Calcium sensing receptor mutations implicated in pancreatitis and idiopathic epilepsy syndrome disrupt an arginine-rich retention motif

Abstract

Calcium sensing receptor (CaSR) mutations implicated in familial hypocalciuric hypercalcemia, pancreatitis and idiopathic epilepsy syndrome map to an extended arginine-rich region in the proximal carboxyl terminus. Arginine-rich motifs mediate endoplasmic reticulum retention and/or retrieval of multisubunit proteins so we asked whether these mutations, R886P, R896H or R898Q, altered CaSR targeting to the plasma membrane. Targeting was enhanced by all three mutations, and Ca(2+)-stimulated ERK1/2 phosphorylation was increased for R896H and R898Q. To define the role of the extended arginine-rich region in CaSR trafficking, we independently determined the contributions of R890/R891 and/or R896/K897/R898 motifs by mutation to alanine. Disruption of the motif(s) significantly increased surface expression and function relative to wt CaSR. The arginine-rich region is flanked by phosphorylation sites at S892 (protein kinase C) and S899 (protein kinase A). The phosphorylation state of S899 regulated recognition of the arginine-rich region; S899D showed increased surface localization. CaSR assembles in the endoplasmic reticulum as a covalent disulfide-linked dimer and we determined whether retention requires the presence of arginine-rich regions in both subunits. A single arginine-rich region within the dimer was sufficient to confer intracellular retention comparable to wt CaSR. We have identified an extended arginine-rich region in the proximal carboxyl terminus of CaSR (residues R890 - R898) which fosters intracellular retention of CaSR and is regulated by phosphorylation. Mutation(s) identified in chronic pancreatitis and idiopathic epilepsy syndrome therefore increase plasma membrane targeting of CaSR, likely contributing to the altered Ca(2+) signaling characteristic of these diseases.

Fig. 1

Arginine-rich region mutations identified in patients enhance surface expression and function of CaSR. A. wt CaSR or R886P, R896H or R898Q mutants were immunoprecipitated from HEK293 cells with anti-FLAG antibody; blot was probed with anti-CaSR LRG antibody. Monomer (m) and dimer (d) regions of the western blot are indicated. B. ERK phosphorylation supported by arginine-rich region mutants. HEK293 cells expressing wt CaSR, R886P, R896H or R898Q mutants were stimulated with 5 mM extracellular Ca²⁺ for 10 min and lysates were analyzed for ERK1/2 phosphorylation as described in Methods. C. ELISA assay of arginine-rich region mutations was performed as described in Methods. Plasma membrane (black bars) and total (white bars) protein abundance were normalized to wt CaSR under the same fixation conditions (wt CaSR = 100%, dashed line). Results are the average ± S.E.M. of 3-4 independent experiments. Significant differences between surface and total protein expression for a given receptor mutant are indicated (* p<0.05).

Fig. 2

Plasma membrane relative to total receptor abundance of carboxyl terminal truncations of CaSR. A. The human CaSR carboxyl terminus extends from residue K863 to S1078. Truncations were generated over the range from CaSRΔ868 through CaSRΔ1052. ELISA assays were performed on replicate samples fixed in either 4% paraformaldehyde (plasma membrane, black bars) or methanol (total abundance, white bars) as described in Methods. Results were normalized to the abundance of wt CaSR under the same fixation conditions (wt CaSR = 100%, indicated with dashed line). B. Truncations between CaSRΔ886 and CaSRΔ908 were analyzed to resolve importance of arginine-rich region residues. For both A and B, significant differences between plasma membrane and total abundance for a given truncation are indicated (* p<0.05), and results are average ± S.E.M. of 5-8 independent experiments.

Fig. 3

Disruptions of arginine-rich region motifs alter CaSR plasma membrane abundance. A. Arginine to alanine mutations were generated in the arginine-rich region (R890A/R891A = 2A; R896A/K897A/R898A = 3A; and R890A/R891A/R896A/ K897A/R898A = 5A) in either full length CaSR or the CaSRΔ898 truncation as described in Methods. B. HEK293 cells transfected with the FLAG-tagged constructs of arginine-rich region mutants were immunoprecipitated with anti-FLAG antibody, and blots were probed with anti-CaSR LRG antibody as described in Methods. Monomer (m) and dimer (d) regions of the blot are indicated. C. HEK293 cells were transfected with full length CaSR, CaSRΔ898 or arginine mutants and stimulated with 5 mM extracellular Ca²⁺ for 10 min as described in Methods. Lysates were run on 4-15 % gradient gels, blotted to nitrocellulose, probed for phosphorylated ERK1/2, stripped, and reprobed for total ERK. D. Arginine mutants (2A, 3A, 5A) of full length CaSR were expressed in HEK293 cells and assayed by ELISA at 3 days after transfection. All data were normalized to the abundance of wt CaSR under comparable fixation conditions, as described in Fig. 1 and Methods. Black bars, plasma membrane abundance; white bars, total abundance; dashed line indicates wt CaSR (= 100%). Results are average ± S.E.M. Significant differences between plasma membrane or total abundance for a given receptor mutant are indicated (* p<0.05).

Fig. 4

A single arginine-rich domain is sufficient for retention equivalent to wt CaSR. A. wt CaSR or CaSR(5A) were coexpressed at a 1:2 cDNA ratio with pcDNA3.1, wt CaSR, CaSRΔ868 or CaSRΔ886-EGFP to facilitate heterodimerization. B. ELISA assays were performed on day 3 after transfection as described in A, and plasma membrane (black bars) and total (white bars) protein abundance were expressed relative to wt CaSR under the same fixation conditions (=100%). Left portion of graph indicates wt CaSR and right portion indicates CaSR(5A) mutant experiments. Results are average ± S.E.M. for 3-5 experiments. Significant differences between plasma membrane and total protein abundance for a given receptor mutant are indicated (* p<0.05).

Fig. 5

Phosphorylation sites flanking the arginine-rich region regulate recognition. A. Serine to alanine or serine to aspartic acid mutants at residues S892 or S899 or both (SS/AA or SS/DD) were transfected in HEK293 cells and immunoprecipitated after 3 days using anti-FLAG antibody. Western blots were probed with anti-CaSR LRG antibody. B. ERK phosphorylation supported by wt CaSR or phosphorylation site mutants. HEK293 cells expressing wt CaSR or S892 or S899 mutants were stimulated with 5 mM extracellular Ca²⁺ for 10 min and lysates were analyzed for ERK1/2 phosphorylation as described in Methods. C. ELISA assay performed as described in Methods and Figure 1 after 3 days of transfection for phosphorylation site mutants described in A. Plasma membrane (black bars) and total (white bars) protein abundance relative to wt CaSR under the same fixation conditions (wt CaSR = 100%, dashed line) are plotted. Results are average ± S.E.M. of 5-7 independent experiments. Significant differences between surface and total protein expression for a given receptor mutant are indicated (* p<0.05).

Fig. 6

The immature form of CaSR coimmunoprecipitates with 14-3-3 proteins. HEK293 cells transiently expressing wt CaSR, CaSR(5A), or truncations CaSRΔ898(5A) or CaSRΔ868, or the phosphorylation site mutants, S892A/D and S899A/D were subjected to immunoprecipitation with anti-14-3-3 antibody (A) or anti-FLAG antibody (B) as described in methods. Blots from both immunoprecipitations were probed with anti-CaSR LRG antibody or anti-CaSR ADD antibody. C. CaSR immunoreactivity from anti-14-3-3 blot was normalized to that of the same sample from the anti-FLAG blot and plotted as % of wt CaSR. Results are average ± S.E.M. of 3-5 independent transfections.