A Naturally Occurring Isoform Inhibits Parathyroid Hormone Receptor Trafficking and Signaling

Parathyroid hormone (PTH) regulates calcium homeostasis and bone remodeling through its cognitive receptor (PTHR). We describe here a PTHR isoform harboring an in-frame 42-bp deletion of exon 14 (Δe14-PTHR) that encodes transmembrane domain 7. Δe14-PTHR was detected in human kidney and buccal epithelial cells. We characterized its topology, cellular localization, and signaling, as well as its interactions with PTHR. The C-terminus of the Δe14-PTHR is extracellular, and cell surface expression is strikingly reduced compared with the PTHR. Δe14-PTHR displayed impaired trafficking and accumulated in endoplasmic reticulum. Signaling and activation of cAMP and ERK by Δe14-PTHR was decreased significantly compared with PTHR. Δe14-PTHR acts as a functional dominant-negative by suppressing the action of PTHR. Cells cotransfected with both receptors exhibit markedly reduced PTHR cell membrane expression, colocalization with Δe14-PTHR in endoplasmic reticulum, and diminished cAMP activation and ERK phosphorylation in response to challenge with PTH. Δe14-PTHR forms heterodimers with PTHR, which may account for cytoplasmic retention of PTHR in the presence of Δe14-PTHR. Analysis of the PTHR heteronuclear RNA suggests that base-pair complementarity in introns surrounding exon 14 causes exon skipping and accounts for generation of the Δe14-PTHR isoform. Thus Δe14-PTHR is a poorly functional receptor that acts as a dominant-negative of PTHR trafficking and signaling and may contribute to PTH resistance. © 2011 American Society for Bone and Mineral Research.


Introduction
T ype I parathyroid hormone (PTH) and PTH-related peptide receptor (PTHR) belong to family B, subfamily 1, of G proteincoupled receptors (GPCRs). Other members include receptors for secretin, vasoactive intestinal peptide, growth hormonereleasing hormone, glucagon, glucagon-like peptide, pituitary adenylyl cyclase-activating peptide, corticotropin-releasing hormone, and calcitonin (CTR). (1) The PTHR is expressed predominantly in kidney and bone, where it mediates PTH actions on calcium and phosphate homeostasis and bone turnover, respectively. (2) In humans, the PTHR gene contains 15 exons ÃÃ coding a 593amino-acid, 7-transmembrane-domain (TMD) receptor. (3,4) Family B1 GPCRs are characterized by an exon-intron organiza-tion that permits alternative splicing of specific critical domains that have been shown in some instances to alter the function of the resulting isoform. (5) Some of these family B isoforms are characterized by the deletion of regions encoding the seventh TMD (TMD7). (5)(6)(7)(8) The biologic role of these isoforms is largely unexplored, but studies with corticotropin-releasing hormone receptor (CRHR) variants suggest that they could be cellular response modulators affecting CRHR signaling. (6) Several PTHR isoforms, or transcripts consistent with receptor isoforms, have been described. (9)(10)(11) It has been suggested that presumptive nonfunctional PTHR isoforms could be the source of pathologies associated with PTH dysfunction, including some cases of pseudohypoparathyroidism type Ib (PHPIb). (12) Analysis of the exon coding structure and promoter regions of the PTHR gene or its mRNA, however, failed to disclose mutations. (13)(14)(15)(16) The biologic behavior and functional consequence of alternatively spliced PTHR forms on signaling and trafficking and their effects on PTHR action are unknown. We now show the existence of a PTHR isoform lacking TMD7, which is encoded by exon 14 (De14-PTHR), in human renal epithelial cells. We characterized De14-PTHR and its actions as a modulator of PTHR. De14-PTHR expression is primarily cytoplasmic, where it interacts with the PTHR in endoplasmic reticulum, thereby reducing delivery of the wild-type receptor to the cell membrane and simultaneously promoting PTHR downregulation. Nonetheless, some De14-PTHR is expressed at the plasma membrane, but the absence of TMD7 results in extracellular localization of C-terminal receptor tail. Signaling via cAMP formation and p44/42 MAP kinase [extracellular signal-regulated kinase (ERK)] phosphorylation were decreased in response to PTH. De14-PTHR also decreases cAMP and ERK responses when coexpressed with the fully active PTHR. We conclude that De14-PTHR acts as a dominant-negative of PTHR and causes PTH resistance.
pBudCE4.1þ-Flag-PTHR-His and HA-De14-PTHR-His were obtained in the following manner: Flag-PTHR and HA-De14-PTHR were amplified using the forward primer with NotI restriction site (AGAAGAAGAAAGCGGCCGCATGGGGACCGCCC-GGATC), and the reverse primer with BstBI restriction site (CGGAGGAGAATTTCGAACATGACTGTCTCCCACTC). Purified PCR fragments were cut by NotI and BstBI and subcloned into the pBudCE4.1 before a polyhistidine-expressing region.

Transient transfection
Cells were grown to 50% to 60% confluence and transfected, as indicated with 1 mg of DNA per well in 6-well plates with HA-PTHR, Flag-PTHR, HA-De14-PTHR, Flag-De14-PTHR, and EPAC, Rab 5, Rab 7, Rab 11, and Arf 1 (22) (kindly provided by Dr J-P Vilardaga) using FuGENE 6 (Roche, Indianapolis, IN, USA) according to the manufacturer's protocol. Experiments involving transfection of PTHR isoforms, Rabs or Arf, alone or in combination, were performed with constant amounts of each cDNA and adding empty-vector DNA (pcDNA3.1) when only one was expressed to keep constant the total amount of DNA. All experiments were performed 48 hours after transfection.

Immunoblot analysis
Transiently transfected cells with different combinations of PTHR isoforms were lysed with Nonidet P40 (50 mM Tris, 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P40) supplemented with protease inhibitor mixture I and incubated for 30 minutes on ice. Lysates were centrifuged for 20 minutes at 14,000g at 48C.
Total lysate proteins were analyzed by SDS-PAGE and transferred to Immobilon-P membranes (Millipore, Billerica, MA, USA) using the semidry method (BioRad, Hercules, CA, USA). Nonspecific binding was blocked by incubating the membranes in 5% nonfat milk in Tris-buffered saline plus 0.1% Tween-20 (TBST) for 1 hour at room temperature, followed by overnight incubation with the indicated antibodies (monoclonal anti-Flag and anti-HA antibodies, polyclonal anti-phospho p42/ 44 and anti-p42/44 antibodies at 1:1000) at 48C. The membranes then were washed and incubated at room temperature for 1 hour in horseradish peroxidase (HRP)-conjugated goat antirabbit IgG or sheep antimouse IgG diluted 1:2000. Protein bands were visualized with a luminol-based enhanced chemiluminescence substrate.

Receptor binding
Receptor binding was measured as described previously (19,23) (24) Immunofluorescence confocal microscopy Cells were seeded on coverslips and allowed to settle overnight. Then 100 nM PTH(1-34) was added for the indicated times, and cells were fixed with 4% paraformaldehyde. Permeabilized samples were treated for 10 minutes with 0.1% Triton X-100 in PBS. Nonspecific binding was blocked with 5% goat serum in PBS for 1 hour at room temperature. Polyclonal anti-HA and anti-LAMP-2 and monoclonal anti-Flag or anti-His antibodies were added for 1 hour at room temperature.
After three PBS washes, samples were incubated with Alexa-Fluor 488 or Alexa-Fluor 546 (1:1000) for 1 hour at room temperature. 4',6-Diamidino-2-phenylindole (DAPI) was used to stain the cell nucleus in some samples. Slides were mounted with aqueous mounting medium and examined by confocal microscopy using an Olympus FluoView 1000 (Olympus Corp., Lake Success, NY, USA).

Receptor internalization
PTHR internalization was measured in cells transiently transfected with HA-PTHR, HA-De14-PTHR, or HA-PTHR plus Flag-De14-PTHR. Cells were seeded on poly-D-lysine-coated 24-well plates. Confluent cells were treated with PTH and fixed with 3.7% paraformaldehyde at room temperature. After 3 washes with PBS, cells were blocked with 1% bovine serum albumin (BSA) for 45 minutes and incubated with polyclonal anti-HA antibody for 1 hour at room temperature. Cells then were washed with PBS, reblocked with 1% BSA for 15 minutes, and incubated with anti-IgG conjugated with alkaline phosphatase (ELISA protocol) or antirabbit Alexa Fluor 680 nm (flow cytometry protocol) for 1 hour at room temperature. After washing, alkaline phosphatase substrate was added for 30 minutes, 100 mL of the reaction mixture was transferred to a 96-well plate, and absorbance was measured at 405 nm (ELISA protocol).

Fluorescence resonance energy transfer (FRET)
HEK-293 cells were transiently transfected with the cAMP biosensor EPAC. (25) Cells plated on poly-D-lysine-coated glass 25-mm coverslips were maintained in HEPES/BSA buffer. Coverslips were mounted on the stage of an Olympus IX 71 microscope equipped with a 60Â oil-immersion objective and a monochromator (TILL Photonics, Gräfelfing, Germany). FRET was monitored as the emission ratio of YFP and CFP with SlideBook (Intelligent Imaging Innovations, Inc., Denver, CO, USA). FRET was calculated and normalized as described previously. (26) Results are shown as the normalized mean (nFRET) AE SEM.
Semiquantitative RT-PCR Total cell RNA was isolated with TRIZOL. Then 400 ng of RNA was reverse-transcribed, and the resulting cDNA was amplified using a commercial kit (Titanium One-Step RT-PCR, Clontech, Palo Alto, CA, USA) with the primers GTCCAGATGCACTATGAG (forward) and GACATTGGTCACACTTGT (reverse), corresponding to nucleotides 1315 to 1332 and 1507 to 1524, respectively, in the human PTHR gene (GenBank Accession Number NM 000316). GAPDH primers GAGTCAACGGATTGGTCGT (forward) and TTGATTTTG-GAGGGATCTCG (reverse) were used for GAPDH coamplification as an internal control. PCR products were separated on 2% agarose gels, and bands were visualized by ethidium bromide staining. Quantitative PCR (qPCR) experiments used the same primers. TaqMan MGB probes were obtained by Assay-by-Design (Applied Biosystems). PTHR VIC-TCGCAATCATATACTGTTTCT-GCAA-TAMRA and De14-PTHR 6FAM-TCAACTCCTTCCAGG-TACAAGCTGAGA-TAMRA cDNA was synthesized using AccuScript High Fidelity RT-PCR System (Stratagene, La Jolla, CA, USA) with random hexamer primers, and qPCR was carried out with an ABI PRISM 7500 System (Applied Biosystems) following the manufacturer's instructions.

Image analysis
Colocalization of De14-PTHR within cytoplasmic compartments was analyzed with ImageJ (27) to calculate the Pearson coefficient, which is defined here as the ratio of the covariance of the red and green color images divided by the product of the standard deviation of the normalized image intensities.

Statistics
Data are presented as the mean AE SE, where n indicates the number of independent experiments. Multiple comparisons were evaluated by one-or two-way analysis of variance with posttest repeated measures analyzed by the Bonferroni procedure (Prism, GraphPad). Differences greater than p .05 were assumed to be significant.

Expression of De14-PTHR in human cells
Previous data from family B1 GPCRs suggested the possibility of an alternatively spliced form of the PTHR lacking TMD7. (5)(6)(7)(8)12) To identify a PTHR isoform with these characteristics in human cells, mRNA from renal tubule cells collected from urine and/or buccal epithelial cells was analyzed. Amplification by RT-PCR generated a fragment of the expected 217 bp indicating PTHR gene expression (Fig. 1A). Notably, an additional smaller product of 171 bp was detected in renal and in some buccal mRNA samples (Fig. 1A), consistent with the size of small PTHR transcripts reported in rat kidney cells. (9) The smaller band was sequenced and corresponds to the PTHR mRNA with an in-frame 42-bp deletion corresponding to exon 14, which encodes most of TMD7 (data not shown). No mutations were noted in the coding regions or in the corresponding donor and acceptor splice sites.
HK-2 renal tubular epithelial cells expressed both PTHR and De14-PTHR forms of the receptor, whereas HKC-8 cells expressed only wild-type PTHR. Full-length and truncated PTH receptors specifically designed were transfected in HEK-293 as a control (Fig. 1A, bottom). The presence of De14-PTHT was corroborated by qPCR using probes specific for this alternatively spliced variant (Fig. 1B).

De14-PTHR topology
We analyzed the predicted topology of the De14-PTHR and compared it with the wild-type receptor using the TMHMM algorithm (http://workbench.sdsc.edu), which predicts transmembrane helices and inverted-loop regions based on a hidden Markov model. (28) Whereas the PTHR displayed the expected heptahelical protein conformation with an intracellular C-terminus, the De14-PTHR folds with 100% probability as a 6-transmembrane-spanning receptor without TMD7 and with the C-terminus located extracellularly ( Fig. 2A). To test this prediction, we generated De14-PTHR with a polyhistidine (6Â His) tag at the C-terminus. The localization of De14-PTHR was determined by confocal microscopy with CHO-N10 cells. In nonpermeabilized cells, PTHR was undetectable, consistent with the inaccessible C-terminal epitope tag in the cytoplasm (Fig. 2B). Under the same conditions, distinct De14-PTHR cell surface fluorescence is present. In permeabilized cells, both De14-PTHR and PTHR immunofluorescence are observed (Fig. 2B). These findings are compatible with an extracellular localization of the C-terminus of De14-PTHR.
Cytoplasmic De14-PTHR expression To assess the subcellular distribution of De14-PTHR, we transiently transfected HEK-293 cells with truncated or fulllength PTH receptors. Confocal microscopy shows that HA-PTHR clearly localizes to the cell membrane (Fig. 3A). Similar results were obtained in CHO-N10 cells and with Flag-PTHR or GFP-PTHR (images not shown). In contrast, Flag-De14-PTHR exhibited conspicuously lower cell surface expression but intense cytoplasmic abundance (Fig. 3A). HA-De14-PTHR also was predominantly cytoplasmic with little plasma membrane expression (image not shown).
We next characterized the influence of De14-PTHR on PTHR distribution. Truncated and full-length receptors were cotransfected in HEK-293 and CHO-N10 cells. Whereas PTHR is not normally observed in cytoplasm (Figs. 2B and 3), strong cytoplasmic colocalization of GFP-PTHR and Flag-De14-PTHR was observed in HEK-293 cells (Fig. 3A). Similar results were obtained in CHO-N10 cells (images not shown). These findings suggest that De14-PTHR causes retention of PTHR in the cytoplasm.
To determine if the interference by De14-PTHR of membrane targeting is specific to the PTHR, we examined the effect of De14-PTHR on the localization of the calcitonin receptor (CTR), a family B receptor with a helix 7 isoform, and the b 2 -adrenergic receptor, a prototype family A receptor. HA-CTR or GFP-b 2 -adrenergic receptors were cotransfected with Flag-De14-PTHR. Both HA-CTR and GFP-b 2 -adrenergic receptors localized to the plasma membrane and did not colocalize with De14-PTHR in the cytoplasm (Fig. 3A). When cotransfected with GFP-PTHR, the HA-CTR showed no effects on PTHR expression at the cell membrane (data not shown). Thus the retention of PTHR in the cytoplasm is specific for De14-PTHR.
In the presence of De14-PTHR, cell surface expression of PTHR decreased by 56% (0.7 Â 10 6 receptors/cell). These findings confirm that De14-PTHR suppresses PTHR membrane expression. Considering the effects of the TMD7 on PTHR topology, we turned our attention to whether this truncation affects inherent affinity for PTH. Scatchard analysis of ligand-binding showed K d values of 5 nM for PTHR, 40 nM for De14-PTHR, and 12 nM when both receptors were cotransfected.
We performed coimmunoprecipitation experiments to determine directly whether De14-PTHR and PTHR interact. Immunoprecipitation of the full-length receptor and immunodetection of PTHR or De14-PTHR showed that both receptors homo-or heterodimerize, respectively (Fig. 3C). The reverse experiment, where the truncated De14-PTHR was immunoprecipitated and the PTHR or De14-PTHR was immunoblotted exhibited compar-able results (data not shown). In addition to De14-PTHR and PTHR heterodimerization, we also observed PTHR homodimerization (Fig. 3C). Together these results show that De14-PTHR interacts directly with PTHR.
To determine the dynamic behavior of De14-PTHR and PTHR and their trafficking response to PTH, we analyzed receptor internalization by an ELISA assay using nonpermeabilized HEK-293 cells. As shown in Fig. 3D, the PTHR was efficiently internalized 30 minutes after PTH . De14-PTHR membrane expression was conspicuously lower than that of the PTHR and did not appreciably internalize on PTH stimulation (Fig. 3D).
We next examined De14-PTHR effects on PTH-induced internalization of the PTHR. De14-PTHR decreased PTHR membrane-delimited expression by 52% (Fig. 3D). PTH induced proportionately similar PTHR internalization in the presence or absence of De14-PTHR (Fig. 3D). Similar results were obtained by flow cytometry (data not shown). These findings suggest that De14-PTHR does not affect internalization of the reduced subset of membrane-delimited PTHR.

Retention of De14-PTHR in the endoplasmic reticulum
The difference between PTHR and De14-PTHR subcellular localization led us to investigate the intracellular compartmentalization of De14-PTHR. We performed confocal microscopy to determine the identity of endosomes containing De14-PTHR in HEK-293 cells transfected with either green fluorescent protein (GFP)-tagged Rab5, -7, or -11 or Arf 1, GTPases that control trafficking of early and late, recycling, and Golgi network endosomes, respectively. Modest levels of De14-PTHR were found in Rab11 þ and Arf1 þ compartments, corresponding to pericentriolar recycling endosomes and the trans-Golgi network ( Fig. 4 and Table 1). No significant localization of De14-PTHR was observed with Rab5 þ or -7 þ early and late endosomes, respectively ( Fig. 4 and Table 1). To determine if De14-PTHR is targeted to the endocytic degradative pathway or endoplasmic reticulum (ER), we used a lysosomal-associated membrane protein (LAMP-2) antibody or a fluorescent ER-Tracker, respectively, in HEK-293 cells transfected with HA-De14-PTHR. Although De14-PTHR was not found in LAMP-2 þ lysosomes, extensive De-14PTHR was observed within ER ( Fig. 4 and Table 1). These results, along with the previous findings showing limited De14-PTHR expression at the cell surface, suggest an early impairment of De14-PTHR trafficking to the membrane and retention within the ER. In contrast, PTHR is not detectable in Rab5, -7, or -11, Arf 1, LAMP-2-positive compartments or in ER (Supplemental Fig. S2). Thus, under resting conditions, the PTHR is found only at the cell membrane. However, in the presence of De14-PTHR, considerable ER accumulation of PTHR is observed (Fig. 5A).

De14-PTHR decreases PTHR protein expression
Decreased cell membrane De14-PTHR expression combined with cytoplasmic accumulation raised the possibility that these effects could be due to decreased protein synthesis alone or in combination with increased receptor degradation. Indeed, we observed decreased De14-PTHR protein expression levels compared with PTHR (Fig. 5B). Moreover, cotransfection of De14-PTHR impaired PTHR expression (Fig. 5B). Notably, no differences in PTHR mRNA expression were observed in cells co- transfected with De14-PTHR (Fig. 5B). Similar data were obtained in HEK-293 and COS-7 cells and by PCR (data not shown).
Net receptor protein expression is a balance between synthesis and degradation. To test the hypothesis that proteasome-or lysosome-dependent degradative mechanisms contribute to diminished De14-PTHR protein levels, HEK-293 cells transfected with De14-PTHR were treated with MG-132 or chloroquine, proteasome and lysosome inhibitors, respectively. De14-PTHR protein expression rebounded after proteasome blockade (t 1/2 ¼ 5.39 hours; Fig. 5C). Lysosome inhibition did not affect De14-PTHR degradation (data not shown). Within experimental error, neither proteosomal nor lysosomal degradation of PTHR was detected (data not shown). Thus De14-PTHR is metabolized by ubiquitination and targeted to proteasomes. When De14-PTHR was cotransfected with PTHR, however, PTHR protein levels that were diminished in the presence of De14-PTHR now increased toward basal expression values when pretreated with the proteasome inhibitor (t 1/2 ¼ 2.0 hours; Fig. 5C). Again, lysosomal inhibition was without effect (data not shown).

De14-PTHR inhibits PTHR signaling
As shown earlier, the absence of TMD7 impairs membrane localization of De14-PTHR and alters its subcellular distribution, suggesting that its biologic response to PTH likely would be compromised. We therefore characterized the signaling capability of De14-PTHR by measuring cAMP and ERK responses to PTH, two well-established and independent signaling mechanisms. Using the cAMP FRET biosensor EPAC (exchange protein directly activated by cAMP), we observed a rapid increase of cAMP formation (denoted as the CFP/YFP ratio) triggered by PTH  in HEK-293 cells transfected with PTHR (t 1/2 ¼ 0.42 AE Fig. 4. Internalized De14-PTHR localizes in the endoplasmic reticulum (ER). HEK-293 cells were transiently cotransfected with Flag-De14-PTHR and GFP-Rab 5, GFP-Rab 7, GFP-Rab 11, or GFP-Arf 1 as indicated, grown on glass cover slips for 48 hours, fixed, and permeabilized as described in ''Materials and Methods.'' Flag-tagged De14-PTHR was detected using a specific primary antibody for Flag (1:1000) and Alexa-Fluor 546 (1:2000) (red) or Alexa-Fluor 488 (1:2000) (green). Lysosomes were detected using a rabbit polyclonal anti-LAMP-2 antibody (1:1000) and Alexa-Fluor 488 (1:2000) (green), and the ER was detected using ER-Tracker Red. Right panels show the merged images. Colocalization of the green and red labels is shown in yellow. The cells were examined by confocal microscopy. Representative images of at least three independent experiments are shown. 0.05 minutes; Fig. 6A). The longer t 1/2 of 0.86 AE 0.16 minutes for the De14-PTHR suggests that cAMP signaling is impaired (Fig. 6A). Additionally, we observed limited ERK phosphorylation in response to PTH  in CHO-N10 cells transfected with De14-PTHR compared with PTHR (Fig. 6B).

Discussion
This study reveals the presence of a novel, alternatively spliced PTHR isoform in renal tubular epithelial cells and characterizes its trafficking and signaling, as well as its structural and functional interactions with the full-length PTHR. The low abundance of De14-PTHR at the plasma membrane underscores the importance of the TMD7 for proper receptor targeting and integration at the cell surface and for membrane retention. The structural basis for the critical role of this domain for accurate membrane receptor localization is not well understood. Failure of receptor export or decreased stability at the membrane could account for reduced De14-PTHR cell surface expression. A GFF motif within the conserved region of TMD7 is indispensable for CRHR membrane expression. (8) This motif, which also is present in the PTHR, may be essential to form the seventh hydrophobic helix, and in its absence, the consequent protein misfolding does not allow the receptor to be transported through the endoplasmic reticulum (ER). (8) Other checkpoint motifs described for vasopressin V 2 , angiotensin II, dopamine D 1 , V1b/V3, and b 2adrenergic receptors are necessary for ER-to-Golgi transfer. (29)(30)(31)(32)(33) However, these motifs are absent in the PTHR C-terminus. Alternatively, excision of the De14-PTHR TMD7 could generate a motif that inhibits transit of the truncated receptor to the membrane by unmasking a cryptic retention signal, as observed in g-aminobutyric acid (GABA) receptors. (34) Dimerization is required for some GPCRs to be transported to the plasma membrane. (35) The C-terminus of the GABA B receptor, for instance, is critical to promote receptor dimerization. More specifically, heterodimerization of GABA B receptors uses the C-terminal retention motif RXR(R), (36) which also is present in the PTHR. It is thus possible that the nascent PTHR is formed as a dimer that dissociates in the ER before transport to the plasma membrane. Recent evidence demonstrates that the PTHR is targeted to the plasma membrane as a dimer and dissociates on binding PTH. (37) PTHR-De14-PTHR heterodimers may not be able to dissociate, accounting for the cytoplasmic accumulation of PTHR in the presence of De14-PTHR. Heterodimerization of CTR with its truncated isoform, a process that involves the C-terminus, prevents transport of the receptor to the cell surface. (5) The aberrant orientation of the De14-PTHR C-terminus and protein misfolding could act on the PTHR in a similar manner, causing accumulation in the ER and retention of the full-length PTHR, thereby impairing its transport to the cell membrane.
In addition to lower expression at the cell surface, De14-PTHR exhibits lower affinity for PTH. Thus TMD7 influences PTH binding, as it does calcitonin binding to CTR, (5) although TMD7 is not necessary for agonist binding to CRH-R1d. (6) Hence similar motifs are capable of exerting distinct roles on ligand affinity to family B GPCRs. Compared with their full-length receptor counterparts, CRHR and CTR isoforms lacking the seventh TMD exhibited impaired ligand-stimulated cAMP formation (6,38) or limited coupling to Gs, Gq, Gi, and Go in the case of the CRHR isoform, CRH-R1d. (6) The fact that the t 1/2 for adenylyl cyclase activation by PTH was reduced suggests that De14-PTHR coupling to adenylyl cyclase is compromised. This kinetic manifestation arises as a consequence of decreased activated (receptor-ligand) complex. By contrast, normalizing the extent of cAMP formation to receptor number indicates that there is no change in De14-PTHR intrinsic activity (ie, the magnitude of the response). Similar observations were reported for the truncated isoform of CTR, which failed to mobilize intracellular calcium or phosphorylate ERK. (8) Thus the reduced signaling by De14-PTHR is likely due to a combination of the 10-fold lower expression of De14-PTHR at the cell membrane and diminished ligand affinity.
Several key signaling motifs situated within the PTHR intracellular tail are inaccessible in the De14-PTHR owing to its extracellular location. This also could contribute importantly to the diminished signaling by the De14-PTHR. For instance,  (27) The calculation shows colocalization of De14-PTHR with Arf 1 (Golgi apparatus), Rab 5 (early endosomes), Rab 7 (late endosomes), Rab 11 (recycling endosomes), LAMP-2 (lysosomes), and endoplasmic reticulum (ER). Technical details are described in ''Materials and Methods.'' Ã p < .5 significant positive colocalization. n ¼ 5 to 8 independent observations for each condition. mutations in the juxtamembrane region of the C-tail between amino acids 468 and 491 of the PTHR disrupt Gßg interactions with the receptor, block PTH signaling by phospholipase C and ERK, and markedly reduce cAMP signaling. (39) Furthermore, the PTHR C-terminus contains several proline-rich motifs that are essential to trigger ERK phosphorylation by c-Src and arrestin activation (36) that would not be available in De14-PTHR. Negative and positive regulators of PTHR endocytosis that are present within the upstream region of the PTHR intracellular tail (40) would no longer exert their actions in the De14-PTHR. Finally, cytoplasmic PDZ scaffolding proteins such as NHERF1 that interact with the C-terminus and regulate signaling and PTHR trafficking (19,23,(41)(42)(43) would be incapable of exerting their modulatory actions on the De14-PTHR. Thus the redirected extracellular C-terminus of the De14-PTHR, in combination with limited De14-PTHR expression at the plasma membrane also may contribute to the reduced signaling of this naturally occurring receptor isoform. Protein synthesis is regulated at multiple levels during transcription and translation. Our results show that diminished PTHR expression is not due to downregulation at transcriptional levels because similar PTHR mRNA expression was observed in the presence or absence of De14-PTHR. This suggests possible posttranscriptional modulation of PTHR expression by the truncated receptor. Proteins localized at the plasma membrane usually are degraded by lysosomes, (44) whereas misfolded proteins that accumulate in cytoplasmic compartments such as the ER, the ER/Golgi intermediate compartment (ERGIC), or , and/or Flag-De14-PTHR as indicated were grown on 6-well plates for 48 hours and serum-starved for 2 hours before stimulation with 100 nM PTH(1-34) for 10 minutes. Total lysates were extracted, and immunoblotting was performed as described in ''Materials and Methods.'' Phospho-p44/42, total p44/42, and HA and Flag epitopes were detected using specific primary antibodies (1:1000) and HRPtagged antibodies (1:2000). Upper panels show representative immunoblot images. Data illustrate three independent experiments performed in triplicate. Ã p < .05 versus control.
the Golgi apparatus eventually are targeted for metabolism by the ubiquitination-and proteasome-dependent ER-associated degradation pathway (ERAD) or by mechanisms that remain unknown, respectively. (45,46) De14-PTHR could interact with PTHR in the ER, ERGIC, or Golgi compartments, leading to its retention and subsequent proteolysis by proteasome degradation. The response to PTH, as in HK-2 cells, could be diminished owing to expression of the De14-PTHR compared with other cells that do not express this isoform.
Exon skipping is a common mechanism of genomic combinatorial control of alternative splicing. (47) The introns flanking the skipped exon typically possess specific sequences, in addition to the canonical splice donor and acceptor sequences that regulate where skipping occurs. A G-rich region distal to the 5' splice donor and a C-rich region proximal to the 3' splice acceptor play key roles in this process. (48) These regions form a stem-loop structure in the heteronuclear RNA (hnRNA) that makes it possible to bring, in the case of the PTHR, exons 13 and 15 close together and permit the deletion of exon 14. The small, 42-bp size of exon 14 makes it an ideal candidate for exon skipping. In a stretch of 11 bases, 8 are complementary. Moreover, although there is significant complementarity between the 5' G-rich region upstream of the exon 14 and the C-rich region downstream of exon 14, it is not perfect. This could permit small nuclear ribonuclear proteins (snRNPs) that regulate the splicing process to promote inclusion or exclusion of exon 14.
Pseudohypoparathyroidism type 1b (PHP1b) is characterized by renal PTH resistance accompanied by hypocalcemia, hyperphosphatemia, and elevated serum PTH levels. (49) Defective genomic imprinting of GNAS accounts for most cases of familial PHP1b. However, autosomal dominant inheritance does not explain the majority of cases of PHP1b (50) or a significant portion of PHP1a. (51) Regulated expression of De14-PTHR by snRNPs might affect PTHR abundance in the kidney. Accumulation of De14-PTHR in cells expressing PTHR from different tissues, we propose, inhibits signaling and function of the full-length receptor and could explain PTH resistance in some cases of pseudohypoparathyroidism and perhaps in other forms of PTH or PTH-related protein (PTHrP) resistance of unknown origin.
In summary, De14-PTHR is present in renal tubular epithelial cells, where it exhibits reduced anchorage to the plasma membrane, mislocation of its C-terminus to the extracellular compartment, and accumulation in the ER and displays impaired cAMP and ERK signaling. Moreover, De14-PTHR decreases PTHR cell surface expression and protein levels, forms heterodimers with PTHR, and also inhibits PTHR-mediated cAMP and ERK signaling. Exon 14 deletion may arise from a regulated but as yet poorly understood pattern of hnRNA complementarity common to family B receptors.

Disclosures
All the authors state that they have no conflicts of interest.