The Receptor for Parathyroid Hormone and Parathyroid Hormone-Related Peptide Is Hydrolyzed and Its Signaling Properties Are Altered by Direc
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内分泌学杂志 2005年第5期
Endocrine Unit, Massachusetts General Hospital and Department of Medicine, Harvard Medical School (M.S., M.J.M., G.V.S.), Boston, Massachusetts 02114; and Queen’s University Cancer Research Institute (P.A.G.), Kingston, Ontario, Canada K7L 3N6
Address all correspondence and requests for reprints to: Dr. Gino V. Segre, Endocrine Unit, Wellman 501, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114. E-mail: segre@helix.mgh.harvard.edu.
Abstract
We show calcium-dependent, direct binding between the N-terminal portion of the PTH/PTHrP receptor (PTH1R) C-terminal intracellular tail and the calpain small subunit. Binding requires, but may not be limited to, amino acids W474, S475, and W477. The wild-type, full-length rat (r) PTH1R, but not rPTH1R with W474A/W477A substitutions, copurifies with the endogenous calpain small subunit in HEK293 cells. Calpain hydrolyzes Nt-rPTH1R, a receptor with a 156-amino acid N-terminal deletion, in a calcium-dependent manner in vitro and in intact cells. Most importantly, PTH stimulation increases the cleavage of Nt-rPTH1R and rPTH1R-yellow fluorescent protein in HEK293 cells, and of talin in HEK293 cells expressing rPTH1R-yellow fluorescent protein and in ROS17/2.8 osteoblast-like cells that express rPTH1R endogenously. The absence of calpain in Capn4-null embryonic fibroblasts and the lowered calpain activity in MC3T3-E1 osteoblastic cells due to stable expression of the calpain inhibitor, calpastatin, reduce PTH-stimulated cAMP accumulation. The calpain small subunit is the second protein, in addition to the sodium-hydrogen exchanger regulatory factor, and the first enzyme that binds the PTH1R; PTH1R bound to both of these proteins results in altered PTH signaling.
Introduction
THE RECEPTOR FOR PTH and PTHrP (PTH1R) belongs to the class II G protein-coupled receptor (GPCR) superfamily (1). Stimulation of the PTH1R by PTH and PTHrP increases the intracellular levels of second messenger molecules, including cAMP, inositol triphosphate, diacylglycerol, and calcium (Ca2+) (2, 3). PTH functions primarily to maintain calcium homeostasis through direct actions in the kidneys, where it increases the resorption of calcium and inhibits the resorption of phosphate (4, 5, 6). It also acts directly on bone formation and indirectly on bone resorption through its actions on cells of the osteoblastic lineage (4, 7). PTHrP normally functions primarily, if not exclusively, as an autocrine/paracrine regulator of cellular growth and differentiation in many organ systems, especially the endochondral skeleton (8, 9). Its aberrant expression and secretion by malignancies are responsible for most cases of tumor-associated hypercalcemia (9, 10).
Our group recently demonstrated that direct binding of the scaffolding proteins, sodium-hydrogen exchanger regulatory factor 1 (NHERF1) and NHERF2, to the intracellular C-terminal portion (C-tail) of the PTH1R markedly affects receptor signaling. NHERF2 assembles phospholipase C? (PLC?) and the PTH1R through PDZ (PSD 95, discs large protein, ZO1) 1 and 2 domains, respectively. PTH stimulation of the NHERF2-PTH1R complex remarkably activates inhibitory G proteins, Gi/o, resulting in dissociation of ?-subunits from -subunits. The ?-subunits then activate PLC?, and the -subunit blocks activation of adenylyl cyclase in a cell- and membrane-specific manner (11). NHERF1 assembles a signaling complex in the apical domain of polarized opossum kidney cells that contains PTH1R, PLC?, and actin cytoskeleton (12), and it also assembles a complex containing the type IIa sodium-phosphate cotransporter (13, 14, 15). These studies extend the paradigm, first described with arrestin (16, 17, 18, 19), that molecules other than G proteins bind directly to GPCRs and markedly alter receptor functions in discrete compartments adjacent to the plasma membrane.
The yeast two-hybrid screen that revealed NHERF-PTH1R binding also showed binding between PTH1R and the calpain small subunit. Calpains are a family of Ca2+-dependent intracellular cysteine proteases that include the ubiquitously expressed μ- and m-calpains (20). Both μ- and m-calpains form heterodimers, consisting of a large catalytic subunit (80 kDa) encoded by the genes Capn1 and Capn2, respectively, and a common small subunit (28 kDa) encoded by the gene Capn4. Calpains are inactive in the absence of Ca2+, but upon Ca2+ binding, they undergo a conformational change, which is essential for activation of the protease (21, 22, 23).
Several lines of evidence suggest that PTH increases calpain activity (24, 25), and that calpains play a crucial role in PTH-mediated cellular retraction in osteoblastic cell lines (24, 25, 26, 27). The molecular mechanism underlying PTH/PTHrP-mediated regulation of calpain, however, has not been identified. In this study we demonstrate that the calpain small subunit binds to a highly conserved region in the N-terminal portion of the PTH1R’s intracellular tail in vitro and in intact cells; that calpain hydrolyzes both the C-tail of the rat PTH1R (rPTH1R) and talin, an actin cytoskeletal protein in a Ca2+- and ligand-dependent manner; and that the absence and reduction of calpain activity critically affect PTH1R signaling properties.
Materials and Methods
Materials
Recombinant rat m-calpain and calpain inhibitors, N-acetyl-Leu-Leu-Met-CHO (ALLM), antipain, and calpastatin were purchased from Calbiochem (La Jolla, CA). FuGene6 was purchased from Roche (Indianapolis, IN). Rat [Nle8, 21,Tyr34]PTH-(1–34)-NH2 (PTH) was prepared by the MGH Biopolymer Synthesis Facility (Boston, MA). cAMP RIA kits and [125I]antirabbit IgG were obtained from PerkinElmer (Boston, MA).
Cell culture and transfection
Capn4-null embryonic fibroblasts (28), HEK293, and ROS17/2.8 cells were cultured in DMEM (Cellgro, Mediatech, Inc., Herndon, VA), supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT) and 1% antibiotic/antimycotic (Invitrogen Life Technologies, Inc., Grand Island, NY) at 37 C in a humidified atmosphere containing 95% air and 5% CO2. MC3T3-E1 cells were cultured in -MEM (Invitrogen Life Technologies, Inc.), 10% FBS, and 1% antibiotic/antimycotic. Various rPTH1R cDNAs were transiently transfected into HEK293 cells using FuGene6. Full-length human calpastatin cDNA (gift from Dr. M. Maki, Nagoya University, Nagoya, Japan) (29) was subcloned into the pcDNA3.1/HYGRO vector (Invitrogen Life Technologies, Inc.) and transfected into MC3T3-E1 cells using FuGene6, and cells expressing human calpastatin were selected with 300 μg/ml hygromycin B (Calbiochem) (30). Two clonal cell lines expressing the highest calpastatin levels were further examined.
Antibodies
The monoclonal antibody, 1D4, which recognizes the rhodopsin epitope, TETSQVAPA (31), was purchased from the National Cell Culture Center (Minneapolis, MN). G48, a polyclonal antiserum raised in a sheep, contains antibodies that recognize epitopes within sequences of the rPTH1R, YPESKENKDVPTGSRRRGRPC, FCNGEVQAEIRKSWSRWTLAL, and SGLDEEASGSARPPPLLQEGWETVM. The first sequence is located in the N-terminal ectodomain, and the last two sequences are located in the intracellular C-tail of the receptor. Monoclonal antibodies to the calpain small subunit and human calpastatin were purchased from Chemicon International (Temecula, CA). S-Protein-horseradish peroxidase (HRP) and monoclonal antibodies to green fluorescent protein (GFP; B34) and talin (8D4) were purchased from Novagen (Madison, WI), Convance (Berkeley, CA), and Sigma-Aldrich Corp. (St. Louis, MO), respectively. Species-specific secondary antibodies conjugated with HRP were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
cDNA constructs
N-Terminal glutathione-S-transferase (GST) fusion proteins of the rPTH1R C-tail were generated by PCR using Taq polymerase (Amersham Biosciences, Piscataway, NJ). PCR fragments corresponding to the full-length C-tail [amino acids (aa) 468–591], N5 (aa 473–591), N10 (aa 478–591), and N15 (aa 483–591) were ligated into pGEX-5X1 (Amersham Biosciences). rPTH1R C-tail point mutations (S473A, W474A, S475A, R476A, and W477A) were generated by incorporation of the appropriate mutation within the forward PCR primers and ligated into pGEX-5X1. The rPTH1R lacking most of the extracellular N-terminal region (aa 26–181; Nt-rPTH1R) was a gift from Dr. T. Gardella of this laboratory (32). The rPTH1R carrying a double tryptophan mutation, rPTH1R-W474A/W477A, was generated by PCR, and the DNA fragment was subcloned into pcDNA3.1 (Invitrogen Life Technologies, Inc.; Nt-rPTH1R-W474A/W477A). The 27-mer encoding the rhodopsin epitope (rho) recognized by 1D4 was inserted in-frame immediately upstream of the stop codon at the C terminus of the Nt-rPTH1R and Nt-rPTH1R-W474A/W477A sequences (Nt-rPTH1R-rho and Nt-rPTH1R-W474A/W477A-rho, respectively); yellow fluorescent protein (YFP) was inserted between aa 579 and 580 of the rPTH1R (rPTH1R-YFP) (12), and the rhodopsin epitope was inserted at the C terminus of rPTH1R-YFP (rPTH1R-YFP-rho). All were ligated into pcDNA3.1 and expressed in HEK293 cells.
Full-length rPTH1R was cloned into a modified pENTR vector (Invitrogen Life Technologies, Inc.) using NheI and XbaI sites. The pENTR-rPTH1R was packaged into an adenovirus using the ViraPower Adenoviral Expression System (Invitrogen Life Technologies, Inc.), and rPTH1R cDNA was introduced into the adenoviral genome via recombination between pENTR-PTH1R and the pAd/CMV/V5-DEST vector using LR Clonase (Invitrogen Life Technologies, Inc.), following the manufacturer’s guidelines. The accuracy of the DNA encoding the GST fusion proteins and the various rPTH1Rs was confirmed by sequencing (Tufts University, Sequencing Core Facility, Boston, MA).
Yeast two-hybrid system
The cytoplasmic C-tail of the rPTH1R was screened for protein interactions using the Matchmaker yeast two-hybrid system (BD Clontech, Palo Alto, CA). Briefly, the C-tail of the rPTH1R (aa 468–591) was cloned in-frame with the galactosidase-4 (GAL4) DNA-binding domain in the pAS2.1 vector using Pfu polymerase (Promega Corp., Madison, WI). The GAL4-C-tail fusion was screened against a Matchmaker cDNA library fused to the GAL4 activation domain from human kidney (BD Clontech) in the CG-1945 yeast strain. His+ clones were isolated on selective medium and reintroduced into the Y190 yeast strain in the absence or presence of the rPTH1R C-tail. Interactions were assessed using the lacZ reporter gene and the Galacton-star chemiluminescent ?-galactosidase assay (BD Clontech). Clones expressing the highest levels of ?-GAL activity in the presence of the rPTH1R C-tail were isolated and identified by sequencing.
GST pull-down assays
The recombinant fusion proteins (GST-rPTH1R C-tail) expressed in BL21-CodonPlus(DE3)-RP competent cells (Stratagene, La Jolla, CA) were purified in one step using a glutathione-Sepharose column and were detected by immunoblotting with G48. Clone 95A, which encodes the calpain small subunit, was subcloned into a bacterial expression vector, pET30 (Novagen, Madison, WI), that incorporates six histidines (6xhis) and an S-tag to the N terminus of expressed proteins. Recombinant proteins were expressed in Escherichia coli and purified using immobilized metal affinity chromatography (Qiagen, Valencia, CA). Three micrograms of purified GST alone and GST fused to the rPTH1R C-tail (GST C-tail) were coupled to a glutathione-Sepharose column, then incubated with the purified calpain small subunit (3 μg) in a buffer containing 25 mM HEPES (pH 7.4), 20% glycerol, 50 mM NaCl, and 1 mM dithiothreitol (interaction buffer) with and without 3 mM Ca2+ at 4 C for 4 h, and complexes were eluted by competition with 5 mM glutathione. Eluates were applied to SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane, followed by immunoblotting using S-protein-HRP.
Coimmunoprecipitation
HEK293 cells transiently expressing either wild-type (WT) rPTH1R or rPTH1R-W474A/W477A, were lysed in a buffer containing 25 mM HEPES (pH 7.4), 50 mM NaCl, 20% glycerol, and 0.5% Triton X-100 (lysis buffer) with protease inhibitors (Sigma-Aldrich Corp.). Cell lysates (500 μg total protein) were immunoprecipitated with G48 (1:100) for 4 h at 4 C. Immune complexes were pelleted with protein A/G plus agarose (Santa Cruz Biotechnology, Inc.), washed with the lysis buffer, and eluted with sodium dodecyl sulfate buffer. Eluates were applied to SDS-PAGE and transferred to a PVDF membrane, followed by immunoblotting using anticalpain small subunit monoclonal antibody.
Cleavage of rPTH1R in vitro and in intact cells
GST fused with the full-length C-tail of rPTH1R (aa 468–591) (GST-C tail) was expressed in competent cells and purified as described above. The immobilized GST-C tail fusion protein (3 μg/sample) was pretreated with either vehicle or ALLM (100 μg/ml) in the interaction buffer (0 C, 15 min) before incubation with and without 20 μg/ml m-calpain and 3 mM Ca2+ at 37 C for 0 and 60 min. The complex was eluted by competition with 5 mM glutathione and run on SDS-PAGE, and proteins were stained with Coomassie blue. rPTH1R-YFP-rho and Nt-rPTH1R-rho were expressed in HEK293 cells and solubilized using the lysis buffer and coupled to 1D4-Sepharose at 4 C for 30 min (33). Immobilized receptors purified from an equal amount of cell lysates per sample in each experiment were pretreated with either vehicle or calpain inhibitors in the interaction buffer (0 C, 15 min) before incubation with and without 20 μg/ml m-calpain and 3 mM Ca2+ at 37 C for 0 and 60 min. Receptors were then eluted with a buffer containing sodium dodecyl sulfate, loaded onto SDS-PAGE, and transferred to PVDF membranes, followed by immunoblot analysis using anti-GFP and 1D4 antibodies, respectively.
HEK 293 cells expressing Nt-rPTH1R-rho and Nt-rPTH1R-W474A/W477A-rho were lysed in Ca2+-free buffer, and the cell lysates (300 μg total protein/sample) were pretreated with either vehicle or calpain inhibitors at 0 C for 15 min before incubation with and without 3 mM Ca2+ at 37 C for 0 and 60 min. The reaction was stopped by the addition of 5 mM EGTA and protease inhibitors. Nt-rPTH1R-rho and Nt-rPTH1R-W474A/W477A-rho were then immunopurified using 1D4-Sepharose. In brief, the reaction mixture was immunopurified by coupling to 1D4-Sepharose at 4 C for 30 min. After extensive washing with PBS, Nt-rPTH1R-rho and Nt-rPTH1R-W474A/W477A-rho were eluted with a buffer containing sodium dodecyl sulfate, applied to SDS-PAGE, and transferred to PVDF membranes, followed by immunoblot analysis using 1D4.
HEK293 cells expressing Nt-rPTH1R-rho were treated with ionomycin (0, 5, and 10 μM) at 37 C for 0 and 60 min, then lysed with a buffer containing 3 mM EGTA and protease inhibitors. In some experiments, purified plasmid containing the Nt-rPTH1R-rho cDNA was transfected into 70–80% confluent HEK293 cells in two 15-cm plates using FuGene6, and the cells were harvested and replated in several 3-cm dishes the next day. After incubation for an additional 48 h, HEK293 cells expressing Nt-rPTH1R-rho were pretreated with either vehicle or ALLM at 37 C for 15 min before incubation with and without PTH (1 μM) at 37 C for 0 and 15 min, then lysed with a buffer containing 3 mM EGTA and protease inhibitors. Receptors were immunopurified from the cell lysates (300 μg total protein/sample), applied to SDS-PAGE, transferred to PVDF membranes, and immunoblotted using 1D4. HEK293 cells expressing rPTH1R-YFP and ROS17/2.8 cells were pretreated with cycloheximide (Sigma-Aldrich Corp.) and either vehicle or ALLM at 37 C for 15 min before incubation with and without PTH (1 μM) at 37 C for 5 and 15 min. Cells were then lysed; the lysates (30 μg/lane) were applied to SDS-PAGE and transferred to PVDF membranes, followed by immunoblotting using monoclonal antibodies to GFP and talin.
Adenoviral infection
293A cells were infected with pAd/CMV/rPTH1R after it was linearized with PacI. Viral particles were isolated by three freeze-thaw cycles and amplified by reinfection of 293A cells at a concentration of approximately 1 x 1012 colony-forming units/ml.
Quantification of rPTH1R expression
HEK293 and MC3T3-E1 cells were washed with 500 μl binding buffer (95% PBS/5% FBS), incubated first with G48 antiserum in 250 μl binding buffer (1:500) for 2 h, then incubated with the secondary antibody, rabbit antisheep IgG (1:2000; Kirkegaard & Perry Laboratories, Gaithersburg, MD) for 1 h, and finally incubated with [125I]antirabbit IgG (100,000 cpm/well) for 1 h. All incubations were performed at room temperature. After intensive washing, cells were lysed with 1 N NaOH, and radioactivity was counted with a Wallac 1470 -counter (Gaithersburg, MD).
Measurement of intracellular cAMP
Intracellular cAMP accumulation was measured as described previously (30, 33).
Measurement of calpain activity
MC3T3-E1 cells expressing empty vector (pcDNA3.1/Hygro) and human calpastatin were grown in six-well plates. Cells were washed with ice-cold PBS twice and lysed in Ca2+-free buffer. Cell lysates (30 μg/sample) were incubated with 100 μM calpain substrate, N-succinyl-Leu-Leu-Val-Tyr-7 amino-4-methylcoumarin, (Suc-LLVY-AMC, Bachem, King of Prussia, PA) in a buffer containing 10 mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, and 3 mM Ca2+ at 37 C for 15 min (34). Fluorescence was measured at 460 nm with excitation at 360 nm using a PTI Deltascan dual wavelength fluorometer (Photon Technologies, Inc., Lawrenceville, NJ). 7-Amino-4-methylcoumarine, purchased from Bachem, was used as a standard.
Statistical analysis
Data were calculated from two to five independent experiments and expressed as the mean ± SE of triplicate determinations. Statistical significance was determined by ANOVA with Fisher’s protected least significant difference (PLSD) test.
Results
Isolation of the calpain small subunit
To identify novel proteins that bind the intracellular C-terminal portion of the rPTH1R, the C-tail (aa 468–591) was used in the yeast two-hybrid system to screen a human kidney cDNA library. Six of 40 clones that were sequenced encoded portions of the calpain small subunit. One clone, 95A, lacks 21 N-terminal aa and includes the five EF-hand motifs that are essential for Ca2+ binding and heterodimerization with the catalytic domain of both μ- and m-calpains. A particularly strong and specific interaction was seen in the yeast reporter strain (Y189) when it was cotransformed with the C-tail of rPTH1R (aa 468–591) and clone 95A (data not shown). Studies of a series of deletion mutants of the C-tail of rPTH1R lacking N-terminal aa (N5, 473–591; N10, 478–591; N15, 483–591) or lacking C-terminal aa (C20, 468–571; C40, 468–551) showed that only N10 and N15 failed to bind clone 95A. Three double substitutions of this region of the rPTH1R (S473A/W474A, S475A/R476A, and W477A/T478A) failed to bind clone 95A, indicating that the SWSRW sequence (aa 473–477) of the rPTH1R is critical for its interaction with the calpain small subunit, at least in yeast (data not shown).
rPTH1R binds the calpain small subunit in vitro
The GST pull-down assay was used to investigate binding between the rPTH1R C-tail and the calpain small subunit. A GST full-length rPTH1R C-tail bound the purified calpain small subunit in a Ca2+-dependent manner (Fig. 1A). The rPTH1R mutant lacking five N-terminal aa of the C-tail (N5, 473–591) also bound the calpain small subunit; however, importantly, N10 (478–591) and N15 (483–591) did not (Fig. 1B). The intensity of bands corresponding to the calpain small subunit pulled down with GST full-length rPTH1R C-tail (468–591) was reduced compared with those pulled down with GST-N5 (473–591), because expression levels of GST-rPTH1R C-tail (468–591) were lower than those of GST-N5. Of five single alanine substitutions of the GST-C-tail fusion protein (S473A, W474A, S475A, R476A, and W477A), receptors with W474A, S475A, and W477A interacted poorly, if at all, with the calpain small subunit, although receptors with S473A and R476A interacted at least as strongly as the GST full-length C-tail of the rPTH1R (Fig. 1C). These results indicate that at least residues W474, S475, and W477 of the rPTH1R are critical for binding the calpain small subunit.
FIG. 1. A–C, Pull-down assays show direct and Ca2+-dependent interactions between the intracellular C-tail of the rPTH1R and the calpain small subunit. A, The interaction between the C-tail of the rPTH1R and the calpain small subunit is Ca2+ dependent. Lanes 1 and 3, GST alone; lanes 2 and 4, GST fused with full-length C-tail of the rPTH1R (aa 468–591). The Ca2+ concentration in the interaction buffer is: lanes 1 and 2, 0 mM; lanes 3 and 4, 1 mM. B, The binding of the calpain small subunit to the N terminus of the rPTH1R C-tail requires some or all of aa 473–477. Lane 1, GST alone; lane 2, GST full-length C-tail; lanes 3–5, GST C-tail with truncations of five (473–591; lane 3), 10 (478–591; lane 4), and 15 (483–591; lane 5) aa from the N-terminal region of the rPTH1R C-tail. C, The interaction between the C-tail of rPTH1R and the calpain small subunit does not occur or occurs to a lesser extent when tryptophans (W) 474 and 477 and serine (S) 475 are replaced with alanine (A). Lane 1, GST alone; lane 2, GST full-length C-tail of the rPTH1R; lanes 3–7, mutant C-tails containing single alanine substitutions S473A (lane 3), W474A (lane 4), S475A (lane 5), R476A (lane 6), and W477A (lane 7). The arrow indicates the calpain small subunit pulled down with the GST-rPTH1R-C-tail fusion protein. After immunoblotting, the membranes were stained with Coomassie blue in A–C. D, The calpain small subunit copurifies with rPTH1R in HEK293 cells. Extracts from HEK293 cells expressing WT-rPTH1R or rPTH1R with W474A/W477A substitutions were precipitated with G48. Immunoprecipitates were blotted onto a PVDF membrane and probed with the calpain small subunit antibody. The arrow indicates the endogenous calpain small subunit copurified with the rPTH1R.
rPTH1R and the calpain small subunit copurify in mammalian cells
The endogenous calpain small subunit and WT rPTH1R copurified when the latter was expressed in HEK293 cells, but not at all when these cells expressed the rPTH1R-W474A/W477A mutant (Fig. 1D). The expressions of WT and W474A/W477A mutant rPTH1Rs, as determined by binding of G48 and immunoblotting using G48, were similar (data not shown). Thus, the rPTH1R and the calpain small subunit directly interact in mammalian cells.
Cleavage of the rPTH1R by calpain in vitro and in intact cells is Ca2+ dependent
The GST-C tail was cleaved when incubated with m-calpain and Ca2+ to yield a fragment slightly larger than GST alone (Fig. 2A). Full-length rPTH1R-YFP was modified by addition of the rhodopsin epitope (rho) at the C terminus (rPTH1R-YFP-rho). The use of rPTH1R-YFP-rho and rPTH1R-YFP has two advantages; they are as well expressed as rPTH1R and are efficiently stimulated by PTH, presumably because the N-terminal extracellular ligand-binding domain is intact (data not shown), and insertion of YFP at a site relatively near the C terminus makes it likely that a fragment of the receptor that results from cleavage by calpain would be sufficiently large to be detected by immunoblotting with anti-GFP antibody after SDS-PAGE. Addition of the rhodopsin epitope enables detection of the receptor by the monoclonal antibody, 1D4, and immunopurification using 1D4-Sepharose. Figure 2B shows that rPTH1R-YFP-rho was cleaved when it was incubated with m-calpain and Ca2+ to yield an approximately 38-kDa fragment containing YFP (indicated by the arrowhead); the intensity of the intact rPTH1R-YFP-rho band of approximately 110 kDa (indicated by the star) was correspondingly reduced. Importantly, the intensity of the approximately 38-kDa band was markedly diminished and that of an approximately 110-kDa band was restored when immobilized rPTH1R-YFP-rho was preincubated with ALLM and calpastatin. (The light chain of IgG runs as a band that is slightly larger than the PTH1R fragment and is heavily stained.) Calpastatin is a potent endogenous calpain-specific inhibitor of μ- and m-calpains (35, 36), whereas ALLM and antipain are less specific; ALLM is cell permeable and also inhibits cathepsin B and L (37, 38), and antipain inhibits serine proteases, papain, and some cysteine proteases in addition to μ- and m-calpains (39).
FIG. 2. A, Calpain hydrolyzes GST fused with full-length rPTH1R C-tail (aa 468–591; GST-C-tail) in vitro in the presence of Ca2+. GST-C-tail was expressed in the competent cells and purified using a glutathione-Sepharose column. Immobilized GST-C-tail was pretreated with either vehicle or ALLM in the interaction buffer at 0 C for 15 min before incubation with and without 20 μg/ml m-calpain and 3 mM Ca2+ at 37 C for 0 and 60 min, then eluted by competition with 5 mM glutathione and run on SDS-PAGE, followed by Coomassie blue staining. B, Calpain hydrolysis of rPTH1R-YFP-rho in vitro after expression and extraction from HEK293 cells depends on the presence of m-calpain and Ca2+. rPTH1R-YFP-rho was expressed in HEK293 cells, solubilized, and immunopurified using 1D4-Sepharose. rPTH1R-YFP-rho immobilized on 1D4-Sepharose was pretreated with either vehicle or calpain inhibitors (ALLM, 100 μg/ml; calpastatin, 10 μM) at 0 C for 15 min before incubation with and without 20 μg/ml m-calpain and 3 mM Ca2+ at 37 C for 0 or 60 min. Receptors were eluted with a buffer containing sodium dodecyl sulfate, and the eluates were electrophoresed by SDS-PAGE, followed by immunoblot analysis using anti-GFP monoclonal antibody. The arrowhead and star indicate a fragment of rPTH1R-YFP-rho and a band of diminished intensity corresponding to full-length rPTH1R-YFP-rho, respectively. N.S., Nonspecific band.
Next, Nt-rPTH1R and Nt-rPTH1R-W474A/W477A, which are well expressed, lack most of the extracellular N-terminal region (aa 26–181) including the four potential sites for glycosylation, and are activated by PTH and its analogs (40, 41), were modified by the addition of a rhodopsin epitope at their C termini (Nt-rPTH1R-rho and Nt-rPTH1R-W474A/W477A-rho, respectively). Deletion of the glycosylation sites facilitated immunoblot analysis, because the receptor migrated as a tight approximately 49-kDa band on SDS-PAGE. The intensity of the Nt-rPTH1R-rho bands was markedly reduced by incubation with m-calpain in the presence, but not in the absence, of 3 mM Ca2+; this effect was blocked by pretreating samples with calpain inhibitors, ALLM, calpastatin, and antipain (Fig. 3A). HEK293 cells transiently expressing Nt-rPTH1R-rho and Nt-rPTH1R-W474A/W477A-rho were lysed with Ca2+-free buffer; the lysates were incubated with and without 3 mM Ca2+ at 37 C for 0 and 60 min, immunopurified with 1D4-Sepharose, and analyzed by SDS-PAGE and immunoblotting with 1D4. The intensity of the Nt-rPTH1R-rho band was diminished only when the incubation buffer contained 3 mM Ca2+; this effect was reversed by ALLM and calpastatin (Fig. 3B). Importantly, the intensity of the Nt-rPTH1R-W474A/W477A-rho band remained unchanged even when the incubation buffer contained Ca2+ (Fig. 3C). In addition, treatment of intact HEK293 cells expressing Nt-rPTH1R-rho with ionomycin resulted in cleavage of Nt-rPTH1R-rho in a dose-dependent manner (Fig. 3D). Thus, calpain cleaves full-length rPTH1R in vitro and in intact cells in a Ca2+-dependent manner.
FIG. 3. Calpain hydrolyzes Nt-rPTH1R-rho in vitro and in intact cells. A, Calpain hydrolysis of the Nt-rPTH1R-rho in vitro after expression and extraction from HEK293 cells depends on the presence of m-calpain and Ca2+. Nt-rPTH1R-rho was expressed in HEK293 cells, solubilized, and immunopurified using 1D4-Sepharose. Immobilized receptors were then pretreated with either vehicle or calpain inhibitors (ALLM, 100 μg/ml; calpastatin, 10 μM; antipain, 50 μg/ml) in the interaction buffer at 0 C for 15 min before incubation with and without 20 μg/ml m-calpain and 3 mM Ca2+ at 37 C for 0 and 60 min as indicated. Receptors were eluted with a buffer containing SDS, and the eluates were electrophoresed by SDS-PAGE, followed by immunoblot analysis using 1D4. B and C, Calpain hydrolyzes Nt-rPTH1R-rho (B), but not Nt-rPTH1R-W474A/W477A-rho (C), in a Ca2+-dependent manner in cell lysates. HEK293 cells expressing endogenous μ- and m-calpains and transfected with Nt-rPTH1R-rho or Nt-rPTH1R-W474A/W477A-rho were lysed in Ca2+-free buffer. Cell lysates were then pretreated with either vehicle or calpain inhibitors (ALLM, 100 μg/ml; calpastatin, 10 μM) at 0 C for 15 min before incubation with and without 3 mM Ca2+ at 37 C for 0 and 60 min. The reaction was stopped by the addition of 5 mM EGTA and protease inhibitors. Nt-rPTH1R-rho and Nt-rPTH1R-W474A/W47A-rho were then immunopurified using 1D4-Sepharose and eluted with a buffer containing SDS. The eluates were electrophoresed by SDS-PAGE, followed by immunoblot analysis using 1D4. D, Nt-rPTH1R-rho is cleaved by treatment with ionomycin in a dose-dependent manner. HEK293 cells expressing Nt-rPTH1R-rho were treated with ionomycin (0, 5, and 10 μM) at 37 C for 0 or 60 min, then lysed with a buffer containing 3 mM EGTA and protease inhibitors. The samples were immunopurified and analyzed by SDS-PAGE and immunoblotting using 1D4. The closed arrows in A, B, and D indicate immunopurified Nt-rPTH1R-rho, and the open arrow in C indicates Nt-rPTH1R-W474A/W477A-rho.
PTH increases cleavage of rPTH1R and talin in intact cells
Next, the effect of PTH on cleavage of Nt-rPTH1R-rho and rPTH1R-YFP when expressed in HEK293 cells was assessed. HEK293 cells transiently expressing Nt-rPTH1R-rho cDNA in 15-cm plates were harvested and replated in several 3-cm dishes. When reaching confluence, the cells were pretreated with either vehicle or ALLM at 37 C for 15 min before incubation with and without PTH (1 μM) at 37 C for 0 and 15 min, then lysed in a buffer containing 3 mM EGTA and protease inhibitors. Cell lysates containing the same amount of total protein from each group were immunopurified and analyzed by SDS-PAGE and immunoblotting using 1D4. The intensity of the Nt-rPTH1R-rho band was significantly reduced by 20% when cells expressing Nt-rPTH1R-rho were incubated with PTH for 15 min compared with that when cells were incubated without PTH, and the effect was reversed by pretreatment of the cells with ALLM (Fig. 4A). HEK293 cells expressing rPTH1R-YFP were incubated with and without PTH for 5 and 15 min, then lysed and analyzed by SDS-PAGE and immunoblotting using anti-GFP monoclonal antibody. When cells were incubated with PTH for 15 min, the intensity of a band corresponding to intact rPTH1R-YFP (open arrow) was reduced to 70% compared with that of the receptor when the cells were incubated without PTH. The approximately 38-kDa receptor fragment containing YFP (arrow) was 1.4- and 1.7-fold more intense after cells were incubated with PTH for 5 and 15 min, respectively, than when cells were incubated in the absence of PTH for 5 min (Fig. 4B).
FIG. 4. PTH-mediated cleavage of Nt-rPTH1R-rho, rPTH1R-YFP, and talin in intact cells. A, PTH mediates cleavage of Nt-rPTH1R-rho by calpain. HEK293 cells expressing Nt-rPTH1R-rho were pretreated with either vehicle or ALLM (100 μg/ml) at 37 C for 15 min before incubation with and without PTH (1 μM) at 37 C for 0 and 15 min as indicated. Cells were lysed with a buffer containing 3 mM EGTA and protease inhibitors, immunopurified on 1D4-Sepharose, and analyzed by SDS-PAGE and immunoblot analysis using 1D4. The density of the bands corresponding to Nt-rPTH1R-rho was measured using an Imager 2200 (Alpha Innotech Co., San Leando, CA). The data are presented as a percentage of the control value obtained by incubating cells in the absence of PTH and ALLM for 15 min. B, PTH mediates cleavage of rPTH1R-YFP. HEK293 cells expressing rPTH1R-YFP were incubated with and without PTH at 37 C for 5 and 15 min. Cell lysates were analyzed by SDS-PAGE and immunoblotting using anti-GFP monoclonal antibody (upper panel). The open and closed arrows indicate intact rPTH1R-YFP and a fragment of rPTH1R-YFP, respectively. The density of the bands corresponding to the rPTH1R-YFP fragment was measured, and the data are presented as a percentage of the control value obtained by incubating cells in the absence of PTH for 5 min (lower panel). C and D, PTH enhances calpain cleavage of endogenous talin. C, HEK293 cells expressing rPTH1R-YFP were pretreated with either vehicle or ALLM (100 μg/ml) at 37 C for 15 min before incubation with and without PTH at 37 C for 5 and 15 min. Cell lysates were analyzed by SDS-PAGE and immunoblotting using antitalin monoclonal antibody. The arrowhead indicates a fragment of talin (upper panel). The density of the bands corresponding to an approximately 190-kDa talin fragment was measured, and the data are presented as a percentage of the control value obtained by incubating cells in the absence of PTH and ALLM for 5 min (lower left) and by incubating cells with only PTH (lower right). D, Confluent ROS17/2.8 cells were pretreated with either vehicle or ALLM (50 μg/ml) at 37 C for 15 min before incubation with and without PTH at 37 C for 5 min. The arrowhead indicates a fragment of talin. The data in A–C are expressed as the mean ± SE of four replicate samples. a, P < 0.05; b, P < 0.005. N.D., No difference between the two groups.
To support the idea that PTH treatment increases calpain activity in HEK293 cells expressing rPTH1R-YFP, the membranes were probed with antitalin monoclonal antibody. The approximately 190-kDa talin fragment was 1.2- and 1.4-fold more intense when cells expressing rPTH1R-YFP were incubated with PTH than when cells were incubated in the absence of PTH for 5 and 15 min, respectively (Fig. 4C, left), and the effect was blocked 60% by pretreatment of the cells with ALLM (Fig. 4C, right). The PTH-mediated cleavage of talin by calpain was also examined in confluent ROS17/2.8 cells, which endogenously express PTH1R. Confluent ROS17/2.8 cells were treated with and without PTH for 5 and 15 min, then lysed and analyzed by SDS-PAGE and immunoblotting using antitalin monoclonal antibody. The approximately 190-kDa talin fragment was markedly increased when ROS17/2.8 cells were incubated in the presence of PTH for 5 and 15 min, and the effect was blocked by pretreatment of the cells with ALLM (Fig. 4D). The result after 15-min treatment with PTH was similar to that shown in Fig. 4D (data not shown).
Calpain activity modulates PTH-mediated cAMP accumulation
PTH-mediated intracellular cAMP accumulation was assessed in Capn4+/+ and Capn4–/– embryonic fibroblasts infected with adenoviruses carrying WT-rPTH1R and expressing indistinguishable levels of receptor, as determined by binding of G48 (Fig. 5A). PTH-stimulated cAMP accumulation was significantly reduced in Capn4–/– cells expressing WT-rPTH1R compared with Capn4+/+ cells expressing WT-rPTH1R (Fig. 5B).
FIG. 5. A and B, The absence of Capn4 significantly reduces PTH-mediated cAMP accumulation in embryonic fibroblasts infected with adenoviruses carrying rPTH1R. A, Cell surface expression of rPTH1R was determined by binding of G48 to Capn4+/+ and Capn4–/– embryonic fibroblasts. B, PTH-mediated intracellular cAMP accumulation in Capn4+/+ and Capn4–/– embryonic fibroblasts. EV, Empty vector. a, P < 0.05. C and D, The inhibition of calpain activity reduces intracellular cAMP accumulation in MC3T3-E1 cells. C. Calpain activity in MC3T3-E1 cells stably expressing empty vector or human calpastatin. Calpain activity was measured as described in Materials and Methods. D, PTH-mediated intracellular accumulation of cAMP in MC3T3-E1 cells was assessed 14 d after plating, when the cells were confluent. a, P < 0.05; b, P < 0.005 [compared with both stable cell lines expressing empty vector (MC3T3EV-7 and -9)]. The data are expressed as the mean ± SE of triplicate samples. The experiments were performed three to five times with similar results.
PTH-stimulated cAMP accumulation was examined in MC3T3-E1 osteoblastic cells stably expressing human calpastatin. Two independent cell lines, MC3T3hCalp-1 and -9, with levels of calpain activity that were 73% and 64%, respectively, those in MC3T3-E1 cell lines expressing empty vector (MC3T3EV-7 and -9), were used (Fig. 5C). PTH-stimulated cAMP accumulation was significantly reduced to less than 20% of that in the controls in both MC3T3hCalp-1 and -9 (Fig. 5D) compared with that in MC3T3EVs, which expressed similar levels of rPTH1R. Thus, calpain plays a crucial role(s) in PTH-mediated cAMP accumulation in both embryonic fibroblasts expressing rPTH1R and MC3T3-E1 cells.
Discussion
We demonstrated that 1) PTH1R binds the calpain small subunit through an N-terminal portion of the intracellular C-tail that requires, but may not be limited to, aa W474, S475, and W477 of the rPTH1R; 2) PTH1R and the calpain small subunit copurify in intact cells; 3) cleavage of PTH1R and talin by calpain is Ca2+ dependent, as expected, but also is increased by PTH; and 4) PTH-mediated cAMP accumulation is markedly reduced in the absence of calpain and when calpain activity is blocked by calpastatin. The last finding may provide a mechanistic explanation for our previous observation that PTH1R with a deletion of 111 aa of the intracellular C-tail has increased PTH-mediated cAMP accumulation (42).
The site of interaction between rPTH1R and the calpain small subunit, the W-R[W] sequence, located in the N-terminal portion of the C-tail, is highly conserved among PTH1Rs in different species and among other type II GPCR, such as receptors for pituitary adenylate cyclase-activating polypeptide, vasoactive intestinal peptide, secretin, and calcitonin. Interestingly, the crystal structure of rhodopsin reveals that the N-terminal portion of the C-tail forms an eighth -helix (43, 44). If this also proves to be true for rPTH1R, the substitutions, W474A, S475A, and W477A and the double mutant W474A/W477A of rPTH1R may well interfere with formation of this eighth helical structure at the N terminus of the rPTH1R C-tail, which, in turn, may obviate or lessen the interaction between PTH1R and the calpain small subunit.
Calpains are cysteine proteases that participate in a variety of cellular functions by cleaving substrate proteins (20). Protein targets for calpains typically have a PEST sequence, so named because it is rich in proline (P), glutamic acid (E), serine (S), and threonine (T), flanked by clusters of positively charged aa (45, 46, 47). Interspecies comparison of the C-tail of PTH1Rs reveals one highly conserved PEST domain in the rat, mouse, and dog receptors, and two in the human, pig, and rabbit receptors (http://emb1.bcc.univie.ac.at/embnet/tools/bio/PESTfind). A PEST domain at the C terminus of the rPTH1R exists between aa 538–557 (KPGAPATETETLPVTMAVPK; PEST score = –0.08; scores above –5 are significant). We showed that the rPTH1R C-tail is a calpain target and that cleavage occurs only in the presence of Ca2+ and is increased by treatment of the cells with PTH. Cleavage of Nt-rPTH1R-rho could be appreciated only as diminished intensity of the intact receptor band, because the size of the predicted approximately 4- to 6-kDa fragment was too small to be resolved on SDS-PAGE. However, when YFP was introduced close to the C terminus of the rPTH1R, an approximately 38-kDa band that included YFP was apparent in vitro and was markedly increased when the cells were treated with PTH.
PTH-induced osteoblastic retraction is thought to be due to calpain-dependent proteolytic modification of the cytoskeletal organization (24, 25, 26, 27). Osteoblastic retraction is critical for providing osteoclasts direct access to mineralized bone surface that is known to contain and release chemoattractants (48, 49). The finding that cleavage of talin, a key cytoskeletal protein, is disrupted in Capn4-null embryonic fibroblasts and then restored when Capn4 was reintroduced (28) led us to examine the effect of PTH on cleavage of talin in HEK293 cells expressing PTH1R-YFP and in ROS17/2.8 cells. Our finding that cleavage of talin is increased by treatment of cells expressing rPTH1R with PTH and is reduced by pretreatment of the cells with a calpain inhibitor implies that PTH-mediated cleavage of talin may play a crucial role in PTH-mediated cell retraction in osteoblasts.
The mechanism by which activation of PTH1R leads to increased calpain activity is unknown, but may reflect PTH-induced modifications of the C-tail, such as phosphorylation, and/or PTH-induced allosteric changes in PTH1R. Perhaps, PTH induces a highly localized increase in intracellular calcium that, in turn, may facilitate calpain binding to PTH1R and/or calpain activation.
Thus, binding between rPTH1R and the calpain small subunit represents the second example of a protein, other than G proteins and their direct regulators, that interacts with PTH1R. In both instances, signaling by PTH is profoundly altered. Stimulation of the NHERF-PTH1R complex markedly lowers PTH-stimulated cAMP accumulation by favoring the activation of Gi/o (11), whereas PTH stimulation of the calpain small subunit-PTH1R complex leads to C-terminal shortening of PTH1R, a form of the receptor that more readily activates adenylyl cyclase, and is also associated with cleavage of talin, a critical regulator of the cytoskeleton.
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Address all correspondence and requests for reprints to: Dr. Gino V. Segre, Endocrine Unit, Wellman 501, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114. E-mail: segre@helix.mgh.harvard.edu.
Abstract
We show calcium-dependent, direct binding between the N-terminal portion of the PTH/PTHrP receptor (PTH1R) C-terminal intracellular tail and the calpain small subunit. Binding requires, but may not be limited to, amino acids W474, S475, and W477. The wild-type, full-length rat (r) PTH1R, but not rPTH1R with W474A/W477A substitutions, copurifies with the endogenous calpain small subunit in HEK293 cells. Calpain hydrolyzes Nt-rPTH1R, a receptor with a 156-amino acid N-terminal deletion, in a calcium-dependent manner in vitro and in intact cells. Most importantly, PTH stimulation increases the cleavage of Nt-rPTH1R and rPTH1R-yellow fluorescent protein in HEK293 cells, and of talin in HEK293 cells expressing rPTH1R-yellow fluorescent protein and in ROS17/2.8 osteoblast-like cells that express rPTH1R endogenously. The absence of calpain in Capn4-null embryonic fibroblasts and the lowered calpain activity in MC3T3-E1 osteoblastic cells due to stable expression of the calpain inhibitor, calpastatin, reduce PTH-stimulated cAMP accumulation. The calpain small subunit is the second protein, in addition to the sodium-hydrogen exchanger regulatory factor, and the first enzyme that binds the PTH1R; PTH1R bound to both of these proteins results in altered PTH signaling.
Introduction
THE RECEPTOR FOR PTH and PTHrP (PTH1R) belongs to the class II G protein-coupled receptor (GPCR) superfamily (1). Stimulation of the PTH1R by PTH and PTHrP increases the intracellular levels of second messenger molecules, including cAMP, inositol triphosphate, diacylglycerol, and calcium (Ca2+) (2, 3). PTH functions primarily to maintain calcium homeostasis through direct actions in the kidneys, where it increases the resorption of calcium and inhibits the resorption of phosphate (4, 5, 6). It also acts directly on bone formation and indirectly on bone resorption through its actions on cells of the osteoblastic lineage (4, 7). PTHrP normally functions primarily, if not exclusively, as an autocrine/paracrine regulator of cellular growth and differentiation in many organ systems, especially the endochondral skeleton (8, 9). Its aberrant expression and secretion by malignancies are responsible for most cases of tumor-associated hypercalcemia (9, 10).
Our group recently demonstrated that direct binding of the scaffolding proteins, sodium-hydrogen exchanger regulatory factor 1 (NHERF1) and NHERF2, to the intracellular C-terminal portion (C-tail) of the PTH1R markedly affects receptor signaling. NHERF2 assembles phospholipase C? (PLC?) and the PTH1R through PDZ (PSD 95, discs large protein, ZO1) 1 and 2 domains, respectively. PTH stimulation of the NHERF2-PTH1R complex remarkably activates inhibitory G proteins, Gi/o, resulting in dissociation of ?-subunits from -subunits. The ?-subunits then activate PLC?, and the -subunit blocks activation of adenylyl cyclase in a cell- and membrane-specific manner (11). NHERF1 assembles a signaling complex in the apical domain of polarized opossum kidney cells that contains PTH1R, PLC?, and actin cytoskeleton (12), and it also assembles a complex containing the type IIa sodium-phosphate cotransporter (13, 14, 15). These studies extend the paradigm, first described with arrestin (16, 17, 18, 19), that molecules other than G proteins bind directly to GPCRs and markedly alter receptor functions in discrete compartments adjacent to the plasma membrane.
The yeast two-hybrid screen that revealed NHERF-PTH1R binding also showed binding between PTH1R and the calpain small subunit. Calpains are a family of Ca2+-dependent intracellular cysteine proteases that include the ubiquitously expressed μ- and m-calpains (20). Both μ- and m-calpains form heterodimers, consisting of a large catalytic subunit (80 kDa) encoded by the genes Capn1 and Capn2, respectively, and a common small subunit (28 kDa) encoded by the gene Capn4. Calpains are inactive in the absence of Ca2+, but upon Ca2+ binding, they undergo a conformational change, which is essential for activation of the protease (21, 22, 23).
Several lines of evidence suggest that PTH increases calpain activity (24, 25), and that calpains play a crucial role in PTH-mediated cellular retraction in osteoblastic cell lines (24, 25, 26, 27). The molecular mechanism underlying PTH/PTHrP-mediated regulation of calpain, however, has not been identified. In this study we demonstrate that the calpain small subunit binds to a highly conserved region in the N-terminal portion of the PTH1R’s intracellular tail in vitro and in intact cells; that calpain hydrolyzes both the C-tail of the rat PTH1R (rPTH1R) and talin, an actin cytoskeletal protein in a Ca2+- and ligand-dependent manner; and that the absence and reduction of calpain activity critically affect PTH1R signaling properties.
Materials and Methods
Materials
Recombinant rat m-calpain and calpain inhibitors, N-acetyl-Leu-Leu-Met-CHO (ALLM), antipain, and calpastatin were purchased from Calbiochem (La Jolla, CA). FuGene6 was purchased from Roche (Indianapolis, IN). Rat [Nle8, 21,Tyr34]PTH-(1–34)-NH2 (PTH) was prepared by the MGH Biopolymer Synthesis Facility (Boston, MA). cAMP RIA kits and [125I]antirabbit IgG were obtained from PerkinElmer (Boston, MA).
Cell culture and transfection
Capn4-null embryonic fibroblasts (28), HEK293, and ROS17/2.8 cells were cultured in DMEM (Cellgro, Mediatech, Inc., Herndon, VA), supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT) and 1% antibiotic/antimycotic (Invitrogen Life Technologies, Inc., Grand Island, NY) at 37 C in a humidified atmosphere containing 95% air and 5% CO2. MC3T3-E1 cells were cultured in -MEM (Invitrogen Life Technologies, Inc.), 10% FBS, and 1% antibiotic/antimycotic. Various rPTH1R cDNAs were transiently transfected into HEK293 cells using FuGene6. Full-length human calpastatin cDNA (gift from Dr. M. Maki, Nagoya University, Nagoya, Japan) (29) was subcloned into the pcDNA3.1/HYGRO vector (Invitrogen Life Technologies, Inc.) and transfected into MC3T3-E1 cells using FuGene6, and cells expressing human calpastatin were selected with 300 μg/ml hygromycin B (Calbiochem) (30). Two clonal cell lines expressing the highest calpastatin levels were further examined.
Antibodies
The monoclonal antibody, 1D4, which recognizes the rhodopsin epitope, TETSQVAPA (31), was purchased from the National Cell Culture Center (Minneapolis, MN). G48, a polyclonal antiserum raised in a sheep, contains antibodies that recognize epitopes within sequences of the rPTH1R, YPESKENKDVPTGSRRRGRPC, FCNGEVQAEIRKSWSRWTLAL, and SGLDEEASGSARPPPLLQEGWETVM. The first sequence is located in the N-terminal ectodomain, and the last two sequences are located in the intracellular C-tail of the receptor. Monoclonal antibodies to the calpain small subunit and human calpastatin were purchased from Chemicon International (Temecula, CA). S-Protein-horseradish peroxidase (HRP) and monoclonal antibodies to green fluorescent protein (GFP; B34) and talin (8D4) were purchased from Novagen (Madison, WI), Convance (Berkeley, CA), and Sigma-Aldrich Corp. (St. Louis, MO), respectively. Species-specific secondary antibodies conjugated with HRP were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
cDNA constructs
N-Terminal glutathione-S-transferase (GST) fusion proteins of the rPTH1R C-tail were generated by PCR using Taq polymerase (Amersham Biosciences, Piscataway, NJ). PCR fragments corresponding to the full-length C-tail [amino acids (aa) 468–591], N5 (aa 473–591), N10 (aa 478–591), and N15 (aa 483–591) were ligated into pGEX-5X1 (Amersham Biosciences). rPTH1R C-tail point mutations (S473A, W474A, S475A, R476A, and W477A) were generated by incorporation of the appropriate mutation within the forward PCR primers and ligated into pGEX-5X1. The rPTH1R lacking most of the extracellular N-terminal region (aa 26–181; Nt-rPTH1R) was a gift from Dr. T. Gardella of this laboratory (32). The rPTH1R carrying a double tryptophan mutation, rPTH1R-W474A/W477A, was generated by PCR, and the DNA fragment was subcloned into pcDNA3.1 (Invitrogen Life Technologies, Inc.; Nt-rPTH1R-W474A/W477A). The 27-mer encoding the rhodopsin epitope (rho) recognized by 1D4 was inserted in-frame immediately upstream of the stop codon at the C terminus of the Nt-rPTH1R and Nt-rPTH1R-W474A/W477A sequences (Nt-rPTH1R-rho and Nt-rPTH1R-W474A/W477A-rho, respectively); yellow fluorescent protein (YFP) was inserted between aa 579 and 580 of the rPTH1R (rPTH1R-YFP) (12), and the rhodopsin epitope was inserted at the C terminus of rPTH1R-YFP (rPTH1R-YFP-rho). All were ligated into pcDNA3.1 and expressed in HEK293 cells.
Full-length rPTH1R was cloned into a modified pENTR vector (Invitrogen Life Technologies, Inc.) using NheI and XbaI sites. The pENTR-rPTH1R was packaged into an adenovirus using the ViraPower Adenoviral Expression System (Invitrogen Life Technologies, Inc.), and rPTH1R cDNA was introduced into the adenoviral genome via recombination between pENTR-PTH1R and the pAd/CMV/V5-DEST vector using LR Clonase (Invitrogen Life Technologies, Inc.), following the manufacturer’s guidelines. The accuracy of the DNA encoding the GST fusion proteins and the various rPTH1Rs was confirmed by sequencing (Tufts University, Sequencing Core Facility, Boston, MA).
Yeast two-hybrid system
The cytoplasmic C-tail of the rPTH1R was screened for protein interactions using the Matchmaker yeast two-hybrid system (BD Clontech, Palo Alto, CA). Briefly, the C-tail of the rPTH1R (aa 468–591) was cloned in-frame with the galactosidase-4 (GAL4) DNA-binding domain in the pAS2.1 vector using Pfu polymerase (Promega Corp., Madison, WI). The GAL4-C-tail fusion was screened against a Matchmaker cDNA library fused to the GAL4 activation domain from human kidney (BD Clontech) in the CG-1945 yeast strain. His+ clones were isolated on selective medium and reintroduced into the Y190 yeast strain in the absence or presence of the rPTH1R C-tail. Interactions were assessed using the lacZ reporter gene and the Galacton-star chemiluminescent ?-galactosidase assay (BD Clontech). Clones expressing the highest levels of ?-GAL activity in the presence of the rPTH1R C-tail were isolated and identified by sequencing.
GST pull-down assays
The recombinant fusion proteins (GST-rPTH1R C-tail) expressed in BL21-CodonPlus(DE3)-RP competent cells (Stratagene, La Jolla, CA) were purified in one step using a glutathione-Sepharose column and were detected by immunoblotting with G48. Clone 95A, which encodes the calpain small subunit, was subcloned into a bacterial expression vector, pET30 (Novagen, Madison, WI), that incorporates six histidines (6xhis) and an S-tag to the N terminus of expressed proteins. Recombinant proteins were expressed in Escherichia coli and purified using immobilized metal affinity chromatography (Qiagen, Valencia, CA). Three micrograms of purified GST alone and GST fused to the rPTH1R C-tail (GST C-tail) were coupled to a glutathione-Sepharose column, then incubated with the purified calpain small subunit (3 μg) in a buffer containing 25 mM HEPES (pH 7.4), 20% glycerol, 50 mM NaCl, and 1 mM dithiothreitol (interaction buffer) with and without 3 mM Ca2+ at 4 C for 4 h, and complexes were eluted by competition with 5 mM glutathione. Eluates were applied to SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane, followed by immunoblotting using S-protein-HRP.
Coimmunoprecipitation
HEK293 cells transiently expressing either wild-type (WT) rPTH1R or rPTH1R-W474A/W477A, were lysed in a buffer containing 25 mM HEPES (pH 7.4), 50 mM NaCl, 20% glycerol, and 0.5% Triton X-100 (lysis buffer) with protease inhibitors (Sigma-Aldrich Corp.). Cell lysates (500 μg total protein) were immunoprecipitated with G48 (1:100) for 4 h at 4 C. Immune complexes were pelleted with protein A/G plus agarose (Santa Cruz Biotechnology, Inc.), washed with the lysis buffer, and eluted with sodium dodecyl sulfate buffer. Eluates were applied to SDS-PAGE and transferred to a PVDF membrane, followed by immunoblotting using anticalpain small subunit monoclonal antibody.
Cleavage of rPTH1R in vitro and in intact cells
GST fused with the full-length C-tail of rPTH1R (aa 468–591) (GST-C tail) was expressed in competent cells and purified as described above. The immobilized GST-C tail fusion protein (3 μg/sample) was pretreated with either vehicle or ALLM (100 μg/ml) in the interaction buffer (0 C, 15 min) before incubation with and without 20 μg/ml m-calpain and 3 mM Ca2+ at 37 C for 0 and 60 min. The complex was eluted by competition with 5 mM glutathione and run on SDS-PAGE, and proteins were stained with Coomassie blue. rPTH1R-YFP-rho and Nt-rPTH1R-rho were expressed in HEK293 cells and solubilized using the lysis buffer and coupled to 1D4-Sepharose at 4 C for 30 min (33). Immobilized receptors purified from an equal amount of cell lysates per sample in each experiment were pretreated with either vehicle or calpain inhibitors in the interaction buffer (0 C, 15 min) before incubation with and without 20 μg/ml m-calpain and 3 mM Ca2+ at 37 C for 0 and 60 min. Receptors were then eluted with a buffer containing sodium dodecyl sulfate, loaded onto SDS-PAGE, and transferred to PVDF membranes, followed by immunoblot analysis using anti-GFP and 1D4 antibodies, respectively.
HEK 293 cells expressing Nt-rPTH1R-rho and Nt-rPTH1R-W474A/W477A-rho were lysed in Ca2+-free buffer, and the cell lysates (300 μg total protein/sample) were pretreated with either vehicle or calpain inhibitors at 0 C for 15 min before incubation with and without 3 mM Ca2+ at 37 C for 0 and 60 min. The reaction was stopped by the addition of 5 mM EGTA and protease inhibitors. Nt-rPTH1R-rho and Nt-rPTH1R-W474A/W477A-rho were then immunopurified using 1D4-Sepharose. In brief, the reaction mixture was immunopurified by coupling to 1D4-Sepharose at 4 C for 30 min. After extensive washing with PBS, Nt-rPTH1R-rho and Nt-rPTH1R-W474A/W477A-rho were eluted with a buffer containing sodium dodecyl sulfate, applied to SDS-PAGE, and transferred to PVDF membranes, followed by immunoblot analysis using 1D4.
HEK293 cells expressing Nt-rPTH1R-rho were treated with ionomycin (0, 5, and 10 μM) at 37 C for 0 and 60 min, then lysed with a buffer containing 3 mM EGTA and protease inhibitors. In some experiments, purified plasmid containing the Nt-rPTH1R-rho cDNA was transfected into 70–80% confluent HEK293 cells in two 15-cm plates using FuGene6, and the cells were harvested and replated in several 3-cm dishes the next day. After incubation for an additional 48 h, HEK293 cells expressing Nt-rPTH1R-rho were pretreated with either vehicle or ALLM at 37 C for 15 min before incubation with and without PTH (1 μM) at 37 C for 0 and 15 min, then lysed with a buffer containing 3 mM EGTA and protease inhibitors. Receptors were immunopurified from the cell lysates (300 μg total protein/sample), applied to SDS-PAGE, transferred to PVDF membranes, and immunoblotted using 1D4. HEK293 cells expressing rPTH1R-YFP and ROS17/2.8 cells were pretreated with cycloheximide (Sigma-Aldrich Corp.) and either vehicle or ALLM at 37 C for 15 min before incubation with and without PTH (1 μM) at 37 C for 5 and 15 min. Cells were then lysed; the lysates (30 μg/lane) were applied to SDS-PAGE and transferred to PVDF membranes, followed by immunoblotting using monoclonal antibodies to GFP and talin.
Adenoviral infection
293A cells were infected with pAd/CMV/rPTH1R after it was linearized with PacI. Viral particles were isolated by three freeze-thaw cycles and amplified by reinfection of 293A cells at a concentration of approximately 1 x 1012 colony-forming units/ml.
Quantification of rPTH1R expression
HEK293 and MC3T3-E1 cells were washed with 500 μl binding buffer (95% PBS/5% FBS), incubated first with G48 antiserum in 250 μl binding buffer (1:500) for 2 h, then incubated with the secondary antibody, rabbit antisheep IgG (1:2000; Kirkegaard & Perry Laboratories, Gaithersburg, MD) for 1 h, and finally incubated with [125I]antirabbit IgG (100,000 cpm/well) for 1 h. All incubations were performed at room temperature. After intensive washing, cells were lysed with 1 N NaOH, and radioactivity was counted with a Wallac 1470 -counter (Gaithersburg, MD).
Measurement of intracellular cAMP
Intracellular cAMP accumulation was measured as described previously (30, 33).
Measurement of calpain activity
MC3T3-E1 cells expressing empty vector (pcDNA3.1/Hygro) and human calpastatin were grown in six-well plates. Cells were washed with ice-cold PBS twice and lysed in Ca2+-free buffer. Cell lysates (30 μg/sample) were incubated with 100 μM calpain substrate, N-succinyl-Leu-Leu-Val-Tyr-7 amino-4-methylcoumarin, (Suc-LLVY-AMC, Bachem, King of Prussia, PA) in a buffer containing 10 mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, and 3 mM Ca2+ at 37 C for 15 min (34). Fluorescence was measured at 460 nm with excitation at 360 nm using a PTI Deltascan dual wavelength fluorometer (Photon Technologies, Inc., Lawrenceville, NJ). 7-Amino-4-methylcoumarine, purchased from Bachem, was used as a standard.
Statistical analysis
Data were calculated from two to five independent experiments and expressed as the mean ± SE of triplicate determinations. Statistical significance was determined by ANOVA with Fisher’s protected least significant difference (PLSD) test.
Results
Isolation of the calpain small subunit
To identify novel proteins that bind the intracellular C-terminal portion of the rPTH1R, the C-tail (aa 468–591) was used in the yeast two-hybrid system to screen a human kidney cDNA library. Six of 40 clones that were sequenced encoded portions of the calpain small subunit. One clone, 95A, lacks 21 N-terminal aa and includes the five EF-hand motifs that are essential for Ca2+ binding and heterodimerization with the catalytic domain of both μ- and m-calpains. A particularly strong and specific interaction was seen in the yeast reporter strain (Y189) when it was cotransformed with the C-tail of rPTH1R (aa 468–591) and clone 95A (data not shown). Studies of a series of deletion mutants of the C-tail of rPTH1R lacking N-terminal aa (N5, 473–591; N10, 478–591; N15, 483–591) or lacking C-terminal aa (C20, 468–571; C40, 468–551) showed that only N10 and N15 failed to bind clone 95A. Three double substitutions of this region of the rPTH1R (S473A/W474A, S475A/R476A, and W477A/T478A) failed to bind clone 95A, indicating that the SWSRW sequence (aa 473–477) of the rPTH1R is critical for its interaction with the calpain small subunit, at least in yeast (data not shown).
rPTH1R binds the calpain small subunit in vitro
The GST pull-down assay was used to investigate binding between the rPTH1R C-tail and the calpain small subunit. A GST full-length rPTH1R C-tail bound the purified calpain small subunit in a Ca2+-dependent manner (Fig. 1A). The rPTH1R mutant lacking five N-terminal aa of the C-tail (N5, 473–591) also bound the calpain small subunit; however, importantly, N10 (478–591) and N15 (483–591) did not (Fig. 1B). The intensity of bands corresponding to the calpain small subunit pulled down with GST full-length rPTH1R C-tail (468–591) was reduced compared with those pulled down with GST-N5 (473–591), because expression levels of GST-rPTH1R C-tail (468–591) were lower than those of GST-N5. Of five single alanine substitutions of the GST-C-tail fusion protein (S473A, W474A, S475A, R476A, and W477A), receptors with W474A, S475A, and W477A interacted poorly, if at all, with the calpain small subunit, although receptors with S473A and R476A interacted at least as strongly as the GST full-length C-tail of the rPTH1R (Fig. 1C). These results indicate that at least residues W474, S475, and W477 of the rPTH1R are critical for binding the calpain small subunit.
FIG. 1. A–C, Pull-down assays show direct and Ca2+-dependent interactions between the intracellular C-tail of the rPTH1R and the calpain small subunit. A, The interaction between the C-tail of the rPTH1R and the calpain small subunit is Ca2+ dependent. Lanes 1 and 3, GST alone; lanes 2 and 4, GST fused with full-length C-tail of the rPTH1R (aa 468–591). The Ca2+ concentration in the interaction buffer is: lanes 1 and 2, 0 mM; lanes 3 and 4, 1 mM. B, The binding of the calpain small subunit to the N terminus of the rPTH1R C-tail requires some or all of aa 473–477. Lane 1, GST alone; lane 2, GST full-length C-tail; lanes 3–5, GST C-tail with truncations of five (473–591; lane 3), 10 (478–591; lane 4), and 15 (483–591; lane 5) aa from the N-terminal region of the rPTH1R C-tail. C, The interaction between the C-tail of rPTH1R and the calpain small subunit does not occur or occurs to a lesser extent when tryptophans (W) 474 and 477 and serine (S) 475 are replaced with alanine (A). Lane 1, GST alone; lane 2, GST full-length C-tail of the rPTH1R; lanes 3–7, mutant C-tails containing single alanine substitutions S473A (lane 3), W474A (lane 4), S475A (lane 5), R476A (lane 6), and W477A (lane 7). The arrow indicates the calpain small subunit pulled down with the GST-rPTH1R-C-tail fusion protein. After immunoblotting, the membranes were stained with Coomassie blue in A–C. D, The calpain small subunit copurifies with rPTH1R in HEK293 cells. Extracts from HEK293 cells expressing WT-rPTH1R or rPTH1R with W474A/W477A substitutions were precipitated with G48. Immunoprecipitates were blotted onto a PVDF membrane and probed with the calpain small subunit antibody. The arrow indicates the endogenous calpain small subunit copurified with the rPTH1R.
rPTH1R and the calpain small subunit copurify in mammalian cells
The endogenous calpain small subunit and WT rPTH1R copurified when the latter was expressed in HEK293 cells, but not at all when these cells expressed the rPTH1R-W474A/W477A mutant (Fig. 1D). The expressions of WT and W474A/W477A mutant rPTH1Rs, as determined by binding of G48 and immunoblotting using G48, were similar (data not shown). Thus, the rPTH1R and the calpain small subunit directly interact in mammalian cells.
Cleavage of the rPTH1R by calpain in vitro and in intact cells is Ca2+ dependent
The GST-C tail was cleaved when incubated with m-calpain and Ca2+ to yield a fragment slightly larger than GST alone (Fig. 2A). Full-length rPTH1R-YFP was modified by addition of the rhodopsin epitope (rho) at the C terminus (rPTH1R-YFP-rho). The use of rPTH1R-YFP-rho and rPTH1R-YFP has two advantages; they are as well expressed as rPTH1R and are efficiently stimulated by PTH, presumably because the N-terminal extracellular ligand-binding domain is intact (data not shown), and insertion of YFP at a site relatively near the C terminus makes it likely that a fragment of the receptor that results from cleavage by calpain would be sufficiently large to be detected by immunoblotting with anti-GFP antibody after SDS-PAGE. Addition of the rhodopsin epitope enables detection of the receptor by the monoclonal antibody, 1D4, and immunopurification using 1D4-Sepharose. Figure 2B shows that rPTH1R-YFP-rho was cleaved when it was incubated with m-calpain and Ca2+ to yield an approximately 38-kDa fragment containing YFP (indicated by the arrowhead); the intensity of the intact rPTH1R-YFP-rho band of approximately 110 kDa (indicated by the star) was correspondingly reduced. Importantly, the intensity of the approximately 38-kDa band was markedly diminished and that of an approximately 110-kDa band was restored when immobilized rPTH1R-YFP-rho was preincubated with ALLM and calpastatin. (The light chain of IgG runs as a band that is slightly larger than the PTH1R fragment and is heavily stained.) Calpastatin is a potent endogenous calpain-specific inhibitor of μ- and m-calpains (35, 36), whereas ALLM and antipain are less specific; ALLM is cell permeable and also inhibits cathepsin B and L (37, 38), and antipain inhibits serine proteases, papain, and some cysteine proteases in addition to μ- and m-calpains (39).
FIG. 2. A, Calpain hydrolyzes GST fused with full-length rPTH1R C-tail (aa 468–591; GST-C-tail) in vitro in the presence of Ca2+. GST-C-tail was expressed in the competent cells and purified using a glutathione-Sepharose column. Immobilized GST-C-tail was pretreated with either vehicle or ALLM in the interaction buffer at 0 C for 15 min before incubation with and without 20 μg/ml m-calpain and 3 mM Ca2+ at 37 C for 0 and 60 min, then eluted by competition with 5 mM glutathione and run on SDS-PAGE, followed by Coomassie blue staining. B, Calpain hydrolysis of rPTH1R-YFP-rho in vitro after expression and extraction from HEK293 cells depends on the presence of m-calpain and Ca2+. rPTH1R-YFP-rho was expressed in HEK293 cells, solubilized, and immunopurified using 1D4-Sepharose. rPTH1R-YFP-rho immobilized on 1D4-Sepharose was pretreated with either vehicle or calpain inhibitors (ALLM, 100 μg/ml; calpastatin, 10 μM) at 0 C for 15 min before incubation with and without 20 μg/ml m-calpain and 3 mM Ca2+ at 37 C for 0 or 60 min. Receptors were eluted with a buffer containing sodium dodecyl sulfate, and the eluates were electrophoresed by SDS-PAGE, followed by immunoblot analysis using anti-GFP monoclonal antibody. The arrowhead and star indicate a fragment of rPTH1R-YFP-rho and a band of diminished intensity corresponding to full-length rPTH1R-YFP-rho, respectively. N.S., Nonspecific band.
Next, Nt-rPTH1R and Nt-rPTH1R-W474A/W477A, which are well expressed, lack most of the extracellular N-terminal region (aa 26–181) including the four potential sites for glycosylation, and are activated by PTH and its analogs (40, 41), were modified by the addition of a rhodopsin epitope at their C termini (Nt-rPTH1R-rho and Nt-rPTH1R-W474A/W477A-rho, respectively). Deletion of the glycosylation sites facilitated immunoblot analysis, because the receptor migrated as a tight approximately 49-kDa band on SDS-PAGE. The intensity of the Nt-rPTH1R-rho bands was markedly reduced by incubation with m-calpain in the presence, but not in the absence, of 3 mM Ca2+; this effect was blocked by pretreating samples with calpain inhibitors, ALLM, calpastatin, and antipain (Fig. 3A). HEK293 cells transiently expressing Nt-rPTH1R-rho and Nt-rPTH1R-W474A/W477A-rho were lysed with Ca2+-free buffer; the lysates were incubated with and without 3 mM Ca2+ at 37 C for 0 and 60 min, immunopurified with 1D4-Sepharose, and analyzed by SDS-PAGE and immunoblotting with 1D4. The intensity of the Nt-rPTH1R-rho band was diminished only when the incubation buffer contained 3 mM Ca2+; this effect was reversed by ALLM and calpastatin (Fig. 3B). Importantly, the intensity of the Nt-rPTH1R-W474A/W477A-rho band remained unchanged even when the incubation buffer contained Ca2+ (Fig. 3C). In addition, treatment of intact HEK293 cells expressing Nt-rPTH1R-rho with ionomycin resulted in cleavage of Nt-rPTH1R-rho in a dose-dependent manner (Fig. 3D). Thus, calpain cleaves full-length rPTH1R in vitro and in intact cells in a Ca2+-dependent manner.
FIG. 3. Calpain hydrolyzes Nt-rPTH1R-rho in vitro and in intact cells. A, Calpain hydrolysis of the Nt-rPTH1R-rho in vitro after expression and extraction from HEK293 cells depends on the presence of m-calpain and Ca2+. Nt-rPTH1R-rho was expressed in HEK293 cells, solubilized, and immunopurified using 1D4-Sepharose. Immobilized receptors were then pretreated with either vehicle or calpain inhibitors (ALLM, 100 μg/ml; calpastatin, 10 μM; antipain, 50 μg/ml) in the interaction buffer at 0 C for 15 min before incubation with and without 20 μg/ml m-calpain and 3 mM Ca2+ at 37 C for 0 and 60 min as indicated. Receptors were eluted with a buffer containing SDS, and the eluates were electrophoresed by SDS-PAGE, followed by immunoblot analysis using 1D4. B and C, Calpain hydrolyzes Nt-rPTH1R-rho (B), but not Nt-rPTH1R-W474A/W477A-rho (C), in a Ca2+-dependent manner in cell lysates. HEK293 cells expressing endogenous μ- and m-calpains and transfected with Nt-rPTH1R-rho or Nt-rPTH1R-W474A/W477A-rho were lysed in Ca2+-free buffer. Cell lysates were then pretreated with either vehicle or calpain inhibitors (ALLM, 100 μg/ml; calpastatin, 10 μM) at 0 C for 15 min before incubation with and without 3 mM Ca2+ at 37 C for 0 and 60 min. The reaction was stopped by the addition of 5 mM EGTA and protease inhibitors. Nt-rPTH1R-rho and Nt-rPTH1R-W474A/W47A-rho were then immunopurified using 1D4-Sepharose and eluted with a buffer containing SDS. The eluates were electrophoresed by SDS-PAGE, followed by immunoblot analysis using 1D4. D, Nt-rPTH1R-rho is cleaved by treatment with ionomycin in a dose-dependent manner. HEK293 cells expressing Nt-rPTH1R-rho were treated with ionomycin (0, 5, and 10 μM) at 37 C for 0 or 60 min, then lysed with a buffer containing 3 mM EGTA and protease inhibitors. The samples were immunopurified and analyzed by SDS-PAGE and immunoblotting using 1D4. The closed arrows in A, B, and D indicate immunopurified Nt-rPTH1R-rho, and the open arrow in C indicates Nt-rPTH1R-W474A/W477A-rho.
PTH increases cleavage of rPTH1R and talin in intact cells
Next, the effect of PTH on cleavage of Nt-rPTH1R-rho and rPTH1R-YFP when expressed in HEK293 cells was assessed. HEK293 cells transiently expressing Nt-rPTH1R-rho cDNA in 15-cm plates were harvested and replated in several 3-cm dishes. When reaching confluence, the cells were pretreated with either vehicle or ALLM at 37 C for 15 min before incubation with and without PTH (1 μM) at 37 C for 0 and 15 min, then lysed in a buffer containing 3 mM EGTA and protease inhibitors. Cell lysates containing the same amount of total protein from each group were immunopurified and analyzed by SDS-PAGE and immunoblotting using 1D4. The intensity of the Nt-rPTH1R-rho band was significantly reduced by 20% when cells expressing Nt-rPTH1R-rho were incubated with PTH for 15 min compared with that when cells were incubated without PTH, and the effect was reversed by pretreatment of the cells with ALLM (Fig. 4A). HEK293 cells expressing rPTH1R-YFP were incubated with and without PTH for 5 and 15 min, then lysed and analyzed by SDS-PAGE and immunoblotting using anti-GFP monoclonal antibody. When cells were incubated with PTH for 15 min, the intensity of a band corresponding to intact rPTH1R-YFP (open arrow) was reduced to 70% compared with that of the receptor when the cells were incubated without PTH. The approximately 38-kDa receptor fragment containing YFP (arrow) was 1.4- and 1.7-fold more intense after cells were incubated with PTH for 5 and 15 min, respectively, than when cells were incubated in the absence of PTH for 5 min (Fig. 4B).
FIG. 4. PTH-mediated cleavage of Nt-rPTH1R-rho, rPTH1R-YFP, and talin in intact cells. A, PTH mediates cleavage of Nt-rPTH1R-rho by calpain. HEK293 cells expressing Nt-rPTH1R-rho were pretreated with either vehicle or ALLM (100 μg/ml) at 37 C for 15 min before incubation with and without PTH (1 μM) at 37 C for 0 and 15 min as indicated. Cells were lysed with a buffer containing 3 mM EGTA and protease inhibitors, immunopurified on 1D4-Sepharose, and analyzed by SDS-PAGE and immunoblot analysis using 1D4. The density of the bands corresponding to Nt-rPTH1R-rho was measured using an Imager 2200 (Alpha Innotech Co., San Leando, CA). The data are presented as a percentage of the control value obtained by incubating cells in the absence of PTH and ALLM for 15 min. B, PTH mediates cleavage of rPTH1R-YFP. HEK293 cells expressing rPTH1R-YFP were incubated with and without PTH at 37 C for 5 and 15 min. Cell lysates were analyzed by SDS-PAGE and immunoblotting using anti-GFP monoclonal antibody (upper panel). The open and closed arrows indicate intact rPTH1R-YFP and a fragment of rPTH1R-YFP, respectively. The density of the bands corresponding to the rPTH1R-YFP fragment was measured, and the data are presented as a percentage of the control value obtained by incubating cells in the absence of PTH for 5 min (lower panel). C and D, PTH enhances calpain cleavage of endogenous talin. C, HEK293 cells expressing rPTH1R-YFP were pretreated with either vehicle or ALLM (100 μg/ml) at 37 C for 15 min before incubation with and without PTH at 37 C for 5 and 15 min. Cell lysates were analyzed by SDS-PAGE and immunoblotting using antitalin monoclonal antibody. The arrowhead indicates a fragment of talin (upper panel). The density of the bands corresponding to an approximately 190-kDa talin fragment was measured, and the data are presented as a percentage of the control value obtained by incubating cells in the absence of PTH and ALLM for 5 min (lower left) and by incubating cells with only PTH (lower right). D, Confluent ROS17/2.8 cells were pretreated with either vehicle or ALLM (50 μg/ml) at 37 C for 15 min before incubation with and without PTH at 37 C for 5 min. The arrowhead indicates a fragment of talin. The data in A–C are expressed as the mean ± SE of four replicate samples. a, P < 0.05; b, P < 0.005. N.D., No difference between the two groups.
To support the idea that PTH treatment increases calpain activity in HEK293 cells expressing rPTH1R-YFP, the membranes were probed with antitalin monoclonal antibody. The approximately 190-kDa talin fragment was 1.2- and 1.4-fold more intense when cells expressing rPTH1R-YFP were incubated with PTH than when cells were incubated in the absence of PTH for 5 and 15 min, respectively (Fig. 4C, left), and the effect was blocked 60% by pretreatment of the cells with ALLM (Fig. 4C, right). The PTH-mediated cleavage of talin by calpain was also examined in confluent ROS17/2.8 cells, which endogenously express PTH1R. Confluent ROS17/2.8 cells were treated with and without PTH for 5 and 15 min, then lysed and analyzed by SDS-PAGE and immunoblotting using antitalin monoclonal antibody. The approximately 190-kDa talin fragment was markedly increased when ROS17/2.8 cells were incubated in the presence of PTH for 5 and 15 min, and the effect was blocked by pretreatment of the cells with ALLM (Fig. 4D). The result after 15-min treatment with PTH was similar to that shown in Fig. 4D (data not shown).
Calpain activity modulates PTH-mediated cAMP accumulation
PTH-mediated intracellular cAMP accumulation was assessed in Capn4+/+ and Capn4–/– embryonic fibroblasts infected with adenoviruses carrying WT-rPTH1R and expressing indistinguishable levels of receptor, as determined by binding of G48 (Fig. 5A). PTH-stimulated cAMP accumulation was significantly reduced in Capn4–/– cells expressing WT-rPTH1R compared with Capn4+/+ cells expressing WT-rPTH1R (Fig. 5B).
FIG. 5. A and B, The absence of Capn4 significantly reduces PTH-mediated cAMP accumulation in embryonic fibroblasts infected with adenoviruses carrying rPTH1R. A, Cell surface expression of rPTH1R was determined by binding of G48 to Capn4+/+ and Capn4–/– embryonic fibroblasts. B, PTH-mediated intracellular cAMP accumulation in Capn4+/+ and Capn4–/– embryonic fibroblasts. EV, Empty vector. a, P < 0.05. C and D, The inhibition of calpain activity reduces intracellular cAMP accumulation in MC3T3-E1 cells. C. Calpain activity in MC3T3-E1 cells stably expressing empty vector or human calpastatin. Calpain activity was measured as described in Materials and Methods. D, PTH-mediated intracellular accumulation of cAMP in MC3T3-E1 cells was assessed 14 d after plating, when the cells were confluent. a, P < 0.05; b, P < 0.005 [compared with both stable cell lines expressing empty vector (MC3T3EV-7 and -9)]. The data are expressed as the mean ± SE of triplicate samples. The experiments were performed three to five times with similar results.
PTH-stimulated cAMP accumulation was examined in MC3T3-E1 osteoblastic cells stably expressing human calpastatin. Two independent cell lines, MC3T3hCalp-1 and -9, with levels of calpain activity that were 73% and 64%, respectively, those in MC3T3-E1 cell lines expressing empty vector (MC3T3EV-7 and -9), were used (Fig. 5C). PTH-stimulated cAMP accumulation was significantly reduced to less than 20% of that in the controls in both MC3T3hCalp-1 and -9 (Fig. 5D) compared with that in MC3T3EVs, which expressed similar levels of rPTH1R. Thus, calpain plays a crucial role(s) in PTH-mediated cAMP accumulation in both embryonic fibroblasts expressing rPTH1R and MC3T3-E1 cells.
Discussion
We demonstrated that 1) PTH1R binds the calpain small subunit through an N-terminal portion of the intracellular C-tail that requires, but may not be limited to, aa W474, S475, and W477 of the rPTH1R; 2) PTH1R and the calpain small subunit copurify in intact cells; 3) cleavage of PTH1R and talin by calpain is Ca2+ dependent, as expected, but also is increased by PTH; and 4) PTH-mediated cAMP accumulation is markedly reduced in the absence of calpain and when calpain activity is blocked by calpastatin. The last finding may provide a mechanistic explanation for our previous observation that PTH1R with a deletion of 111 aa of the intracellular C-tail has increased PTH-mediated cAMP accumulation (42).
The site of interaction between rPTH1R and the calpain small subunit, the W-R[W] sequence, located in the N-terminal portion of the C-tail, is highly conserved among PTH1Rs in different species and among other type II GPCR, such as receptors for pituitary adenylate cyclase-activating polypeptide, vasoactive intestinal peptide, secretin, and calcitonin. Interestingly, the crystal structure of rhodopsin reveals that the N-terminal portion of the C-tail forms an eighth -helix (43, 44). If this also proves to be true for rPTH1R, the substitutions, W474A, S475A, and W477A and the double mutant W474A/W477A of rPTH1R may well interfere with formation of this eighth helical structure at the N terminus of the rPTH1R C-tail, which, in turn, may obviate or lessen the interaction between PTH1R and the calpain small subunit.
Calpains are cysteine proteases that participate in a variety of cellular functions by cleaving substrate proteins (20). Protein targets for calpains typically have a PEST sequence, so named because it is rich in proline (P), glutamic acid (E), serine (S), and threonine (T), flanked by clusters of positively charged aa (45, 46, 47). Interspecies comparison of the C-tail of PTH1Rs reveals one highly conserved PEST domain in the rat, mouse, and dog receptors, and two in the human, pig, and rabbit receptors (http://emb1.bcc.univie.ac.at/embnet/tools/bio/PESTfind). A PEST domain at the C terminus of the rPTH1R exists between aa 538–557 (KPGAPATETETLPVTMAVPK; PEST score = –0.08; scores above –5 are significant). We showed that the rPTH1R C-tail is a calpain target and that cleavage occurs only in the presence of Ca2+ and is increased by treatment of the cells with PTH. Cleavage of Nt-rPTH1R-rho could be appreciated only as diminished intensity of the intact receptor band, because the size of the predicted approximately 4- to 6-kDa fragment was too small to be resolved on SDS-PAGE. However, when YFP was introduced close to the C terminus of the rPTH1R, an approximately 38-kDa band that included YFP was apparent in vitro and was markedly increased when the cells were treated with PTH.
PTH-induced osteoblastic retraction is thought to be due to calpain-dependent proteolytic modification of the cytoskeletal organization (24, 25, 26, 27). Osteoblastic retraction is critical for providing osteoclasts direct access to mineralized bone surface that is known to contain and release chemoattractants (48, 49). The finding that cleavage of talin, a key cytoskeletal protein, is disrupted in Capn4-null embryonic fibroblasts and then restored when Capn4 was reintroduced (28) led us to examine the effect of PTH on cleavage of talin in HEK293 cells expressing PTH1R-YFP and in ROS17/2.8 cells. Our finding that cleavage of talin is increased by treatment of cells expressing rPTH1R with PTH and is reduced by pretreatment of the cells with a calpain inhibitor implies that PTH-mediated cleavage of talin may play a crucial role in PTH-mediated cell retraction in osteoblasts.
The mechanism by which activation of PTH1R leads to increased calpain activity is unknown, but may reflect PTH-induced modifications of the C-tail, such as phosphorylation, and/or PTH-induced allosteric changes in PTH1R. Perhaps, PTH induces a highly localized increase in intracellular calcium that, in turn, may facilitate calpain binding to PTH1R and/or calpain activation.
Thus, binding between rPTH1R and the calpain small subunit represents the second example of a protein, other than G proteins and their direct regulators, that interacts with PTH1R. In both instances, signaling by PTH is profoundly altered. Stimulation of the NHERF-PTH1R complex markedly lowers PTH-stimulated cAMP accumulation by favoring the activation of Gi/o (11), whereas PTH stimulation of the calpain small subunit-PTH1R complex leads to C-terminal shortening of PTH1R, a form of the receptor that more readily activates adenylyl cyclase, and is also associated with cleavage of talin, a critical regulator of the cytoskeleton.
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