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Originally published In Press as doi:10.1074/jbc.M303377200 on June 3, 2003

J. Biol. Chem., Vol. 278, Issue 33, 31167-31176, August 15, 2003
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m-Calpain Colocalizes with the Calcium-sensing Receptor (CaR) in Caveolae in Parathyroid Cells and Participates in Degradation of the CaR*

Olga Kifor {ddagger}, Imre Kifor, Francis D. Moore, Jr., Robert R. Butters, Jr. and Edward M. Brown

From the Endocrine-Hypertension Division, Membrane Biology Program and Departments of Medicine and Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, April 2, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The calcium-sensing receptor (CaR) is a G protein-coupled, seven-transmembrane receptor and resides within caveolin-rich membrane domains in bovine parathyroid cells. The proenzyme of calpain 2 (m-calpain) is a heterodimeric calcium-dependent cysteine protease consisting of catalytic and regulatory subunits. The effects of calcium on the enzyme include activation, autolysis, and subunit dissociation. Here, we examine the potential role of caveolin-1 and caveolae in regulating the cellular distribution and function of m-calpain in parathyroid cells. We show that the inactive heterodimeric forms of m-calpain are concentrated in caveolin-rich membrane fractions prepared from parathyroid cells incubated with low extracellular calcium (Ca2+o). In contrast, in cells incubated with 3 mM Ca2+o, which activates the CaR and increases intracellular calcium, there is a reduction in m-calpain in association with an increase in CaR protein and phosphorylated protein kinase C {alpha} and {beta} in caveolin-rich fractions. To assess the impact of activation of calpain on CaR protein in caveolar fractions, we analyzed the effects of m-calpain on the CaR. Activation of the CaR with high Ca2+o induced the release of lower molecular weight fragments of the receptor into the cell culture medium, and calpain inhibitors blocked this effect. Moreover, the fragments of the CaR as well as caveolin-1, m-calpain, and alkaline phosphatase were localized in membrane vesicles shed by parathyroid cells, supporting the association of these proteins in living cells. Treatment of CaR proteins in vitro with m-calpain also resulted in the appearance of lower molecular weight fragments of the CaR. Our data suggest that localization of m-calpain within caveolae may contribute to maintenance of the enzyme in an inactive state and that m-calpain may also contribute to the regulation of CaR levels.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Compartmentalization through protein-protein or protein-lipid interactions has now been recognized as a fundamental mechanism for efficient and organized cellular responses to external stimuli (1). The plasma membrane of most cell types contains specialized microdomains with distinct lipid and protein compositions, referred to as lipid raft microdomains and caveolae (1–4). Caveolins, which are 21–24-kDa integral membrane proteins, have been identified as principal components of caveolar membranes and exist as several isoforms (caveolin-1, -2, and -3) (1–4). It has been suggested that caveolae may function as subcellular compartments that store inactive signaling molecules for regulated activation by appropriate receptors and facilitate cross-talk between distinct signaling cascades (1–3). Caveolin-1 interacts with and negatively regulates the activity of some signaling molecules such as the {alpha} subunit of G proteins, Ras, Src, nitric oxide synthase, growth factors, and G protein-coupled receptors (GPCRs),1 protein kinase C (PKC), and adenylyl cyclase (5–14). The region of caveolin-1 responsible for these various inhibitory effects is located in the so-called caveolin scaffolding domain (residues 82–101) (2). Upon activation, some GPCRs (e.g. endothelin-A, bradykinin-B2, and {beta}2 adrenergic and muscarinic m2 receptors) translocate to caveolae where they bind to caveolin (9–11). Recent studies suggest that caveolae can modulate specific signaling events that depend on calcium. Ca2+-ATPase, inositol 1,4,5-trisphosphate (IP3) receptors, calmodulin, and the extracellular calcium (Ca2+o)-sensing receptor (CaR) have all been localized to caveolae (5–7). Isshiki et al. have shown that the signaling machinery regulating Ca2+ entry is functionally organized in the caveolae of living cells (8).

The calpain family is composed of at least 14 different forms of the enzyme (15). Major calpain species are calpain 1 or µ-calpain and calpain 2 or m-calpain, which are activated at micromolar and millimolar concentrations of Ca2+, respectively, and distributed ubiquitously. Localization of calpain has been described in many cell types (15–20). In erythrocytes, calpain translocates to the plasma membrane from the cytosol when activated by Ca2+ in the presence of ionophore A23187 [GenBank] (16). It has been suggested that interaction of the small subunit of m-calpain with membranes could lead to activation of the enzyme at intracellular levels of calcium (17). Recent studies have reported the existence of calpain in the extracellular spaces of various tissues. In skeletal muscle, m-calpain is distributed on extracellular collagen fibrils, and m-calpain has also been found in the synovial fluid of the knee joint (21). Plasma membrane vesiculation has been described to occur spontaneously in normal and tumor cells in culture (22). The released vesicles are enriched in sphingomyelin, cholesterol, gangliosides, and several glycosylphosphatidylinositol (GPI)-anchored proteins (23). Ganglioside GD3 and caveolin-1 are enriched in shed membrane vesicles of CABA I human ovarian carcinoma cells (23). MC3T3-EI cells produce both matrix vesicles and media vesicles, and m-calpain was co-secreted into the culture medium with these vesicles (21).

µ-Calpain and m-calpain have distinct large subunits but share the same small subunit. The large (80–82 kDa) catalytic subunit is divided into domains I-IV, and the small (28–30 kDa) regulatory subunit is divided into domains V and VI (15–20). It is generally agreed that calpains are proenzymes (16). The small and large subunits are present in a 1:1 molar ratio and comigrate through various chromatographic and non-denaturing electrophoretic procedures (16). The two subunits have been chemically crosslinked, and the resulting product has a molecular weight of ~110,000 and contains immunoreactive epitopes for each of the two subunits (16).

The calpains undergo autolytic processing in the presence of calcium to form the fully active proteolytic isozymes (19). The effects of Ca2+ on the enzyme include activation, aggregation, and autolysis. In the absence of Ca2+, the protease is inactive. µ-Calpain is enzymatically active at 10–50 µM Ca2+, whereas m-calpain is active at higher Ca2+ concentrations (740–780 µM) in reconstituted assay systems in vitro (24, 25). The binding of Ca2+ to the protease presumably induces a conformational change that, in turn, promotes an intramolecular cleavage of the small subunit and results in activation of the protease (18, 19). The crystal structure of calpain reveals the structural basis for Ca2+-dependent protease activity and a novel mode of enzyme activation (25). Crawford et al. have suggested that the activated enzyme is released from the membrane surface, because the glycine-rich sequence at the N terminus is removed by autoproteolysis during activation (26).

Calpains cleave target proteins in a restricted manner to modify their properties rather than digest them entirely (19). Calpains have been implicated in several important cellular functions, including signal transduction, cell differentiation, apoptosis, and cytoskeletal reorganization (15, 16). At the pathological level, excessive activation of calpain as well as mutations abrogating calpain activity have been implicated in muscular dystrophy, cardiac and cerebral ischemia, platelet aggregation, neurodegenerative diseases, Alzheimer's disease, cataract formation, and rheumatoid arthritis (27–29). Activated and membrane-translocated calpains are able to cleave many substrate proteins such as PKC, phospholipase C (PLC), tyrosine kinases, the epidermal growth factor (EGF) and N-methyl-D-aspartate (NMDA) receptors, heterotrimeric G-proteins, insulin, angiotensin II, parathyroid hormone (PTH), and cytoskeletal proteins including filamin, talin, fibronectin, and vimentin (15, 16, 30–33). Bovine parathyroid (PT) cells express µ-calpain and m-calpain (33). Investigation of PT glands using an immunohistochemical method revealed that calpain is abundant in the chief cell cytoplasm (33). The use of a calpain inhibitor in bovine PT cells in culture increased the release of intact PTH at high Ca2+o levels and reversed the inhibitory effect of phorbol myristate acetate (PMA) on PTH release at low levels of Ca2+o (33).

The CaR is a GPCR that plays key roles in extracellular calcium homeostasis (34, 35). CaR activation mediates the inhibition of PTH secretion in PT cells and the inhibition of Ca2+ reabsorption in the kidney. Mutations affecting the CaR that make it either less or more sensitive to Ca2+o cause various clinical disorders (34, 35). Recent studies have documented that the expression of CaR mRNA and/or protein can change in a variety of circumstances (34, 35). The ligand binding and signaling properties of a number of GPCRs are modified as a result of receptor dimerization, suggesting a functional relevance of this phenomenon. The CaR forms disulfide-linked dimers, and intermolecular interactions between CaR monomers are important for its normal function (36–38). Previously, we have shown that bovine PT cells contain caveolin-1 protein and that the CaR resides within caveolae (7). Other signaling proteins, including Gq/11, endothelial nitric oxide synthase (eNOS), and several PKC isoforms ({alpha}, {delta}, and {zeta}), are also present in PT caveolae, and activation of the CaR by high Ca2+o increases the tyrosine phosphorylation of caveolin-1 (7). In the present study, we investigated the subcellular distribution of m-calpain in bovine and human PT cells. Our results indicate that, at low Ca2+o, m-calpain is present in PT caveolae in its inactive heterodimeric, presumably proenzyme form. Short-term activation of the CaR with high calcium causes a reduction of m-calpain in a caveolin-rich fraction and increases of CaR protein and phosphorylated PKCs ({alpha} and {beta}). Similar to PTH, the CaR is also a substrate for m-calpain in longer term incubations with high Ca2+o.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Monoclonal and polyclonal anti-caveolin-1 antibodies were purchased from BD Biosciences Transduction Laboratories. Polyclonal anti-caveolin-1 antibody and protein A/G agarose beads were obtained from Santa Cruz Biotechnology. A polyclonal antiserum against the m-calpain (Domain III) large subunit as well as monoclonal anti-calpastatin and peroxidase-conjugated anti-mouse and anti-rabbit antibodies were purchased from Sigma. Monoclonal anti-calpain small subunit (µ- and m-calpain) and monoclonal anti-calpastatin were obtained from Chemicon International (Temecula, CA). Polyclonal antibodies to phospho-pPKC (pan) and phospho-PKCs {alpha} and {beta} were obtained from Cell Signaling Technology (Beverly, MA). CaR-specific polyclonal antisera were raised in rabbits against peptides based on the CaR sequence. Antiserum 4637, raised against amino acids 345–359, and antiserum 4641 to amino acids 215–237 as well as CaR-transfected HEK-293 cells (HEKCaR) were generous gifts of NPS Pharmaceuticals, Inc. (Salt Lake City, UT). Goat anti-mouse IgG coupled to Alexa 568 and goat anti-rabbit IgG coupled to Alexa 488 were purchased from Molecular Probes (Eugene, OR). The TSATM Cyanine 5 system was purchased from PerkinElmer Life Sciences. Biotinylated anti-mouse IgG and Vectashield mounting medium were from Vector Labs (Burlingame, CA).

Cell Preparation and Incubation—PT cells were prepared by collagenase and DNase digestion of bovine or human PT glands as described previously (7, 39) and were used immediately as acutely dispersed cells or cultured overnight in Dulbecco's modified Eagle's medium F12 with penicillin and streptomycin. For immunocytochemistry, the cells were seeded on glass coverslips and allowed to attach for 1 h in a culture incubator. The cells were then washed with Eagle's minimal essential medium containing 0.5 mM Ca2+o, 0.5 mM Mg2+o, and 0.2% bovine serum albumin (standard medium) and subsequently incubated in standard medium at 37 °C with varying levels of Ca2+o (detailed under "Results"). HEK-293 cells stably transfected with the human CaR cDNA were grown in 24-well plates in Dulbecco's modified Eagle's medium (without sodium pyruvate) with 10% fetal calf serum (39). Bovine PT cells media vesicles were obtained by a sedimentation method (21). Bovine PT cells were incubated with varying levels of Ca2+o for 24 h. At the end of incubation, the culture medium was removed and centrifuged for 10 min at 10,000 x g. To pellet the media vesicles, the resulting supernatant was centrifuged at 100,000 x g for 30 min. The resulting pellet was stained for alkaline phosphatase (AP) activity by using an AP substrate kit (nitro blue tetrazolium/5-bromo-4-chloro-3-indoyl phosphate) from Invitrogen (7) or solubilized with SDS sample buffer containing DTT and subjected to SDS-PAGE.

Immunocytochemistry—Glass slides with adherent PT cells were washed with ice-cold phosphate-buffered saline (PBS) and fixed for 10 min with 3.7% formaldehyde in PBS at room temperature without permeabilization with detergent. After washing and inhibition of endogenous peroxidase, the slides were immunostained with antibody to caveolin-1 and the appropriate peroxidase-conjugated secondary antibodies, and immunostaining was visualized with the DAKO AEC Substrate System (DAKO Corp.) as described (7). For immunofluorescence staining, the cells were permeabilized by incubation for 10 min with 0.5% Triton X-100 in PBS. They were then incubated with 0.1 M glycine, pH 7.4, for 10 min (to quench intrinsic fluorescence) and blocking solution (1% bovine serum albumin and 1% non-immune goat serum in PBS) for 15 min. For triple immunofluorescence staining, the slides were incubated with a mixture of three primary antibodies (i.e. monoclonal antibody to caveolin-1, sheep anti-PTH antiserum, and rabbit polyclonal m-calpain antiserum) in blocking solution overnight at 4 °C in a humidified chamber. The next day the cells were washed and incubated with a mixture of secondary antibodies (i.e. goat anti-mouse IgG tagged with Alexa 568, donkey anti-sheep IgG tagged with Alexa 488, and biotinylated anti-rabbit IgG) in PBS containing 1% bovine serum albumin for 30 min at 37 °C. After being washed, the slides were incubated with the TSATM cyanine 5 system, washed again, and mounted in Vectashield to prevent photo-bleaching. Fluorescence images were obtained using the 100x objective of a BioRad MRC 1024/2P multi-photon microscope equipped with krypton and argon lasers at the Brigham and Women's Hospital Confocal Microscopy Core facility. Alexa 568 produces red, Alexa 488 produces green, and the cyanine 5 tyramide system produces blue signals, respectively. When the red, blue, and green components are merged, colocalization of all three colors results in a white color. Autofluorescence of the samples was minimal and was subtracted from the values obtained during measurements.

Electrophoresis and Immunoblots—Western blot analysis was performed essentially as described (7, 39). Cells were rinsed with ice-cold PBS and lysed in ice-cold lysis buffer containing 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1.0 mM EGTA, 0.25 M sucrose, 1% Triton X-100, and protease inhibitors (10 µg/ml each aprotinin, leupeptin, and calpain inhibitor and 100 µg/ml pefabloc). Nuclei and cell debris were removed by low-speed centrifugation (1000 x g for 10 min), and the resultant cell lysate in the supernatant was used for Western blot analysis. Equal amounts of supernatant proteins were mixed with 2x SDS-Laemmli buffer, separated on 7.5 or 10% SDS-PAGE gels or linear 4–10% SDS gradients gels, and then transferred to nitrocellulose filters (Schleicher and Schuell). The blots were subsequently incubated with blocking solution (PBS with 0.25% Triton X-100 and 5% nonfat dry milk) for 1 h at room temperature. The membranes were incubated overnight with primary antiserum and, after washing, with secondary antibodies. The bands were visualized by chemiluminescence (Renaissance ECL system, PerkinElmer Life Sciences). Protein concentrations were measured using the Micro BCA protein kit (Pierce Chemical). Quantitative comparisons of the CaR protein under various experimental conditions were performed using an ImageQuant Personal Densitometer (Amersham Biosciences).

Immunoprecipitation—Cells were washed with ice-cold PBS and lysed with immunoprecipitation buffer containing 150 mM NaCl, 10 mM Tris, pH 7.4, 1% glycerol, 1 mM EDTA, 1 mM EGTA, 1 mM sodium o-vanadate, protease inhibitors (as described above), and 1% Triton X-100 (7, 39). The cell lysates were centrifuged at 10,000 x g for 10 min. Supernatant proteins (300 µg of total lysate) were incubated with 2–4 µg of monoclonal antibodies to caveolin-1 or irrelevant mouse IgG or antibodies to calpain for 3 h at 4 °C. Protein A/G-agarose beads (Santa Cruz Biotechnology) were then added for an additional hour at 4°. Bound immunocomplexes were washed three times with immunoprecipitation buffer. The pellet was eluted with 2x Laemmli sample buffer by boiling for 5 min. After SDS-PAGE, immunoblot analysis was performed as described above. For assessment of calpain activity in vitro, immunoprecipitated calpains (10 µl of packed protein A/G agarose beads) were resuspended in reaction mixtures containing 200 µl of 20 mM sodium-HEPES (pH 7.4), 3 mM CaCl2, and 1 mM dithiothreitol.

Purification of Caveolin-rich Membrane Fractions—Fractions enriched in caveolin were prepared by a detergent-free or detergent-based method using MES-buffered saline (MBS) containing 25 mM MES, pH 6.5, and 0.15 M NaCl (13, 14). In the detergent-free method the cells were homogenized with 2 ml of 500 mM sodium carbonate, pH 11, and protease inhibitors (as described above). The homogenate was then adjusted to 40% sucrose by adding an equal volume of 80% sucrose prepared in MBS. The mixture was placed at the bottom of an ultracentrifuge tube, overlaid with a discontinuous 5–35% sucrose gradient in MBS containing 250 mM sodium carbonate, and sedimented at 39,000 rpm for 18 h in an SW40 rotor (Beckman). Fractions were removed sequentially from the top and designated fractions 1–9 (fractions 3–4 and 6–9 are considered to be caveolar and non-caveolar in origin, respectively). In the detergent method the tissue or cells were homogenized with 2 ml of MBS buffer containing protease inhibitors and 1% Triton X-100. The samples were mixed with an equal amount of 80% sucrose (prepared in MBS), transferred to a 12-ml ultracentrifuge tube, overlaid with a discontinuous gradient, and centrifuged and fractionated as described above (14).

Protocol—The studies using human parathyroid adenomas were carried out under a protocol approved by the local institutional review board for the use of discarded human material.

Statistics—The data are presented as mean ± S.E. of the indicated number of experiments. Sigmastat statistical software was used to analyze the results. One-way analysis of variance (ANOVA) with the Student Newman Keuls Method was used. A p value of <0.05 was considered to indicate a statistically significant difference.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Autolysis of m-Calpain in the Presence of Varying Levels of Ca2+o and PMA in Bovine PT Cells—Bovine PT cells were treated with 0.5, 3, or 5 mM Ca2+o in the presence or absence of 1 µM PMA for 10 min. Total cell lysates were prepared by adding boiling lysis buffer containing 1% SDS, 1 mM sodium o-vanadate vanadate, and 10 mM Tris-HCl, pH 7.4, directly to PT cells. The cell lysates were then analyzed for m-calpain by Western blot analysis using an antibody to the large subunit of m-calpain, which reacts with bands of 80 and 58 kDa and a series of further cleaved, active forms of the enzyme as described by The Sigma-RBI Handbook. We observed autolysis of cellular m-calpain even at 0.5 mM Ca2+o. Immunoblot analysis demonstrated the presence of 80-kDa, 58-kDa, and further cleaved lower molecular mass forms of m-calpain (Fig. 1A). There were no consistent changes in the relative amounts of the activated forms of m-calpain in the presence of PMA or the various concentrations of Ca2+o in these total cellular lysates.



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  		FIG. 1.
Presence of m-calpain and calpastatin in PT cells and the association of caveolin with CaR and m-calpain. A, bovine PT cells were incubated with 0.5, 3, or 5 mM Ca2+o in the presence or absence of 1 µM PMA. The cells were lysed with Laemmli buffer, and proteins were separated on SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies specific for the large subunits of m-calpain (m-Calp-c) as described under "Experimental Procedures." B, comparative analyses of calpastatin (CPS) and calpain small subunits (Calp-r) in HEKCaR cells and bovine PT cells. C, tissue lysates were subjected to subcellular fractionation after homogenization in MBS buffer containing sodium carbonate (see "Experimental Procedures"). Samples of caveolar fractions were immunoblotted with anti-caveolin-1 (Cav-1), anti-CaR (4637), or an antibody to the large subunits of m-calpain. Results are representative of those obtained in two separate preparations. D, bovine PT (BPT) and human PT (HPT) cell lysates were prepared and immunoprecipitated (IP) with monoclonal antibodies to caveolin-1. Immunoprecipitates were resolved by SDS-PAGE and subjected to immunoblot analysis using a rabbit polyclonal antibody to the large subunits of m-calpain.

 

Expression of Calpastatin in PT Cells and HEKCaR Cells— The level of the endogenous calpain inhibitor, calpastatin, was examined using immunoblot analysis. Western blot analysis of total cell lysates (10 µg of protein) demonstrated that calpastatin is present in HEKCaR cells (Fig. 1B), but, in bovine PT cells, calpastatin could only be detected weakly after 30 s of exposure of the films. Very weak immunoreactivity was also found in human PT cells prepared from adenomatous PT glands using two different antibodies to calpastatin (not shown). The antibody against the small subunit of calpains reacts with the regulatory subunits of both µ- and m-calpain. In HEKCaR cells, less calpain protein was detected with an antibody to the regulatory subunits than in bovine PT cells (Fig. 1B).

CaR and m-Calpain Are Present in Caveolin-rich Protein Fractions Prepared from Bovine PT Cells—Bovine PT glands were homogenized in lysis buffer containing 500 mM sodium carbonate and fractionated by discontinuous sucrose gradient centrifugation. Equal amounts of protein from each fraction were subjected to SDS-PAGE and then immunostained with antibodies to caveolin-1, the CaR, and the large subunit of m-calpain (Fig. 1C). The bulk of the caveolin-1 and CaR immunoreactivity was found in fractions 3 and 4, which exclude >90% of total cellular protein (i.e. cytosolic, plasma membrane proteins). As shown in Fig. 1C, the antibody to m-calpain recognizes a band of ~110 kDa in fractions 3 and 4, which is consistent with the predicted molecular masses of the proenzyme forms of calpain. In contrast, the activated, autolyzed, and degraded forms of m-calpain were found in high-density fractions (fractions 5–9). These results demonstrate that the inactive proenzyme form of calpain is present in caveolin-rich membrane fractions in bovine PT cells. Human PT cells prepared from adenomatous tissues express flotillin and a high level of caveolin-2 but variably reduced levels of caveolin-1.2 In this study we included only caveolin-1-expressing human PT cells prepared from PT adenomas. To verify an interaction between caveolin-1 and m-calpain, an immunoprecipitation experiment was performed. Cell lysates were prepared from bovine and human PT cells and then immunoprecipitated with a caveolin-1 antibody and subjected to Western blot analysis with an antibody to m-calpain. As shown in Fig. 1D, caveolin-1 co-immunoprecipitates with m-calpain in bovine and human PT cells.

Effect of Varying Ca2+o Concentrations on m-Calpain in Caveolar Fractions—We next analyzed the effect of 3 mM Ca2+o on m-calpain in bovine and in human PT cells. The cells were incubated with 0.5 or 3 mM Ca2+o in standard medium for 30 min and homogenized in the presence of 1% Triton X-100 and protease inhibitors; low density membrane fractions were then isolated using sucrose gradient centrifugation. Equal amounts of protein from each of the nine fractions were subjected to SDS-PAGE, and Western blot analysis was performed for m-calpain and caveolin-1 in bovine (Fig. 2) and human PT cells (Fig. 3). Using a rabbit polyclonal antibody to the m-calpain large subunit and a mouse monoclonal IgG to the small subunit, we observed a calpain enzyme that was immunoreactive with both antibodies at 110-kDa bands in the Triton X-100-insoluble, caveolin-1-rich fraction (fraction 4) prepared from both bovine and human PT cells incubated with 0.5 mM Ca2+o (Figs. 2 and 3). In fraction 5, the antibody against the small subunit recognizes bands of 110 and 30–25 kDa, and the antibody against the large subunits reacts with bands of 110 and weakly with a band at 80 kDa (Fig. 2). Treatment of PT cells with 3 mM Ca2+o results in a reduction in high molecular weight m-calpain that is immunoreactive with both antibodies in fraction 4 (Figs. 2 and 3). In fraction 5, prepared from 3 mM Ca2+o-treated bovine PT cells, the antibody to the large subunits reacts very weakly with a band at 80 kDa, and the antibody against the small subunit reacts with bands of ~36, 25, and 18 kDa. In Triton X-100-soluble fractions (fractions 7, 8, and 9) prepared from both 0.5 and 3 mM Ca2+o-treated bovine PT cells, the antibody to the large subunits reacts with bands of 80, 78, and 76 kDa, and the antibody against the small subunit recognizes bands of ~ 36, 25, and 18 kDa (Fig. 2). In fraction 5 and the Triton-soluble fractions prepared from human PT cells, the antibody to the small subunits reacts weakly with bands of 110 kDa and intensely with those of 30 and 18 kDa, and the antibody to the large subunits reacts with bands of 110, 80, and 76 kDa. Thus, caveolar fractions from PT cells incubated at low Ca2+o contain predominantly inactive, pro-enzyme forms of m-calpain.



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  		FIG. 2.
Association of caveolin-1 with m-calpain in bovine PT cells. Bovine PT cells were incubated with 0.5 or 3 mM Ca2+o in standard medium at 37 °C for 30 min. Cell lysates were subjected to subcellular fractionation after homogenization in MBS buffer containing 1% Triton X-100. Equal amounts of proteins from each of the nine fractions were immunoblotted with anti-caveolin-1 antibody (Cav-1) or polyclonal antibody to the large catalytic subunits of m-calpain (m-Calp-c). Then the m-Calp-c immunoblot was stripped and reprobed with mouse monoclonal IgG to the small regulatory subunit (Calp-r). The data are representative of two separate experiments.

 


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  		FIG. 3.
Association of caveolin-1 with m-calpain in human PT cells. Dispersed human PT cells were incubated with 0.5 and 3 mM Ca2+o for 30 min. Cell lysates were subjected to subcellular fractionation using the detergent-based method. Equal amounts of proteins from each of the nine fractions were subjected to SDS-PAGE and immunoblotted with antibodies to caveolin-1 (Cav-1) or the catalytic subunits of m-calpain (m-Calp-c). After stripping, the m-Calp-c immunoblot was reprobed with mouse monoclonal IgG to the regulatory subunit (Calp-r). The data are representative of two separate experiments.

 

Agonist-induced Targeting of CaR to Caveolin-enriched Fractions—High Ca2+o induces changes in several second messengers and suppresses the PTH secretion in PT cells. Previously, we showed that the CaR and its dimeric forms are located primarily in the caveolae of bovine PT cells (7). In the present study, we tested whether incubation with 3 mM Ca2+o modulates the level of the CaR in PT caveolae. Fig. 4A shows that the intensity of the band immunostained with the anti-CaR antibody was greater in detergent-insoluble caveolar fractions (2, 3, 4) prepared from bovine PT cells incubated with 3 mM Ca2+o than in those incubated with 0.5 mM Ca2+o. High Ca2+o significantly increased CaR association with caveolar fractions (Fig. 4B).



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  		FIG. 4.
Agonist-induced targeting of the CaR and pPKC{alpha} and {beta} to caveolin-1-enriched fractions in bovine PT cells. A, bovine PT cells were incubated with 0.5 or 3 mM Ca2+o in standard medium for 30 min; the cells were then homogenized in the presence of 1% Triton X-100 in MBS buffer, and caveolin-1-rich, low density membrane fractions were isolated using sucrose gradient centrifugation (see Fig. 2). Proteins fractionated in Triton X-100-insoluble and -soluble fractions from Fig. 2 were used for the analysis in Fig. 4. Equal amounts of proteins from each of the nine fractions were mixed with 2x Laemmli buffer without DTT (CaR)or2x Laemmli buffer containing 100 mM DTT (pPKC) and separated on linear 4–10% gradient gels. The proteins were transferred to nitrocellulose filters and probed with antibodies to the CaR (CaR), the phospho-PKC (pan) ({alpha}, {beta}, {zeta}, {epsilon}, and {delta}), or the phospho-PKC ({alpha} and {beta}). The graph (B) shows the density of the CaR in caveolar fraction of bovine PT cells incubated with 0.5 and 3 mM Ca2+o. High Ca2+o significantly stimulated the association of the CaR with caveolae; p = 0.00197. Results are expressed as means ± S.E. of the results from three separate experiments, each performed in duplicate.

 

Subcellular Distribution of Phosphorylated PKC Isoenzymes in Bovine PT Cells—One of the best known examples of the function of calpain in signal transduction is the activation and subsequent down-regulation of PKC enzymes (15). The effect of treating bovine PT cells with 3 mM Ca2+o on PKC phosphorylation was, therefore, investigated. Fig. 4A shows that incubation with 3 mM Ca2+o increases the phosphorylation of PKC enzymes in the Triton X-100-insoluble, caveolin-rich fraction (fraction 4) as assessed with an anti-phospho-PKC (pan) polyclonal antiserum, which recognizes the {alpha}, {beta}, {zeta}, {epsilon}, {eta}, and {delta} forms of the enzyme. The phosphorylation of PKC enzymes detected with this pan-PKC antibody was not significantly different in Triton X-100-soluble non-caveolar fractions (fractions 7, 8, and 9) prepared from PT cells treated with 0.5 or 3 mM Ca2+o. To further investigate which PKC subtypes are phosphorylated in the presence of high Ca2+o, an anti-phospho-PKC {alpha}, {beta}-specific polyclonal antibody was used. Fig. 4 A shows that incubation with 3 mM Ca2+o increases the phosphorylation of PKC {alpha} and {beta} in the caveolar fraction (Fraction 4), whereas little or none of these PKC isoforms was detected in non-caveolar fractions. These results illustrate that high Ca2+o activates/phosphorylates calcium-regulated PKC isoforms (PKC {alpha} and {beta}) in bovine PT cells but that most of these enzymes were located in caveolar fractions, which contain low levels of activated m-calpain.

Co-localization of Caveolin-1, m-Calpain, and PTH in Bovine PT Cells—Dispersed bovine PT cells were incubated with 0.5 or 3 mM Ca2+o in standard medium for 30 min, and the subcellular localization of caveolin-1, m-calpain, and PTH was then studied by indirect immunofluorescence using a confocal microscope (Fig. 5). PT cells incubated with 0.5 mM Ca2+o and immunostained with sheep anti-PTH antibody revealed punctate green fluorescence close to the cell surface (Fig. 5A, sections 4µ, 4.5µ, and 5µ). Immunostaining with a monoclonal, anti-caveolin-1 antibody and an Alexa 568-labeled secondary antibody produced a red signal (Fig. 5, B2 and C2). To observe the distribution of m-calpain, a rabbit polyclonal antiserum produced against the large subunits was applied to the same PT cell preparation using TSATM cyanine 5, which produces a blue signal (Fig. 5, B3 and C3). Superimposition of the images obtained for the three colors individually revealed colocalization of caveolin-1 with m-calpain and PTH at or close to the surface of bovine PT cells, as reflected by the white fluorescence observed when the fluorophores were in close proximity (Fig. 5A, sections 5.5µ, 6µ, and 6.5µ, and B4). Thus, there was clear co-localization of caveolin-1, m-calpain and PTH at or close to the plasma membrane in the presence of 0.5 mM Ca2+o. In Fig. 5, optical sections 7µ and 7.5µ, most of the colocalization was of caveolin-1 with m-calpain. Parathyroid chief cells are polarized in intact parathyroid glands (40). In epithelial cells, large caveolin-1 homo-oligomers accumulate at the apical surface, and apical exocytosis can be inhibited with caveolin-1 antibody (41). Analysis of triple immunofluorescence experiments in bovine PT cells treated with 3 mM Ca2+o indicated redistribution of PTH, caveolin-1, and some of the m-calpain to one edge of the cells (Fig. 5, C1, C2, and C3), which is probably the apical pole of the cells. The magenta color in the merged image C4 (Fig. 5) indicates caveolin-1 and m-calpain co-localization; however, there was minimal co-localization with PTH, as demonstrated by the lack of white color.



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  		FIG. 5.
Immunofluorescent detection of caveolin-1, m-calpain, and PTH in bovine PT cells. Dispersed bovine PT cells were incubated with 0.5 (A, sections 4µ–7.5µ and B1–B4) and 3 mM Ca2+o (C1–C4) for 30 min, fixed on coverslips, and stained for PTH (B1 and C1), caveolin-1 (B2 and C2), or m-calpain (B3 and C3) as detailed under "Experimental Procedures." The distribution of PTH was detected by immunostaining with sheep antibody to PTH and visualized with fluorescein-conjugated secondary antibody (B1 and C1, green); caveolin-1 was detected with the monoclonal antibody to caveolin-1 and Alexa 568-labeled secondary antibody (B2 and C2, red); and, finally, m-calpain was visualized with an antibody produced against the large subunits and with TSATM cyanine 5 (B3 and C3, blue). In A, optical sections are presented from the tops of the cells to the bases that were adherent to the coverslip (from 4µ to 7.5 µ). In B and C, a combination of optical sections from 5µ to 6.5µ are presented. B4 is the merged image analysis of B1, B2, and B3, and C4 is the overlay of C1, C2, and C3. Magnification is 1000x.

 

CaR Is an Endogenous Substrate of m-Calpain—During overnight incubation with varying concentrations of Ca2+o, bovine PT cells released into the medium substantial amounts of fragments of the CaR (~80-kDa molecular mass) containing epitopes from the extracellular amino-terminal domain (ECD) of the receptor. The release of these CaR fragments is Ca2+o-dependent (Fig. 6, A and B) and could be detected with two different antibodies (4641 and 4637) produced to two different peptides from within the CaR ECD. The Ca2+o dependence of this proteolytic processing suggests that calpain may be responsible for the cleavage of the CaR. To examine whether cleavage of the CaR was produced by Ca2+o-activated calpain, bovine PT cells were incubated with low and high Ca2+o in the presence or absence of calpain inhibitors (Fig. 6, C and D). Cell-permeable calpain inhibitors reduced the fragmentation of the CaR observed at 3 mM Ca2+o in cell lysates as well as in the medium (Fig. 6, C and D). In total lysates prepared from bovine PT cells incubated with high Ca2+o, Western blot analysis using non-reducing conditions revealed higher and lower molecular weight forms of the CaR (Fig. 6C). In contrast, in cell lysates prepared from bovine PT cells incubated with low Ca2+o or cells incubated with high Ca2+o in the presence of calpain inhibitors, we observed only high molecular weight forms of the CaR.



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  		FIG. 6.
Effect of Ca2+o treatment on the CaR, and localization of caveolin-1 immunoreactivity and alkaline phosphatase activity in bovine PT cells. A and B, bovine PT cells were incubated overnight with varying concentrations of Ca2+o. Cell-free medium was centrifuged at 100,000 x g for 30 min, and then the sediment was solubilized with SDS-sample buffer containing DTT and subjected to SDS-PAGE using antibody 4641 to the ECD of the CaR (A). B, the antibody (4641) was pre-absorbed with specific peptide. C, D, and E, bovine PT cells were incubated with 0.5 or 3 mM Ca2+o in the presence (0.5+ and 3+) or absence (0.5– and 3–) of 2.0 µM calpeptin overnight at 37 °C. Cell lysates (C) and cell-free medium (D and E) were prepared, mixed with SDS-Laemmli buffer containing no DTT, and subjected to SDS-PAGE. The CaR in whole cell lysate (C) and medium (D) was detected with an antibody to the CaR (4637), and the presence of caveolin-1 and calpain in the medium (E) was documented with the respective antibodies. In panel E, calpain was detected with an antibody to the large catalytic subunits (m-calp-c) or an antibody to the regulatory subunit (calp-r). In panel F, the PT cells (without permeabilization with detergent) were stained with monoclonal antibody to caveolin-1 followed by a peroxidase-coupled, anti-mouse IgG. In panel G, PT cells were stained for AP activity using an AP substrate kit. Punctate staining for caveolin-1 and AP was clearly observed on the cell surface. Membrane vesicles shed by PT cells were isolated from conditioned medium as detailed under "Experimental Procedures" and stained for AP activity (H). Photomicrographs were taken at magnifications of 1000x.

 

Immunolocalization of caveolin-1 in COS-7 cells requires detergent permeabilization to allow specific IgG access to the cytoplasm (42). It is interesting, therefore, that immunocytochemical analysis of bovine PT cells shows abundant punctate staining for caveolin-1 on the cell surface and around the cells without detergent permeabilization (Fig. 6F). Cytochemical analysis shows that the PT cells are surrounded by AP-containing granules (Fig. 6G). Plasma membrane vesiculation occurs spontaneously in normal and tumor cells in culture (22). To determine whether PT cells spontaneously release caveolin-1 and AP-containing microdomains, we utilized the sedimentation method (21). Membrane vesicles shed by PT cells were isolated and stained for AP activity in Fig. 6H. Western blot analysis shows the presence of caveolin-1, m-calpain, and fragments of the CaR in the isolated medium vesicles (Fig. 6, D and E). Fig. 6E shows that incubation with high Ca2+o caused increased secretion of caveolin-1 and calpain into the medium and that calpain inhibitors reduced this secretion.

Calpain activation results in truncation of the C-terminal domains of several glutamate receptor subunits (31). In bovine PT cells, the secreted active calpain likely cleaves the N-terminal ECD of the CaR. To further verify that calpain was capable of cleaving the CaR, we purified m-calpain from PT cells by immunoprecipitation (Fig. 7A). We obtained the CaR from caveolar fractions from bovine PT cells (which contain CaR dimer and mostly the inactive forms of the calpain) as well as from total lysates from HEKCaR cells. Incubation of the CaR-containing cell lysates prepared from PT and HEKCaR cells with purified m-calpain in the presence of 3 mM calcium and 1 mM DTT produced lower molecular weight fragments of the CaR in both cell types (Fig. 7B).



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  		FIG. 7.
m-Calpain cleaves CaR in HEKCaR and bovine PT cell lysates. A, bovine PT cell lysates were prepared in non-denaturing conditions (in the presence of Triton X-100) and immunoprecipitated with non-immune mouse IgG (Control), polyclonal antibody to m-calpain catalytic subunits (m-Calp-c), or mouse antibody to the calpain regulatory subunit (Calp-r) as detailed under "Experimental Procedures." Immunoprecipitates were resolved by SDS-PAGE and subjected to immunoblot analysis for m-calpain using antibody to the catalytic subunits (A) or used for incubation with HEKCaR and bovine PT cell extracts (B). Protein extracts from HEKCaR and bovine PT cells were incubated in the presence of 3 mM Ca2+o and 1 mM dithiothreitol for 30 min at room temperature in the absence (Control) or presence of m-calpain that was immunoprecipitated using an antibody to the catalytic subunits (m-Calp-c) or an antibody to the regulatory subunit (Calp-r) (B). After digestion, the extracts were fractionated by 7% SDS-PAGE under nonreducing conditions (in the absence of DTT) and subjected to immunoblot analysis for the CaR using the 4637 antiserum. The data are representative of two separate experiments.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The importance of calpains has been demonstrated in transgenic mice that lack the calpain regulatory subunit, eliminating detectable calpain activity. These mice die during embryonic development due to defects in vascular development (43). There is evidence that calpain is tightly regulated post-transcriptionally by many mechanisms, including its endogenous inhibitor calpastatin, varying requirements for calcium, and autoproteolytic cleavage (reviewed in Refs. 15–19). Stimulation of cells with EGF activates calpain via the ERK/MAPK signaling pathway (20). Phosphorylation of m-calpain by ERK may increase its proteolytic activity (20). Available evidence suggests that active calpain is found predominantly at the plasma membrane and that this membrane localization may be important for the regulation of calpain activity. EGF receptor signaling events that lead to calpain activation are confined to the plasma membrane (20). These findings suggest that there are factors in the cytoplasm such as calpastatin that may prevent activation of calpain or that additional factors (phospholipids) required for activation are localized at the plasma membrane (15, 16, 24).

The µ- and m-calpains are highly expressed in bovine PT cells and may play a role in the Ca2+-dependent degradation of PTH to secreted C-terminal fragments (33). Watson et al. showed that calpain is capable of Ca2+o -dependent, limited proteolysis of PTH in vitro (33). It was reported that the m-calpain (the millimolar Ca2+o-requiring protease) autolyzed when incubated in vitro with 750–780 µM free Ca2+o (24). In response to a rise in Ca2+o from 0.5 to 2 mM, intracellular Ca2+ (Ca2+i) in PT cells is transiently elevated to 1–3 µM and maintains a sustained level of 0.6–0.8 µM (44). It has also been shown that calpain is activated by PMA (16). Therefore, we analyzed the effects of Ca2+o and PMA on m-calpain in bovine PT cells incubated in the presence of low or high Ca2+o with or without PMA. We observed autolysis of m-calpain even at 0.5 mM Ca2+o. Calpastatin, the endogenous calpain inhibitor, may function in protecting cellular substrates from calpain-mediated proteolysis (15, 16). Therefore, we next determined the protein level of calpastatin by Western blot. We detected calpastatin in HEKCaR cells, but in bovine and human PT cells there was a discrepancy between the levels of calpain and calpastatin, with low amounts of calpastatin relative to those of calpain. Thus, the results presented here suggest that the role of calpastatin in PT cells is of limited importance and that other mechanism(s) are needed for protection of substrate proteins from calpain-mediated proteolysis.

Caveolin-1 has been described as an inhibitor of various signaling pathways (2); thus we next analyzed whether caveolin-1 has any protective effect on activated m-calpain in PT cells. To examine the potential association of m-calpain with caveolin-1, subcellular fractionation of bovine PT cells was performed. We found that both m-calpain and the CaR localize in the caveolin-1-rich fraction. Interestingly, we detected the heterodimeric, proenzyme forms of m-calpain mostly in caveolar fractions. In the non-caveolar fractions, we observed less of the high molecular weight forms of m-calpain and more of the lower molecular weight forms, which were probably the dissociated, autolyzed forms of m-calpain. Immunoprecipitation showed that this heterodimeric 110-kDa form of m-calpain could be immunoprecipitated with antibodies to either the catalytic or the regulatory subunit, and co-immunoprecipitation showed that this form of m-calpain is associated with caveolin-1 in PT cells.

To obtain further information about the functional significance of interactions between caveolin-1 and m-calpain, we analyzed the effect of 3 mM Ca2+o on m-calpain in PT cells. The heterodimeric forms of m-calpain localize in caveolae in bovine and human PT cells incubated with low Ca2+o. Incubation of the cells with 3 mM Ca2+o, in contrast, produced a reduction in the 110-kDa forms of m-calpain in caveolin-1-containing, detergent-insoluble fractions. In the Triton X-100-soluble, non-caveolar fractions, we detected separately both the large catalytic subunits and the small regulatory subunit. Our immunofluorescence data also indicate that m-calpain and caveolin-1 are colocalized in bovine PT cells incubated with 0.5 mM Ca2+o, indicating that m-calpain is resident in PT caveolae. There was clear co-localization of caveolin-1 with m-calpain, and, interestingly we observed that these two proteins colocalized with PTH. PTH is a substrate of m-calpain (33), supporting our observation that m-calpain in its inactive, proenzyme forms associates with caveolin-1 and PTH in bovine PT cells incubated with 0.5 mM Ca2+o, which minimizes the degradation of cellular PTH. In contrast, we observed that the presence of high Ca2+o, which increases intracellular degradation of PTH, reduced the levels of PTH and minimized the co-localization of the three proteins.

The CaR mediates the responses of cells to changes in the concentration of Ca2+o in their external environmental and is expressed in a wide variety of tissues, including PT, kidney, brain, and other cell types (34, 35). In the PT, the CaR has been implicated in the regulation of PTH release and cell proliferation (34, 35). However, the molecular mechanism(s) by which the receptor regulates these pathways has not yet been clearly defined, and we propose a role for caveolae in this process. Caveolin may function as a scaffolding protein to organize and concentrate inactive signaling molecules within caveolae membranes for regulated activation by appropriate receptors. Recent observations demonstrate the ligand-dependent association of endothelin A, bradykinin B2, and m2 muscarinic acetylcholine receptors with caveolae (9–11). In the present study we demonstrate that high Ca2+o promotes translocation of the CaR into low density fractions in bovine PT cells. However, the cellular mechanism(s) through which the CaR inhibits PTH secretion at high Ca2+o remain unresolved. Activators of PKC such as PMA substantially blunt the high Ca2+o-elicited increases in inositol phosphates and Ca2+i in bovine PT cells (45, 46). Previous studies using enzymatic activity to assess the translocation of PKC between particulate and soluble fractions of PT cells have provided evidence for a reduction in PKC activity at high Ca2+o levels despite the concomitant increases in the levels of diacylglycerols and Ca2+i, but these studies did not investigate the plasma membrane domains in which the enzyme was located (47, 48). The presence of predicted PKC phosphorylation sites within the intracellular domains of the CaR suggests that PKC may modulate the receptor's function by phosphorylating one or more of these sites (34, 35, 49). Indeed, PKC phosphorylation of threonine at position 888 in the C-terminal tail of the CaR inhibits its coupling to Ca2+ store release (49).

Calpain cleaves substrate proteins localized near membranes and the cytoskeleton in a limited manner and, thus, is thought to function as a biomodulator of cell physiology (15). Activated and membrane-associated calpains are able to cleave many substrate proteins such as PKC. Conventional calcium/phospholipid/diacylglycerol-dependent PKC enzymes ({alpha}, {beta}, and {gamma}) are composed of regulatory and catalytic domains connected by a variable region (V3). In vitro, calpain cleaves PKC enzymes at the inter-domain V3 and produces active protein kinase M (PKM) (15, 50, 51). Recent analysis of the crystal structures of calpain revealed the presence of a C2-like structure in domain III that shares structural characteristics with the calcium/phospholipid binding C2 domain of PKC (15, 25). Both calpain and PKC are expected to translocate to the plasma membrane after activation (15). Caveolar localization of PKC isoforms as well as regulation of caveolar function by PKC are well known (2, 3, 52, 53). Calpains can cleave PKC{alpha}, {beta}, {gamma}, {delta}, {epsilon}, and {zeta} isoforms in vitro, but they cleave only the activated forms of PKC in living cells (15). To demonstrate a functional role of calpain, we analyzed phosphorylated forms of PKC present in caveolar and non-caveolar fractions in PT cells. We found that high Ca2+o increases the translocation of the CaR to caveolae and that phosphorylated forms of PKC {alpha} and {beta} are resident in bovine PT caveolae but not in Triton X-100-soluble fractions. Our results demonstrate that activated calpain, which is capable of cleaving activated PKC, is present in a Triton X-100-soluble fraction. Parathyroid caveolae could be the location for CaR activation and inactivation. Previously, we observed that the CaR activates MAPK through a PKC-dependent mechanism, presumably through Gq/11-mediated activation of the phosphatidylinositol-specific phospholipase C (PI-PLC) in bovine PT cells (39) and that activated, phosphorylated ERK1/2 localizes in PT caveolae.2 Co-localization of activated, phosphorylated PKC and phosphorylated ERK1/2 in PT caveolae suggest the importance of compartmentalized signaling in PT caveolae. Moreover, co-localization of the CaR with activated, phosphorylated PKC {alpha} and {beta} in PT cells in the presence of high Ca2+o could produce phosphorylation of threonine at position 888 in the CaR C-terminal tail, and this phosphorylation could inhibit its coupling to Ca2+ store release. In freshly prepared bovine PT cells, high Ca2+o-stimulated ERK1/2 phosphorylation peaked at 10 min, producing a 7-fold increase in activity, but decreased to the basal level after 1 h and then remained at that level (39). Inactivation of the MAPK cascade could be the result of CaR phosphorylation.

Many proteins lead a dual existence as both membranebound and soluble isoforms. The soluble forms of receptors have been shown to consist of their extracellular domains (54). In cases where the soluble forms are produced by proteolytic release, the cells are desensitized through the loss of their cell-surface receptors (54). Activated calpains are able to cleave many receptors such as the EGF receptor, the PDGF receptor, and the {alpha}-adrenergic receptor (15, 16). Calpain-mediated truncation of the C-terminal domains of several subunits of the {alpha}-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) and N-methyl-D-aspartate receptors modulates their structures and other features of these receptors (31). The CaR principally exists as a dimer on the cell surface of HEKCaR cells where it is covalently linked by disulfide bonds (36–38). Previously, we observed that the dimeric form of the CaR protein is highly enriched in PT cell caveolae (7). In the present study, our results demonstrate that the CaR can be cleaved by m-calpain in vitro and that the CaR may also be a substrate for calpains in living cells. On Western blot analysis, we demonstrated that an ~80-kDa band recognized by two antibodies produced against the ECD of the CaR is generated and released into the conditioned medium of bovine PT cells. The CaR ECD is involved in ligand binding and receptor activation. Certain residues within loops I and IV are critical for Ca2+o activation of the receptor (55). The residues in loop II are critical for maintaining the inactive state of the CaR. Deletion of the entire loop II (residues 117–137) abolished receptor expression and function (53). Cleavage of the ECD of the CaR by m-calpain would be expected to impair ligand binding and receptor activation.

Shedding of membrane vesicles by viable cells takes place in vitro (56) and probably occurs in vivo as well, because membrane vesicles have been found in bodily fluids such as serum (57). Matrix vesicles with the capacity to mineralize have been isolated from a variety of mammalian bone sources and the media of cultured cells (21). Many other cells, including erythrocytes, fibroblasts, platelets, and tumor cells are also capable of vesiculating, and GPI-anchored proteins are enriched in the released vesicles (57). Our experiments also show that the CaR fragment, m-calpain, caveolin-1, and GPI-anchored alkaline phosphatase were associated with vesicles isolated from PT cell-conditioned medium. The association of these proteins in media vesicles produced by PT cells supports the presence of pre-existing caveolin-1, GPI-anchored alkaline phosphatase, CaR, and m-calpain-containing domains in living cells. Additional studies will be necessary to evaluate the function of media vesicles in PT metabolism and function.

We hypothesize that the mechanism of activation of calpain by the CaR is as follows. In the presence of low Ca2+o, m-calpain is present in caveolae in its inactive heterodimeric form. Stimulation of the CaR by high Ca2+o causes translocation of the CaR to caveolae and increases phosphatidylinositol turnover, thereby producing increased levels of diacylglycerol and Ca2+i followed by activation of calcium-dependent PKCs ({alpha} and {beta}) in PT caveolae and ERK1/2 phosphorylation. This could lead to activation of m-calpain as a consequence of activation of the ERK cascade within the immediate microenvironment. Increased Ca2+i produces a conformational change in calpain, followed by autolysis at the N termini of the catalytic and regulatory subunits, dissociation of the two subunits, and translocation of the subunits from the caveolae. Activated calpains cleave the activated forms of PKCs in the Triton X-100-soluble fraction but not the caveolar fraction. The presence of activated, phosphorylated PKC{alpha} and {beta} in caveolar fractions prepared from bovine PT cells incubated at high Ca2+o indicates translocation of the activated calpain from the caveolae. Activated calpain could also produce truncation of the CaR and release CaR fragments into the medium.

In conclusion, our results help to characterize caveolae as the plasma membrane microdomain in which the CaR is compartmentalized along with different signaling molecules, including m-calpain. Although the functional significance of this interaction is not at present fully understood, the apparently negative regulation of m-calpain activity by caveolin-1 may be relevant to the control of the various signaling pathways in which m-calpain is involved. Most importantly, it will be interesting to understand the biological as well as the functional implications of the interactions between the CaR, m-calpain, and caveolin-1 in PT cells and probably in other cells as well. Further experiments will be needed to establish the functional role of the cleavage of the CaR. Moreover, additional studies will therefore be necessary to elucidate the full signal transduction pathway culminating in the inverse regulation of PTH secretion by extracellular calcium.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants DK41415, DK48330, and DK52005 and grants from NPS Pharmaceuticals and the St. Giles Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Endocrine-Hypertension Division, Brigham and Women's Hospital, 221 Longwood Ave, Boston, MA 02115. Tel.: 617-732-5661; Fax: 617-732-5764; E-mail: okifor{at}rics.bwh.harvard.edu.

1 The abbreviations used are: GPCR, G protein-coupled receptor; AP, alkaline phosphatase; Ca2+i, intracellular calcium; Ca2+o, extracellular calcium; CaR, Ca2+o-sensing receptor; DTT, dithiothreitol; ECD, extracellular amino-terminal domain; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; GPI, glycosylphosphatidylinositol; HEK, human embryonic kidney (cells); HEKCaR, CaR-transfected HEK cells; MAPK, mitogen-activated protein kinase; MES, 4-morpholineethanesulfonic acid; MBS, MES-buffered saline; PBS, phosphate-buffered saline; PKC, protein kinase C; PMA, phorbol myristate acetate; PT, parathyroid; PTH, parathyroid hormone. Back

2 O. Kifor, I. Kifor, F. D. Moore, Jr., R. R. Butters, Jr., T. Cantor, P. Gao, and E. M. Brown, unpublished observations. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Kurzchalia, T. V., and Parton, R. G. (1999) Curr. Opin. Cell Biol. 11, 424–431[CrossRef][Medline] [Order article via Infotrieve]
  2. Okamoto, T., Schlegel, A., Scherer, P. E., and Lisanti, M. P. (1998) J. Biol. Chem. 273, 5419–5422[Free Full Text]
  3. Anderson, R. G. W. (1998) Annu. Rev. Biochem. 67, 199–223[CrossRef][Medline] [Order article via Infotrieve]
  4. Liu, J., Razani, B., Tang, S., Terman, B. I., Ware, J. A., and Lisanti, M. P. (1999) J. Biol. Chem. 274, 15781–15785[Abstract/Free Full Text]
  5. Isshiki, M., and Anderson, R. G. W. (1999) Cell Calcium 26, 201–208[CrossRef][Medline] [Order article via Infotrieve]
  6. Mitchel, J. B., Feron, O., Sacks, D., and Mitchel, T. (1997) J. Biol. Chem. 272, 18522–18525[Abstract/Free Full Text]
  7. Kifor, O, Diaz, R., Butters, R. R., Kifor, I., and Brown, E. M. (1998) J. Biol. Chem. 273, 21708–21713[Abstract/Free Full Text]
  8. Isshiki, M., Ying, Y., Fujita, T., and Anderson, R. G. W. (2002) J. Biol. Chem. 277, 43389–43398[Abstract/Free Full Text]
  9. Murthy, K. S., and Makhlouf, G. M. (2000) J. Biol. Chem. 275, 30211–30219[Abstract/Free Full Text]
  10. de Weerd, W. F. C., and Leeb-Lundberg, L. M. F. (1997) J. Biol. Chem. 272, 17858–17866[Abstract/Free Full Text]
  11. Feron, O., Smith, T. W., Michel, T., and Kelly, R. A. (1997) J. Biol. Chem. 272, 17744–17748[Abstract/Free Full Text]
  12. Razani, B., Rubin, C. S., and Lisanti, M. P. (1999) J. Biol. Chem. 274, 26353–26360[Abstract/Free Full Text]
  13. Rybin, V. O., Xu, X., Lisanti, M. P., and Steinberg, S. F. (2000) J. Biol. Chem. 275, 41447–41457[Abstract/Free Full Text]
  14. Lisanti, M. P., Tang, Z., Scherer, P. E., and Sargiacomo, M. (1995) Methods Enzymol. 250, 655–668[Medline] [Order article via Infotrieve]
  15. Sato, K., and Kawashima, S. (2001) Biol. Chem. 382, 743–751[CrossRef][Medline] [Order article via Infotrieve]
  16. Croall, D. E., and DeMartino, G. N. (1991) Physiol. Rev. 71, 813–847[Free Full Text]
  17. Daman, A., Harris, F., Biswas, S., Wallace, J., and Phoenix D. A. (2001) Mol. Cell. Biochem. 223, 159–163[CrossRef][Medline] [Order article via Infotrieve]
  18. Perrin, B. J., and Huttenlocher, A. (2002) Int. J. Biochem. Cell Biol. 34, 722–725[CrossRef][Medline] [Order article via Infotrieve]
  19. Suzuki, K., and Sorimachi, H. (1998) FEBS Lett. 433, 1–4[CrossRef][Medline] [Order article via Infotrieve]
  20. Glading. A., Uberall, F., Keyse, S, M., Lauffenburger, D. A., and Wells, A. (2001) J. Biol. Chem. 276, 23341–23348[Abstract/Free Full Text]
  21. Nishihara, H., Nakagawa, Y., Ishikawa, H., Ohba, M., Shimizu, K., and Nakamura, T. (2001) Biochem. Biophys. Res. Commun. 285, 845–853[CrossRef][Medline] [Order article via Infotrieve]
  22. Civenni, G., Test, S, T., Brodbeck, U., and Butikofer, P. (1998) Blood 91, 1784–1792[Abstract/Free Full Text]
  23. Dolo, V., Li, R., Dillinger, M., Flati, S., Manela, J., Taylor, B. J., Pavan, A., and Ladisch, S. (2000) Biochim. Biophys. Acta. 1486, 265–274[Medline] [Order article via Infotrieve]
  24. Cong, Y., Goll, D. E., Peterson, A. M., and Kapprell, H. P. (1989) J. Biol. Chem. 264, 10096–10103[Abstract/Free Full Text]
  25. Hosfield, C. M., Elce, J. S., Davies, P. L., and Jia, Z. (1999) EMBO J. 18, 6880–6889[CrossRef][Medline] [Order article via Infotrieve]
  26. Crawford, C., Brown, N. R., and Willis, A. C. (1990) Biochem. J. 265, 575–579[Medline] [Order article via Infotrieve]
  27. Carafoli, E., and Molinari, M. (1998) Biochem. Biophys. Res. Commun. 247, 193–203[CrossRef][Medline] [Order article via Infotrieve]
  28. Khorchid, A., and Ikura, M. (2002) Nat. Struct. Biol. 9, 239–241[CrossRef][Medline] [Order article via Infotrieve]
  29. Huang, Y., and Wang, K. K. W. (2001) Trends Mol. Med. 7, 355–362[CrossRef][Medline] [Order article via Infotrieve]
  30. Kishimoto, A., Mikawa, K., Hashimoto, K., Yasuda, I., Tanaka, S., Tominaga, M., Kuroda, T., and Nishizuka, Y. (1989) J. Biol. Chem. 264, 4088–4092[Abstract/Free Full Text]
  31. Rong, Y., Lu, X., Bernard A, Khrestchatisky, M., and Baudry, M. (2001) J. Neurochem. 79, 382–390[CrossRef][Medline] [Order article via Infotrieve]
  32. Carragher, N. O., Westhoff, M. A., Riley, D., Potter, D. A., Dutt, P., Elce, J. S., Greer, P. A., and Frame, M. C. (2002) Mol. Cell. Biol. 22, 257–269[Abstract/Free Full Text]
  33. Watson, P. H., Mortimer, S. T., Wang, K. K. W., Croall, D. E., and Hanley, D. A. (1995) J. Mol. Endocrinol. 15, 61–71[Abstract/Free Full Text]
  34. Brown, E. M., Vassilev, P. M., Quinn, S., and Hebert, S. C. (1999) Vitam. Horm. 55, 1–70[Medline] [Order article via Infotrieve]
  35. Brown, E. M., and MacLeod, R. J. (2001) Physiol. Rev. 31, 239–297
  36. Bai, M., Trivedi, S., and Brown, E. M. (1998) J. Biol. Chem. 273, 23605–23610[Abstract/Free Full Text]
  37. Bai, M., Trivedi, S., Kifor, O., Quinn, S. J., and Brown, E. M. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 2834–2839[Abstract/Free Full Text]
  38. Pace, A. J., Gama, L., and Breitwieser G. E. (1999) J. Biol. Chem. 274, 11629–11634[Abstract/Free Full Text]
  39. Kifor, O., MacLeod, R. J., Diaz, R., Bai, M., Yamaguchi, T., Yao, T., Kifor, I., and Brown, E. M. (2001) Am. J. Physiol. Renal Physiol. 280, F291–F302[Abstract/Free Full Text]
  40. Wild, P., and Setoguti, T. (1995) Microsc. Res. Tech. 32, 120–128[CrossRef][Medline] [Order article via Infotrieve]
  41. Scheiffele, P., Verkade, P., Fra, A. M., Virta, H., Simons, K., and Ikonen, E. (1998) J. Cell Biol. 140, 795–806[Abstract/Free Full Text]
  42. Tang, Z., Okamoto, T., Boontrakulpoontawee, P., Katada, T., Otsuka, A., and Lisanti, M. P. (1997) J. Biol. Chem. 272, 2437–2445[Abstract/Free Full Text]
  43. Arthur, J. S. C., Elce J. S., Hegadorn, C., Williams, K., and Greer, P. A. (2000) Mol. Cell. Biol. 20, 4474–4481[Abstract/Free Full Text]
  44. Nemeth, E. F., and Scarpa, A. (1987) J. Biol. Chem. 262, 5188–5196[Abstract/Free Full Text]
  45. Kifor, O., Congo, D., and Brown E, M. (1990) J. Bone Miner. Res. 5, 1003–1011[Medline] [Order article via Infotrieve]
  46. Racke, F. K., and Nemeth, E. F. (1994) Am. J. Physiol. 267, E429–E438[Medline] [Order article via Infotrieve]
  47. Brown, E. M. (1991) Physiol. Rev. 71, 371–411[Free Full Text]
  48. Morrissey, J. J. (1988) Am. J. Physiol. Endocrinol. Metab. 254, E63–E70[Abstract/Free Full Text]
  49. Bai, M., Trivedi, S., Lane, C. R., Yang, Y., Quinn, S. J., and Brown, E. M. (1988) J. Biol. Chem. 273, 21267–21275
  50. Nishizuka, Y. (1989) J. Am. Med. Assoc. 262, 1826–1833[Abstract/Free Full Text]
  51. Hug, H., and Sarre, T. F. (1993) Biochem. J. 291, 329–343[Medline] [Order article via Infotrieve]
  52. Oka, N., Yamamoto, M, Schwencke, C., Kawabe, J., Ebina, T., Ohno, S., Couet, J., Lisanti, M. P., and Ishikawa, Y. (1997) J. Biol. Chem. 272, 33416–33421[Abstract/Free Full Text]
  53. Das, K., Lewis, R. Y., Scherer, P. E., and Lisanti, M. P. (1999) J. Biol. Chem. 274, 18721–18728[Abstract/Free Full Text]
  54. Ehlers, M. R. W., and Riordan, J. F. (1991) Biochemistry 30, 10065–10074[CrossRef][Medline] [Order article via Infotrieve]
  55. Reyes-Cruz, G., Hu, J., Goldsmith, P. K., Steibach, P. J., and Spiegel, A. M. (2001) J. Biol. Chem. 276, 32145–32151[Abstract/Free Full Text]
  56. Taylor, D. D., Chou, I.-N., and Black, P. H. (1983) Biochem. Biophys. Res. Commun. 113, 470–476[CrossRef][Medline] [Order article via Infotrieve]
  57. Xie, M., and Low, M. G. (1995) Biochem. J. 305, 529–537[Medline] [Order article via Infotrieve]

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