Fluorescent ligand-directed co-localization of the parathyroid hormone 1 receptor with the brush-border scaffold complex of the proximal tubule reveals hormone-dependent changes in ezrin immunoreactivity consistent with inactivation.

Through binding to parathyroid hormone (PTH), PTH1R interacts with kidney-specific scaffold proteins, including the sodium hydrogen exchanger regulatory factors 1 and 2 (NHERFs), and ezrin. To facilitate in vivo localization, tetramethylrhodamine-labeled PTH (PTH-TMR) was used as a fluorescent probe. In mice, PTH-TMR localizes to luminal surfaces of tubular S1 segments that overlap PTH1R immunostaining, but does not directly overlap with megalin-specific antibodies. PTH-TMR staining directly overlaps with Npt2a in nascent, endocytic vesicles, marking the location of transporter regulation. PKA substrate antibodies display marked staining increases in segments labeled with PTH-TMR, demonstrating a functional effect. In the presence of secondary hyperparathyroidism, PTH-TMR staining is markedly reduced and shifts to co-localizing with megalin. At 15min post-injection, PTH-TMR-labeled vesicles do not co-localize with either NHERF or ezrin, suggesting PTH1R dissociation from the scaffold complex. At the 5min time point, PTH-TMR stains the base of microvilli where it localizes with both NHERF2 and ezrin, and only partially with NHERF1. Strikingly, the bulk of ezrin protein becomes undetectable with the polyclonal, CS3145 antibody, revealing a PTH-induced conformational change in the scaffold. A second ezrin antibody (3C12) is capable of detecting the altered ezrin protein. The CS3145 antibody only binds to the active form of ezrin and fails to recognize the inactive form, while the 3C12 reagent can detect either active or inactive ezrin. Here we show that the PTH1R is part of the ezrin scaffold complex and that acute actions of PTH suggest a rapid inactivation of ezrin in a spatially defined manner.

Vectors bicistronically linking a gene of interest to the SV40 large T antigen in combination with the SV40 origin of replication enhance transient protein expression and luciferase reporter activity.

The simian virus 40 large T antigen (SVLT) induces replication of plasmids bearing the SV40 origin of replication (SV40 ori) within mammalian cells. The internal ribosomal entry site (IRES) is an element that allows for the cotranslation of proteins from one polycistronic mRNA. Through the combination of these elements, IRES-dependent coexpression of a protein of interest and the SVLT, either constitutive or regulated, on plasmids bearing the SV40 ori generates a positive feedback loop, resulting in enhanced expression. A vector linking red fluorescent protein (RFP) to the IRES-SVLT element enhances fluorescence ~10-fold over that demonstrated from a vector lacking this element. In transfection-resistant CV-1 cells, the RFP-IRES-SVLT vector substantially increases the number of cells expressing detectable levels of RFP. Furthermore, inclusion of the IRES-SVLT/SV40 ori elements in standard luciferase-based reporter gene constructs and associated effectors results in marked increases in luminescent output and sensitivity, using the ß-catenin/TCF pathway and the mammalian two-hybrid assay as models. Ultimately, vector systems combining these well-established elements (IRES-SVLT/SV40 ori) will increase the utility of transient transfection for the production of recombinant proteins, the use of transfection-resistant cell lines, and the effectiveness of luciferase-based high-throughput screening assays.

pHluorin2: an enhanced, ratiometric, pH-sensitive green florescent protein.

Green florescent protein (GFP) variants that are sensitive to changes in pH are invaluable reagents for the analysis of protein dynamics associated with both endo- and exocytotic vesicular trafficking. Ratiometric pHluorin is a GFP variant that displays a bimodal excitation spectrum with peaks at 395 and 475 nm and an emission maximum at 509 nm. Upon acidification, pHluorin excitation at 395 nm decreases with a corresponding increase in the excitation at 475 nm. GFP2, a GFP variant that contains mammalianized codons and the folding enhancing mutation F64L, displays ~8-fold higher florescence compared to pHluorin upon excitation at 395 nm. Using GFP2 as a template, an enhanced ratiometric pHluorin (pHluorin2) construct was developed to contain fully mammalianized codons, the F64L mutation and ten of the thirteen pHluorin-specific mutations. As a result, pHluorin2 displays markedly higher florescence when compared to pHluorin while maintaining the ratiometric pH-sensitivity. Unlike native pHluorin, pHluorin2 expressed in the ligand-binding domain of the parathyroid hormone 1 receptor is readily detectable by confocal microscopy and displays a marked increase in florescence upon ligand-induced endocytosis to intracellular vesicles. Thus, pHluorin2's enhanced florescence while sustaining ratiometric pH-sensitivity represents a significant improvement for this methodological approach.

Apical membrane segregation of phosphatidylinositol-4,5-bisphosphate influences parathyroid hormone 1 receptor compartmental signaling and localization via direct regulation of ezrin in LLC-PK1 cells.

The parathyroid hormone 1 receptor (PTH1R), a primary regulator of mineral ion homeostasis, is expressed on both the apical and basolateral membranes of kidney proximal tubules and in the LLC-PK1 kidney cell line. In LLC-PK1 cells, apical PTH1R subpopulations are far more effective at signaling via phospholipase (PLC) than basolateral counterparts, revealing the presence of compartmental signaling. Apical PTH1R localization is dependent upon direct interactions with ezrin, an actin-membrane cross-linking scaffold protein. Ezrin undergoes an activation process that is dependent upon phosphorylation and binding to phosphatidylinositol-4,5-bisphosphate (PIP2), a lipid that is selectively concentrated to apical surfaces of polarized epithelia. Consistently, the intracellular probe for PIP2, GFP-PLCδ1-PH, localizes to the apical membranes of LLC-PK1 cells, directly overlapping ezrin and PTH1R expression. Activation of the apical PTH1R shifts the GFP-PLCδ1-PH probe from the apical membrane to the cytosol and basolateral membranes, reflecting domain-specific activation of PLC and hydrolysis of PIP2. This compartmental signaling is likely due to the polarized localization of PIP2, the substrate for PLC. PIP2 degradation using a membrane-directed phosphatase shifts ezrin localization to the cytosol and induces ezrin de-phosphorylation, processes consistent with inactivation. PIP2 degradation also shifts PTH1R expression from brush border microvilli to basolateral membranes and markedly blunts PTH-elicited activation of the MAPK pathway. Transient expression of ezrin in HEK293 cells shifts PTH1R expression from the plasma membrane to microvilli-like surface projections that also contain PIP2. As a result, ezrin enhances PTH mediated activation of the PLC pathway in this cell model with increasing total receptor surface expression. Collectively, these findings demonstrate that the apical segregation of PIP2 to the apical domains not only promotes the activation of ezrin and the subsequent formation of the PTH1R containing scaffold, but also ensures the presence of ample substrate for propagating the PLC pathway.

Acute down-regulation of sodium-dependent phosphate transporter NPT2a involves predominantly the cAMP/PKA pathway as revealed by signaling-selective parathyroid hormone analogs.

The parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTHR1) in cells of the renal proximal tubule mediates the reduction in membrane expression of the sodium-dependent P(i) co-transporters, NPT2a and NPT2c, and thus suppresses the re-uptake of P(i) from the filtrate. In most cell types, the liganded PTHR1 activates Gα(S)/adenylyl cyclase/cAMP/PKA (cAMP/PKA) and Gα(q/11)/phospholipase C/phosphatidylinositol 1,4,5-trisphosphate (IP(3))/Ca(2+)/PKC (IP(3)/PKC) signaling pathways, but the relative roles of each pathway in mediating renal regulation P(i) transport remain uncertain. We therefore explored the signaling mechanisms involved in PTH-dependent regulation of NPT2a function using potent, long-acting PTH analogs, M-PTH(1-28) (where M = Ala(1,12), Aib(3), Gln(10), Har(11), Trp(14), and Arg(19)) and its position 1-modified variant, Trp(1)-M-PTH(1-28), designed to be phospholipase C-deficient. In cell-based assays, both M-PTH(1-28) and Trp(1)-M-PTH(1-28) exhibited potent and prolonged cAMP responses, whereas only M-PTH(1-28) was effective in inducing IP(3) and intracellular calcium responses. In opossum kidney cells, a clonal cell line in which the PTHR1 and NPT2a are endogenously expressed, M-PTH(1-28) and Trp(1)-M-PTH(1-28) each induced reductions in (32)P uptake, and these responses persisted for more than 24 h after ligand wash-out, whereas that of PTH(1-34) was terminated by 4 h. When injected into wild-type mice, both M-modified PTH analogs induced prolonged reductions in blood P(i) levels and commensurate reductions in NPT2a expression in the renal brush border membrane. Our findings suggest that the acute down-regulation of NPT2a expression by PTH ligands involves mainly the cAMP/PKA signaling pathway and are thus consistent with the elevated blood P(i) levels seen in pseudohypoparathyroid patients, in whom Gα(s)-mediated signaling in renal proximal tubule cells is defective.

Potent constitutive cyclic AMP-generating activity of XLαs implicates this imprinted GNAS product in the pathogenesis of McCune-Albright syndrome and fibrous dysplasia of bone.

Patients with McCune-Albright syndrome (MAS), characterized primarily by hyperpigmented skin lesions, precocious puberty, and fibrous dyslasia of bone, carry postzygotic heterozygous mutations of GNAS causing constitutive cAMP signaling. GNAS encodes the α-subunit of the stimulatory G protein (Gsα), as well as a large variant (XLαs) derived from the paternal allele. The mutations causing MAS affect both GNAS products, but whether XLαs, like Gsα, can be involved in the pathogenesis remains unknown. Here, we investigated biopsy samples from four previously reported and eight new patients with MAS. Activating mutations of GNAS (Arg201 with respect to the amino acid sequence of Gsα) were present in all the previously reported and five of the new cases. The mutation was detected within the paternally expressed XLαs transcript in five and the maternally expressed NESP55 transcript in four cases. Tissues carrying paternal mutations appeared to have higher XLαs mRNA levels than maternal mutations. The human XLαs mutant analogous to Gsα-R201H (XLαs-R543H) showed markedly higher basal cAMP accumulation than wild-type XLαs in transfected cells. Wild-type XLαs demonstrated higher basal and isoproterenol-induced cAMP signaling than Gsα and co-purified with Gß1γ2 in transduced cells. XLαs mRNA was measurable in mouse calvarial cells, with its level being significantly higher in undifferentiated cells than those expressing preosteoblastic markers osterix and alkaline phosphatase. XLαs mRNA was also expressed in murine bone marrow stromal cells and preosteoblastic MC3T3-E1 cells. Our findings are consistent with the possibility that constitutive XLαs activity adds to the molecular pathogenesis of MAS and fibrous dysplasia of bone.

The parathyroid hormone 1 receptor directly binds to the FERM domain of ezrin, an interaction that supports apical receptor localization and signaling in LLC-PK1 cells.

PTH 1 receptor (PTH1R) regulates mineral ion homeostasis. Both apical and basolateral PTH1R subpopulations exist within the renal proximal tubule. The purpose of this research was to examine determinants within the PTH1R that direct apical localization. When expressed in LLC-PK1 cells, a proximal tubule cell model, the PTH1R localizes to both apical and basolateral membranes. The C terminus of the PTH1R contains a psd-95, discs large, ZO-1 domain interaction motif that binds the sodium-hydrogen exchanger regulatory factor 1 (NHERF-1), a renal tubule scaffold protein. Receptors lacking the psd-95, discs large, ZO-1 domain interaction motif (PTH1R-CDelta4) partly localize to apical membranes, suggesting that additional factors may be involved. Ezrin, a membrane-cytoskeleton linking protein, directly binds NHERF-1 and thus links assembled complexes to actin. In vitro, subdomain C of the ezrin band 4.1, ezrin, radixin, domain interacts with the C-terminal tail of the PTH1R on a site that is mutually exclusive from the NHERF-1 interaction domain, suggesting the presence of a ternary complex. Mutating the lysine-arginine-lysine motif within the juxtamembrane region of the PTH1R C-terminal tail to alanines markedly disrupts interactions with the band 4.1, ezrin, radixin, domain of ezrin both in vitro and within cells. Inclusion of these mutations in the context of the full-length PTH1R disrupts apical localization with no effect on basolateral expression. Expression of a dominant-negative ezrin selectively disrupts apical expression and signaling of the PTH1R. However, dominant-negative ezrin does not affect expression or signaling of the basolateral PTH1R subpopulation. These findings reveal that direct ezrin interactions promote PTH1R apical localization and signaling in LLC-PK1 cells.

Ezrin promotes functional expression and parathyroid hormone-mediated regulation of the sodium-phosphate cotransporter 2a in LLC-PK1 cells.

The sodium-phosphate cotransporter 2a (NPT2a) is the principal phosphate transporter expressed in the brush border of renal proximal tubules and is downregulated by parathyroid hormone (PTH) through an endocytic mechanism. Apical membrane expression of NPT2a is dependent on interactions with the sodium-hydrogen exchanger regulatory factor 1 (NHERF-1). An LLC-PK1 renal cell line stably expressing the PTH receptor (PTH1R) and NHERF-1, termed B28-N1, fails to functionally express NPT2a. In B28-N1 cells, NHERF-1 and NPT2a are inappropriately localized to the cytoplasm. Ezrin, in the activated state, is capable at linking NHERF-1-assembled complexes to the actin cytoskeleton. Early-passage LLC-PK1 cells stably transfected with either empty vector or wild-type ezrin express a comparable level of the active, T567 phosphorylated form of ezrin and are capable of functionally expressing NPT2a. Colocalization of the PTH1R, NPT2a, and ezrin exists and is prominently associated with actin-containing microvilli in apical domains of these cells. Upon PTH treatment, the PTH1R, NPT2a, NHERF-1, and ezrin colocalize to endocytic vesicles and NPT2a-dependent phosphate uptake is markedly inhibited. LLC-PK1 cells expressing the constitutively active ezrin (T567D) display enhanced NPT2a functional expression and PTH-mediated regulation of phosphate. Expression of a dominant-negative ezrin, consisting of the NH(2)-terminal half of the protein, markedly disrupts NPT2a-dependent phosphate uptake. PTH does not appear to alter ezrin phosphorylation at T567. Instead, PTH perhaps initiates NPT2a endocytosis by inducing reorganization of the actin-containing microvilli in a process that is blocked by the actin-stabilizing compound jasplakinolide.

Coding GNAS mutations leading to hormone resistance impair in vitro agonist- and cholera toxin-induced adenosine cyclic 3',5'-monophosphate formation mediated by human XLalphas.

Most loss of function mutations of GNAS identified in different forms of pseudohypoparathyroidism disrupt not only the stimulatory G protein alpha-subunit (Gsalpha), but also its paternally expressed variant, XLalphas. However, the possibility that XLalphas deficiency contributes to disease pathogenesis has remained unexplored. We therefore examined the signaling property of human XLalphas and the effects of one novel (XLalphas(H704P) or Gsalpha(H362P)) and two previously described (XLalphas(DelI724) and XLalphas(Y733X) or Gsalpha(DelI382) and Gsalpha(Y391X), respectively) GNAS mutations on either XLalphas or Gsalpha activity. Confocal immunofluorescence microscopy detected human XLalphas immunoreactivity at the plasma membrane of transduced mouse embryonic fibroblasts null for endogenous Gsalpha and XLalphas (Gnas(E2-/E2-) cells). Cholera toxin- and isoproterenol-induced cAMP accumulation in Gnas(E2-/E2-) cells transiently expressing wild-type human XLalphas was similar to that in cells transiently expressing wild-type Gsalpha. Human XLalphas, like Gsalpha, mediated PTH-induced cAMP accumulation in Gnas(E2-/E2-) cells coexpressing PTH receptor type 1 and either of these proteins. Moreover, overexpression of human XLalphas or Gsalpha markedly enhanced the PTH-induced cAMP accumulation in opossum kidney cells that endogenously express PTH receptor type 1. In contrast, each XLalphas mutant failed to mediate isoproterenol- and PTH-induced cAMP accumulation in transduced Gnas(E2-/E2-) cells. XLalphas(DelI724) showed a reduced cholera toxin response over the basal level compared with wild-type XLalphas, and XLalphas(H704P) completely failed to respond to cholera toxin. These findings were comparable to those observed with each corresponding Gsalpha mutant transiently expressed in Gnas(E2-/E2-) cells. Thus, mutations that typically inactivate Gsalpha also impair XLalphas activity, consistent with a possible role for XLalphas deficiency in diseases caused by paternal GNAS mutations.

A docking site for G protein ßγ subunits on the parathyroid hormone 1 receptor supports signaling through multiple pathways.

The G protein-coupled receptor for PTH and PTH-related protein (PTH1R) signals via many intracellular pathways. The purpose of this work is to investigate a G protein binding site on an intracellular domain of the PTH1R. The carboxy-terminal, cytoplasmic tail of the PTH1R fused to glutathione-S-transferase interacts with Gi/o proteins in vitro. All three subunits of the heterotrimer interact with the receptor C-tail. Activation of the heterotrimeric complex with GTPgammaS has no effect on Gbetagamma interactions, but markedly disrupts binding of the Galphai/o subunits to the receptor tail, suggesting that direct Gbetagamma binding indirectly links Galpha subunits to this region of the receptor. Gbetagamma subunits alone bind the C-tail with an affinity that is comparable to the heterotrimeric G protein complex. G protein complexes consisting of Galphashis6-beta1gamma2 and Galphaqhis6-beta1gamma2 also interact with the PTH1R tail in vitro. The Gbetagamma interaction domain is located on the juxta-membrane region of the tail between amino acids 468 and 491. Mutations that disrupt Gbetagamma interactions block PTH signaling via phospholipase Cbeta/[Ca2+]i and MAPK and markedly reduce signaling via adenylyl cyclase/cAMP. Herein, we define a domain on the PTH1R that is capable of binding G protein heterotrimeric complexes via direct Gbetagamma interactions.

Mechanisms of ligand binding to the parathyroid hormone (PTH)/PTH-related protein receptor: selectivity of a modified PTH(1-15) radioligand for GalphaS-coupled receptor conformations.

Mechanisms of ligand binding to the PTH/PTHrP receptor (PTHR) were explored using PTH fragment analogs as radioligands in binding assays. In particular, the modified amino-terminal fragment analog, (125)I-[Aib(1,3),Nle8,Gln10,homoarginine11,Ala12,Trp14,Tyr15]rPTH(1-15)NH2, (125)I-[Aib(1,3),M]PTH(1-15), was used as a radioligand that we hypothesized to bind solely to the juxtamembrane (J) portion of the PTHR containing the extracellular loops and transmembrane helices. We also employed (125)I-PTH(1-34) as a radioligand that binds to both the amino-terminal extracellular (N) and J domains of the PTHR. Binding was examined in membranes derived from cells expressing either wild-type or mutant PTHRs. We found that the binding of (125)I-[Aib(1,3),M]PTH(1-15) to the wild-type PTHR was strongly (approximately 90%) inhibited by guanosine 5'-O-(3-thio)triphosphate (GTPgammaS), whereas the binding of (125)I-PTH(1-34) was only mildly (approximately 25%) inhibited by GTPgammaS. Of these two radioligands, only (125)I-[Aib(1,3),M]PTH(1-15) bound to PTHR-delNt, which lacks most of the receptor's N domain, and again this binding was strongly inhibited by GTPgammaS. Binding of (125)I-[Aib(1,3),M]PTH(1-15) to the constitutively active receptor, PTHR-H223R, was only mildly (approximately 20%) inhibited by GTPgammaS, as was the binding of (125)I-PTH(1-34). In membranes prepared from cells lacking Galpha(S) via knockout mutation of Gnas, no binding of (125)I-[Aib(1,3),M]PTH(1-15) was observed, but binding of (125)I-[Aib(1,3),M]PTH(1-15) was recovered by virally transducing the cells to heterologously express Galpha(S). (125)I-PTH(1-34) bound to the membranes with or without Galpha(S). The overall findings confirm the hypothesis that (125)I-[Aib(1,3),M]PTH(1-15) binds solely to the J domain of the PTHR. They further show that this binding is strongly dependent on coupling of the receptor to Galpha(S)-containing heterotrimeric G proteins, whereas the binding of (125)I-PTH(1-34) can occur in the absence of such coupling. Thus, (125)I-[Aib(1,3),M]PTH(1-15) appears to function as a selective probe of Galpha(S)-coupled, active-state PTHR conformations.

The receptor for parathyroid hormone and parathyroid hormone-related peptide is hydrolyzed and its signaling properties are altered by directly binding the calpain small subunit.

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 delta 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 delta 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.

Calmodulin interacts with the cytoplasmic tails of the parathyroid hormone 1 receptor and a sub-set of class b G-protein coupled receptors.

Parathyroid hormone (PTH) binds to its receptor (PTH 1 receptor, PTH1R) and activates multiple pathways. The PTH1R, a class b GPCR, contains consensus calmodulin-binding motifs. The PTH1R cytoplasmic tail interacts with calmodulin in a calcium-dependent manner via the basic 1-5-8-14 motif. Calcium-dependent calmodulin interactions with the cytoplasmic tails of receptors for PTH 2, vasoactive intestinal peptide, pituitary adenylate cyclase activating peptide, corticotropin releasing hormone, calcitonin, and the glucagon-like peptides 1 and 2 are demonstrated. The cytoplasmic tails of the secretin receptor and the growth hormone releasing hormone receptor either interact poorly or not at all with calmodulin, respectively. Fluphenazine, a calmodulin antagonist, enhances PTH-mediated accumulation of total inositol phosphates, suggesting that calmodulin regulates signaling via phospholipase C.

Stimulation by parathyroid hormone of a NHERF-1-assembled complex consisting of the parathyroid hormone I receptor, phospholipase Cbeta, and actin increases intracellular calcium in opossum kidney cells.

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 PLCbeta1 and -beta3 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, PLCbeta, and the actin cytoskeleton. Disruption of this complex blocks the PTH mediated increases of intracellular calcium.

Na+/H+ exchanger-regulatory factor 1 mediates inhibition of phosphate transport by parathyroid hormone and second messengers by acting at multiple sites in opossum kidney cells.

The opossum kidney (OK) line displays PTH-mediated activation of adenylyl cyclase and phospholipase C and inhibition of phosphate (Pi) uptake via regulation of the type IIa sodium-phosphate cotransporter, consistent with effects in vivo. OKH cells, a subclone of the OK cell line, robustly activates PTH-mediated activation of adenylyl cyclase, but is defective in PTH-mediated inhibition of sodium-phosphate cotransport and signaling via phospholipase C. Compared with wild-type OK cells, OKH cells express low levels of the Na+/H+ exchanger regulatory factor 1 (NHERF-1). Stable expression of NHERF-1 in OKH cells (OKH-N1) rescues the PTH-mediated inhibition of sodium-phosphate cotransport. NHERF-1 also restores the capacity of 8-bromo-cAMP and forskolin to inhibit Pi uptake, but the PTH dose-response for cAMP accumulation and inhibition of Pi uptake differ by 2 orders of magnitude. NHERF-1, in addition, modestly restores phorbol ester-mediated inhibition of Pi uptake, which is much weaker than that elicited by PTH. A poor correlation exists between the inhibition of Pi uptake mediated by PTH ( approximately 60%) and the inhibition mediated by phorbol 12-myristate 13-acetate ( approximately 30%) and the ability of these molecules to activate the protein kinase C-responsive reporter gene. Furthermore, we show that NHERF-1 directly interacts with type IIa cotransporter in OK cells. Although, PTH-mediated inhibition of Pi uptake in OK cells is largely NHERF-1 dependent, the signaling pathway(s) by which this occurs is still unclear. These pathways may involve cooperativity between cAMP- and protein kinase C-dependent pathways or activation/inhibition of an unrecognized NHERF-1-dependent pathway(s).

Na(+)/H(+ ) exchanger regulatory factor 2 directs parathyroid hormone 1 receptor signalling.

The parathyroid hormone 1 receptor (PTH1R) is a class II G-protein-coupled receptor. PTH1R agonists include both PTH, a hormone that regulates blood calcium and phosphate, and PTH-related protein (PTHrP), a paracrine/autocrine factor that is essential for development, particularly of the skeleton. Adenylyl cyclase activation is thought to be responsible for most cellular responses to PTH and PTHrP, although many actions appear to be independent of adenylyl cyclase. Here we show that the PTH1R binds to Na(+)/H(+) exchanger regulatory factors (NHERF) 1 and 2 through a PDZ-domain interaction in vitro and in PTH target cells. NHERF2 simultaneously binds phospholipase C beta 1 and an atypical, carboxyl-terminal PDZ consensus motif, ETVM, of the PTH1R through PDZ1 and PDZ2, respectively. PTH treatment of cells that express the NHERF2 PTH1R complex markedly activates phospholipase C beta and inhibits adenylyl cyclase through stimulation of inhibitory G proteins (G(i/o) proteins). NHERF-mediated assembly of PTH1R and phospholipase C beta is a unique mechanism to regulate PTH signalling in cells and membranes of polarized cells that express NHERF, which may account for many tissue- and cell-specific actions of PTH/PTHrP and may also be relevant to signalling by many G-protein-coupled receptors.