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

JCS03469 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Characterisation of a HEK cell line stably expressing rat CaSR. (A). CaSR immunofluorescence. WT or CaSR-HEK cells were fixed and stained for CaSR and nuclei using an anti-CaSR antibody and DAPI, respectively. The staining was performed in the presence or absence of the antigenic peptide. Note that the GPCR is not expressed in HEK WT cells but is present in all CaSR-HEK cells and that this fluorescence disappears when the antigenic peptide is added, demonstrating the specificity of the labelling. (B) The CaSR is surface expressed. Total and surface CaSR were assessed using biotinylation assays followed by immunoblotting using an anti-CaSR antibody as described (Bouschet et al., 2005). (C) Stimulation of the ERK1/2 pathway. CaSR-HEK cells were treated with 5 mM CaCl₂ or 10 μM NPS R-467 for the indicated periods of time and lysed directly in Laemmli’s buffer. Samples were then subjected to immunoblotting using antibodies recognizing the diphosphorylated (activated) forms of ERK1/2 and β-tubulin (to ensure equal protein loading).

• Supplemental Figure S2 -

  Fig. S2. Increasing extracellular calcium stimulates CaSR HEK cell chemotaxis. CaSR HEK cells were placed on 8-μm-pore membranes facing control or high calcium solution and allowed to migrate for 5 hours. Cells were removed from the upper chamber, leaving only those that migrated through the membrane pores that were counted (see Materials and methods). Two experiments done in duplicate were performed.

• Movie 1 -

  Movie 1. Increasing Ca²⁺_o from 1 to 5 mM induces PM ruffling of SEP-CaSR-expressing HEK cells. HEK cells expressing SEP-CaSR were stimulated by an increase in Ca²⁺_o and imaged on the temperature-controlled stage of a Zeiss LSM510 confocal laser scanning microscope at 37°C. Single images were captured every 15 seconds for 30 minutes. The movie displays 15 frames per second and Ca²⁺_o was added after 8 frames.

• Movie 2 -

  Movie 2. 3D projection from z-stack of phalloidin-staining of an unstimulated CaSR-HEK cell.

• Movie 3 -

  Movie 3. 3D projection from z-stack of phalloidin-staining of a CaSR-HEK cell stimulated with 5 mM calcium for 10 minutes.

• Movie 4 -

  Movie 4. Relocation of GFP-β-arrestin 1 to protrusions of cells stimulated with calcium. HEK cells co-expressing CaSR and GFP-β-arrestin 1 were stimulated by an increase in Ca²⁺_o and imaged on the temperature-controlled stage of a Zeiss LSM510 confocal laser scanning microscope at 37°C. Single images were captured every 10 seconds. The movie displays 8 frames per second.

• Movie 5 -

  Movie 5. Relocation of GFP-β-arrestin 1 to protrusions of cells stimulated with NPS R-467. HEK cells co-expressing CaSR and GFP-β-arrestin 1 were stimulated by 10 μM NPS R-467 and imaged on the temperature-controlled stage of a Zeiss LSM510 confocal laser scanning microscope at 37°C. Single images were captured every 10 seconds. The movie displays 8 frames per second.