Title: Interaction of the Calcium-sensing Receptor and Filamin, a Potential Scaffolding Protein
Abstract: In many cases, the biologic responses of cells to extracellular signals and the specificity of the responses cannot be explained solely on the basis of the interactions of known signaling proteins. Recently, scaffolding and adaptor proteins have been identified that organize signaling proteins in cells and that contribute to the nature and specificity of signaling pathways. In an effort to identify proteins that might organize the signaling system(s) activated by the extracellular Ca2+ receptor (CaR), we used a bait construct representing the intracellular C terminus of the human CaR and the yeast two hybrid system to screen a human kidney cDNA library. We identified a clone representing the C-terminal 1042 amino acids (aa) of the cytoskeletal protein filamin (ABP-280). Analysis of truncation and deletion constructs of the CaR C terminus and the filamin cDNA clone demonstrated that the CaR and filamin interact via regions containing aa 907–997 of the CaR C terminus and aa 1566–1875 of filamin. Interaction of the two proteins in mammalian HEK-293 cells was demonstrated by co-immunoprecipitation and colocalization of them using immunofluorescence microscopy. The functional importance of their interaction was documented by transiently expressing the CaR in M2 melanoma cells that lack filamin, or in A7 melanoma cells that stably express filamin, and demonstrating that the CaR activated ERK only in the presence of filamin. Co-expression of the CaR with a peptide derived from the region of the CaR C terminus that interacts with filamin reduced the ability of the CaR to activate p42ERK in a dose-dependent manner, but did not inhibit the ability of the ETA receptor to activate ERK. The fact that filamin interacts with the CaR and other cell signaling proteins including mitogen-activated protein kinases and small GTPases, indicates that it may act as a scaffolding protein to organize cell signaling systems involving the CaR. In many cases, the biologic responses of cells to extracellular signals and the specificity of the responses cannot be explained solely on the basis of the interactions of known signaling proteins. Recently, scaffolding and adaptor proteins have been identified that organize signaling proteins in cells and that contribute to the nature and specificity of signaling pathways. In an effort to identify proteins that might organize the signaling system(s) activated by the extracellular Ca2+ receptor (CaR), we used a bait construct representing the intracellular C terminus of the human CaR and the yeast two hybrid system to screen a human kidney cDNA library. We identified a clone representing the C-terminal 1042 amino acids (aa) of the cytoskeletal protein filamin (ABP-280). Analysis of truncation and deletion constructs of the CaR C terminus and the filamin cDNA clone demonstrated that the CaR and filamin interact via regions containing aa 907–997 of the CaR C terminus and aa 1566–1875 of filamin. Interaction of the two proteins in mammalian HEK-293 cells was demonstrated by co-immunoprecipitation and colocalization of them using immunofluorescence microscopy. The functional importance of their interaction was documented by transiently expressing the CaR in M2 melanoma cells that lack filamin, or in A7 melanoma cells that stably express filamin, and demonstrating that the CaR activated ERK only in the presence of filamin. Co-expression of the CaR with a peptide derived from the region of the CaR C terminus that interacts with filamin reduced the ability of the CaR to activate p42ERK in a dose-dependent manner, but did not inhibit the ability of the ETA receptor to activate ERK. The fact that filamin interacts with the CaR and other cell signaling proteins including mitogen-activated protein kinases and small GTPases, indicates that it may act as a scaffolding protein to organize cell signaling systems involving the CaR. G protein-dependent signaling systems are composed of three basic sets of proteins: receptors, heterotrimeric G proteins, and effector molecules that may be enzymes, RGS proteins, or ion channels (1Hamm H.E. J. Biol. Chem. 1998; 273: 669-672Abstract Full Text Full Text PDF PubMed Scopus (943) Google Scholar). The traditional model of signaling by these systems suggests that the various components, receptors, membrane-associated G proteins, and effector proteins, interact preferentially in their activated states, and that the specificity of these interactions is determined primarily by the structures of the proteins. However, this model cannot explain the specificity or character of these signaling systems that is foundin vivo (2Vojtek A.B. Der C.J. J. Biol. Chem. 1998; 273: 19925-19928Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). For example, in the renal collecting duct, both vasopressin (AVP) and β-adrenergic receptors are coupled to adenylyl cyclase via Gαs, but only AVP receptors stimulate H2O transport. In intact platelets, AVP and platelet-activating factor activate phospholipase C (PLC), but do not inhibit stimulation of adenylyl cyclase, whereas in platelet membranes, AVP and platelet-activating factor activate PLC and inhibit stimulation of adenylyl cyclase (3Brass L.F. Woolkalis M.J. Manning D.R. J. Biol. Chem. 1988; 263: 5348-5355Abstract Full Text PDF PubMed Google Scholar). These data suggest that cell structure or additional proteins such as "scaffolding" proteins may place constraints on cell signaling systems that contribute to their specificity (4Bockaert J. Pin J.P. EMBO. 1999; 18: 1723-1729Crossref PubMed Scopus (1233) Google Scholar, 5Zuker C.S. Ranganthan R. Science. 1999; 283: 650-651Crossref PubMed Scopus (26) Google Scholar). In eukaryotic cells, signaling complexes can be organized by proteins that make use of structural motifs to recognize and position components of signaling cascades (2Vojtek A.B. Der C.J. J. Biol. Chem. 1998; 273: 19925-19928Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar, 6Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1904) Google Scholar). Signaling pathways involving receptor tyrosine kinases utilize multiple structural motifs and adaptor proteins to generate complex signals (7Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4245) Google Scholar). Examples of organization of signaling pathways by structural proteins include STE-5, which coordinates members of the MAP kinase cascade inSaccharomyces cerevisiae; INA-D, a pentavalent PDZ domain-containing protein in the Drosophila retina that coordinates rhodopsin, PLC, protein kinase C (PKC), and Ca2+ channels; NHE-RF, a PDZ domain-containing protein that couples the β-adrenergic receptor to NHE-3; Homer, a protein that couples the M5 metabotropic glutamate receptor to intracellular Ca2+ stores; and Jip-1 (Jun kinase interacting protein) that coordinates members of the Jun kinase cascade in mammalian cells (5Zuker C.S. Ranganthan R. Science. 1999; 283: 650-651Crossref PubMed Scopus (26) Google Scholar, 6Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1904) Google Scholar, 8Symons M. Trends Biochem. Sci. 1996; 21: 178-181Abstract Full Text PDF PubMed Scopus (260) Google Scholar, 9Elion E.A. Science. 1998; 281: 1625-1626Crossref PubMed Scopus (121) Google Scholar, 10Gomperts S.N. Cell. 1996; 84: 659-662Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). The cytoskeletal actin-binding protein filamin (actin-binding protein 280, ABP-280, FLN1) may act as a scaffolding protein for signaling cascades because it binds a number of cell surface receptors and intracellular signaling molecules, and disruption of the interactions of these proteins with filamin interferes with signaling by them (11Fox J.W. Lamperti E.D. Eksioglu Y.Z. Hong S.E. Feng Y. Graham D.A. Scheffer I.E. Dobyns W.B. Hirsch B.A. Radtke R.A. Berkovic S.F. Huttenlocher P.R. Walsh C.A. Neuron. 1998; 21: 1315-1325Abstract Full Text Full Text PDF PubMed Scopus (708) Google Scholar, 12Marti A. Luo Z. Cunningham C. Ohta Y. Hartwig J. Stossel T.P. Kryakis J.M. Avruch J. J. Biol. Chem. 1997; 272: 2620-2628Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 13Leonardi A. Ellinger-Ziegelbauer H. Franzoso G. Brown K. Siebenlist U. J. Biol. Chem. 2000; 275: 271-278Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 14Li M. Bermak J.C. Wang Z.W. Zhou Q.Y. Mol. Pharmacol. 2000; 57: 446-452Crossref PubMed Scopus (113) Google Scholar). Filamin is a ubiquitously expressed homodimer composed of 2647 aa (280 kDa) monomers, each of which contains 24 immune globulin-like repeats, two hinge regions, a dimerization domain at the C terminus, and an actin-binding domain at the N terminus (15Gorlin J.B. Yamin R. Egan S. Stewart M. Stossel T.P. Kwiatkowski D.J. Hartwig J.H. J. Cell Biol. 1990; 111: 1105Crossref Scopus (434) Google Scholar). Filamin cross-links actin in orthogonal arrays and directly links cell surface receptors to the cytoskeleton. Filamin interacts directly and specifically with the dopamine D2 receptor (a G protein-coupled receptor), the Fcγ receptor, platelet glycoprotein Ibα, β1 and β2 integrins, tissue factor, MAP kinases, the tumor necrosis factor receptor-associated factor-2 (TRAF2), Rho GTPases, and Smad proteins (12Marti A. Luo Z. Cunningham C. Ohta Y. Hartwig J. Stossel T.P. Kryakis J.M. Avruch J. J. Biol. Chem. 1997; 272: 2620-2628Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 13Leonardi A. Ellinger-Ziegelbauer H. Franzoso G. Brown K. Siebenlist U. J. Biol. Chem. 2000; 275: 271-278Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 16Ohta Y. Stossel T.P. Hartwig J.H. Cell. 1991; 67: 282Abstract Full Text PDF Scopus (81) Google Scholar, 17Glogauer M. Arora P. Chou D. Janmey P.A. Downey G.P. McCullouch C.A.G. J. Biol. Chem. 1998; 273: 1689-1698Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 18Ohta Y. Suzuki N. Nakamura S. Hartwig J.H. Stossel T.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2122-2128Crossref PubMed Scopus (376) Google Scholar, 19Loo D.T. Kanner S.B. Aruffo A. J. Biol. Chem. 1998; 273: 23304-23312Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 20Muller M. Albrecht S. Golfert F. Hofer A. Funk R.H.W. Magdolen V. Flossel C. Luther T. Exp. Cell. Res. 1999; 248: 136-147Crossref PubMed Scopus (54) Google Scholar, 21Sasaki A. Masuda Y. Ohta Y. Ikeda K. Watanabe K J. Biol. Chem. 2001; 276: 17871-17877Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Filamin also interacts directly with caveolin, a protein component of caveolae, structures that contain many signaling molecules including the CaR and G proteins (22Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1727) Google Scholar, 23Stahlhut M. Van Deurs B. Mol. Biol. Cell. 2000; 11: 325-337Crossref PubMed Scopus (266) Google Scholar). Filamin is involved in protein trafficking and contributes to localization and cycling of proteins in the cell (14Li M. Bermak J.C. Wang Z.W. Zhou Q.Y. Mol. Pharmacol. 2000; 57: 446-452Crossref PubMed Scopus (113) Google Scholar, 24Liu G. Thomas L. Warren R.A. Enns C.A. Cunningham C.C. Hartwig J.H. Thomas G. J. Cell Biol. 1997; 137: 1719-1733Crossref Scopus (121) Google Scholar). These results suggest that filamin may serve as a scaffolding protein to co-localize and organize signaling molecules from a variety of signaling systems. The extracellular calcium-sensing receptor, a G protein-coupled receptor that responds to extracellular Ca2+ and other polycations, was recently cloned by expression (25Brown E.M. Gamba G. Riccardi D. Lombardi M. Butters R. Kifor O. Sun A. Hediger M. Lytton J. Hebert S.C. Nature. 1993; 366: 575-580Crossref PubMed Scopus (2384) Google Scholar, 26Brown E.M. MacLeod R.J. Physiol. Rev. 2001; 81: 239-297Crossref PubMed Scopus (1240) Google Scholar). This receptor is expressed in parathyroid cells where it is the Ca2+sensor that regulates PTH secretion, the kidney where it contributes to Ca2+, Na+, Cl−, and H2O balance, and in the brain, gastrointestinal tract, skin, and other epithelial tissues where its role is less clear (27Chattopadhyay N. Vassilev P.M. Brown E.M. Biol. Chem. 1997; 378: 759-768PubMed Google Scholar). Recent studies indicate that the CaR may participate in paracrine signaling (28Hofer A.M. Curci S. Doble M.A. Brown E.M. Soybel D.I. Nat. Cell Biol. 2000; 2: 392-398Crossref PubMed Scopus (124) Google Scholar, 29Thomas A.P. Nat. Cell Biol. 2000; 2: E126-E128Crossref PubMed Scopus (16) Google Scholar). In various tissues, the CaR regulates numerous second messengers and signaling proteins including cAMP (inhibition), inositol 1,4,5-trisphosphate, diacylglycerol, phosphatidic acid, arachidonic acid metabolites, ion channels, and MAP kinases through at least two G proteins, Gαi and Gαq (30Brown E.M. Pollak M. Hebert S.C. Annu. Rev. Med. 1998; 49: 15-29Crossref PubMed Scopus (192) Google Scholar, 31Handlogten M.E. Huang C. Shiraishi N. Awata H. Miller R.T. J. Biol. Chem. 2001; 276: 13941-13948Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). However, these second messenger systems are not sufficient to explain all of the biologic functions of the CaR (32Brown E.M. Physiol. Rev. 1991; 71: 371-411Crossref PubMed Scopus (641) Google Scholar). In an effort to identify proteins that interact with the CaR and that could potentially organize the signaling proteins through which it acts, we used the yeast two-hybrid cloning approach with the C terminus of the CaR as bait to screen an adult kidney cDNA library. We identified a number of interacting proteins, one of which was filamin. Chemicals were purchased from Sigma or Fisher Scientific unless specified otherwise. Tissue culture medium and serum were obtained from Life Technologies, Inc., plasticware was from Falcon, the HEK-293 cells were from the American Tissue Culture Collection, restriction and DNA-modifying enzymes were purchased from Promega, and Superfect and plasmid preparation kits were from Quaigen. The cDNA coding for the human CaR was a gift from Drs. E. M. Brown and M. Bai (Harvard University, Boston, MA), and the full-length filamin cDNA was from Dr. John Hartwig (Harvard Medical School, Boston, MA). The M2 and A7 melanoma cells, originally described by Cunningham et al. (33Cunningham C.C. Gorlin J.B. Kwiatkowski D.J. Hartwig J.H. Janmey P.A. Byers R. Stossel T.P. Science. 1992; 255: 325-327Crossref PubMed Scopus (498) Google Scholar) were from Dr. Fred Southwick (University of Florida, Gainesville, FL). A cDNA representing the C-terminal 219 aa of the human calcium receptor (aa 861–1078) in the bait plasmid pAS2.1 was used to screen a human adult kidney cDNA library in pACT2 (Invitrogen). The S. cerevisiae strain PJ69-4A was obtained from John Aris (University of Florida, Gainesville, FL) and maintained on minimal medium supplemented with histidine, leucine, tryptophan, and adenine (34James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar). The bait plasmid and cDNA library were co-transformed into the yeast, using the method of Geitz et al. (33Cunningham C.C. Gorlin J.B. Kwiatkowski D.J. Hartwig J.H. Janmey P.A. Byers R. Stossel T.P. Science. 1992; 255: 325-327Crossref PubMed Scopus (498) Google Scholar), and plated on medium deficient in leucine, tryptophan, and histidine. The plates were replicated on media lacking histidine, tryptophan, leucine, and adenine. The colonies that grew under these conditions were tested for β-galactosidase activity. Yeast expressing the library plasmid pACT2 without an insert and the pACT2 vector with β-galactosidase were used as controls. Colonies in which the β-galactosidase activity exceeded the activity of the pACT2 vector without an insert by a factor of 2 were analyzed further by purifying the plasmids and sequencing the inserts from either end. The yeast bait constructs representing the predicted C terminus of the human CaR (aa 861–1078) and regions of it were prepared using PCR with the wild type human cDNA as a template. All primer pairs contained NdeI sites at the 5′ end of the 5′ oligonucleotide to allow in-frame cloning of the construct into the expression vector, pAS2.1. The 3′ primers contained TAA stop codons in frame and SalI sites to facilitate subcloning of the construct. The full-length C terminus of the CaR (aa 861–1078) was produced with the primer pairs AGA TCC ATA TGA TTC TCT TCA AG (containing an NdeI site at the 5′ end) and GCC CAG TCG ACT CCT TCC ATT (3′ SalI site). The fragment corresponding to aa 860–991 was produced by cutting the full-length C terminus with NdeI and BamHI, and cloning the fragment in frame into pAS2.1 The fragment corresponding to aa 860–908 was made by cutting the full-length C terminus with NdeI and Bam-HI, and cloning it in frame into pAS2.1. The fragment corresponding to aa 907–997 was produced by cutting the C terminus (above) withBamHI and SalI and cloning it in frame into pAS2.1. The fragment corresponding to aa 987–1078 was produced using a sense primer (TTG GAT CCG GCA CGG TCA CCT TC) that contained anNdeI site, and an antisense primer (GCC CAG TCG ACT CCT TCC ATT) that contained a SalI site. The PCR fragments were cloned into the TA cloning vector pCR2.2-TOPO (Invitrogen) and then subcloned in frame into pAS2.1 (Invitrogen) using the NdeI and SalI sites. The sequences of these fragments were verified by dideoxy sequencing. The filamin constructs were produced in a similar manner by PCR using the human cDNA clone as a template (15Gorlin J.B. Yamin R. Egan S. Stewart M. Stossel T.P. Kwiatkowski D.J. Hartwig J.H. J. Cell Biol. 1990; 111: 1105Crossref Scopus (434) Google Scholar). The fragment corresponding to aa 1566–1875 was produced using the sense primer containingEcoRI/SalI sites (GCC GAA TTC GTC GAC GTG GAG TTC ACC ATC G) and the antisense primer (TGA CAG ATC TCC GAG TTG ACT TAA TCC ACA TAG AA) that contained XhoI and BglII sites. The fragment corresponding to aa 2021–2647 was produced using the sense primer that contained BamHI/KpnI sites (CAC GGT ACC AGC AGC CGG ATC CCG GTG GTG AT) and the antisense primer that contained an XbaI site (TTC TCT CTA GAC CAG TCT TCT CC). The fragment corresponding to aa 2021–2209 was produced using the sense primer into which BamHI and KpnI sites had been incorporated (CAC GGT ACC AGC AGC CGG ATC CCG GTG GTG AT), and the antisense primer (CCC AGA TCT CGA GGG GTC ACA CGG TGA ACT GGA A) into which XhoI and BglII sites had been incorporated. The fragment corresponding to aa 2402–2647 was produced using a sense primer containing BamHI and KpnI sites (ACC CGG ATC CCT GGT ACC CCC TTC AA), and an antisense primer containing anXbaI site (TTC TCT CTA GAC CAG TCT TCT CC). The PCR products were cloned into pCR2.2-TOPO, amplified, and then subcloned into pACT2 in frame. The sequences were verified using dideoxy sequencing. For the mammalian expression studies, the fragment representing aa 907–1024 was subcloned from pBluescript into pCruzMycB using the KpnI and BglII sites. The monoclonal antibody against the CaR was produced in mice by standard techniques in the University of Florida hybridoma core facility. A synthetic peptide (WHSSAYGPDQRAQ) that corresponds to amino acids 15–29 in the extracellular N terminus of the CaR was synthesized by the University of Florida biotechnology core facility and injected into mice, and hybridomas were prepared. Supernatants were screened by enzyme-linked immunosorbent assay for reactivity against the peptide. Positive clones were tested against membranes from HEK 293 cells that express an HA-tagged CaR and membranes prepared from the medulla and cortex of mouse kidneys. The monoclonal antibody to the CaR (6D4) identified specific bands of 125 and 140 kDa that were also identified by the anti-HA antibody in the membranes from the cells that expressed the HA-tagged CaR. HEK293 cells that express the CaR were grown to ∼80% confluence on polylysine-coated coverslips. The cells were washed with PBS and fixed in 4% paraformaldehyde for 20 min, washed three times in PBS, quenched for 30 min in PBS with 50 mm glycine, and washed again in PBS. The cells were blocked and permeabilized by incubating them for 30 min in PBS containing 3% BSA, 5% normal goat serum (NGS), and 0.1% Triton X-100. The cells were incubated in primary antibody (10 μg/ml CaR 6D4 or 10 μg/ml filamin) and 1 μg/ml rhodamine phalloidin in PBS containing 3% BSA, 5% NGS, and 0.1% Triton X-100 for 2 h at room temperature. The slides were then washed three times with PBS and incubated in secondary antibody (1:200 FITC-conjugated goat anti-mouse IgG) in PBS containing 3% BSA, 5% NGS, and 0.1% Triton for 2 h at room temperature. The cells were then washed in PBS and attached to slides with a solution that contained 4,6-diamidino-2-phenylindole to visualize nuclei. Images of the cells were obtained with a Zeiss Axiophot fluorescence microscope, and images were stored using a digital Spot camera (Diagnostic Instruments, Inc.). cDNAs were transfected into HEK293 cells using the CaPO4co-precipitation technique (35). A total of 3 μg of DNA (a combination of cDNAs coding for proteins of interest with empty vector and carrier DNA) were added to 60-mm dishes and allowed to precipitate. The medium was changed after 18–20 h, and experiments were performed 48 h after transfection. For stable expression of the CaR in HEK-293 cells, stable clones were selected with G-418. cDNAs were transfected into M2 and A7 melanoma cells using Superfect according to the manufacturer's instructions. Experiments were performed 48 h after transfection. Cells were lysed on ice in immunoprecipitation (IP) buffer containing 125 mm NaCl, 62.5 mm NaH2PO4, pH 7.2, 0.625% C12Ε10 (Lubrol), and protease inhibitors. The lysates were centrifuged at 13,500 rpm in a microcentrifuge for 30 min at 4 °C and incubated with an equal volume (500 μl) of the hybridoma supernatant from clone 6D4 (anti-CaR monoclonal antibody) or the anti-filamin antibody (45 μg) at 4 °C by slow rotation overnight. The samples were then incubated with 7.5 μl of rabbit anti-mouse antibody for 30 min at 4 °C. The precipitations were performed by incubating the extracts with Pansorbin cells (S. aureus, Calbiochem) for 30 min on ice and then centrifuging them at top speed in a microcentrifuge at 4 °C. The pellet was resuspended in 1× IP buffer, layered over 0.9 ml of 20% sucrose in IP buffer, and centrifuged at top speed in a microcentrifuge at 4 °C for 6 min. The pellet was resuspended in PBS and centrifuged for 3 min at top speed. That pellet was resuspended in 1× electrophoresis buffer, heated to 90 °C, subjected to SDS-PAGE, and processed for immunoblotting with the antibody indicated. HEK-293, M2, and A7 melanoma cells were serum-deprived overnight before experiments. At the time of experiments, the medium was replaced with a solution containing 150 mm NaCl, 5 mm KCl, 10 mm HEPES, pH 7.4, 0.5 mm CaCl2, and 0.5 mmMgCl2. At time 0, reagents were added at the concentrations indicated and incubated with the cells at 37 °C for the times indicated. Commonly, the cells were exposed to activators of the receptor for 5 min, at which time the reactions were stopped by rinsing the cells at 4 °C in buffer containing 50 mm NaF, 100 mm NaCl, 0.1 mm sodium orthovanadate, and 20 mm NaH2PO4, pH 7.4–7.5, and placing the dishes on a dry ice and ethanol bath. The cells were scraped in iced buffer that contained 50 mm Tris, pH 7.5, 50 mm NaCl, 5 mm EDTA, 1 mm EGTA, 1 mm sodium orthovanadate, 40 mmβ-glycerophosphate, 50 mm NaF, 50 nm okadaic acid, 5 mm sodium pyrophosphate, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 40 mm p-nitrophenyl phosphate, 4 μg/ml pepstatin, 4 μg/ml aprotinin, 4 μg/ml leupeptin, and 1 mmphenylmethylsulfonyl fluoride. The cell lysates were centrifuged at 15,000 × g for 10 min in a refrigerated microcentrifuge. Triton-soluble extracts were normalized for protein, size-fractionated using SDS-PAGE, and processed for immunoblotting. Proteins were detected by enhanced chemiluminescence. The anti-active ERK antibody was obtained from Promega, and the anti-filamin monoclonal antibody was obtained from Serotec. The monoclonal antibodies to the HA epitope, 12CA5, and the Myc epitope, 9E10, were obtained from the University of Florida hybridoma core. In order to identify proteins that interact with the CaR and that could act as scaffolding proteins to organize the signaling molecules regulated by the CaR, we used the C-terminal 219 aa (aa 861–1078) of the human CaR representing the cytoplasmic tail as bait in the pAS2 vector (Invitrogen) to screen a human adult kidney cDNA library in pACT2 (Invitrogen) using the yeast two-hybrid system (36Gietz R.D. Triggs-Raine B. Robbins A. Graham K.C. Woods R.A. Mol. Cell. Biochem. 1997; 172: 67-79Crossref PubMed Scopus (128) Google Scholar). We used the yeast strain PJ69-4A, which offers triple genetic selection and a reduced rate of false positive clones (34James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar). The bait plasmid and the cDNA library were co-transformed into the yeast using the lithium acetate/single-stranded DNA/polyethylene glycol method of Geitz et al. (37Gietz R.D. Schiestl R.H. Willems A.R. Woods A.R. Yeast. 1995; 11: 335-360Crossref Scopus (1712) Google Scholar), and plated on medium deficient in Leu, Trp, and His. The plates were replicated on media lacking His, Trp, Leu, and adenine, and the surviving colonies were tested for β-galactosidase activity. Positive clones were sequenced from either end. Four positive clones were isolated and sequenced, one of which was filamin 1 (ABP-280). The filamin clone was 3326 nucleotides in length and represented the C-terminal 1082 aa (aa 1566–2647), which includes the C-terminal 61 aa of repeat 14, repeats 15–24, and the two hinge regions (15Gorlin J.B. Yamin R. Egan S. Stewart M. Stossel T.P. Kwiatkowski D.J. Hartwig J.H. J. Cell Biol. 1990; 111: 1105Crossref Scopus (434) Google Scholar, 38Patrosso M.C. Repetto M. Villa A. Milanes L. Frattini A. Faranda S. Mancini M. Maestrini E. Toniolo D. Vezzoni P. Genomics. 1994; 21: 71-76Crossref PubMed Scopus (26) Google Scholar). Further analysis of the CaR C terminus in yeast (Fig.1, Table I) demonstrated that the first 48 aa of the C terminus distal to the seventh membrane-spanning domain (aa 860–908), a region of many receptors that interacts with G proteins and that is required for signaling by the CaR, does not interact with filamin (39Ray K. Fan G.-F. Goldsmith P.K. Spiegel A.M. J. Biol. Chem. 1997; 272: 31355-31361Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 40Gamma L. Breitwieser G.E. J. Biol. Chem. 1998; 273: 29712-29718Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). The interacting region includes aa 907–997, but may also include additional amino acids because the full-length C terminus interacts with filamin more strongly based on growth and β-galactosidase assays (Table I). The interacting 91-aa region is rich in glutamine and proline residues but does not contain sequences that correspond to functional domains that mediate protein-protein interactions such as Src homology regions 2 and 3, or PDZ domains (Fig. 1,bottom).Table IInteraction of CaR C-terminal constructs with two filamin constructsCaR constructFilamin 1566–2647 growthFilamin 1566–2647Filamin 1566–1647 growthFilamin 1566–1647β-Gal unitsβ-Gal unitsVector (pAS2.1)−5−4.2860–1078++14.3++15.5860–991+10.1+11.0860–908−4.5−5.1907–997+12.6+12.3987–1078−7.3−6.2 Open table in a new tab Fig. 2 shows a map of the filamin cDNA isolated from the human kidney cDNA library with a diagram of filamin showing the locations of the repeats and hinge regions. The truncation mutants used to map the region of filamin that interacts with the CaR are also shown with numbers corresponding to the first and last amino acids of each construct. The full-length filamin clone (aa 1566–2647) and a shorter construct (aa 1566–1875) corresponding to the last 61 aa of repeat 14, repeat 15, the 25-aa hinge region, repeat 16, and 15 aa of repeat 17, referred to as "repeats 15–16," both interacted with the full-length CaR C terminus and the construct corresponding to CaR aa 907–997 (see Fig. 1) (15Gorlin J.B. Yamin R. Egan S. Stewart M. Stossel T.P. Kwiatkowski D.J. Hartwig J.H. J. Cell Biol. 1990; 111: 1105Crossref Scopus (434) Google Scholar, 38Patrosso M.C. Repetto M. Villa A. Milanes L. Frattini A. Faranda S. Mancini M. Maestrini E. Toniolo D. Vezzoni P. Genomics. 1994; 21: 71-76Crossref PubMed Scopus (26) Google Scholar). The full-length C terminus interacts with both filamin constructs more strongly than the shorter construct (aa 907–997), further suggesting that other regions of the CaR C terminus, probably containing amino acids C-terminal of aa 997, contribute to the interaction of the CaR with filamin (Table II). These results indicate that the CaR and filamin interact directly via peptides containing aa 907–997 in the CaR C terminus, and aa 1566–1875 corresponding to a region of filamin that contains most of repeat 14 through repeat 16.Table IIInteraction of filamin constructs with two CaR C-terminal constructsFilamin constructCaR 860–1078 growthCaR 860–1078CaR 907–997 growthCaR 907–997β-Gal unitsβ-Gal unitsVector (pACT2)−7.1−61566–2647++18.2+12.11566–1875++21.3+13.82021–2647−8−72021–2029−6−8.22402–2647−4.9−5.9 Open table in a new tab In order to determine if the CaR and filamin interact in mammalian cells, we coimmunoprecipitated the two proteins using either the anti-CaR antibody, 6D4, or the anti-filamin antibody. Fig.3 shows co-immunoprecipitation of the CaR and filamin from HEK-293 cells that stabl