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Stimulation by Parathyroid Hormone of a NHERF-1-assembled Complex Consisting of the Parathyroid Hormone I Receptor, Phospholipase Cβ, and Actin Increases Intracellular Calcium in Opossum Kidney Cells

2004; Elsevier BV; Volume: 279; Issue: 22 Linguagem: Inglês

10.1074/jbc.m313229200

1083-351X

Matthew J. Mahon, Gino V. Segre,

Protein Kinase Regulation and GTPase Signaling

Parathyroid hormone (PTH) binds its cognate G-protein-coupled receptor (PTH1R) and signals through both adenylyl cyclase and phospholipase C (PLC). C-terminal determinants of the PTH1R interact with the Na+/H+ exchanger regulatory factor 1 (NHERF-1) by binding the first of two PDZ (psd95, discs-large, ZO-1) domains. Compared with wild-type opossum kidney (OK) cells, OKH cells, a sub-clone, do not display PTH-mediated increases of [Ca2+]i and express NHERF-1 at markedly lower levels. Stable expression of NHERF-1 in the OKH parent (OKH-N1) restores the PTH-mediated increase of [Ca2+]i that arises from an influx of extracellular calcium and is both PLC-dependent and pertussis toxin-sensitive. From a morphological perspective, NHERF-1 and the PTH1R co-localize to apical patches of OKH-N1 cells, an expression pattern that is absent in OKH cells and depends on a direct NHERF-1-PTH1R interaction in OKH-N1 cells. Actin and PLCβ1 and -β3 co-localize with NHERF-1 and the PTH1R in OKH-N1 cell apical patches. Actin is also an integral component of the NHERF-1-assembled complex because cytochalasin D disrupts apical localization of both NHERF-1 and the PTH1R and inhibits the PTH-mediated increase of [Ca2+]i. Expression of the first PDZ domain of NHERF-1 acts as a dominant-negative interactor by blocking apical localization of the PTH1R and inhibiting PTH-elicited increases of [Ca2+]i. Thus, NHERF-1 assembles a signaling complex in the apical domains of OK cells that contains the PTH1R, PLCβ, and the actin cytoskeleton. Disruption of this complex blocks the PTH mediated increases of intracellular calcium. Parathyroid hormone (PTH) binds its cognate G-protein-coupled receptor (PTH1R) and signals through both adenylyl cyclase and phospholipase C (PLC). C-terminal determinants of the PTH1R interact with the Na+/H+ exchanger regulatory factor 1 (NHERF-1) by binding the first of two PDZ (psd95, discs-large, ZO-1) domains. Compared with wild-type opossum kidney (OK) cells, OKH cells, a sub-clone, do not display PTH-mediated increases of [Ca2+]i and express NHERF-1 at markedly lower levels. Stable expression of NHERF-1 in the OKH parent (OKH-N1) restores the PTH-mediated increase of [Ca2+]i that arises from an influx of extracellular calcium and is both PLC-dependent and pertussis toxin-sensitive. From a morphological perspective, NHERF-1 and the PTH1R co-localize to apical patches of OKH-N1 cells, an expression pattern that is absent in OKH cells and depends on a direct NHERF-1-PTH1R interaction in OKH-N1 cells. Actin and PLCβ1 and -β3 co-localize with NHERF-1 and the PTH1R in OKH-N1 cell apical patches. Actin is also an integral component of the NHERF-1-assembled complex because cytochalasin D disrupts apical localization of both NHERF-1 and the PTH1R and inhibits the PTH-mediated increase of [Ca2+]i. Expression of the first PDZ domain of NHERF-1 acts as a dominant-negative interactor by blocking apical localization of the PTH1R and inhibiting PTH-elicited increases of [Ca2+]i. Thus, NHERF-1 assembles a signaling complex in the apical domains of OK cells that contains the PTH1R, PLCβ, and the actin cytoskeleton. Disruption of this complex blocks the PTH mediated increases of intracellular calcium. Multiple signals emanate from the activated parathyroid hormone (PTH) 1The abbreviations used are: PTH, parathyroid hormone; PTH1R, parathyroid hormone 1 receptor; PCT, proximal convoluted tubule; AC, adenylyl cyclase; Npt2, type IIa sodium-phosphate co-transporter; OK, opossum kidney; PLC, phospholipase C; NHERF, sodium-hydrogen exchanger regulatory factor; PDZ, psd95, discs large, and ZO-1; GST, glutathione S-transferase; HRP, horseradish peroxidase; YFP, yellow fluorescent protein; MERM, merlin-ezrin-radixin-moesin.1The abbreviations used are: PTH, parathyroid hormone; PTH1R, parathyroid hormone 1 receptor; PCT, proximal convoluted tubule; AC, adenylyl cyclase; Npt2, type IIa sodium-phosphate co-transporter; OK, opossum kidney; PLC, phospholipase C; NHERF, sodium-hydrogen exchanger regulatory factor; PDZ, psd95, discs large, and ZO-1; GST, glutathione S-transferase; HRP, horseradish peroxidase; YFP, yellow fluorescent protein; MERM, merlin-ezrin-radixin-moesin. and parathyroid hormone-related protein receptor (PTH1R) (1Abou-Samra A.B. Jüppner H. Force T. Freeman M.W. Kong X.F. Schipani E. Urena P. Richards J. Bonventre J.V. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2732-2736Crossref PubMed Scopus (992) Google Scholar, 2Bringhurst F.R. Juppner H. Guo J. Urena P. Potts Jr., J.T. Kronenberg H.M. Abou-Samra A.B. Segre G.V. Endocrinology. 1993; 132: 2090-2098Crossref PubMed Google Scholar). Activation of the various pathways and subsequent downstream processes mediated by the PTH1R depends on the cellular environment. PTH1R actions in the proximal convoluted tubule (PCT) of the kidney, for example, illustrate that its signaling depends not only on the cell type but also on the presence of the receptor on a specific membrane surface.The PTH1R is abundantly expressed in the PCT, where it mediates PTH signaling primarily via activation of adenylyl cyclase (AC) and phospholipase C and by increasing intracellular calcium ([Ca2+]i). PTH-mediated inhibition of phosphate uptake, which is caused by internalization and degradation of the type IIa sodium-phosphate co-transporter (Npt2), is an often-studied response in opossum kidney (OK) cells (3Pfister M.F. Lederer E. Forgo J. Ziegler U. Lotscher M. Quabius E.S. Biber J. Murer H. J. Biol. Chem. 1997; 272: 20125-20130Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 4Pfister M. Ruf I. Stange G. Ziegler U. Lederer E. Biber J. Murer H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1909-1914Crossref PubMed Scopus (163) Google Scholar, 5Pfister M.F. Forgo J. Ziegler U. Biber J. Murer H. Am. J. Physiol. 1999; 276: F720-F725PubMed Google Scholar) and the PCT (6Lotscher M. Kaissling B. Biber J. Murer H. Kempson S.A. Levi M. Kidney Int. 1996; 49: 1010-1011Abstract Full Text PDF PubMed Scopus (25) Google Scholar, 7Traebert M. Volkl H. Biber J. Murer H. Kaissling B. Am. J. Physiol. 2000; 278: F792-F798Crossref PubMed Google Scholar). It has recently become appreciated that in polarized epithelia, such as the PCT, discrete microdomains are formed in the apical and basolateral compartments, a process that arises from targeted expression of specialized proteins (8Fanning A.S. Anderson J.M. J. Clin. Investig. 1999; 103: 767-772Crossref PubMed Scopus (400) Google Scholar, 9Fanning A.S. Anderson J.M. Curr. Opin. Cell Biol. 1999; 11: 432-439Crossref PubMed Scopus (271) Google Scholar, 10Brown D. Am. J. Physiol. 2000; 278: F192-F201Crossref Google Scholar). The actin cytoskeleton and associated proteins maintain cell polarity by blocking lateral movement and mixing of apical- and basolateral-membrane constituents. Several lines of evidence suggest that the PTH1R, although expressed in both apical and basolateral compartments of the PCT (11Amizuka N. Lee H.S. Kwan M.Y. Arazani A. Warshawsky H. Hendy G.N. Ozawa H. White J.H. Goltzman D. Endocrinology. 1997; 138: 469-481Crossref PubMed Scopus (70) Google Scholar, 12Ba J. Brown D. Friedman P.A. Am. J. Physiol. 2003; 285: F1233-F1243Crossref PubMed Scopus (139) Google Scholar), is differentially coupled to signaling pathways. First, the PTH1R in isolated basolateral membranes activates AC in vitro, whereas receptors expressed in apical membranes do not (13Kaufmann M. Muff R. Stieger B. Biber J. Murer H. Fischer J.A. Endocrinology. 1994; 134: 1173-1178Crossref PubMed Scopus (45) Google Scholar). Second, PTH administered to the apical side of OK cells, grown on membrane filters, displays an EC50 (5 pm) for the inhibition of phosphate uptake that is 100-fold more sensitive than when PTH is administered to the basolateral surface (500 pm) (14Reshkin S.J. Forgo J. Murer H. Pfluegers Arch. Eur. J. Physiol. 1990; 416: 624-631Crossref PubMed Scopus (35) Google Scholar, 15Reshkin S.J. Forgo J. Murer H. J. Membr. Biol. 1991; 124: 227-237Crossref PubMed Scopus (32) Google Scholar). Third, PTH mediates Npt2 internalization and degradation via cAMP/protein kinase A when applied to the basolateral surface and via PLC/protein kinase C when applied to the luminal surface of isolated PCT segments (7Traebert M. Volkl H. Biber J. Murer H. Kaissling B. Am. J. Physiol. 2000; 278: F792-F798Crossref PubMed Google Scholar). Last, sequential perfusion of PCT cells in primary culture with PTH at 20-min intervals transiently increases [Ca2+]i to the same amplitude but results in a progressive decrease in the activation of AC (16Goligorsky M.S. Loftus D.J. Hruska K.A. Am. J. Physiol. 1986; 251: F938-F944PubMed Google Scholar). Although these results might be compatible with a model involving two PTH receptors, publication of several mammalian genomes have not revealed relevant PTH1R isoforms, suggesting that cell-specific factors are responsible for diverse signaling by this receptor. Cell-specific signaling by the PTH1R is obviously dependent on the repertoire of effector molecules expressed in a given cell.We have recently shown that signaling properties of the PTH1R are profoundly affected by its direct binding of a "scaffolding" protein; the Na+/H+ exchanger regulatory factor 2 (NHERF-2) (17Mahon M.J. Donowitz M. Yun C.C. Segre G.V. Nature. 2002; 417: 858-861Crossref PubMed Scopus (269) Google Scholar). NHERF-2 switches PTH1R signaling from AC to PLC through a direct psd95, discs large, ZO1 (PDZ)-domain-specific interaction between the more C-terminal of two PDZ domains and the C terminus of the receptor (17Mahon M.J. Donowitz M. Yun C.C. Segre G.V. Nature. 2002; 417: 858-861Crossref PubMed Scopus (269) Google Scholar). NHERF-2 and the closely related protein, NHERF-1 (also called EBP50), are PDZ domain-containing proteins that also indirectly bind actin via interactions with MERM (merlin, ezrin, radixin, moesin) proteins (18Reczek D. Berryman M. Bretscher A. J. Cell Biol. 1997; 139: 169-179Crossref PubMed Scopus (516) Google Scholar, 19Bretscher A. Chambers D. Nguyen R. Reczek D. Annu. Rev. Cell Dev. Biol. 2000; 16: 113-143Crossref PubMed Scopus (323) Google Scholar). Like NHERF-2, NHERF-1 also interacts with the PTH1R in vitro and in mouse kidney extracts, but this seems to involve the more N-terminal of the two PDZ domains (data shown herein).NHERF-1-null mice display a complex phenotype, which includes renal phosphate wasting, hypocalcemia, and osteopenia (20Shenolikar S. Voltz J.W. Minkoff C.M. Wade J.B. Weinman E.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11470-11475Crossref PubMed Scopus (279) Google Scholar). Npt2 localizes to intracellular sites in PCT lacking NHERF-1, demonstrating that NHERF-1 targets and/or anchors the transporter to apical domains (20Shenolikar S. Voltz J.W. Minkoff C.M. Wade J.B. Weinman E.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11470-11475Crossref PubMed Scopus (279) Google Scholar). We recently reported that OKH cells, a sub-clone of wild-type OK cells that do not display PTH-mediated inhibition of Npt2, markedly lack NHERF-1 expression compared with wild-type cells (21Mahon M.J. Cole J.A. Lederer E.D. Segre G.V. Mol. Endocrinol. 2003; 17: 2355-2364Crossref PubMed Scopus (79) Google Scholar). It is noteworthy that stable expression of NHERF-1 in OKH cells (OKH-N1) restored the PTH-mediated inhibition of phosphate uptake (21Mahon M.J. Cole J.A. Lederer E.D. Segre G.V. Mol. Endocrinol. 2003; 17: 2355-2364Crossref PubMed Scopus (79) Google Scholar). Together with data that show binding of NHERF-1 to Npt2 (21Mahon M.J. Cole J.A. Lederer E.D. Segre G.V. Mol. Endocrinol. 2003; 17: 2355-2364Crossref PubMed Scopus (79) Google Scholar, 22Hernando N. Deliot N. Gisler S.M. Lederer E. Weinman E.J. Biber J. Murer H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11957-11962Crossref PubMed Scopus (162) Google Scholar), these findings suggest that NHERF-1 assembles a regulatory complex for Npt2.Herein, we demonstrate that expression of NHERF-1 in the OK cell model is essential for numerous cellular functions. OKH cells also do not respond to PTH with an increase of phosphoinositide hydrolysis or [Ca2+]i and do not manifest apical patches, which are typical of the parental OK cell. When they stably express exogenous NHERF-1, however, OKH-N1 cells now have apical patches and respond to PTH with an increase in [Ca2+]i, which is caused by extracellular influx, rather than a release from internal stores. This influx is both PLC-dependent and pertussis toxin-sensitive and requires the assembly by NHERF-1 of PLCβ, actin, and the PTH1R.EXPERIMENTAL PROCEDURESMaterials—The OK cell lines were a kind gift from Dr. Judith Cole. Plasmids containing cDNAs for PLCβ1 were from Dr. Sue Goo Rhee, for PLCβ2 from Dr. Eva Neer, and for PLCβ3 from Dr. Barbara Sanborn. NHERF-1 polyclonal antibodies were from Dr. Chris Yun. GST-Sepharose was from Amersham Biosciences. Fura-2/acetoxymethyl ester, Alexa Fluor 546 phalloidin, and secondary antibodies conjugated with Alexa Fluor 488 and 546 were from Molecular Probes (Eugene, OR). U73122, SKF96365, cytochalasin D, and thrombin were from Calbiochem. S-protein-HRP and rapid S-tag assay kit were from Novagen (San Diego, CA). Monoclonal antibodies to Na+-K+ATPase were from Upstate Biotechnology (Charlottesville, VA). Pertussis toxin, Flag antibodies, 3,3′,5,5′-tetramethylbenzidine liquid substrate system and general chemicals were from Sigma.Overlay and Plate-binding Assay—The full-length C-terminal tail of the PTH1R (amino acids 463-591), the N-terminal deletions of the C-terminal tail (amino acids: NΔ60, 523-591; NΔ80, 543-591; NΔ100, 563-591; NΔ110, 573-591; and NΔ120, 583-591) and C-terminal tail point mutations (P581A, L582K, Q584A, E585A, EE585/586AA, and EE585/586KK) were cloned into pGEX vector (Amersham Biosciences) using PCR with PfuTurbo polymerase (Stratagene, La Jolla, CA), which generates an N-terminal GST fusion protein. The various GST-PTH1RC-tail constructs were expressed in Bl-21 Escherichia coli and purified using GST-Sepharose. Full-length NHERF-1 (amino acids 1-358), N1-PDZ1short (amino acids 1-127), N1-PDZ2short (amino acids 128-358), N1-PDZ1long (amino acids 1-162), and N1-PDZ2long (amino acids 65-358) were cloned into pET-30b (Novagen), using PCR with Pfu polymerase, which generates an N-terminal S-tag and His6 tag. All clones were verified using automated sequencing. These NHERF-1 constructs were expressed in Bl21(DE3)pLysS E. coli and partially purified using immobilized metal affinity chromatography (nickel-nitrilotriacetic acid; Qiagen, Valencia, CA), following the manufacturer's procedures. Protein concentrations were determined using the S-tag Rapid Assay Kit from Novagen.For overlay assays, 2 μg of purified GST and the various GST PTH1R-C-tails were run on SDS-PAGE and blotted to polyvinylidene difluoride membranes. The membrane was blocked with 5% non-fat dry milk in phosphate-buffered saline containing 0.1% Tween 20 (PBSTw) for 45 min. Full-length NHERF-1 and the various fragments were overlaid on the membrane in 5% milk in PBSTw at a concentration of 200 nm for 1 h at room temperature. Membranes were washed thoroughly and blotted with S-protein-HRP (1:5000) in 5% milk for 20 min and washed a second time; interactions were detected using the Western Lighting Chemiluminescence reagent (PerkinElmer Life and Analytical Sciences, Boston, MA). For the plate-binding assay, 0.5 μg of either GST or the various GST-PTH1R-C-tail proteins were adhered to wells of 96-well plates overnight at 4 °C. The wells were washed with PBST and blocked with 5% milk for 1 h at room temperature. The S-tagged NHERF-1 proteins were incubated in the wells at concentrations ranging from 0.01 nm to 1 μm in 5% milk/PBSTw. The wells were thoroughly washed, incubated with S-protein-HRP (1:5000 in 5% milk), and washed a second time; interactions were detected using the 3,3′,5,5′-tetramethylbenzidine liquid substrate system (Sigma). After 20 min at room temperature, reactions were stopped by the addition of 0.5 m H2SO4 and the reaction was quantified by spectroscopy at 450 nm. Binding to GST alone was subtracted from each value at a given concentration, and the data were reported as a percentage of the maximum binding.Determination of [Ca2+]i—OKH cells were stably transfected with NHERF-1 (OKH-N1), as described previously (21Mahon M.J. Cole J.A. Lederer E.D. Segre G.V. Mol. Endocrinol. 2003; 17: 2355-2364Crossref PubMed Scopus (79) Google Scholar). [Ca2+]i was monitored using the fura-2 method. In brief, cells were cultured in Dulbecco's modified Eagle's medium/Ham's F12 media supplemented with 10% fetal bovine serum and plated into Lab-Tek II chambered cover glasses (Nalge Nunc International, Naperville, IL). Cells were serum-starved for 4-5 h before the assay. Cells were cooled to room temperature and washed with a balanced salt solution (127 mm NaCl, 3.8 mm KCl, 1.2 mm KH2PO4, 1.2 mm CaCl2, 0.8 mm MgCl2, 5 mm glucose, and 10 mm HEPES, pH 7.4). Cells were then loaded with 5 μm fura-2/acetoxymethyl ester with 0.05% Pluronic F-127 (Molecular Probes, Eugene, OR) for 45 min at room temperature, washed in balanced salt solution, and unloaded for 30 min. Fura-2 fluorescence was diffuse without a punctate distribution pattern. Fluorescence was measured in single cells excited at 340 and 380 nm using a PTI Deltascan dual-wavelength fluorimeter (Photon Technologies Incorporated, Lawrenceville, NJ) directed through the stage of an inverted Nikon Diaphot 200 microscope (Melville, NY) with a cut-off filter at 400 nm. Emissions were monitored in real time with a Sensys charge-coupled device camera (Photometrics, Ltd., Tucson, AZ) and analyzed using the Poenie-Tsien ratio with Imagemaster 2 software (Photon Technologies Incorporated). At the end of each experiment, fura-2 fluorescence was calibrated to [Ca2+]i by treating the cells with 5 μm ionomycin to achieve the Ca2+-bound dye ratio at 340/380 nm and fluorescence at 480 (Rmax and Sb2), followed by treatment with 5 mm EGTA to determine Ca2+-free dye ratio at 340/380 and fluorescence at 480 (Rmin and Sf2). These parameters were used to estimate [Ca2+]i using the equation of Grynkiewicz et al. (23Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar), which is provided as a function in the Imagemaster 2 software. Calcium transients monitored in the absence of extracellular calcium are reported as a ratio of fura-2 fluorescence of 340 over 380 nm.Transient Transfection and Immunohistochemistry—Cells were cultured in chambered microscope slides (Nalge Nunc International). The cDNA for the yellow fluorescent protein (YFP; BD Biosciences Clontech) was cloned in-frame with 30 amino acids from the C terminus of the PTH1R using PCR. This region of the C-terminal tail has a low degree of homology among species, and placing YFP in this location does not interfere with NHERF-1 interactions in vitro (data not shown). The Flag epitope (DYKDDDDK) was cloned in-frame on the N terminus of PLCβ1 and -β3 in pcDNA3.1 (Invitrogen). PLCβ2 was already Flag-tagged in pcDNA3.1. Forty-eight hours before analysis, cells were transfected with FuGene 6 (Roche Applied Science) at 0.25 μg of plasmid/well at a FuGene6/DNA ratio of 5:1. Cells then were fixed with 2% paraformaldehyde in phosphate-buffered saline for 20 min, thoroughly washed with phosphate-buffered saline, permeabilized with 0.1% Triton X-100, and blocked with 5% non-fat milk in phosphate-buffered saline plus 0.1% Triton X-100 (PBSTx) for 30-45 min. Primary antibodies were diluted in 5% milk/PBSTx (NHERF-1 polyclonal, 1:2000; Flag monoclonal, 1:1000; Na+/K+-ATPase monoclonal, 1:500) and incubated with the cells for 1 h. Cells then were washed in PBSTx and incubated with species-specific secondary antibodies conjugated with either Alexa Fluor 488 (green) or Alexa Fluor 546 (red). Actin was stained with Alexa Fluor 546 conjugated to phalloidin. Immunostained cells were analyzed using a Radiance 2100 confocal microscope and the associated LaserSharp 2000 operating software (Bio-Rad, Hercules, CA). Basolateral membranes were identified using the Na+/K+-ATPase antibody (data not shown).Analysis of the NHERF-1 Dominant Negative—Amino acids of NHERF-1 corresponding to the PDZ1long (see "Overlay and Plate-binding Assay") and β-galactosidase were cloned in-frame with the Flag epitope (DYKDDDDK) into pcDNA3.1 using PCR. Flag-PDZ1long of NHERF-1 and Flag-β-galactosidase were transiently expressed in OKH-N1 cells using FuGene6 (Roche Applied Science) in cover glass chamber slides, and PTH-elicited increases of [Ca2+]i were determined using the fura-2 method. Efficiency of the transient transfection was estimated to be ∼50% of the cells. Calcium measurements were taken from all cells in the microscopic field and data combined from three independent experiments (n = 10 ± S.E.).Determination of cAMP—PTH-mediated accumulation of cAMP in OKH-N1 cells was determined using a cAMP radioimmunoassay kit (PerkinElmer Life and Analytical Sciences).RESULTSThe PTH1R and NHERF-1 Interactions in Vitro—NHERF-1 interacts with the PTH1R through an atypical PDZ interaction motif consisting of the four C-terminal amino acids of the receptor (17Mahon M.J. Donowitz M. Yun C.C. Segre G.V. Nature. 2002; 417: 858-861Crossref PubMed Scopus (269) Google Scholar). Using overlay assays, full-length NHERF-1 binds the PTH1R tail with high affinity (Fig. 1A). NHERF-1 contains two PDZ domains; one is N-terminal (PDZ1) and the other is centrally located (PDZ2) in the molecule (Fig. 1B). PDZ1 was separated from PDZ2 by dividing the interdomain region in half, yielding PDZ1short and PDZ2short (Fig. 1B). The individual PDZ domains (PDZ1short and PDZ2short) unexpectedly failed to interact with the PTH1R C-tail to any significant degree (Fig. 1A), suggesting that disruption of the interdomain region severely affects interactions of NHERF-1 with target proteins. Additional PDZ domain constructs then were synthesized that included the entire interdomain region (PDZ1long and PDZ2long) (Fig. 1B). Relative binding affinities, determined with a plate-binding assay, show that full-length NHERF-1 and PDZ1long display indistinguishable apparent (Kd) affinities for the PTH1R C-tail of ∼110 to 120 nm (Fig. 1C). On the other hand, PDZ2long does not interact with the PTH1R C-tail to any significant degree (Fig. 1C), demonstrating that PDZ1 of NHERF-1 is the primary binding site for the PTH1R.We next sought to determine whether residues N-terminal to the four C-terminal amino acids that comprise the canonical PDZ interaction motif contribute to the NHERF-1 interaction. As demonstrated by an overlay assay, GST fusions containing N-terminal deletions of the C-tail up to 110 amino acids in length did not affect the NHERF-1-receptor interaction (Fig. 2A). However, deletion of 120 amino acids from the N terminus completely disrupts the NHERF-1-receptor interaction (Fig. 2A), suggesting that the PDZ interaction motif on the PTH1R extends N-terminal to the four terminal residues to include some or all of the C-terminal 18 amino acids. Because the C-tail fused to the large GST moiety (27 kDa) might sterically impair the NHERF-1-receptor interaction, point mutations N-terminal to the PDZ interaction motif were incorporated in the full-length PTH1R C-tail, and its interactions with NHERF-1 were assessed by the plate-binding assay. Incorporation of P581A, L582K, or Q584A point mutations does not affect the NHERF-1-receptor interaction, whereas single and double point mutations of the glutamic acid residues at 585 and 586 markedly disrupt this interaction, suggesting that the acidic side chains in the C-tail stabilize the NHERF-1-receptor interaction (Fig. 2B).Fig. 2Amino acid residues N-terminal to the PDZ interaction motif are required for the PTH1R-NHERF-1 interaction. A, GST or GST fused to N-terminal deletions of the PTH1R C-tail (amino acids: NΔ60, 523-591; NΔ80, 543-591; NΔ100, 563-591; NΔ110, 573-591; NΔ120, 583-591) were blotted to a membrane and overlaid with NHERF-1 (200 nm). Interactions were detected with S-protein-HRP and ECL. Data are representative of three independent experiments. B, PTH1R C-tail containing the following point mutations: P581A (closed squares), L582K (triangles), Q584A (inverted triangles), E585A (diamonds), EE585/586AA (circles) and EE585/586KK (open squares), were interacted with NHERF-1 at the concentrations indicated in the plate-binding assay. Relative binding affinities were determined and data are indicative of two independent experiments (n = 8 ± S.E.).View Large Image Figure ViewerDownload (PPT)NHERF-1 Restores PTH-mediated Increases of Intracellular Calcium in OKH Cells—OKH cells, a sub-clone of the wild-type OK cell line, do not display PTH-mediated inhibition of phosphate uptake or increases of [Ca2+]i (24Miyauchi A. Dobre V. Rickmeyer M. Cole J. Forte L. Hruska K.A. Am. J. Physiol. 1990; 259: F485-F493PubMed Google Scholar), although they do display a robust PTH-mediated activation of AC (25Cole J.A. Forte L.R. Krause W.J. Thorne P.K. Am. J. Physiol. 1989; 256: F672-F679PubMed Google Scholar). We showed previously that OKH cells were deficient in expression of NHERF-1, and stable expression of NHERF-1 in OKH cells restored PTH-mediated inhibition of phosphate uptake (21Mahon M.J. Cole J.A. Lederer E.D. Segre G.V. Mol. Endocrinol. 2003; 17: 2355-2364Crossref PubMed Scopus (79) Google Scholar). Fig. 3A shows that stable expression of NHERF-1 in OKH cells (OKH-N1) also restores the PTH-elicited increase of [Ca2+]i, which is rapid, robust, and transient in nature. OKH-N1 cells in nominally calcium-free buffer plus 0.5 mm EGTA do not display PTH-mediated increases of [Ca2+]i (Fig. 3B), suggesting that the increase in cytoplasmic free calcium is caused by an influx from the extracellular space and not by release from intracellular stores. The receptor-mediated calcium channel blocker SKF96365 (26Fasolato C. Pizzo P. Pozzan T. J. Biol. Chem. 1990; 265: 20351-20355Abstract Full Text PDF PubMed Google Scholar) inhibits the PTH-mediated increase in [Ca2+]i in a dose-dependent manner (Fig. 3C), which supports this conclusion, and is consistent with published data that PTH predominantly mediates an influx of calcium ions in wild-type OK cells (24Miyauchi A. Dobre V. Rickmeyer M. Cole J. Forte L. Hruska K.A. Am. J. Physiol. 1990; 259: F485-F493PubMed Google Scholar) and PCT cultures (16Goligorsky M.S. Loftus D.J. Hruska K.A. Am. J. Physiol. 1986; 251: F938-F944PubMed Google Scholar, 27Hruska K.A. Goligorsky M. Scoble J. Tsutsumi M. Westbrook S. Moskowitz D. Am. J. Physiol. 1986; 251: F188-F198PubMed Google Scholar, 28Tanaka H. Smogorzewski M. Koss M. Massry S.G. Am. J. Physiol. 1995; 268: F330-F337PubMed Google Scholar). The phospholipase C inhibitor U73122 and pertussis toxin also block the PTH-mediated increase in [Ca2+]i (Fig. 3C), indicating that this effect depends on activation of PLC by a member of the Gi/o family of G proteins. The inactive analog of U73122, U73343, did not inhibit the PTH-mediated increase in [Ca2+]i (data not shown). OKE cells, another sub-clone of the OK cell line, express abundant amounts of NHERF-1, display responses to PTH similar to those of the parental OK cells, and display PTH-mediated calcium responses that are indistinguishable from that displayed by OKH-N1 cells (data not shown).Fig. 3NHERF-1 rescues the PTH-mediated, PLC-dependent influx of calcium ions into OKH cells. A, OKH and OKH-N1 cells, loaded with fura-2, were treated with 10 nm PTH (1-34) and single cells monitored for changes in [Ca2+]i, as described under "Experimental Procedures." B, OKH-N1 cells, loaded with fura-2, in the absence of added extracellular calcium and the presence of 0.5 mm EGTA were treated with 10 nm PTH (1-34), and changes in the fura-2 340/380 nm ratio were monitored. C, OKH-N1 cells, loaded with fura-2, in the presence of either vehicle, 25 μm, or 50 μm SKF96365 (SKF), were treated with 10 nm PTH (1-34). and [Ca2+]i was monitored. D, OKH-N1 cells, incubated with either U73122 (1 μm) for 15 min or pertussis toxin (PTX; 100 ng/ml) for 16 h, were treated with 10 nm PTH (1-34) and [Ca2+]i monitored. Traces are representative of individual cellular responses in a given microscopic field from at least three independent experiments.View Large Image Figure ViewerDownload (PPT)OK cells respond to thrombin with an increase in [Ca2+]i, a response that has been attributed to an increase of inositol trisphosphates (29Malmstrom K. Stange G. Murer H. Biochem. J. 1988; 251: 207-213Crossref PubMed Scopus (58) Google Scholar). To control for potential depletion of intracellular calcium in OKH-N1 cells when incubated in calcium-free media or an unexpected block of calcium release from intracellular stores by SKF96365, we assessed the response of these treatments to thrombin. Depletion of extracellular calcium (Fig. 4A) or inclusion of SKF96365 (Fig. 4B) does not inhibit the thrombin-mediated increase of [Ca2+]i in OKH-N1 cells, demonstrating that thrombin induces a release of calcium from intracellular stores. These data demonstrate that depletion of extracellular calcium or SKF96365 treatment do not introduce confounding artifacts and thus supports the assertion that PTH increases cytoplasmic-free calcium by increasing influx from outside the OKH-N1 cell.Fig. 4Thrombin stimulates a release of [Ca2+]i in OKH-N1 cells. A, OKH-N1 cells, loaded with fura-2, in the presence of calcium or the absence of added extracellular calcium and the presence of 0.5 mm EGTA, were treated with thrombin (THR; 2 units/ml), and changes in [Ca2+]i were monitore