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Calcium-sensing receptor modulates cell adhesion and migration via integrins - PubMed

  • ️Sat Jan 01 2011

Calcium-sensing receptor modulates cell adhesion and migration via integrins

Sujeenthar Tharmalingam et al. J Biol Chem. 2011.

Abstract

The calcium-sensing receptor (CaSR) is a family C G protein-coupled receptor that is activated by elevated levels of extracellular divalent cations. The CaSR couples to members of the G(q) family of G proteins, and in the endocrine system this receptor is instrumental in regulating the release of parathyroid hormone from the parathyroid gland and calcitonin from thyroid cells. Here, we demonstrate that in medullary thyroid carcinoma cells, the CaSR promotes cellular adhesion and migration via coupling to members of the integrin family of extracellular matrix-binding proteins. Immunopurification and mass spectrometry, co-immunoprecipitation, and co-localization studies showed that the CaSR and β1-containing integrins are components of a macromolecular protein complex. In fibronectin-based cell adhesion and migration assays, the CaSR-positive allosteric modulator NPS R-568 induced a concentration-dependent increase in cell adhesion and migration; both of these effects were blocked by a specific CaSR-negative allosteric modulator. These effects were mediated by integrins because they were blocked by a peptide inhibitor of integrin binding to fibronectin and β1 knockdown experiments. An analysis of intracellular signaling pathways revealed a key role for CaSR-induced phospholipase C activation and the release of intracellular calcium. These results demonstrate for the first time that an ion-sensing G protein-coupled receptor functionally couples to the integrins and, in conjunction with intracellular calcium release, promotes cellular adhesion and migration in tumor cells. The significance of this interaction is further highlighted by studies implicating the CaSR in cancer metastasis, axonal growth, and stem cell attachment, functions that rely on integrin-mediated cell adhesion.

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Figures

FIGURE 1.
FIGURE 1.

Co-immunoprecipitation (IP) of the CaSR and β1 integrin from rMTC 44-2 cells. Samples of solubilized (Sol) rMTC 44-2 cells were immunoprecipitated using three anti-CaSR-specific antibodies (ADD, 4640, or 4641) targeting the extracellular domain of the CaSR and two different antibodies targeting the C or N terminus of β1 integrin. The samples were subjected to immunoblotting (IB) using the ADD anti-CaSR monoclonal antibody and anti-β1 integrin antibodies. A, deglycosylation of the CaSR with peptide:N-glycosidase (PNGase) F showing that the upper band in the doublet is the fully glycosylated form of the receptor, and the lower band is the core-glycosylated form; B and C, co-immunoprecipitation of CaSR and β1 integrin using the CaSR ADD antibody and β1 integrin-specific N-terminal antibody. D and E, co-immunoprecipitation of CaSR and β1 integrin using CaSR-specific 4640 and 4641 antibodies, and β1 integrin-specific C-terminal antibody. The β1 integrin N-terminal antibody recognized primarily the immature 88-kDa form of the protein (C), although the C-terminal β1 antibody recognized primarily the fully glycosylated 130-kDa form (E). The blots are representative of two experiments with similar results.

FIGURE 2.
FIGURE 2.

Cellular localization and cell surface expression of the CaSR and β1 integrin in rMTC 44-2 cells. A, cells grown in subthreshold (1.8 m

m

) Ca2+ were immunolabeled with extracellular epitope mouse ADD anti-CaSR antibody (1:500) and a rabbit anti-β1 integrin antibody (1:400) and were viewed using a confocal microscope (×100). CaSR expression was present in discrete puncta distributed throughout the cell (green). β1 integrin was also expressed throughout the cell (red) and showed extensive overlap with the CaSR expression (yellow). Photomicrographs are representative of similar results obtained in three experiments. B, cells were preincubated with 0.5 m

m

Ca2+ for 5 h prior to addition of buffer (control) or 10 m

m

Ca2+ for 10 min, and cell surface proteins were covalently biotinylated and subjected to streptavidin-agarose chromatography. Cytoplasmic proteins appeared in the Flow Through fraction, whereas cell surface proteins were eluted in the Eluate. The CaSR, β1 integrin, and α3 subunits of the Na+/K+-ATPase (plasma membrane marker) were found in the cell surface fraction, whereas IP3 receptor (intracellular membrane marker) was found in the intracellular fractions. The CaSR immunoblot analysis (using the ADD monoclonal antibody) showed that the 130-kDa mature form of the CaSR was recovered primarily in the cell surface fraction as expected, whereas the 120-kDa immature form was recovered in the “flow-through” (n = 3). C, band intensities present in the eluted fractions of the CaSR, β1 integrin, and α3 subunit blots were analyzed and are presented as a percent of control (n = 3).

FIGURE 3.
FIGURE 3.

CaSR-positive allosteric modulator NPS R-568 potentiated fibronectin mediated cell adhesion. A, rMTC 44-2 cells were incubated on fibronectin-coated wells (0–15 μg/ml) in the absence or presence of 0.02% DMSO (control), 6 μ

m

NPS R-568, or 6 μ

m

NPS 89636. Treatment of rMTC 44-2 cells with 6 μ

m

NPS R-568 significantly increased cell adhesion over control levels, whereas 6 μ

m

of the CaSR antagonist NPS 89636 did not significantly affect cell adhesion. Each condition from individual experiments was performed in triplicate and expressed as a percentage of “input,” which represents control wells that were not subjected to washes. Data points were normalized to maximal cell adhesion level obtained under the control condition (i.e. in the absence of R-568 or 89636) (n = 5). B, NPS R-568 increased cell adhesion in a concentration-dependent manner. rMTC 44-2 cells were incubated on 1 μg/ml fibronectin in the presence of various concentrations of NPS R-568 (1 n

m

to 10 μ

m

) or DMSO control (3 × 10−6 to 3 × 10−3%). The data points (mean ± S.E.) were normalized by standardizing basal cell adhesion levels to 0% and maximal adhesion levels to 100% by analyzing the NPS R-568-treated data using GraphPad software. At 1 μg/ml fibronectin, NPS R-568 stimulated cell adhesion in a dose-dependent manner with an EC50 = 111 ± 29 n

m

and Hill slope = 1.9 ± 0.8 (n = 7). C, at 2.5 μg/ml fibronectin, NPS R-568 displayed a more potent dose-dependent stimulation on cell adhesion, with EC50 at 38 ± 12 n

m

and Hill slope at 1.6 ± 0.5 (n = 7). The DMSO vehicle controls showed essentially no effect on cell adhesion.

FIGURE 4.
FIGURE 4.

GRGDSP peptide inhibited NPS R-568-mediated cell adhesion. rMTC 44-2 cells were incubated on 1 μg/ml fibronectin or 2.5 μg/ml fibronectin in the presence of 300 n

m

R-568 with varying concentrations of the integrin-inhibiting peptide, GRGDSP, or a control peptide, GRADSP, that does not inhibit integrins. A, at 1 μg/ml fibronectin, GRGDSP peptide significantly inhibited NPS R-568-mediated cell adhesion at peptide concentrations greater than 100 μ

m

compared with the control GRADSP peptide (n = 2–4). B, at 2.5 μg/ml fibronectin, 500 μ

m

GRGDSP peptide was required to significantly reduce the potentiation effects of 300 n

m

NPS R-568 on cell adhesion compared with GRADSP peptide (n = 4). The control GRADSP peptide showed a small nonsignificant degree of inhibition on cell adhesion at all concentrations. Data points represent the mean ± S.E. normalized to cell adhesion levels obtained by stimulation with 300 n

m

NPS R-568 in the absence of peptides. *, p < 0.05.

FIGURE 5.
FIGURE 5.

NPS R-568 increased fibronectin-mediated cell migration via integrin activation. rMTC 44-2 cells added to the upper surface of transwell filters migrated to the lower chamber coated with 10 μg/ml fibronectin. 1 μ

m

NPS R-568 induced a 2-fold increase in cell migration toward the fibronectin matrix as compared with control conditions. The effects of NPS R-568 on cell migration were inhibited in the presence of 500 μ

m

GRGDSP or 1 μ

m

NPS 89636 but not by 500 μ

m

of the GRADSP control peptide. 1 μ

m

NPS 89636 treatment alone did not inhibit cell migration. Each data point represents the mean ± S.E. of 3–6 independent experiments. *, p < 0.005.

FIGURE 6.
FIGURE 6.

shRNA-mediated knockdown of β1 integrins reduces CaSR-stimulated cell migration. Cells were treated with one of three shRNA constructs as follows: a scrambled control and two β1 integrins shRNAs (#1 and #2). A, Western blot probed with anti-β1 integrin antibody and anti-GAPDH antibody (loading control). B, effects of shRNA on cell adhesion (1 μg/ml fibronectin); C, effects of shRNA on cell migration (10 μg/ml fibronectin). B and C, each column is the average ± S.E. of 2–3 experiments. *, p < 0.05.

FIGURE 7.
FIGURE 7.

Effects of intracellular signaling inhibitors on Ca2+ release, ERK activation, and CaSR-mediated cell adhesion and migration. A, effects of the PLC inhibitor U73122 and the inactive isomer U73343 on CaSR-induced increase in [Ca2+]i. B, effects of U73122 on NPS R-568 potentiation of cell adhesion. C, effects of U73122 on cell migration. D, BAPTA inhibition of [Ca2+]i release. E, effects of BAPTA on NPS R-568 enhancement of cell adhesion. F, BAPTA inhibition of the NPS R-568-mediated potentiation of cell migration. G, effects of the ERK inhibitor PD 98059 on ERK activation. H, effects of PD 98059 on cell adhesion. I, PD 98059 enhancement of NPS R-568 potentiation of cell migration. J, ROCK inhibitor Y-27632 had no effect on cell adhesion. K, effects of Y-27632 on cell migration. Each bar represents the mean ± S.E. of 3–5 independent experiments. *, p < 0.05.

FIGURE 8.
FIGURE 8.

Proposed mechanistic scheme for CaSR-integrin interactions. The figure depicts one possible scenario where the CaSR may be associated with integrin heterodimers in a signaling complex. However, the actual protein-protein interaction may be indirect and involve additional proteins. Left, complex prior to CaSR stimulation; right, elevated extracellular cation concentrations activate the CaSR causing its Venus flytrap domain to undergo a conformational change. This protein domain movement together with increased [Ca2+]i from Gαq activation of PLC and the IP3 receptor could induce the integrin to flip it into an activated state promoting binding to the ECM resulting in enhanced cell adhesion and migration. The X in the right panel denotes a hypothetical protein that may participate in facilitating the CaSR-mediated activation of integrins subsequent to increased [Ca2+]i.

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