binding modules (6). It therefore remains possible that filamin A targets critical signaling molecules to the vicinity of CaR even when productive high affinity interactions between filamin A and CaR have been inhibited (by dominant negative peptide inhibitors (4,5) or deletions of the critical binding domain (this study)). The mechanistic details and the pathways involved in CaR signaling to the MAPK pathway have not been well established, and therefore the critical proteins targeted to CaR by filamin A are not known. Recent studies suggest that signaling may be complex because both a dependence on dynamin-independent CaR internalization (38) and triple span signaling through the epidermal growth factor receptor have been suggested (39,40).
In summary, we have defined two independent roles for filamin A in support of CaR-mediated MAPK signaling. First, high affinity interactions with filamin A increase the level of total cellular CaR as well as plasma membrane-localized CaR by attenuating CaR degradation. Second, filamin A facilitates signaling to the MAPK pathway even by CaR truncations (CaR⌬886) or deletion mutants (CaR972⌬ or CaR962⌬) that cannot engage in high affinity interactions with filamin A, suggesting the targeting of critical signaling proteins to CaR. Further studies are needed to identify the critical components in the CaR-filamin A signaling complex.