Role of receptor activity modifying protein 1 in function of the calcium sensing receptor in the human TT thyroid carcinoma cell line

Abstract

The Calcium Sensing Receptor (CaSR) plays a role in calcium homeostasis by sensing minute changes in serum Ca(2+) and modulating secretion of calciotropic hormones. It has been shown in transfected cells that accessory proteins known as Receptor Activity Modifying Proteins (RAMPs), specifically RAMPs 1 and 3, are required for cell-surface trafficking of the CaSR. These effects have only been demonstrated in transfected cells, so their physiological relevance is unclear. Here we explored CaSR/RAMP interactions in detail, and showed that in thyroid human carcinoma cells, RAMP1 is required for trafficking of the CaSR. Furthermore, we show that normal RAMP1 function is required for intracellular responses to ligands. Specifically, to confirm earlier studies with tagged constructs, and to provide the additional benefit of quantitative stoichiometric analysis, we used fluorescence resonance energy transfer to show equal abilities of RAMP1 and 3 to chaperone CaSR to the cell surface, though RAMP3 interacted more efficiently with the receptor. Furthermore, a higher fraction of RAMP3 than RAMP1 was observed in CaSR-complexes on the cell-surface, suggesting different ratios of RAMPs to CaSR. In order to determine relevance of these findings in an endogenous expression system we assessed the effect of RAMP1 siRNA knock-down in medullary thyroid carcinoma TT cells, (which express RAMP1, but not RAMP3 constitutively) and measured a significant 50% attenuation of signalling in response to CaSR ligands Cinacalcet and neomycin. Blockade of RAMP1 using specific antibodies induced a concentration-dependent reduction in CaSR-mediated signalling in response to Cinacalcet in TT cells, suggesting a novel functional role for RAMP1 in regulation of CaSR signalling in addition to its known role in receptor trafficking. These data provide evidence that RAMPs traffic the CaSR as higher-level oligomers and play a role in CaSR signalling even after cell surface localisation has occurred.

Figure 1. Spatial localization of CaSR and RAMPs in COS-7 cells.

(A) COS-7 cell transfected with CaSR-citrine alone showing intracellular localisation of CaSR (red circle) in the absence of RAMP expression. (B) COS-7 cells co-transfected with RAMP-cerulean (left column) and CaSR-citrine (column 2) were imaged by confocal microscopy 48 hr post transfection. 50 pixel dot ROI was manually drawn around the cell membrane of the CaSR-citrine image to measure cell-surface FRET (column 3). Scale bar 10µm. Red arrows on the FRET images (right column) indicate areas of co-localization between CaSR and RAMP on the cell-surface, which are shown magnified in the insets.

Figure 2. Cell-surface FRET efficiencies of CaSR+RAMPs and fraction of receptor components involved in FRET complex.

(A) Cell-surface FRET efficiencies of individual RAMPs with CaSR compared among themselves and also with respective negative control Citrine alone+RAMP-Cerulean. Data are expressed as a percentage of a positive control comprising cells expressing a Citrine-Cerulean fusion protein. *** p<0.001 (2-way ANOVA, Bonferroni post-test) ***p<0.001 (Kruskal-Wallis test, Dunn's multiple comparison test) (B) and (C) Stoichiometric analysis of fraction of donor RAMP (Fd) and acceptor CaSR (Fa) present in FRET complex on the cell-surface, respectively.* p<0.05 Mann Whitney test. The graph represents data from three independent experiments.

Figure 3. Cell surface expression of non-tagged CaSR.

(A) Western blot of membrane and total cell lysate preparations from COS-7 cells transfected with CaSR alone (lane 1), CaSR and RAMP1 (lane 2), and CaSR and RAMP3 (lane 3), incubated with antibody to the CaSR, demonstrating the ability of RAMPs 1 and 3 to traffic the CaSR to the cell surface. (B) FACS analyses of non-permeabilised COS-7 cells expressing CaSR with either RAMPs 1, 2 or 3 showing shift from the IgG control in number of cells with surface fluorescence in RAMP1 and RAMP3 expressing cells, but not RAMP2 expressing cells.

Figure 4. Expression of CaSR and RAMP1 in TT cells.

(A) mRNA expression levels of CaSR and RAMPs in control TT cells. (B) Representative western blot showing protein expression of CaSR and RAMP1 in TT cell membranes. (C) Concentration-dependent Cinacalcet-induced increase in intracellular calcium release in TT cells in presence of 1.5 mM CaCl₂ (Ec₅₀ 503±1.29 nM). The data are combined from three independent experiments with a total of n = 50 cells analysed at -4.5, -5, -5.5, -6 and -11 M; 20 cells analysed at −6.5, −7, −7.5 and −8 M; and 30 cells analysed for −9 and −10 M concentrations. (D) Concentration-dependent Neomycin-induced response in presence of 2 mM CaCl₂ (Ec₅₀ 91±1.45 µM). The data are combined from three independent experiments with a total of n =  43, 48, 45, 46, 88, 74, 23 and 29 cells analysed per concentration respectively, going from high to low doses.

Figure 5. Effect of RAMP1 mRNA knockdown on TT cells.

(A) mRNA expression levels of RAMP1, RAMP2 and CaSR in TT cells transfected with RAMP1 or scrambled siRNA, 72 hr post-transfection expressed as fold change normalised to Actβ. (B) Representative images from immunofluorescent staining for RAMP1 expression in cells transfected with scrambled siRNA (top panel) and RAMP1 siRNA(bottom panel), 72 hr after transfection. (C) Intracellular calcium response of the RAMP1 siRNA cells to 1µM Cinacalcet in presence of 1.5 mM CaCl₂ and (D) 100µM Neomycin in presence of 2 mM CaCl₂ The responses were decreased by ∼42% and ∼50% respectively compared to scrambled siRNA transfected cells. The data are combined from five independent experiments with a total of 241, 231 and 154 cells analysed in (C), and 354, 354 and 118 cells analysed in (D) for knock-down, control and normal conditions respectively. **** p<0.0001 analysed by two-tailed Mann-Whitney test.

Figure 6. Attenuation of CaSR response in TT cells by RAMP1 antibodies.

(A) Concentration-dependant decrease in Cinacalcet-induced intracellular calcium release in presence of 1.5 mM CaCl₂, by RAMP1 antibodies in TT cells. Total cells analysed combined from five independent experiments are 272 (1µM Cinacalcet), 261 (0.0125µg/µl control IgG), 240 (0.025µg/µl control IgG), 252 (0.0025µg/µl RAMP1 Ab), 272 (0.0125µg/µl RAMP1 Ab) and 250 (0.025µg/µl RAMP1 Ab). (B) 0.025µg/µl RAMP1 antibody caused a significant inhibition of 100µM Neomycin response in presence of 2 mM CaCl₂ compared to control IgG. Total cells analysed combined from five independent experiments are 192 (100µM Neomycin), 177 (0.025µg/µl control IgG) and 166 (0.025µg/µl RAMP1 Ab). *** p<0.001 determined by Kruskal-Wallis test, Dunn's multiple comparison post-test, ** p<0.01 two-tailed Mann-Whitney test.