Gerda E. Breitwieser

Heterodimerization of Calcium Sensing Receptors with Metabotropic Glutamate Receptors in Neurons

Calcium sensing (CaR) and Group I metabotropic glutamate receptors exhibit overlapping expression patterns in brain, and share common signal transduction pathways. To determine whether CaR and Group I metabotropic glutamate receptors (mGluRs) (mGluR1α and mGluR5) can form heterodimers, we immunoprecipitated CaR from bovine brain and observed co-precipitation of mGluR1α. CaR and mGluR1α co-localize in hippocampal and cerebellar neurons, but are expressed separately in other brain regions. In vitro transfection studies in HEK-293 cells established the specificity and disulfide-linked nature of the CaR:mGluR1α (CaR:mGluR5) interactions. CaR:mGluR1α (CaR:mGluR5) heterodimers exhibit altered trafficking via Homer 1c when compared with CaR:CaR homodimers. CaR becomes sensitive to glutamate-mediated internalization when present in CaR:mGluR1α heterodimers. These results demonstrate cross-family covalent heterodimerization of CaR with Group I mGluRs, and increase the potential role(s) for CaR in modulating neuronal function.

Ca2+-sensing receptors in intestinal epithelium

Expression of Ca2+-sensing receptors (CaR) was demonstrated in several human intestinal epithelial cell lines (T84, HT-29, and Caco-2) and in rat intestinal epithelium by both reverse transcriptase-polymerase chain reaction (PCR) and Northern blotting of RNA. Restriction patterns of the PCR products were of the sizes predicted by the human and rat sequences. CaR agonists (Ca2+, poly-l-arginine, protamine) mediated an increase in intracellular Ca2+ in HT-29-18-C1 cells (monitored by changes in fura 2 fluorescence), which was dependent on release from thapsigargin-sensitive stores. U-73122, an inhibitor of phosphatidylinositol-phospholipase C, eliminated the CaR agonist-mediated rise in intracellular Ca2+, whereas its inactive analog, U-73343, had no effect. Pertussis toxin pretreatment had no effect on CaR agonist-mediated modulation of intracellular Ca2+. Taken together, these studies demonstrate that CaR are expressed in intestinal epithelial cells and couple to mobilization of intracellular Ca2+. The presence of CaR in intestinal epithelial cells presents a new locus for investigations into the role(s) of extracellular Ca2+ in modulating intestinal epithelial cell differentiation and transepithelial Ca2+ transport.

Dimerization of the Calcium-sensing Receptor Occurs within the Extracellular Domain and Is Eliminated by Cys → Ser Mutations at Cys101 and Cys236

Calcium-sensing receptors are present in membranes as dimers that can be reduced to monomers with sufhydryl reagents. All studies were carried out on the human calcium-sensing receptor tagged at the carboxyl terminus with green fluorescent protein (hCaR-GFP) to permit identification and localization of expressed proteins. Truncations containing either the extracellular agonist binding domain plus transmembrane helix 1 (ECD/TMH1-GFP) or the transmembrane domain plus the intracellular carboxyl terminus (TMD/carboxyl terminus-GFP) were used to identify the dimerization domain. ECD/TMH1-GFP was a dimer in the absence of reducing reagents, whereas TMD/carboxyl-terminal GFP was a monomer in the absence or presence of reducing agents, suggesting that dimerization occurs via the ECD. To identify the residue(s) involved in dimerization within the ECD, cysteine → serine point mutations were made in residues that are conserved between hCaR and metabotropic glutamate receptors. Mutations at positions 60 and 131 were expressed at levels comparable to wild type in HEK 293 cells, had minimal effects on hCaR function, and did not eliminate dimerization, whereas mutations at positions 101 and 236 greatly decreased receptor expression and resulted in significant amounts of monomer in the absence of reducing agents. The double point mutant hCaR(C101S/C236S)-GFP was expressed more robustly than either C101S or C236S and covalent dimerization was eliminated. hCaR(C101S/C236S)-GFP had a decreased affinity for extracellular Ca2+ and slower response kinetics upon increases or decreases in agonist concentration. These results suggest that covalent, disulfide bond-mediated dimerization of the calcium-sensing receptor contributes to stabilization of the ECD and to acceleration of the transitions between inactive and active receptor conformations.

A Carboxyl-terminal Domain Controls the Cooperativity for Extracellular Ca2+ Activation of the Human Calcium Sensing Receptor A STUDY WITH RECEPTOR-GREEN FLUORESCENT PROTEIN FUSIONS

Calcium sensing receptors are part of a growing G protein-coupled receptor family, which includes metabotropic glutamate, γ-aminoisobutyric acid, and pheromone receptors. The distinctive structural features of this family include large extracellular domains that bind agonist and large intracellular, carboxyl-terminal domains of as yet undefined function(s). We have explored the contribution(s) of the carboxyl terminus of the human calcium sensing receptor (CaR) by assessing extracellular Ca2+-mediated changes in intracellular Ca2+ in individual HEK-293 cells transfected with CaR clones. In-frame fusion of EGFP to the carboxyl terminus of CaR had no effect on either the dose response for extracellular Ca2+ activation or CaR desensitization. Carboxyl-terminal truncations, fused in-frame with EGFP (CaRΔ1024-EGFP, CaRΔ908-EGFP, CaRΔ886-EGFP, and CaRΔ868-EGFP), were assessed for alterations in Ca2+-dependent activation or desensitization. Significant effects on the dose-response relation for extracellular Ca2+ were observed only for the CaRΔ868 truncation, which exhibited a decreased affinity for extracellular Ca2+ and a decrease in the apparent cooperativity for Ca2+-dependent activation. The alterations in extracellular Ca2+ affinity and cooperativity observed with CaRΔ868 were recapitulated by a point mutation, T876D, in the full-length CaR-EGFP background. All truncations with wild type dose-response relations exhibited desensitization time courses that were comparable to the full-length CaR, whereas the CaRΔ868 receptor desensitized completely after two exposures to 10 mmCa2+. Interestingly, the CaR point mutation T876D exhibited desensitization comparable to wild type CaR, suggesting that this mutation specifically modifies CaR cooperativity. In conclusion, these studies suggest that amino acid residues between 868 and 886 are critical to the apparent cooperativity of Ca2+-mediated activation of G proteins and to CaR desensitization.

Rescue of Calcium-sensing Receptor Mutants by Allosteric Modulators Reveals a Conformational Checkpoint in Receptor Biogenesis

The calcium-sensing receptor (CaR), a member of G protein-coupled receptor family C, regulates systemic calcium homeostasis by activating Gq- and Gi-linked signaling in the parathyroid, kidney, and intestine. CaR is ubiquitinated by the E3 ligase dorfin and degraded via the endoplasmic reticulum-associated degradation pathway (Huang, Y., Niwa, J., Sobue, G., and Breitwieser, G. E. (2006) J. Biol. Chem. 281, 11610–11617). Here we provide evidence for a conformational or functional checkpoint in CaR biogenesis using two complementary approaches. First we characterized the sensitivity of loss- or gain-of-function CaR mutants to proteasome inhibition by MG132. The stabilization of loss-of-function mutants and insensitivity of gain-of-function mutants to MG132 suggests that receptor sensitivity to calcium influences susceptibility to proteasomal degradation. Second, we used the allosteric activator NPS R-568 and antagonist NPS 2143 to promote the active and inactive conformations of wild type CaR, respectively. Overnight culture in NPS R-568 increased expression of CaR, whereas NPS 2143 had the opposite effect. NPS R-568 and NPS 2143 differentially regulated maturation and cell surface expression of wild type CaR, directly affecting maximal signaling responses. NPS R-568 rescued expression of loss-of-function CaR mutants, increasing plasma membrane expression and ERK1/2 phosphorylation in response to 5 mm Ca2+. Disorders of calcium homeostasis caused by CaR mutations may therefore result from altered receptor biogenesis independent of receptor function, i.e. a protein folding disorder. The allosteric modulators NPS R-568 and NPS 2143 not only alter CaR sensitivity to calcium and hence signaling but also modulate receptor expression.

Agonist-Driven Maturation and Plasma Membrane Insertion of Calcium-Sensing Receptors Dynamically Control Signal Amplitude

Activation of a G protein–coupled receptor increases its own surface abundance to enhance its signaling. Linking Activity to Abundance Many G protein–coupled receptors decrease their signaling output when activated by ligands. However, the calcium-sensing receptor (CaSR), which maintains the serum concentration of calcium within a narrow range, is constantly exposed to calcium, leading Grant et al. to investigate how CaSR-mediated signaling increases in response to increases in the concentration of extracellular calcium. Activation of CaSRs by calcium or other agonists resulted in mobilization of CaSRs to the plasma membrane from a large intracellular pool, thereby enabling increased and sustained CaSR signaling. Mutations in CASR can result in hypocalcemia or hypercalcemia; thus, understanding its regulation could help in the development of drugs to treat these conditions. In addition, this mechanism might regulate the signaling output of other receptors that are also constantly exposed to ligands. Calcium-sensing receptors (CaSRs) regulate systemic calcium homeostasis in the parathyroid gland, kidney, intestine, and bone and translate fluctuations in serum calcium into peptide hormone secretion, cell signaling, and regulation of gene expression. The CaSR is a G protein (heterotrimeric guanosine triphosphate–binding protein)–coupled receptor that operates in the constant presence of agonist, sensing small changes with high cooperativity and minimal functional desensitization. Here, we used multiwavelength total internal reflection fluorescence microscopy to demonstrate that the signaling properties of the CaSR result from agonist-driven maturation and insertion of CaSRs into the plasma membrane. Plasma membrane CaSRs underwent constitutive endocytosis without substantial recycling, indicating that signaling was determined by the rate of insertion of CaSRs into the plasma membrane. Intracellular CaSRs colocalized with calnexin in the perinuclear endoplasmic reticulum and formed complexes with 14-3-3 proteins. Ongoing CaSR signaling resulted from agonist-driven trafficking of CaSR through the secretory pathway. The intracellular reservoir of CaSRs that were mobilized by agonist was depleted when glycosylation of newly synthesized receptors was blocked, suggesting that receptor biosynthesis was coupled to signaling. The continuous, signaling-dependent insertion of CaSRs into the plasma membrane ensured a rapid response to alterations in the concentrations of extracellular calcium or allosteric agonist despite ongoing desensitization and endocytosis. Regulation of CaSR plasma membrane abundance represents a previously unknown mechanism of regulation that may be relevant to other receptors that operate in the chronic presence of agonist.

Calcium-sensing Receptor Ubiquitination and Degradation Mediated by the E3 Ubiquitin Ligase Dorfin

Calcium-sensing receptors (CaR) contribute to regulation of systemic calcium homeostasis by activation of Gq- and Gi-linked signaling pathways in the parathyroids, kidney, and intestine. Little is known about the mechanisms regulating CaR synthesis and degradation. Screening of a human kidney yeast two-hybrid library identified the E3 ubiquitin ligase dorfin as a binding partner for the intracellular carboxyl terminus of CaR. Interaction between CaR and dorfin was confirmed by coimmunoprecipitation from HEK293 cells. Ubiquitination of CaR was observed in the presence of the proteasomal inhibitor MG132; mutation of all putative intracellular loop and carboxyl-terminal lysine residues abolished ubiquitination of CaR. Coexpression with dorfin decreased the amount of total CaR protein and increased CaR ubiquitination, whereas a dominant negative fragment of dorfin had opposite effects. The AAA-ATPase p97/valosin-containing protein associates with both CaR and dorfin in HEK293 cells. Treatment with tunicamycin, an inhibitor of N-linked glycosylation, induced the appearance of the unglycosylated 115-kDa CaR form, which was further increased by exposure to MG132, or upon transfection with a dorfin dominant negative construct, suggesting that dorfin-mediated proteasomal degradation of immature CaR occurs from the endoplasmic reticulum. Because endogenous CaR in Madin-Darby canine kidney cells is also subject to degradation from the endoplasmic reticulum, dorfin-mediated ubiquitination may contribute to a general mechanism for CaR quality control during biosynthesis.

Minireview: The Intimate Link Between Calcium Sensing Receptor Trafficking and Signaling: Implications for Disorders of Calcium Homeostasis

The calcium-sensing receptor (CaSR) regulates organismal Ca(2+) homeostasis. Dysregulation of CaSR expression or mutations in the CASR gene cause disorders of Ca(2+) homeostasis and contribute to the progression or severity of cancers and cardiovascular disease. This brief review highlights recent findings that define the CaSR life cycle, which controls the cellular abundance of CaSR and CaSR signaling. A novel mechanism, termed agonist-driven insertional signaling (ADIS), contributes to the unique hallmarks of CaSR signaling, including the high degree of cooperativity and the lack of functional desensitization. Agonist-mediated activation of plasma membrane-localized CaSR increases the rate of insertion of CaSR at the plasma membrane without altering the constitutive endocytosis rate, thereby acutely increasing the maximum signaling response. Prolonged CaSR signaling requires a large intracellular ADIS-mobilizable pool of CaSR, which is maintained by signaling-mediated increases in biosynthesis. This model provides a rational framework for characterizing the defects caused by CaSR mutations and the altered functional expression of wild-type CaSR in disease states. Mechanistic dissection of ADIS of CaSR should lead to optimized pharmacological approaches to normalize CaSR signaling in disorders of Ca(2+) homeostasis.

Cellular calcium dynamics in lactation and breast cancer: from physiology to pathology

Breast cancer is the second leading cause of cancer mortality in women, estimated at nearly 40,000 deaths and more than 230,000 new cases diagnosed in the U.S. this year alone. One of the defining characteristics of breast cancer is the radiographic presence of microcalcifications. These palpable mineral precipitates are commonly found in the breast after formation of a tumor. Since free Ca(2+) plays a crucial role as a second messenger inside cells, we hypothesize that these chelated precipitates may be a result of dysregulated Ca(2+) secretion associated with tumorigenesis. Transient and sustained elevations of intracellular Ca(2+) regulate cell proliferation, apoptosis and cell migration, and offer numerous therapeutic possibilities in controlling tumor growth and metastasis. During lactation, a developmentally determined program of gene expression controls the massive transcellular mobilization of Ca(2+) from the blood into milk by the coordinated action of calcium transporters, including pumps, channels, sensors and buffers, in a functional module that we term CALTRANS. Here we assess the evidence implicating genes that regulate free and buffered Ca(2+) in normal breast epithelium and cancer cells and discuss mechanisms that are likely to contribute to the pathological characteristics of breast cancer.

Calcium-sensing receptor biosynthesis includes a cotranslational conformational checkpoint and endoplasmic reticulum retention.

Metabolic labeling with [35S]cysteine was used to characterize early events in CaSR biosynthesis. [35S]CaSR is relatively stable (half-life ∼8 h), but maturation to the final glycosylated form is slow and incomplete. Incorporation of [35S]cysteine is linear over 60 min, and the rate of [35S]CaSR biosynthesis is significantly increased by the membrane-permeant allosteric agonist NPS R-568, which acts as a cotranslational pharmacochaperone. The [35S]CaSR biosynthetic rate also varies as a function of conformational bias induced by loss- or gain-of-function mutations. In contrast, [35S]CaSR maturation to the plasma membrane was not significantly altered by exposure to the pharmacochaperone NPS R-568, the allosteric agonist neomycin, or the orthosteric agonist Ca2+ (0.5 or 5 mm), suggesting that CaSR does not control its own release from the endoplasmic reticulum. A CaSR chimera containing the mGluR1α carboxyl terminus matures completely (half-time of ∼8 h) and without a lag period, as does the truncation mutant CaSRΔ868 (half-time of ∼16 h). CaSRΔ898 exhibits maturation comparable with full-length CaSR, suggesting that the CaSR carboxyl terminus between residues Thr868 and Arg898 limits maturation. Overall, these results suggest that CaSR is subject to cotranslational quality control, which includes a pharmacochaperone-sensitive conformational checkpoint. The CaSR carboxyl terminus is the chief determinant of intracellular retention of a significant fraction of total CaSR. Intracellular CaSR may reflect a rapidly mobilizable “storage form” of CaSR and/or may subserve distinct intracellular signaling roles that are sensitive to signaling-dependent changes in endoplasmic reticulum Ca2+ and/or glutathione.