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First published online 10 July 2007
doi: 10.1242/jcs.03469

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The calcium-sensing receptor changes cell shape via a -arrestin-1–ARNO–ARF6–ELMO protein network

Tristan Bouschet^1,*, Stéphane Martin¹, Venkateswarlu Kanamarlapudi^2,, Stuart Mundell² and Jeremy M. Henley^1,

¹ Department of Anatomy, MRC Centre for Synaptic Plasticity, University of Bristol, University Walk, Bristol, BS8 1TD, UK
² Department of Pharmacology, School of Medical Sciences, University of Bristol, University Walk, Bristol, BS8 1TD, UK

View larger version (47K): [in this window] [in a new window]   Fig. 1. (A) CaSR stimulation generates PM ruffling. HEK cells were transfected with SEP-CaSR. 48 hours post-transfection, cells were challenged (bottom) or not (top) with 5 mM CaCl2 for 10 minutes, fixed and stained for SEP-CaSR (green) and F-actin (Alexa-Fluor-568–phalloidin, red). Colocalisation areas appear in yellow in the merge panels. Cells exhibiting PM ruffles are shown by empty arrowheads while cells that did not change shape are indicated by filled arrowheads. Bars, 10 µm. (B) Representative micrographs of CaSR-agonist-induced cytoskeleton reorganisation in SEP-CaSR-expressing HEK cells before (untreated) and after 5 mM CaCl2 or 10 µM NPS R-467 stimulation for 10 minutes. Colocalisation of SEP-CaSR (green) and F-actin (red) appears in yellow in the merge panels. The right panels show fourfold magnified views of the boxed regions. Note the presence of SEP-CaSR at the edge of protrusions. The PM ruffles are shown by arrowheads. Bars, 5 µm. Pictures shown are representative of six independent experiments. (C) Quantification of the effects of CaSR agonists on PM ruffling of cells. These results are the mean±s.e.m. of three to six independent experiments. *P0.001 compared with controls. (D) Extracellular-calcium-mediated PM ruffling is CaSR-dependent. Representative confocal images of F-actin staining of wild-type (WT) or stably expressing CaSR HEK cells challenged with 5 mM CaCl2 for 10 minutes, in the presence (+) or absence (–) of CaSR antagonist (NPS 89636 at 1 µM, pre-incubated for 10 minutes). Ruffling cells are indicated by arrowheads. Bars: 10 µm. (E) Quantification of cells exhibiting PM ruffling in the conditions described in (D). The results shown are the mean± s.e.m. of three independent experiments. *P0.001 compared with untreated. (F) Time course of CaSR-induced PM ruffling. The percentage of SEP-CaSR-expressing cells exhibiting PM ruffling after different times of CaCl2 stimulation was determined. All time points of stimulation were significantly different (P0.001) from the untreated (time 0 minutes) condition.

View larger version (51K): [in this window] [in a new window]   Fig. 2. -arrestin 1 is required for CaSR-induced PM ruffling. (A) Expression of a dominant negative form of -arrestin 1 impairs CaSR-induced PM ruffling. Representative pictures of HEK cells expressing SEP-CaSR in combination with dominant negative -arrestin 1 (319-418) or wild-type (WT) -arrestin 1. HEK cells were co-tranfected with SEP-CaSR (1 µg) and excess (4 µg) dominant negative -arrestin 1 (319-418), wild-type (WT) -arrestin 1 or CAT (as a control plasmid). Cells were stimulated with CaSR agonists (5 mM CaCl2, 10 µM NPS R-467) for 10 minutes, fixed and stained for SEP-CaSR (green), and F-actin (red). Arrowheads indicate ruffles. The micrographs are representative of three independent experiments. (B) Histogram (expressed as mean±s.e.m.) of CaSR agonist-induced PM ruffling in the conditions presented in A. *P0.001 compared with control plasmid-expressing cells stimulated with agonists (n=3). (C) siRNA-mediated silencing of -arrestin 1 impairs CaSR-induced PM ruffling. HEK cells were transfected with a siRNA duplex targeting -arrestin 1 (or a siRNA control) together with SEP-CaSR. 50 hours post-transfection, cells were stimulated with agonists, imaged and scored. Arrows indicate transfected cells whose morphology was unaffected and arrowheads point to the ruffles. (D) Quantification of data represented in C. *P0.001 compared with control siRNA-expressing cells stimulated with agonists. (n=3). (E) Protein samples prepared in parallel to the ruffling experiments from cells transfected simultaneously with CaSR and siRNA, described in C and D, were probed with CaSR (top panel) and pan -arrestin (A1CT, bottom panel) antibodies, respectively. (F) Pan -arrestin (A1CT) blot of cells transfected with control or -arrestin 1 siRNA alone showing 60% knockdown of -arrestin 1 (see text for details).

View larger version (33K): [in this window] [in a new window]   Fig. 3. Effect of CaSR stimulation on -arrestin 1. (A) Representative frames from live confocal microscopy experiments showing the subcellular redistribution of GFP--arrestin 1 in HEK cells co-expressing CaSR before (0 seconds) and after (30-300 seconds) activation by 5 mM CaCl2. (B) HEK cells co-expressing GFP--arrestin 1 and CaSR (1 and 4 µg, respectively) were stimulated with 5 mM CaCl2 for 2 or 10 minutes, fixed and stained for F-actin. Colocalisation of GFP--arrestin 1 (green) and F-actin (red) appears in yellow. Arrows indicate ruffles. Bar, 5 µm. Data are representative of four independent experiments. (C) The CaSR is constitutively associated with -arrestin 1. HEK293 cells were transfected with SEP-CaSR and Flag--arrestin 1 in the indicated combinations. 48 hours post-transfection, cells were stimulated with 5 mM CaCl2 for different periods of time and lysed. 400 µg of resulting proteins were subjected to immunoprecipitation (IP) using sepharose beads coupled to Flag-M2 antibodies. CaSR present in the immunoprecipitate and total fractions of CaSR and -arrestin 1 (input) were assessed by immunoblotting (IB).

View larger version (44K): [in this window] [in a new window]   Fig. 4. Role of ARNO in CaSR-induced PM ruffling. (A) ARNO(E156K) impairs CaSR-induced PM ruffling. CaSR-HEK cells expressing GFP, Myc-ARNO(E156K) or Myc-WT-ARNO (cells shown in C) were stimulated with 5 mM CaCl2, for 10 minutes, fixed and stained for Myc-ARNO (using 9E10 anti-Myc monoclonal antibody followed by a Cy2-labelled anti-mouse Ab) (green), and F-actin (red). Arrows indicate the cells that did not change shape. Arrows point to transfected cells whose morphology was unaffected and arrowheads point to ruffles. The micrographs are representative of three independent experiments. Bar, 10 µm. (B) Quantification of CaSR-induced PM ruffling illustrated in A. *P0.001. NS, no significant difference between tested conditions (P>0.05); n=3. (C) Myc-ARNO-expressing CaSR-HEK cells were challenged or not with 5 mM CaCl2 for 10 minutes, fixed and stained for Myc-ARNO and F-actin as described in A. Bar, 5 µm. Note ARNO relocation to protrusions following CaSR stimulation.

View larger version (51K): [in this window] [in a new window]   Fig. 5. -arrestin 1 interacts and colocalises with ARNO. (A) Myc-WT-ARNO co-immunoprecipitates with Flag--arrestin 1. CaSR-HEK cells were transfected with plasmids encoding Myc-WT-ARNO ± Flag--arrestin 1. 48 hours post-transfection, cells lysates were prepared and subjected to immnoprecipitation using Flag beads (see Materials and Methods). Retained proteins were eluted with Laemmli's buffer and immunoblotted using anti--arrestins and ARNO antibodies. A total of the lysate (input) was run in parallel. (B) Catalytically inactive Myc-ARNO(E156K) co-immunoprecipitates with Flag--arrestin 1. (C) CaSR stimulation does not affect ARNO association with Flag--arrestin 1. Cells co-expressing WT-ARNO and Flag--arrestin 1 were stimulated with CaCl2 for the indicated periods of time and Flag immunoprecipitation was performed as in A. Total ARNO and ARNO bound to Flag--arrestin 1 are shown. (D) Colocalisation of Myc-WT-ARNO and GFP–-arrestin 1 in protrusions. Transfected CaSR-HEK cells were challenged with 5 mM CaCl2 for 1 or 10 minutes, fixed and stained for GFP--arrestin 1 (green), Myc-ARNO (using 9E10 Ab followed by a Cy5-coupled anti-mouse Ab, shown in blue) and F-actin (Alexa-Fluor-568–phalloidin, red). An inset showing a magnified view for the 1 minute stimulation highlights -arrestin 1 and ARNO colocalisation in protrusions. Bar, 5 µm. (E) GFP-ARNO, RFP--arrestin 1 and Flag-CaSR colocalise in protrusions. Transfected HEK cells were challenged with 5 mM CaCl2 for 10 minutes, fixed and stained for GFP-ARNO (green), mRFP--arrestin 1 (red) and Flag-CaSR (detected by mouse anti-Flag Ab followed by a Cy5-coupled anti-mouse Ab, blue). A magnified view of the PM ruffling area is shown in the bottom panels. Merge areas appear in white. Bar, 5 µm.

View larger version (37K): [in this window] [in a new window]   Fig. 6. ARF6 is involved in CaSR-induced PM ruffling. (A) ARF6(T27N) impairs CaSR-induced PM ruffling. CaSR-HEK cells transfected with GFP, GFP-ARF6T27N or GFP-ARF6WT were treated with 5 mM CaCl2, for 10 minutes, fixed and stained for F-actin (red). The micrographs are representative of three independent experiments. Arrowheads indicate cells exhibiting ruffles and the arrow indicates the cell that did not ruffle. Bars, 10 µm. Right panels are a magnified view of inserts. (B) Quantification of CaSR-induced PM ruffling in cells presented in (A). *P0.001. NS, no significant difference (P>0.05, n=3). (C) Endogenous ARF6 is targeted to PM ruffles. CaSR-HEK cells were challenged with 5 mM CaCl2, for 10 minutes, fixed and stained for endogenous ARF6 (using a rabbit anti-ARF6 Ab followed by a Cy2-coupled anti-rabbit Ab, green) and for F-actin (red). Ruffles are indicated by arrowheads. Colocalisation areas appear in yellow. Bars, 5 µm.

View larger version (43K): [in this window] [in a new window]   Fig. 7. ELMO is involved in CaSR-induced PM ruffling. (A) Expression of GFP-ELMO(T629) but not GFP-WT-ELMO impairs CaSR-induced PM ruffling. CaSR-HEK cells transfected with GFP-ELMO(T629), GFP-WT-ELMO or GFP were stimulated with 5 mM CaCl2 for 10 minutes, fixed and stained for GFP (green) and F-actin (red). The micrographs are representative of three independent experiments. Arrowheads indicate cells exhibiting ruffles and arrows indicate cells not ruffling. Right panels are a magnified view of the boxed regions in the merge panels. (B) Quantification of cells presented in (A). *P0.001. NS, not significantly different (P>0.05) (n=3). (C) GFP-ELMO and Flag-CaSR colocalise in protrusions. HEK cells co-expressing the two plasmids indicated above were challenged with 5 mM CaCl2 for 2 or 10 minutes, fixed and stained for GFP-ELMO (green) and Flag-CaSR (red). Arrowheads show protrusions, and colocalisation areas appear in yellow. Bar, 5 µm.

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