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Am J Physiol Cell Physiol 290: C1543-C1551, 2006. First published January 11, 2006; doi:10.1152/ajpcell.00482.2005
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RECEPTORS AND SIGNAL TRANSDUCTION

Ca2+-sensing receptor induces Rho kinase-mediated actin stress fiber assembly and altered cell morphology, but not in response to aromatic amino acids

Sarah L. Davies,1,* Claire E. Gibbons,1,* Thomas Vizard,2 and Donald T. Ward1

1Faculty of Life Sciences, The University of Manchester, Manchester; and 2Cardiff School of Biosciences, University of Cardiff, Cardiff, United Kingdom

Submitted 27 September 2005 ; accepted in final form 6 January 2006


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The Ca2+-sensing receptor (CaR) is a pleiotropic, type III G protein-coupled receptor (GPCR) that associates functionally with the cytoskeletal protein filamin. To investigate the effect of CaR signaling on the cytoskeleton, human embryonic kidney (HEK)-293 cells stably transfected with CaR (CaR-HEK) were incubated with CaR agonists in serum-free medium for up to 3 h. Addition of the calcimimetic NPS R-467 or exposure to high extracellular Ca2+ or Mg2+ levels elicited actin stress fiber assembly and process retraction in otherwise stellate cells. These responses were ablated by cotreatment with the calcilytic NPS 89636 and were absent in vector-transfected HEK-293 cells. Cotreatment with the Rho kinase inhibitors Y-27632 and H1152 attenuated the CaR-induced morphological change but not intracellular Ca2+ (Cai2+) mobilization or ERK activation, although transfection with a dominant-negative RhoA-binding protein also inhibited calcimimetic-induced actin stress fiber assembly. CaR effects on morphology were unaffected by inhibition of Gq/11 or Gi/o signaling, epidermal growth factor receptor, or the metalloproteinases. In contrast, CaR-induced cytoskeletal changes were not induced by the aromatic amino acids, treatments that also failed to potentiate CaR-induced ERK activation despite inducing Cai2+ mobilization. Together, these data establish that CaR can elicit Rho-mediated changes in stress fiber assembly and cell morphology, which could contribute to the receptor's physiological actions. In addition, this study provides further evidence that aromatic amino acids elicit differential signaling from other CaR agonists.

cytoskeleton; signaling

WHOLE BODY Ca2+ homeostasis is controlled primarily by the action of the extracellular Ca2+-sensing receptor (CaR) on parathyroid hormone (PTH) secretion and renal Ca2+ reabsorption (3). The signaling mechanisms by which the extracellular CaR mediates its physiological functions remain an area of active investigation.

It is well established that the CaR couples to Gq/11 and Gi/o proteins (reviewed in Refs. 3, 34), however, more recent evidence demonstrates additional CaR coupling to G12/13 proteins (14, 26, 28). Receptor-mediated stimulation of G12/13 proteins is associated with activation of the monomeric G protein Rho and its downstream effector Rho kinase, with resulting changes in the actin cytoskeleton and cell architecture (5, 8). Thus, because the CaR has also been reported to associate with the cytoskeletal protein filamin (1, 12), we have investigated whether CaR activation modulates cytoskeletal structure and cell morphology in human embryonic kidney (HEK)-293 cells stably transfected with CaR (CaR-HEK). The CaR responds to type I agonists, such as extracellular Ca2+ (Cao2+) and extracellular Mg2+ (Mgo2+) and to positive allosteric modulators (type II agonists), such as the calcimimetics (e.g., NPS R-467, NPS R-568, cinacalcet, calindol) (20, 22, 23, 25), and can be inhibited by negative allosteric modulators known as calcilytics (e.g., NPS 2143, NPS 89636, Calhex 231) (20, 21, 25). Other type II CaR agonists include the L-aromatic amino acids (6, 7), and interestingly, there is evidence of potency differences or even ligand-specific signaling associated with these agonists, because L-amino acids induce Cai2+ oscillations with frequencies different from those of other CaR agonists (37). Thus the effects of divalent cations, calcimimetics, calcilytics, and L-aromatic amino acids on cell morphology were also tested.

PTH secretion and vasopressin-elicited water reabsorption in renal collecting ducts are two exocytic processes that either have been shown or have been suggested to be inhibited by CaR activation (3, 27, 32). Because changes in the actin filament network can retard vesicular trafficking and exocytosis, it is possible that the CaR interacts functionally with the cytoskeleton to suppress PTH secretion or to antagonize the vasopressin-induced fusion of aquaporin-2-containing vesicles with the collecting duct apical membrane. To test these hypotheses, it was first necessary to establish whether CaR activation can actually affect the cell cytoskeleton or cell morphology; therefore, in the current study, this issue was investigated in CaR-transfected HEK-293 cells treated with CaR-selective compounds.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. The Rho kinase inhibitors Y-27632 and H1152 {(S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]homopiperazine, 2HCl} were purchased from Calbiochem. The calcimimetics NPS R-467 and NPS S-467 and the calcilytic NPS 89636 were a kind gift of Dr. E. Nemeth (NPS Pharmaceuticals, Salt Lake City, UT). Fura-2 AM was obtained from Molecular Probes (Eugene, OR). All other chemicals were purchased from Sigma-Aldrich (Poole, UK).

Cell culture. HEK-293 cells stably transfected with human parathyroid CaR (10) were a gift from Dr. E. F. Nemeth (NPS Pharmaceuticals), and empty vector-transfected HEK-293 cells were a gift from Drs. K. Croskery and R. Prince (University of Manchester, Manchester, UK). Both nontransfected and stably transfected HEK-293 cells were grown in DMEM (Invitrogen, Paisley, UK) supplemented with 10% heat-inactivated FBS (HyClone, Cramlington, UK), and the stably transfected cells were treated routinely with 200 µg/ml hygromycin B (Boehringer-Mannheim, Lewes, UK) until 24 h before use.

Assessment of cell morphology and actin stress fiber assembly. For each treatment, four cell dishes were incubated in serum-free medium in the presence or absence of cotreatments for up to 3 h, and then for each dish, three regions of cells were photographed using an Olympus digital camera attached to an Olympus phase-contrast microscope. Alternatively, changes in cell morphology were observed using an AS MDW Live Cell Imaging System (37°C, 5% CO2; Leica Microsystems, Wetzlar, Germany). To assess actin stress fiber assembly, cells were grown on glass coverslips, treated as described above, and then fixed with paraformaldehyde and stained with phalloidin-TRITC before obtaining images using a Zeiss Axioplan 2 fluorescence microscope equipped with a Hamamatsu digital camera. Cytosolic TRITC fluorescence intensity per unit area was quantified using ImageJ software and was corrected for background levels. In some experiments, CaR-HEK cells were first cotransfected with kinectin domain (KiD)2-enhanced green fluorescent protein (EGFP) (Ref. 33; Dr. E. Vignal, University of Montpellier, Montpellier, France) or EGFP empty vector (Clontech) using FuGENE 6 reagent (Roche Applied Science, Basel, Switzerland).

ERK phosphorylation assay. Cells were grown to 80–90% confluence in 35-mm-diameter culture dishes, and ERK was assayed as described previously (35). Experiments were performed at 37°C before cells were lysed on ice in RIPA buffer supplemented with protease and phosphatase inhibitors, and then phospho-ERK was quantified using semiquantitative immunoblot analysis with a phosphospecific PAb (35).

Intracellular [Ca2+] measurement. Cells were cultured on glass coverslips and loaded with fura-2 AM (either 1 µM for 1–2 h or 5 µM for 20 min) at room temperature in the dark in buffer containing 20 mM HEPES, pH 7.4, 125 mM NaCl, 4 mM KCl, 1.2 mM CaCl2, 0.5 mM MgCl2, 5.5 mM glucose, and 0.1% BSA (Sigma). Unabsorbed fura-2 AM was removed by washing, and cells were equilibrated for 20 min in experimental buffer containing the baseline [Ca2+]o appropriate for the subsequent experiment. Dual-excitation wavelength microfluorometry was then performed using a Nikon Diaphot inverted microscope. The cells were mounted in a perfusion chamber (Warner Instruments, Hamden, CT) and observed through a x40 oil-immersion lens objective microscope. Experiments were performed in buffer containing (in mM) 20 HEPES, pH 7.4, 125 NaCl, 4 KCl, 0.5–3 CaCl2, 0.5 MgCl2, and 5.5 glucose at room temperature.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Incubation of the CaR-HEK cells in serum-free DMEM for up to 3 h induced cell stellation, whereas cotreatment with the calcimimetic NPS R-467 (1 µM) or exposure to elevated Mgo2+ levels (5.8 mM vs. 0.8 mM in control) elicited dose-dependent process retraction (Fig. 1A, i; see also supplemental movie for time-lapse images; http://ajpcell.physiology.org/cgi/content/full/00482.2005/DC1). Cotreatment with the calcilytic (CaR antagonist) NPS 89636 (1 µM) abolished the morphological changes induced by the CaR agonists (Fig. 1A,ii; see also supplemental movie), which were themselves without effect on HEK-293 cells stably transfected with the empty vector (Fig. 1A,iii). The effect in CaR-HEK-293 cells was also observed with increasing concentrations of Cao2+, although it was necessary to use a HEPES buffer rather than DMEM to avoid precipitation (Fig. 1B). Under these conditions, maximal process retraction occurred in the presence of 2.5–3 mM Cao2+, with a half-maximal response at ~2 mM Cao2+. Staining of CaR-HEK cells treated for 3 h with NPS R-467 (in serum-free DMEM) with phalloidin-TRITC revealed increased actin filament formation, an effect that was inhibited by calcilytic cotreatment (Fig. 2A). In HEPES buffer, Cao2+ also elicited dose-dependent actin filament formation (Fig. 2B), which we fitted to a sigmoid dose-response curve (variable slope, GraphPad Prism software; R2 = 0.9985, Hill coefficient nH = 4.1) with EC50 = 1.55 mM.


Figure 1
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  		Fig. 1. The Ca2+-sensing receptor (CaR) agonists induce morphological change in CaR-human embryonic kidney (HEK)-293 (CaR-HEK) cells, but not in calcilytic-cotreated cells or empty vector-transfected cells. A: incubation of CaR-HEK cells in serum-free medium for 3 h induced stellation, whereas cotreatment with the calcimimetic NPS R-467 (1 µM) or exposure to elevated extracellular Mg2+ concentration ([Mg2+]o) levels (5.8 mM final vs. 0.8 mM in medium) elicited dose-dependent cell process retraction (A,i). The responses to NPS R-467 and high [Mg2+]o were ablated by cotreatment with the calcilytic NPS 89636 (1 µM; A,ii), whereas the same treatments were without effect in empty vector-transfected HEK-293 cells (A,iii). B: CaR-HEK were incubated as above using a HEPES-based experimental buffer (see EXPERIMENTAL PROCEDURES) to avoid precipitation after Ca2+ supplementation. The cells were then incubated for 3 h at various (0.5–3 mM) extracellular Ca2+ concentrations ([Ca2+]o). All results are representative of a minimum of 4 independent experiments, with each experiment performed at least in triplicate.

 

Figure 2
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  		Fig. 2. Calcimimetic treatment induces actin filament formation. A: CaR-HEK cells grown on glass coverslips were incubated for 3 h in NPS R-467 (1 µM) ± NPS 89636 (1 µM) in DMEM and then fixed in paraformaldehyde and stained with phalloidin-TRITC as described in EXPERIMENTAL PROCEDURES. The average cytosolic TRITC fluorescence intensity per unit area was determined for each cell imaged and the resulting data are presented histographically. P < 0.001 vs. all other conditions. n ≥ 4. B: alternatively, cells were incubated for 3 h in HEPES buffer containing various [Ca2+]o concentrations (0.5–4 mM) before phalloidin-TRITC staining (n = 3).

 
The calcimimetic effect was stereoselective because NPS S-467 (1 µM) failed to alter CaR-HEK cell morphology (Fig. 3A, i), and these results are consistent with the effects of these compounds on ERK phosphorylation (Fig. 3A,ii). Next, we investigated the concentration-effect relationship of calcimimetic treatment on CaR-HEK cell morphology and found graded but partial changes with 40 and 60 nM, whereas the response to 100 nM NPS R-467 was near-maximal because increasing the concentration to 1 µM did not produce a greater effect (Fig. 3B).


Figure 3
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  		Fig. 3. Changes in CaR-HEK cell morphology induced by calcimimetic treatment are stereoselective as well as time- and dose-dependent. A: CaR-HEK cells were incubated in serum-free medium for 3 h in the presence or absence of NPS R-467 or NPS S-467 (1 µM; A,i) and then assessed morphologically as described in EXPERIMENTAL PROCEDURES. n = 4. Acute treatment with NPS R-467 (1 µM; in presence of 2.5 mM [Ca2+]o) elicited increased ERK phosphorylation, whereas NPS S-467 was without effect (A,ii). n = 3. B: CaR-HEK cells were exposed to various concentrations (40 nM–1 µM) of NPS R-467 for 3 h and then imaged.

 
Calcimimetic-induced CaR-HEK cell shape change was ablated using the Rho kinase inhibitors Y-27632 (10 µM) and H1152 (1 µM) (Fig. 4A; see also supplemental movie). In contrast, neither inhibitor attenuated calcimimetic-induced ERK activation (Fig. 4B) or had any effect on the Cai2+ oscillations induced by 3 mM Cao2+ or 1 µM NPS R-467 (Fig. 4C). To confirm the involvement of the Rho pathway in CaR-induced actin filament assembly, CaR-HEK cells were cotransfected transiently with KiD2, the kinectin domain that specifically binds RhoA, to test whether the sequestration of active RhoA protein by KiD2 could interfere with CaR responses (33). The EGFP tag on KiD2 was used to discriminate KiD2-expressing from KiD2-nonexpressing cells. In cells expressing KiD2-EGFP, little stress fiber assembly was observed inside the cells after calcimimetic treatment (Fig. 5A, i) and was equivalent to levels in untreated KiD2-EGFP-expressing cells (Fig. 5A,iii), although some actin-dense regions were present at membrane ruffle-like protrusions. However, in KiD2-negative cells, marked stress fiber assembly was observed throughout the cell (Fig. 5A,i) at a level comparable to that shown in Fig. 1. Some actin-dense regions were also present at the membrane, but phalloidin staining appeared less intense in these regions than in the KiD2-positive cells. Overexpression of the EGFP empty vector alone failed to interfere with CaR-induced stress fiber assembly (Fig. 5A,ii), and transfection efficiency was equivalent for both EGFP and KiD2-EGFP vectors (Fig. 5B).


Figure 4
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  		Fig. 4. CaR-induced morphological change is mediated by Rho kinase. A: CaR-HEK cells were incubated for 3 h with NPS R-467 (100 nM) in serum-free DMEM as described in Fig. 1. In the presence of the Rho kinase inhibitors H1152 (1 µM) (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]homopiperazin, 2HCl} (A,i) or 10 µM Y-27632 (A,ii), representing an experiment wholly independent from that shown in A,I, the morphological response to calcimimetic treatment was ablated. Identical results were obtained in 3 independent experiments, with each experiment performed in quadruplicate. B: CaR-induced ERK phosphorylation is not inhibited by Y-27632 (Y27) or H1152 (H11). Cells were incubated for 5 min at 37°C in 2.5 mM [Ca2+]o-containing buffer in the presence or absence of 1 µM NPS R-467. C: cells were loaded with fura-2 and then incubated in 1.2 mM [Ca2+]o and exposed to 1 µM NPS R-467 in the presence or absence of 10 µM Y-27632 and intracellular [Ca2+] ([Ca2+]i) assessed as described in EXPERIMENTAL PROCEDURES. The Cai2+ response to NPS R-467 was unaffected by the addition of Y-27632, either during the oscillatory phase or before calcimimetic treatment. The single-cell trace shown is representative of 3 independent experiments. n > 25 cells in each experiment.

 

Figure 5
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  		Fig. 5. Overexpression of the RhoA-binding protein kinectin domain (KiD)2 inhibits calcimimetic-induced stress fiber assembly. CaR-HEK cells were transiently transfected with KiD2-enhanced green fluorescent protein (EGFP) or with EGFP empty vector, seeded onto glass coverslips, and then incubated in serum-free DMEM for 3 h in the presence or absence of 1 µM NPS R-467. Cells were then fixed and stained with phalloidin-TRITC as before. A: after transfection of CaR-HEK cells with KiD2-EGFP vector, expression of the KiD2 was observed in some cells (A,i, bottom right) but not in others (top left). In the KiD2-negative cells, NPS R-467 induced stress fiber assembly as before; however, in the KiD2-expressing cells, few stress fibers were observed, whereas overexpression of EGFP empty vector alone failed to impair calcimimetic-induced stress fiber assembly (A,ii). A,iii, absence of stress fibers in untreated control cells, regardless of KiD2-EGFP expression (+vector, left; –vector, right). B: immunoblot analysis of KiD2-EGFP and empty vector EGFP-transfected cells with anti-EGFP antibody confirmed equivalent EGFP expression in both samples.

 
The CaR is a pleiotropic receptor that couples to Gq and some Gi proteins (in addition to the putative G12/13 association); thus we tested the contribution of these pathways to receptor-mediated process retraction. Neither cotreatment with the PLC-beta inhibitor U-73122 nor overnight pretreatment with pertussis toxin inhibited the CaR-induced morphological changes (Fig. 6A). However, in a parallel experiment (data not shown), pertussis toxin substantially inhibited CaR-induced ERK activation, confirming previous observations (13, 17) and validating its efficacy in these experiments.


Figure 6
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  		Fig. 6. CaR-induced cell morphological changes are unaffected by inhibitors of G{alpha}q, G{alpha}i, and epidermal growth factor receptor (EGFR) signaling. CaR-HEK cells were incubated for 3 h with NPS R-467 (100 nM) in serum-free DMEM as described in Fig. 1. Change in cell morphology induced by NPS R-467 was unaffected by cotreatment with the phosphatidylinositol 4,5-bisphosphate-PLC inhibitor U-73122 (1 µM), overnight pretreatment with 100 ng/ml pertussis toxin (PTX; A), matrix metalloproteinase inhibitor GM 6001 (10 µM; B), or EGFR antagonist AG 1478 (500 nM). n ≥ 4. B,ii, CaR-HEK cells were pretreated for 5 min with or without 10 µM GM 6001 or 500 nM AG 1478 and then exposed to 100 nM NPS R-467 and 2.5 mM [Ca2+]o in the continued presence or absence of the inhibitors (n = 3).

 
Recent reports suggested that CaR-induced ERK activation occurs via a triple-pass mechanism whereby CaR activation elicits matrix metalloproteinase-mediated transactivation of the epidermal growth factor receptor (EGFR) to induce the ERK pathway (19, 36). Indeed, this phenomenon was demonstrated in the present study by inhibiting CaR-induced ERK activation using GM 6001, a matrix metalloproteinase inhibitor, or the EGFR kinase inhibitor tyrphostin AG 1478. However, in the present study, neither agent impaired the CaR-induced cytoskeletal changes described (Fig. 6B).

The aromatic L-amino acids have been shown to act as allosteric activators of the CaR; that is, in the presence of partially activating concentrations of type I CaR agonists, cotreatment with an aromatic amino acid such as L-Phe or L-Trp increases agonist-induced Cai2+ mobilization. In the present study, the effect of aromatic amino acids on CaR-induced cell shape change were investigated. However, at none of the amino acid concentrations tested (up to 10 mM) was there any evidence of increased process retraction or actin filament assembly, whether treated in the presence of DMEM (Fig. 7A) or HEPES buffer containing 1.5 mM CaCl2 (data not shown). Similarly, L-Phe and L-Trp (10 mM) both failed to potentiate the ERK phosphorylation induced by either 30 µM neomycin or 2.5 mM Cao2+(Fig. 7B). The lack of amino acid effects could not be explained by the neomycin/Cao2+ treatments eliciting already maximal effects, because treatment with 100 µM neomycin or 5 mM Cao2+ induced substantially greater ERK phosphorylation. In all experiments, the addition of L-Phe or L-Trp (10 mM) alone had no effect on basal ERK phosphorylation.


Figure 7
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  		Fig. 7. Aromatic amino acids induce Cai2+ mobilization but not CaR-mediated morphological change or ERK activation. A: incubation of CaR-HEK cells in serum-free medium supplemented with 10 mM L-Phe, L-Trp, or L-His failed to mimic the CaR-induced process retraction observed in association with NPS R-467. The same result was obtained in 3 independent experiments, with each experiment performed in quadruplicate. CaR-HEK cells were treated with 30 or 100 µM neomycin (B,i,iii) or with 0.5, 2.5, or 5 mM [Ca2+]o (B,ii,iv) in the presence or absence of 10 mM aromatic amino acid (AA; B,i,ii, L-Phe; B,iii,iv, L-Trp) for 5 min at 37°C and then lysed in RIPA buffer and assayed for ERK phosphorylation as described in EXPERIMENTAL PROCEDURES. Neither amino acid increased the partial responses evoked by moderate neomycin (30 µM) or 2.5 mM [Ca2+]o treatment. Results are from a minimum of 3 independent experiments. C: CaR-HEK cells were loaded with fura-2 AM in buffer containing 1.5 mM [Ca2+]o and then exposed to L-Phe (3 and 10 mM) and 1 µM NPS R-467 in the presence of either 1.2 mM (B,i) or 2.5 mM (B,ii) [Ca2+]o. [Ca2+]i levels were then quantified as described in EXPERIMENTAL PROCEDURES. Exposure of the cells to 3 or 10 mM L-Phe induced Cai2+ oscillations when treated in the presence of high [Ca2+]o (2.5 mM) but not at low [Ca2+]o levels (1.2 mM). In contrast, the calcimimetic NPS R-467 induced oscillations in the presence of 1.2 mM [Ca2+]o but induced a nonoscillatory response in the presence of 2.5 mM [Ca2+]o. When the cells were incubated in DMEM instead of HEPES buffer to replicate conditions shown in A, L-Phe (10 mM) still elicited Cai2+ mobilization. n ≥ 3.

 
In contrast, analysis of Cai2+ concentrations in the CaR-HEK cells using microfluorometry confirmed that the amino acid treatments did elicit Cai2+ mobilization with Ca2+ oscillations (Fig. 7C) as reported previously (6, 37). In the presence of 1.2 mM Cao2+, there was no response to 3 mM L-Phe, whereas 10 mM L-Phe induced a single transient phase of Cai2+ mobilization in ~50% of the cells and NPS R-467 elicited sustained Cai2+ oscillations in all of the cells. When [Ca2+]o was increased to 2.5 mM, L-Phe induced sustained Cai2+ oscillations in most cells at both 3 and 10 mM concentrations, whereas NPS R-467 induced a spike and plateau response. Addition of L-Phe (10 mM) to cells incubated in DMEM containing 1.8 mM CaCl2 also elicited Cai2+ mobilization.


   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we show that CaR activation can elicit actin stress fiber assembly and process retraction and provide evidence that these phenomena are mediated via Rho/Rho kinase. The morphological response was observed both with the primary agonists Cao2+ and Mgo2+ and with the calcimimetics. The calcimimetic response was stereoselective, concentration dependent over a range consistent with CaR activation (23), absent in empty vector-transfected cells, and most important, inhibited by cotreatment with a calcilytic (20, 21), thus proving definitively that these responses are mediated via the CaR. Although CaR activation elicits rapid responses such as Cai2+ mobilization and ERK activation, the shape changes in the current study were observed during the course of several hours rather than within the first few minutes. Interestingly, half-maximal actin filament formation and process retraction was observed at concentrations of ~1.5–2 mM, which is lower than the EC50 values typically obtained for CaR-induced Cai2+ release, i.e., 3.3–4.2 mM Cao2+(2, 7, 9). This could suggest that the cytoskeletal response to CaR activation is more sensitive than the Cai2+ response, although it should be noted that Cai2+ mobilization is usually quantified within seconds, whereas the cytoskeletal changes reported herein were observed after 3 h and, in any case, were only semiquantitative.

Cotreatment with the Rho kinase inhibitors H1152 and Y-27632 prevented CaR-induced cell shape change, although the RhoA-binding protein KiD2 (32) disrupted CaR-induced stress fiber assembly. The effects of the Rho kinase inhibitors on CaR signaling appeared to be specific to the cytoskeleton because Cao2+- and calcimimetic-induced Cai2+ oscillations were unaffected by them. Similarly, H1152 and Y-27632 failed to inhibit calcimimetic-induced ERK activation. Thus, together, these data implicate Rho activity in the cytoskeletal and morphological changes elicited by the CaR.

Because the calcimimetic-induced shape changes were not affected by pertussis toxin pretreatment or by U-73122, it appears that the effects are not mediated via Gi/o signaling or via the Gq/11/PLC pathway, respectively. Therefore, together, these data provide further evidence that the CaR can couple to the Rho pathway, presumably via G12/13 (14, 26, 28). Given the pleiotropic nature of the CaR, it will be interesting to learn whether the relative coupling of Gq/11, Gi/o, and G12/13 proteins to the CaR is determined merely by the stoichiometry of their expression or whether it could be regulated specifically by other signals or, for example, by prior receptor activation. In any case, the current data demonstrate that in future CaR investigations, HEK-293 cell shape changes will represent a convenient, efficient readout for receptor activation and/or antagonism.

Other GPCR agonists known to elicit Rho-mediated cytoskeletal changes (for review, see Ref. 31) include lysophosphatidic acid (LPA) (15), thrombin (16), and cholecystokinin (24). Indeed, LPA is present in serum, and its removal from serum-deprived cells most likely accounts for the stellation observed in the control cells. There is evidence that CaR-induced ERK activation occurs via the matrix metalloproteinase-mediated activation of the EGFR (19), and it has been reported that the EGFR antagonist AG 1478 can prevent LPA-induced stress fiber assembly (11), suggesting a similar mechanism in that case. In addition, there is a known association between growth factors, including EGF and Rho activation (29). However, in the present study, neither matrix metalloproteinase inhibition (GM 6001) nor EGFR antagonism (AG 1478) affected morphological changes, suggesting that the triple-pass mechanism that mediates or at least contributes to CaR-induced ERK activation (19) does not mediate CaR-induced process retraction.

The aromatic amino acids L-Phe and L-Trp elicited CaR-induced Cai2+ mobilization with oscillations as they did for other CaR agonists; yet, they failed to elicit detectable changes in cell morphology or potentiation of ERK activation at the Cao2+ and neomycin concentrations tested. This apparent ligand-specific signaling is consistent with the observations of Rozengurt and co-workers (28, 37, 38), who reported that L-aromatic acids induce CaR-mediated Cai2+ oscillations with a frequency different from Cao2+, possibly by different intracellular signaling pathways, and that the responses to these agonists are differentially affected by Thr888Ala residue mutation. Interestingly, in the present study, calcimimetic treatment induced robust Cai2+ oscillations in the presence of 1.2 mM Cao2+ but a spike and plateau response when cotreated together with 2.5 mM Cao2+, indicating a more potent stimulation of the receptor. Similarly, the L-Phe responses were markedly greater when cotreated with 2.5 mM Cao2+ than with 1.2 mM Cao2+, in which the amino acids had little effect. It should be noted that in contrast to the recent study of Rey et al. (28), who examined the responses to transiently transfected CaR, the amino acid-induced Cai2+ oscillations reported in the present study appeared to be sinusoidal. Indeed, the lack of amino acid response in the morphology and ERK experiments compared with the calcimimetic responses might be explained by their considerably different receptor potency profiles (possibly influenced by their apparently independent binding sites) rather than by differential intracellular signaling. However, if this were the case, then the amino acids would be more likely to induce cytoskeletal change, given the apparently heightened sensitivity of this response to Cao2+. Overall, there is no definitive evidence in the current study to prove whether the differential effects of Cao2+ and the aromatic amino acids are related to potency or to ligand-specific signaling. All that can be stated at present is that under conditions in which Cao2+ and aromatic amino acids induce Cai2+ mobilization, only Cao2+, Mgo2+, or NPS R-467, elicits cytoskeletal alterations.

One possible mechanism for the CaR-mediated acute suppression of PTH secretion could be limitation of the movement of secretory granules through the parathyroid cell cytoskeletal network. The function of cortical actin as a barrier to exocytosis has been described in a number of endocrine cells, such as adrenal chromaffin cells and pancreatic beta-cells (4), although it has yet to be demonstrated in parathyroid cells. Significantly, the cortical actin barrier is not simply a constitutive brake on secretion but can be regulated via extracellular signals causing actin assembly/disassembly (4). Therefore, it is possible that CaR-induced cytoskeletal changes could contribute to the acute inhibition of PTH secretion by impairing secretory granule exocytosis. Furthermore, in the kidney collecting duct, CaR-induced cytoskeletal changes could perhaps contribute to the Cao2+-mediated antagonism of vasopressin-elicited water reabsorption (26, 31). Because the CaR is also expressed in neurons (30), then a role in neurite extension is also possible (18). In fact, Rho activation has been associated with a wide variety of cellular processes, ranging from the regulation of cell adhesion and polarization to cell fate effects such as differentiation and mitogenesis (5, 8) and therefore the potential role of CaR-induced Rho signaling could be significant.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Biotechnology and Biological Sciences Research Council Grant BBSB04986 and Kidney Research UK Grant (NKRF) TF6/2002. D. T. Ward was a recipient of a NKRF Career Development Fellowship.


   ACKNOWLEDGMENTS
 
We thank Drs. David Maldonado-Pérez, Tristan Bouschet, Arthur Conigrave, and Daniela Riccardi for advice and technical assistance and Dr. Ed Nemeth (NPS Pharmaceuticals, Salt Lake City, UT) for supplying calcimimetic-and calcilytic reagents.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. Ward, The Univ. of Manchester, Faculty of Life Sciences, Fl. 2, Core Technology Facility, 46 Grafton St., Manchester, M13 9NT, UK (e-mail: d.ward{at}manchester.ac.uk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* S. L. Davies and C. E. Gibbons contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
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