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Endocrinology Vol. 143, No. 10 3830-3838
Copyright © 2002 by The Endocrine Society

ARTICLE

Calcium-Sensing Receptor Activation of Rho Involves Filamin and Rho-Guanine Nucleotide Exchange Factor

Min Pi, Robert F. Spurney, Qisheng Tu, Todd Hinson and L. Darryl Quarles

Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710

Address all correspondence and requests for reprints to: L. Darryl Quarles, M.D., Duke University Medical Center, Box 3036, Durham, North Carolina 27710. E-mail: quarl001{at}mc.duke.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated the role of G{alpha}q, filamin, Rho, the RhoGEF Lbc, and the C terminus of calcium-sensing receptor (CasR) in CasR signaling. We found that Ca2+, Mg2+, or the calcimimetic R isomer of N-(3-[2-chlorophenyl]propyl)-(R)-{alpha}-methyl-3-methoxybenzylamine (NPS-R568) stimulated serum response element (SRE) activity human embryonic kidney 293 cells transfected with CasR and an SRE-luciferase reporter construct. Coexpression of either the dominant negative G{alpha}q(305–359) minigene, regulators of G protein signaling (RGS)2 or RGS4, inhibited CasR-stimulated SRE activity, consistent with CasR activation of G{alpha}q. The cytoskeletal associated Rho protein is involved CasR activation of SRE, as evidenced by CasR-mediated increase in membrane-associated Rho A and by the ability of Clostridium botulinum C3 (C3) exoenzyme to inhibit both CasR and G{alpha}qQL-stimulated SRE activity. Overexpression of the RhoGEF Lbc, lacking either the Dbl-homology or Pleckstrin homology domain, as well as the filamin peptide (1530–1875) inhibited CasR-mediated activation of SRE. A carboxyl-terminal CasR minigene, CasR(906–980), encoding a filamin binding region, also blocked CasR- and G{alpha}qQL-stimulated SRE activity. Potential interactions between CasR, RhoGEF Lbc, Rho A, G{alpha}q, and filamin were demonstrated by reciprocal coimmunoprecipitation studies. Our results suggest that the C terminus of CasR may interact with filamin to create a cytoskeletal scaffold necessary for the spatial organization of G{alpha}q, RhoGEF Lbc, and Rho signaling pathways upstream of SRE activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE CALCIUM-SENSING receptor (CasR) belongs to the class C G protein-coupled receptor (GPCR) receptors that include metabotropic glutamate receptors, {gamma}-aminobutyric acid type B receptors, pheromone receptors, and several orphan GPCRs (1, 2). CasR is the major molecular target for extracellular calcium regulation of parathyroid function (3, 4, 5, 6). Inactivating mutations of CasR cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism (3), whereas targeted ablation of CasR in mice leads to parathyroid gland abnormalities including excess PTH secretion, hypertrophy, and hyperplasia (4). In contrast, activating mutations of CasR result in hypoparathyroidism (5). In addition to calcium, a diverse group of ligands can activate CasR, including magnesium, gadolinium, neomycin, and phenylalkylamine-derived calcimimetics (2, 6).

The signal transduction pathways mediating CasR effects on parathyroid gland function are currently being elucidated. This receptor acts through at least two G proteins, G{alpha}i and G{alpha}q, to regulate multiple second messengers. These include the inhibition of agonist-induced cAMP accumulation and phosphatidylinositol-phospholipase activation leading to increments in inositol trisphosphate, diacylglycerol, and intracellular calcium (7, 8) as well as phospholipase A2 activation leading to the accumulation of arachidonic acid metabolites (8, 9). In addition, CasR activates phospholipase D, stimulates ERK pathways, and induces transcription from the serum response element (SRE) (7, 8, 9, 10, 11, 12, 13). At present, the precise role of these pathways in CasR regulation of PTH gene transcription, PTH protein secretion, and parathyroid gland growth have not been established.

The Rho family of small molecular weight GTPases, which mediate responses to G{alpha}q/11- and G{alpha}12/13-coupled GPCRs (14, 15, 16), represent other potential pathways for mediating CasR effects on the parathyroid gland. In this regard, Rho proteins have been shown to regulate exocytosis, cell proliferation, and hypertrophy in a variety of tissues and culture systems (14, 15, 16, 17, 18, 19, 20). There also is evidence that the C-terminal region of CasR physically interacts with cytoskeletal element filamin necessary for signaling to Rho (21, 22, 23). Filamin interacts with a Pleckstrin-homology (PH) domain of Rho guanine nucleotide exchange factor (GEF) Lbc (23, 24) to directly link G{alpha}-subunits and Rho A (25, 26). To date, no studies have investigated whether RhoGEFs and/or Rho proteins are downstream effectors in CasR signaling cascades.

In the current study, we examined whether CasR participates in signaling through Rho-dependent pathways via its C terminus. We found that CasR activation of G{alpha}q results in a Rho-dependent stimulation of SRE-mediated transcription that is disrupted by coexpression of a dominant negative RhoGEF Lbc, filamin, or C-terminal CasR constructs. In addition, filamin, Rho A, and RhoGEF Lbc coimmunoprecipitated with CasR. We propose that the C terminus of CasR provides a framework for binding of a RhoGEF/filamin complex that may couple G{alpha}q to Rho A.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
All culture reagents were from Life Technologies, Inc. (Rockville, MD). Human embryonic kidney (HEK)-293 cells were obtained from American Type Culture Collection (Manassas, VA). HEK-293 cells stably expressing rat CasR were created as previously described (27). Aluminum chloride (AlCl3·6 H20) was obtained from Fisher (Springfield, NJ). Gadolinium chloride hexahydrate was purchased from Alcon Laboratories, Inc. (Milwaukee, WI). Calcium chloride, magnesium chloride, neomycin sulfate, PD98059, U46619, and PTH(1–34) were purchased from Sigma (St. Louis, MO), BSA (faction V) was from Roche (Indianapolis, IN). The calcimimetic R isomer of N-(3-[2-chlorophenyl]propyl)-(R)-{alpha}-methyl-3-methoxybenzylamine (NPS-R568) and its inactive isomer NPS-S568 were provided by Amgen, Inc. (Thousand Oaks, CA).

Cell culture
HEK-293 cells were grown in DMEM supplemented with 10% fetal calf serum and 1% penicillin/streptomycin at 37 C in a humidified atmosphere of 95% air and 5% CO2.

Sources and construction of expression plasmids
The rat CasR cDNA was obtained from Drs. A. M. Snowman and S. H. Snyder (28) and subcloned in the mammalian expression vector pcDNA 3 (Invitrogen, Carlsbad, CA) as previously described (27). All of the G protein {alpha}-subunits and Rho A QL, C3-toxin, and Rho A I41 (generously provided by Dr. J. Silvio Gutkind) were in pcDNA I expression vectors (29, 30). RGS2, RGS4, and RGS12 in the p-cytomegalovirus (pCMV) expression vector as previously described (31) were generous gifts of Dr. Dianqing Wu (Department of Pharmacology and Physiology, University of Rochester). The G{alpha}q(305–359) minigene construct that correspond to the COOH-terminal peptide sequence of G{alpha}q residues 305 to 359 was kindly provided by Dr. Robert J. Lefkowitz from Duke University (32). Plasmid constructs containing the full-length Lbc onocoprotein (ONCO-Lbc) that contains the Dbl-homology (DH) and PH domains and FLAG-epitope tagged DH (ONCO-NODH), and PH (ONCO-NOPH) Lbc deletion constructs in the pSR{alpha}Neo expression vector were kindly provided by Dr. Deniz Toksoz from Tufts University (33).

The CasR C-terminal minigene, CasR(906–980), was created as follows. The rat CasR cDNA was amplified by PCR using the forward primer 5'-CTTAAGCTTCCATGGGCTCCACCGGCTCC-3' that contains a HindIII site and the reverse primer 5'-TTAGATCTAGATTACAGAGAGAAGGTGACC-3' that contains an XbaI site. The PCR products were digested with HindIII and XbaI, purified, and ligated into a modified expression vector pSV.SPORT. In addition, we subcloned the C-terminal minigene into pBK.CMV at the BglII and XbaI site. A rat PTH receptor expression vector pBK-CMV-PTHr and the thromboxane A2 (TxA2) receptor expression vector pcDNA-TxA2r were generated as previously described (34, 35). We used the previously described SRE-luciferase plasmid DNA (36). We used the technique of mutually priming oligonucleotides and PCR to insert the FLAG tag in the N terminus of CasR immediately 3' to the signal peptide (34, 27). A derivative of previously described dominant negative filamin construct (21) containing amino acids 1530 through 1875 (designated Filamin 1530–1875) was produced in a similar manner by RT-PCR using the human total RNA as a template. The primer set are the forward primer 5'-CTTAAGCTTCCATGGTACCCCGGAGCCCC-3' that contains a HindIII site and the reverse primer 5'-TTAGATCTAGATTAATCCACATAGAAC-3' that contains an Xba I site. The PCR products were digested with HindIII and XbaI, purified, and ligated into a modified expression vector pSV.SPORT.

Transient and stable transfection
For these studies, all plasmid DNAs as described above, were prepared using the EndoFree plasmid maxi kit (QIAGEN Inc., Valencia, CA). Transient transfections were preformed as follows: 2 x 105 HEK-293 or HEK-293 cells stably transfected with CasR were plated in the six-well plate and incubated overnight at 37 C. A DNA-liposome complex was prepared by mixing DNA of the SRE-luciferase reporter plasmid, pCMV-ß-gal and other expression vector as indicated with TransFast transfection reagent (1:2 DNA/TransFast transfection reagent; Promega Corp., Madison, WI) in Opti-MEM I reduced serum medium (Life Technologies, Inc.). The total plasmid DNA was equalized in each well by adjusting the total amount of DNA to 2 µg/well with the empty vector. The resulting mixtures of plasmid DNAs and TransFast transfection reagent were incubated at room temperature for 15 min and diluted with Opti-MEM I reduced serum medium (1 ml/well) before addition to cells that had been rinsed with Hanks’ balanced saline. After 1 h of incubation at 37 C, 2 ml DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin was added to the medium overlying the transfected cells, and the cells were incubated for an additional 2 d before the described studies.

Assessment of agonist-stimulated SRE activity
Quiescence of transfected cells was achieved in subconfluent cultures by incubation for 24 h in serum-free DMEM/F12 containing 0.1% BSA. After 2 d of transfection, quiescent cells were treated with vehicle or stimulated for the last 8 h with appropriate agonists [Ca2+ (5 mM), Mg2+ (10 mM), NPS-R568 (1 µM), 100 nM PTH(1–34), or 2 µl/ml U46619]as indicated. Luciferase activity was assessed after 8 h of stimulation. The luciferase activity in cell extracts was measured using the luciferase assay system (Promega Corp.) following the manufacturer’s protocol using a BG-luminometer (Gem Biomedical Inc., Hamden, CT).

RhoA activation assay
Western blot analysis of Rho A was preformed as previously published (37) in membrane fractins prepared from HEK-293 cells transfected with empty vector and pcDNA.rCasR. Following incubation with and without magnesium at the indicated concentrations, membrane and cytosolic fractions were isolated by high-speed centrifugation 100,000 x g for 30 min. Protein fractions were separated on 4–12% SDS-PAGE, transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA), and the blots probed with a mouse monoclonal anti-RhoA antibody (clone 26C4, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The signal from immunoreactive bands was detected by ECL kit (Amersham Pharmacia Biotech, Piscataway, NJ).

Measurement of inositol phosphate generation
IPs were measured as previously described using anion exchange chromatography (38). Briefly, HEK-293 cells were plated at a density 2 x 105 cells/well in 6-well plates and transfected with wild-type pcDNA.rCasR or truncated pcDNA.rCasR(1–876). After 48-h culture, the cells were equilibrated overnight in DMEM low inositol medium (Invitrogen) containing 0.1% dialyzed fetal calf serum, penicillin (100 U/ml), and streptomycin (100 µg/ml, all from Invitrogen) containing 5 µCi/ml myo-[3H]-inositol (NEN Life Science Products, Boston, MA). The cultures were washed three times with 2 ml buffer solution containing 20 mM HEPES, 125 mM NaCl, 4 mM KCl, 0.5 mM CaCl2, 0.5 mM MgSO4, and 0.1% dextrose (pH 7.4). Cells were then incubated for 30 min with 1 µM NPS-R568 or their vehicle in 2 ml buffer solution at 37 C containing 20 mM lithium chloride, which was added to inhibit breakdown of inositol phosphates.

Immunoprecipitations (IPs)
HEK-293 cells stably expression FLAG-tagged CasR or FLAG-tagged ONCO-Lbc were rinsed with PBS and lysed in IP buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 0.02 tablet/ml protease inhibitor cocktail. Cell lysates were prewashed with protein A-Sepharose (Sigma) for 1 h at 4 C. Supernatants were incubated with either mouse antihuman filamin monoclonal antibody (Chemicon International, Inc., Temecula, CA) or mouse anti-FLAG M2 monoclonal antibody (Sigma). Filamin, FLAG-tagged CasR, or FLAG-tagged ONCO-Lbc and associated proteins were precipitated with protein A-Sepharose beads (Sigma). For controls, the above procedure was duplicated without the addition of the antibody or using another mouse monoclonal antibody V5 to control for nonspecific precipitation. Following overnight incubation at 4 C, the immunocomplexes were pelleted by centrifugation (3000 xg, 1 min) and were washed three times. The precipitates were separated by SDS-PAGE and electrophoretically transferred to Immobilon-P membranes (Millipore Corp.), and then immunoblotted with anti-FLAG M2 monoclonal antibody (1/500) (Sigma), mouse anti-CasR antibody ADD (1/32000) (NPS Pharmaceuticals, Inc., Salt Lake City, UT), rabbit anti-G{alpha}q (1/1000) (C-19, Santa Cruz Biotechnology, Inc.), or mouse anti-Rho A monoclonal antibody (1/1000) (Santa Cruz Biotechnology). Specific bands were detected by enhanced chemiluminescence (ECL+Plus, Amersham Pharmacia Biotech).

Statistics
We evaluated differences between groups by one-way ANOVA. Values sharing the same superscript are not significantly different at P < 0.05. All computations were performed using the Statgraphic statistical graphics system (STSC, Inc., Rockville, MD).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CasR activates SRE-mediated gene transcription
The SRE reporter construct, consisting of two c-fos-derived SREs (16, 17, 36), was transfected into HEK-293 cells expressing the full-length CasR and stimulated with various CasR agonists. Ca2+ (5 mM), Mg2+ (5 and 10 mM), at concentrations previously shown to activate the receptor (5, 6, 7, 8, 9, 11), significantly increased luciferase activity from the SRE reporter construct above that found under control (unstimulated) conditions (Fig. 1AGo). In contrast, Al3+ (25 µM), which does not activate CasR (27), had no effect on luciferase activity. NPS-R568 also stimulated CasR-induced luciferase activity, achieving maximal activation with 1 µM NPS-R568 in the presence of 1 mM calcium (Fig. 1Go, A and B), whereas the inactive S-isomer did not stimulate CasR-induced luciferase activity (Fig. 1BGo).



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  		Figure 1. CasR induces SRE reporter gene activity through SRE-dependent pathway. A, Cation specificity of CasR activation of the SRE reporter. HEK-293 cells stably transfected with CasR were transiently transfected with the SRE-luciferase reporter gene (0.5 µg) and then stimulated with CasR agonists Ca2+ (5 mM), Mg2+ (10 mM), or NPS-R568 (1 µM) and with Al3+ (25 µM) that is not a CasR agonist. B, The calcimimetic NPS-R568 activates CasR-mediated transcription from the SRE-luciferase reporter gene in a concentration-dependent and stereoselective manner. The CasR-expressing HEK-293 cells were transfected with the SRE-luciferase reporter gene (0.5 µg) and then stimulated with either NPS-R568 or the inactive isomer NPS-S568 in the presence of 1 mM CaCl2. In all of the above studies, values for luciferase activity (expressed as percent of control production) represent the mean ±SEM of a minimum of three separate experiments. Values sharing the same superscript are not significantly different at P < 0.05.

 
CasR activates SRE-mediated gene transcription through G{alpha}q
To determine the G protein subunits that mediate CasR stimulation of SREs, we attempted inhibition of G{alpha}q-mediated signaling in the HEK-293 cells with a G{alpha}q minigene, G{alpha}q(305–359) (Fig. 2AGo). This minigene consists of the COOH-terminal peptide residues 305 to 359 and has previously been shown to uncouple G{alpha}q-coupled receptors without affecting coupling to other classes of G{alpha}-subunits (32). Coexpression of G{alpha}q(305–359) with CasR resulted in significant inhibition of cation-stimulated luciferase activity (Fig. 2AGo). In addition, we tested RGS2, which lacks in vitro GAP activity for G{alpha}i but is a potent inhibitor of G{alpha}q, RGS4, which has GTPase-activating effects for the G{alpha}i and/or G{alpha}q subunits, and RGS12, which inhibits G12 and G13-mediated signaling (31, 39). Coexpression of either RGS 2 and 4 with CasR significantly inhibited cation-induced SRE activation, whereas RGS12 had no effects (Fig. 2BGo), results consistent with recent studies, demonstrating CasR activation of G{alpha}q (40). Next, we cotransfected CasR expressing HEK-293 cells with the SRE reporter gene plasmid and cDNA encoding one of the constitutively active G{alpha} subunits (G{alpha}12 QL, G{alpha}s QL, G{alpha}i2 QL, G{alpha}q QL). We found that cells expressing activated {alpha}-subunit of G{alpha}q produced the highest induction of luciferase activity in the presence of CasR (Fig. 2CGo). The activation of SRE by G{alpha}q was significantly less in the absence of CasR (Fig. 2CGo), indicating that the presence of CasR enhanced the activity of G{alpha}q. In contrast, overexpression of activated G{alpha}12, G{alpha}s, and G{alpha}i2 did not stimulate SRE in these cells. These results indicate that G{alpha}q is likely to be the predominant pathway by which CasR activates SRE transcription in HEK-293 cells. The failure of G{alpha}12 to activate SRE-dependent pathways was a characteristic of HEK-293 cells because overexpression of constitutively active G{alpha}12 strongly activated SRE in NIH3T3 fibroblasts (data not shown), similar to previous reports (27).



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  		Figure 2. CasR is coupled to activation of G{alpha}q. A, CasR activity is inhibited by the expression of G{alpha}q minigene construct, G{alpha}q(305–359). HEK-293 cells were cotransfected with the constructs directing the expression of G{alpha}q(305–359) (1.5 µg) along with the CasR (0.1 µg), the SRE-luciferase reporter gene (0.01 µg) and p-cytomegalovirus-ß-galactosidase (pCMV-ß) (0.015 µg). B, Inhibition of CasR activity by expression of RGS2, 4, 12. HEK-293 cells were cotransfected with expression vectors for RGS2, RGS4, or RGS12 (1 µg) along with CasR (0.1 µg), the SRE-luciferase reporter gene (0.01 µg) and pCMV-ß-gal (0.015 µg). C, Effect of activated mutants of G{alpha}-subunits on the activity of the SRE. HEK-293 cells were cotransfected with 0.5 µg expression vectors for the constitutively activated mutant of G{alpha}12, G{alpha}s, G{alpha}i2, G{alpha}q, and Rho A (G{alpha}12 QL, G{alpha}s QL, G{alpha}i2 QL, G{alpha}q QL, and Rho A QL) along with expression vectors for CasR (0.1 µg) or empty vector, the SRE-luciferase reporter gene (0.01 µg) and pCMV-ß-gal (0.015 µg). Data are shown as relative luciferase activity reported as the percent induction, compared with the activity under nonstimulated conditions and normalized for ß-galactosidase. Values represent the mean ±SEM of at least three experiments. Values sharing the same superscript are not significantly different at P <0.05.

 
Rho A mediates CasR and G{alpha}q stimulation of SRE
We next investigated where Rho is a downstream effector of CasR activation of SRE, as has been reported for other GPCRs (18). Initially, we cotransfected CasR expressing HEK-293 cells with the reporter gene plasmid and a cDNA encoding the constitutively active Rho A (Rho A QL). We found that Rho A QL significantly stimulated SRE activity (Fig. 2CGo). To further examine the role of Rho in CasR signaling, we made use of a DNA construct that expresses the C3 exoenzyme (Fig. 3AGo). This toxin ADP ribosylates Rho A at asparagine 41, thereby preventing the exchange of GDP by GTP and retaining Rho A in its GDP-bound inactive form (16, 37). Cotransfection with a plasmid expressing the C3 toxin inhibits CasR-stimulated SRE activity (Fig. 3AGo). Coexpression of a Rho A mutant (Rho A-I4I), which is insensitive to C3 toxin because of mutating the Asn41 in the catalytic site to Ile (30, 41), attenuated the inhibitory effects of C3 toxin on CasR-induced activation of the SRE. Similarly, C3 coexpression significantly, but partially, inhibited the induction of SRE by G{alpha}q, and this response was significantly restored by coexpression of Rho A-I41 (Fig. 3BGo). To confirm that stimulation of CasR is associated with RhoA activation, we measured membrane-associated Rhoa A in CasR-expressing HEK-293 cells. Stimulation of CasR with magnesium resulted in a marked increase in the amount of RhoA in the membrane fraction (Fig. 3CGo).



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  		Figure 3. Constitutively active G{alpha}q subunits and CasR activate the SRE in a Rho A-dependent manner. A, C3 toxin (C3) blocks CasR stimulation SRE. B, C3 blocks constitutively activated mutant of G{alpha}q (G{alpha}q QL) stimulation of SRE, indicating that Rho A is downstream of G{alpha}q. Cells were treated with vehicle or stimulated for 8 h with 10 mM MgCl2, as indicated. HEK-293 cells were cotransfected with SRE-luciferase reporter gene (0.01 µg) and pCMV-ß-gal (0.015 µg) along with CasR expression plasmid (0.1 µg), expression vectors (0.5 µg) for the constitutively activated mutant of G{alpha}q (G{alpha}q QL), C3, and the C3-insensitive mutant of Rho A (Rho A I41), as indicated. The data in A and B represent luciferase activity normalized by the ß-gal activity present in each cellular lysate, expressed as percentage induction with respect to control cells and are the mean ± SEM of triplicate samples from a typical experiment and repeated at least twice. Values sharing the same superscript are not significantly different at P < 0.05. C, Activation of Rho A by CasR. Magnesium (10 mM) induces membrane association of Rho A in HEK-293 cells transfected with CasR as assessed by Western blot analysis of RhoA in membrane fractions prepared as described in Materials and Methods.

 
C-terminal region of CasR is involved in SRE activation
We next focused on whether the C terminus of CasR, which has recently been reported to bind to filamin (21, 22), is involved in activation of Rho-dependent pathways (42, 43, 44). We compared the ability of the truncated calcium sensing receptor CasR(1–876) to stimulate the SRE reporter construct in HEK-293 cells (Fig. 4AGo). In contrast to the potent stimulation of the full-length CasR (1–1078), Ca2+ (5 mM), Mg2+ (10 mM) and NPS-R568 (1 µM) failed to stimulate luciferase activity in cells expressing the C-terminal truncated CasR. This likely is not due to alterations in membrane expression because previous studies of C-terminal-truncated CasR receptors have shown that this region does not affect receptor expression or desensitization (43, 45). Moreover, the truncated calcium sensing receptor CasR(1–876) retained the ability to stimulate inositol phosphate synthesis similar to the wild-type CasR (Fig. 4BGo).



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  		Figure 4. The C terminus of CasR is necessary for CasR-mediated activation of SRE. A, C-terminal truncated CasR(1–876) lacks the capacity to activate SREs. The data represent luciferase activity normalized by the ß-gal activity and represent the mean ± SEM. Values sharing the same superscript are not significantly different at P < 0.05. B, Effects of NPS-568 on inositol monophosphate production in HEK-293 cells transfected with full-length (CasR) or truncated (CasR 1–876) receptors. Inositol phosphate fractions were separated by an ion exchange chromatography and subsequently quantified via liquid scintillation counting as described in Materials and Methods. NPS-568 stimulated increments in inositol phosphate synthesis in HEK-293 cells stably expressing the full-length CasR as well as the C-terminal truncated CasR (1–876). No stimulation was observed in control HEK-293 cells lacking this receptor, indicating that this response is mediated by the stably transfected CasR constructs. Values represent the mean ± SEM of at least four separate determinations.

 
To further examine functional domains in the C terminus of CasR, we achieved receptor class-specific inhibition of signaling in the HEK-293 cells with a C-terminal minigene, CasR(906–980), consisting of the peptide residues 906 to 980 that have previously been shown to bind to filamin. Coexpression of the dominant negative CasR(906–980) with CasR resulted in significant inhibition of cation-stimulated luciferase activity (Fig. 5AGo). This inhibition of SRE was due to the expression of the 906 to 980 region because another CasR minigene(861–905) had no effect on CasR-stimulated SRE (data not shown). In addition, coexpression of the CasR(906–980) also blocked SRE activation by the constitutively activated mutant of G{alpha}q (Fig. 5BGo), indicating that action of the C-terminal minigene affected a common pathway. The CasR C-terminal minigene, however, appears to be receptor specific because the C-terminal CasR(906–980) minigene failed to inhibit agonist-stimulated luciferase activity in HEK-293 cells cotransfected with PTH and thromboxane receptors (Fig. 5CGo).



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  		Figure 5. Dominant negative effects of a C-terminal CasR minigene on CasR-mediated activation of SRE. A, Overexpression of the C-terminal CasR minigene, CasR (906–980), inhibits CasR-stimulated SRE activity. B, The C-terminal minigene inhibits G{alpha}q-dependent activation of SRE. HEK-293 cells were cotransfected with the expression plasmid for CasR (0.1 µg) or G{alpha}q QL (0.1 µg) and the CasR (906–980) dominant negative minigene (2 µg). C, The dominant negative actions of the CasR (906–980) C-terminal minigene is specific for CasR. HEK-293 cells were cotransfected with the C-terminal truncated pSV-CasR 876R, the full-length pSV-CasR(1–1078) plasmid (1 µg), with the expression plasmid for the PTH receptor (1 µg pBK-CMV-PTHr) or the thromboxane receptor (1 µg pcDNA-TxA2r CasR) with and without the CasR (906–980) dominant negative minigene (2 µg) along with the SRE-luciferase reporter gene (0.01 µg) and pCMV-ß-gal (0.015 µg). The data represent luciferase activity normalized by the ß-gal activity present in each cellular lysate, expressed as percentage induction with respect to control cells, and are the mean ± SEM. Values sharing the same superscript are not significantly different at P < 0.05.

 
The Rho GEF Lbc couples CasR to SRE
Previous reports demonstrate that the Rho GEF Lbc link GPCR to Rho through a physical interaction with G{alpha}q (26, 33). To determine whether this RhoGEF is involved in coupling CasR-dependent activation of G{alpha}q to Rho A and SRE, we cotransfected HEK-293 cells with CasR and ONCO-Lbc, ONCO-NODH, or ONCO-NOPH. HEK-293 cells expressing ONCO-Lbc demonstrated a small increment in CasR-mediated stimulation of SRE luciferase activity, whereas HEK-293 cells coexpressing CasR and either ONCO-NODH or ONCO-NOPH failed to show a CasR-mediated SRE luciferase response after stimulation with magnesium (Fig. 6AGo). These findings indicate that both the DH and PH domains may be functionally involved in modulating CasR signaling.



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  		Figure 6. Role of the RhoGEF Lbc and Filamin in CasR activation of SRE. A, Quiescent HEK-293 cells cotransfected with CasR and either ONCO-Lbc, ONCO-NODH (DH deletion construct), or ONCO-NOPH (PH deletion construct) were stimulated with magnesium as described in Materials and Methods. RhoGEF Lbc constructs lacking either the DH or PH domains inhibit CasR-stimulated SRE. B, HEK-293 cells were cotransfected with the constructs directing the expression of Filamin (1530–1875) (1.5 µg) along with the CasR (0.1 µg), the SRE-luciferase reporter gene (0.01 µg), and pCMV-ß-gal (0.015 µg). The pCMV-ß-gal was used to control for transfection efficiency, and the total amount of DNA was equalized to 2 µg/well by the addition of the empty vector plasmid pSVSPORT. Values represent the mean ± SEM of three separate determinations. Values sharing the same superscript are not significantly different at P < 0.05.

 
A dominant negative filamin peptide disrupts CasR stimulation of SRE
To determine whether CasR signaling through SRE requires filamin, we overexpressed a peptide corresponding to amino acids 1530–1875 of filamin together with CasR and the SRE reporter construct (Fig. 6BGo). This region of filamin is reported to interact with the C terminus of CasR (21, 22). Stimulation of CasR failed to activate SRE in HEK-293 cells coexpressing filamin (1530–1875) (Fig. 6BGo).

Evidence for complex formation among CasR, Rho A, Lbc, and filamin
To determine whether CasR and Rho A associate with filamin, we stably transfected HEK-293 cells with FLAG-tagged CasR and performed IP with an antifilamin antibody. The precipitated CasR and Rho A proteins were analyzed by Western blotting using anti-M2 and anti-Rho antibodies. Both CasR and Rho A were present in filamin precipitates (Fig. 7AGo). To further confirm the specificity of these associations, we examined the ability of CasR to immunoprecipitate filamin and Rho A in reciprocal studies. We found that immunoprecipitaton of FLAG-tagged CasR with anti-M2 also coimmunoprecipitated both Rho A and filamin (data not shown). Finally, to determine whether Rho-GEF Lbc potentially complexes with filamin, Rho A, G{alpha}q, and CasR, we performed additional IP with RhoGEF (ONCO-Lbc) in HEK-293 cells transfected with plasmid encoding ONCO-Lbc that contain FLAG M2 tagged (33). All of the components of the putative scaffolding were coprecipitated by ONCO-Lbc, including filamin, Rho A, G{alpha}q, and CasR (Fig. 7BGo). We did not observe these complexes in the absence of antifilamin and anti-FLAG-M2 antibodies (data not shown). We also failed to observe these complexes using mouse monoclonal antibody not directed to any of these components, which was used as an additional control for nonspecific precipitation (data not shown).



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  		Figure 7. Coimmunoprecipitation of CasR, filamin, and Rho A identify scaffolding and adaptor proteins leading to CasR activation of SRE. A, Coimmunoprecipitation of CasR, Rho A, and filamin. Selective precipitation of CasR and Rho A by IP with an antibody to filamin using HEK-293 cells were stably transfected with plasmid encoding FLAG-tagged CasR (lane 1) and empty vector only (lane 2). CasR and Rho A were respectively detected by Western blot analysis of immunoprecipitates using anti-FLAG-M2 and anti-Rho A antibody probes (arrows). B, Selective precipitation of filamin, CasR, G{alpha}q, and Rho A with RhoGEF (ONCO-Lbc) using HEK-293-CasR cells transiently transfected with plasmid encoding ONCO-Lbc that contained FLAG M2 tagged (lane 1) and empty vector only (lane 2). Immunoprecipitates of ONCO-Lbc were performed with the anti-FLAG-M2. Filamin, CasR, G{alpha}q, and Rho A were detected by Western blot analysis of Onco-Lbc immunoprecipitates using antifilamin, anti-CasR, anti-G{alpha}q, and anti-Rho A antibodies (arrows) as described in described in Materials and Methods. Omission of the antibody or use of a nonspecific antibody in the immunoprecipitation reaction did not result in IP of CasR, RhoA, or filamin.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the current studies, we show that Rho is an integral part of the signaling pathway downstream of CasR-mediated G{alpha}q activation. Several findings support the involvement of G{alpha}q and Rho A in SRE-mediated gene expression by CasR. First, known CasR agonists (Fig. 1Go) as well as activated G{alpha}q QL and Rho A QL (Fig. 2CGo) induced activation of the SRE, which is known to mediate Rho-dependent transactivation-independent of effects on the ternary complex factor (26). Second, CasR-mediated activation of SREs is blocked by overexpression of a carboxyl-terminal peptide of the {alpha} subunit G{alpha}q (Fig. 2AGo) as well as RSG2 and RGS4 (Fig. 2BGo), similar to other G{alpha}q-coupled heptahelical receptors (32, 39). Third, coexpression of a plasmid expressing the botulinum C3 exoenzyme, which inactivates Rho A by ADP ribosylation of asparagine 41, inhibited CasR and G{alpha}q induction of SRE (Fig. 3AGo). Finally, CasR-mediated activation of RhoA was confirmed by the membrane-association of RhoA following magnesium stimulation of CasR in HEK-293 cells (Fig. 3CGo). Our findings that CasR activates G{alpha}q and Rho are similar to those recently reported by Huang et al. (40).

To determine the mechanisms whereby CasR-activation of G{alpha}q leads to Rho A activation, we explored the role of the C terminus of CasR and the involvement of the Rho GEF Lbc (23, 24). We show that the C-terminal region 906–980 of CasR is necessary for its signaling through Rho and SRE-mediated gene transcription. Indeed, C-terminal deletion mutant of CasR, lacking residues beyond amino acid 876, loses its ability to activate SREs, compared with wild-type CasR (Fig. 4AGo), but not its ability to stimulate phosphatidylinositol-phospholipase as measured by IP hydrolysis (Fig. 4BGo). Using CasR with C-terminal tail truncated after residue 895, Chang et al. (46) also found retention of calcium-stimulated IP hydrolysis comparable to wild-type CasR, similar to our findings (Fig. 4BGo). In addition, overexpression of a CasR(906–980) minigene inhibits the ability of CasR to activate SRE in response to extracellular cations and calcimimetic NPS-568 (Fig. 5AGo). This inhibitory action seems to be limited to the region between 906 and 980 because another minigene construct, CasR(861–905), fails to inhibit CasR-induced SRE activation (data not shown), and other investigators have localized the functionally important domains to a similar region of the CasR C terminus (21). The actions of the CasR(906–980) minigene also involves G{alpha}q because the minigene construct also inhibited induction of SRE by the activated form of G{alpha}q (Fig. 5BGo). Finally, the inhibitory actions of the CasR(906–980) minigene appears to be specific for CasR or related receptors because the minigene construct failed to block PTH activation the transfected PTH receptor or U46619 activation of the transfected thromboxane A2 receptor in HEK-293 cells (Fig. 5CGo).

We also found functional evidence for agonist-induced binding of the C terminus of CasR to the scaffolding protein filamin (22), which in turn functions to couple G{alpha}q to Rho via a mechanism likely involving the Lbc RhoGEF adaptor protein (24, 26). Lbc is a RhoGEF that links G subunits to Rho and binds to filamin (23, 24) and G{alpha}q (26). This adapter function of RhoGEF is mediated at the molecular level through RGS-like domain that bind to G{alpha}-subunits, a DH domain that binds to Rho and a PH domain that is responsible for the cellular localization, possibly via interactions with filamin (33). We found that coexpression of Lbc constructs lacking the DH or PH domains inhibits CasR-stimulated SREs, consistent with disruption of the adaptor function of this RhoGEF (Fig. 6AGo). We also found that a dominant negative filamin construct coprecipitates with CasR (40), consistent with filamin binding to the carboxyl-terminal tail (22), and inhibits cation stimulation of SRE activity (Fig. 6BGo).

Potential interactions responsible for these functional responses were identified by complementary IP studies that demonstrated coprecipitation of CasR, filamin, G{alpha}q, RhoGEF Lbc, and Rho A (Fig. 7Go, A and B), consistent with these proteins participate forming a complex responsible for CasR activation of Rho. Although we did not show direct physical interactions among these factors, previous investigations have demonstrated that the C terminus of CasR binds to filamin (22). In addition, our observations are consistent with other investigations showing that G{alpha}q coprecipitates with the RhoGEF Lbc (26) and that a member of the RhoGEF family can interact with filamin (24). Because RhoGEF Lbc also immunoprecipitates filamin, CasR, G{alpha}q, and Rho A (Fig. 7BGo), it is tempting to speculate that the PH domain of the RhoGEF Lbc, which is known to localize RhoGEF Lbc to the cytoskeleton (33), directly binds filamin. If so, the C-terminal CasR may serve as a scaffolding linking CasR to SRE activation via a complex involving filamin binding to the RhoGEF Lbc.

Many GPCRs that induce Rho activation by coupling to G{alpha}q also activate G{alpha}12/13 G{alpha}q (47). Unexpectedly, RGS12, which inhibits the G{alpha}12/13 function, did not inhibit CasR, and the constitutively active G{alpha}12 QL failed to activate SRE in HEK-293 cells in our studies (Fig. 3BGo). This discrepancy may be due to cell type-specific difference in responses as reported by others (26, 36) because we found that overexpression of activated G{alpha}12/13 subunit stimulated SREs in fibroblasts (data not shown). Because RSG4 can inhibit both G{alpha}i as well as G{alpha}q (43), we also cannot exclude a role of G{alpha}i in the coupling of CasR to SREs. Finally, although we show that Rho-dependent pathways are downstream of CasR, there are likely to be other second messengers that link CasR to SRE activation (26) as well as potential coupling of other pathways through the C-terminal tail of CasR (46). One of these could be ERK-dependent pathways, which are activated by CasR (40). Moreover, C3 inhibition of both CasR and G{alpha}q-stimulated SRE activity was partial (Fig. 3Go), indicating that CasR and G{alpha}q likely stimulate other signaling pathways leading to SRE activation in addition to Rho A.

In conclusion, CasR activates SREs through G{alpha}q- and Rho-mediated activation of SREs, at least in part, through its C-terminal domain. The interaction between the C terminus of CasR and filamin could link the activated receptor to a cytoskeletal scaffold that in turn recruits a RhoGEF, such as Lbc, to catalyze the activation of Rho. Given the importance of Rho in mediating exocytosis, hypertrophy, and hyperplasia (12), the existence of CasR activation of Rho-dependent pathways warrants additional studies to assess the role of this pathway in mediating the adaptive changes to the parathyroid gland caused by disease states leading to chronic stimulation of CasR.


   Acknowledgments
 
We thank J. Silvio Gutkind, Dianqing Wu, Deniz Toksoz, and Robert J. Lefkowitz for providing cDNAs.


   Footnotes
 
This work was supported by the National Institutes of Arthritis and Musculoskeletal and Skin Disorders Grant R01-AR-37308 from the NIH.

Abbreviations: CasR, Calcium-sensing receptor; C3, Clostridium botulinum C3; CMV, cytomegalovirus; DH, Dbl homology; ß-gal, ß-galactosidase; GEF, guanine nucleotide exchange factor; GPCR, G protein-coupled receptor;HEK, human embryonic kidney; IP, immunoprecipitation; Lbc, Rho GEF lymphoid blast crisis; NPS-R568, R isomer of N-(3-[2-chlorophenyl]propyl)-(R)-{alpha}-methyl-3-methoxybenzylamine;PH, Pleckstrin homology; PI, phosphoinositol; RGS, regulator of G protein signaling; SRE, serum response element; TXA2, thromboxane A2.

Received February 28, 2002.

Accepted for publication July 1, 2002.


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