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Keywords:

  • PARATHYROID HORMONE;
  • STIMULATORY G PROTEIN;
  • CYCLIC AMP;
  • PSEUDOHYPOPARATHYROIDISM;
  • ALBRIGHT HEREDITARY OSTEODYSTROPHY

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Pseudohypoparathyroid patients have resistance predominantly to parathyroid hormone (PTH), and here we have examined the ability of an alternative Gαs-related protein to inhibit Gαs activity in a hormone-selective manner. We tested whether the GNAS exon A/B-derived NH2-terminally truncated (Tr) αs protein alters stimulation of adenylate cyclase by the PTH receptor (PTHR1), the thyroid-stimulating hormone (TSH) receptor (TSHR), the β2-adrenergic receptor (β2AR), or the AVP receptor (V2R). HEK293 cells cotransfected with receptor and full-length (FL) Gαs ± Tr αs protein expression vectors were stimulated with agonists (PTH [10−7 to 10−9 M], TSH [1 to 100 mU], isoproterenol [10−6 to 10−8 M], or AVP [10−6 to 10−8 M]). Following PTH stimulation, HEK293 cells cotransfected with PTHR1 + FL Gαs + Tr αs had a significantly lower cAMP response than those transfected with only PTHR1 + FL Gαs. Tr αs also exerted an inhibitory effect on the cAMP levels stimulated by TSH via the TSHR but had little or no effect on isoproterenol or AVP acting via β2AR or V2R, respectively. These differences mimic the spectrum of hormone resistance in pseudohypoparathyroidism type 1a (PHP-1a) and type 1b (PHP-1b) patients. In opossum kidney (OK) cells, endogenously expressing the PTHR1 and β2AR, the exogenous expression of Tr αs at a level similar to endogenous FL Gαs resulted in blunting of the cAMP response to PTH, whereas that to isoproterenol was unaltered. A pseudopseudohypoparathyroid patient with Albright hereditary osteodystrophy harbored a de novo paternally inherited M1I Gαs mutation. Similar maternally inherited mutations at the initiation codon have been identified previously in PHP-1a patients. The M1I αs mutant (lacking the first 59 amino acids of Gαs) blunted the increase in cAMP levels stimulated via the PTHR1 in both HEK293 and OK cells similar to the Tr αs protein. Thus NH2-terminally truncated forms of Gαs may contribute to the pathogenesis of pseudohypoparathyroidism by inhibiting the activity of Gαs itself in a GPCR selective manner. © 2011 American Society for Bone and Mineral Research


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Pseudohypoparathyroidism (PHP) involves target-organ resistance primarily to the actions of parathyroid hormone (PTH), leading to hypocalcemia and hyperphosphatemia with reduced serum concentrations of 1,25-dihydroxyvitamin D.1 Serum PTH levels are elevated, and exogenous PTH fails to increase urinary cAMP and phosphate excretion.

There are several different forms of PHP.2 Patients with PHP type 1a (PHP-1a) have somatic defects termed Albright hereditary osteodystrophy (AHO) that include short stature, brachydactyly, round facies, obesity, and ectopic ossification and exhibit resistance to several hormones, including PTH, thyroid-stimulating hormone (TSH), gonadotropins, and growth hormone–releasing hormone.3 Pseudo-PHP (PPHP) patients have AHO without hormone resistance.4 PHP-1b patients are characterized by renal resistance to PTH and mild thyroid resistance to TSH in the absence of other physical or endocrine abnormalities.2

PTH and the other hormones for which resistance has been identified are agonists for G protein–coupled receptors (GPCRs) that couple to the heterotrimeric Gαs/β/γ complex.5 Heterozygous loss-of-function mutations in the exons encoding Gαs at the guanine nucleotide (binding) α subunit (GNAS) locus (at 20q13.3) that are inherited from the mother lead to Gαs deficiency and are expressed as PHP-1a. In contrast, when the mutations are inherited from the father, patients present with PPHP.6 The erythrocyte Gαs function of PHP-1a and PPHP patients is reduced, whereas the erythrocyte Gαs function of PHP-1b patients is normal. Accordingly, PHP-Ib patients do not have mutations in the Gαs-encoding exons. PHP-1b is the result of imprinting defects on the maternal allele at the GNAS locus,7, 8 although the precise mechanisms causing PHP-1b endocrinopathies remain to be elucidated.

GNAS generates multiple mRNA and protein products through the use of four alternative first exons (XL, NESP, A/B [also known as 1A], and 1) that all splice to exon 2 of the 13 exons that encode Gαs.9 The GNAS locus is under complex imprinting control. The major GNAS product, Gαs, which couples GPCRs to stimulation of adenylate cyclase, is generated from the most downstream promoter. The exon 1–derived mRNA is partially imprinted in some human tissues (e.g., pituitary, ovary, and thyroid), being expressed predominantly from the maternal allele10, 11 and biallelically expressed in other tissues.12, 13 In contrast, the XL and A/B transcripts are expressed specifically from the paternal allele, whereas the NESP transcript is derived from the maternal allele.14, 15 XLαs is a large isoform of Gαs that can functionally couple to receptors and activate adenylate cyclase.16–19 The NESP mRNA encodes a chromogranin-like protein20 that has no Gαs sequence because the mRNA stop codon lies within the NESP exon before the site that splices to exon 2. Hence the NESP mRNA uses Gαs exons 2 to 13 as its 3' untranslated region (UTR).

Exon A/B does not have an initiation codon, but it has been shown that translation of the A/B transcript can be initiated at an in-frame ATG start site within exon 2.21, 22 In transfected cells, the transcript encodes an NH2-terminally truncated (∼38 kDa) Gαs isoform lacking the first 59 amino acids of Gαs, a region that contains the βγ-binding site and one of the receptor interacting regions. The rest of the molecule, however, is conserved, including the receptor-interacting region at the COOH-terminus. Therefore, the truncated Gαs isoform potentially could inhibit the activity of Gαs.

In this study we have identified a novel de novo c.3G > A M1I mutation on the paternal allele of a PPHP proband who exhibited the brachydactyly of AHO. In this case, it would be predicted that translation initiation at the in-frame codon 60 ATG in exon 2 would result in an NH2-terminally truncated Gαs protein. We have tested the ability of NH2-terminally truncated Gαs proteins (encoded by transfected constructs for either the A/Bαs transcript, a Tr αs transcript that comprises Gαs exons 2 to 13, or a Gαs transcript incorporating the M1I mutation) to alter stimulation of adenylate cyclase following activation of the PTH receptor (PTHR1), the TSH receptor (TSHR), the β2-adrenergic receptor (β2AR), or the AVP receptor (V2R).

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Patient

The proband (Fig. 1A, individual III-1) was a full-term female baby (2.5 kg, 47 cm) born by C-section for breech presentation to normal parents (II-5, mother, 52.3 kg, 157.5 cm; II-6, father, 77.3 kg, 177.8 cm) and has two healthy siblings (III-2 and III-3). The infant exhibited breath-holding spells (8 months to 2.5 years of age) and suffered a febrile seizure at 15 months of age in association with vaccinations. She had no history of paresthesias or muscle cramps. She did have a history of delayed dental eruption and short stature noted since early childhood and was diagnosed with attention-deficit disorder between 5 and 6 years of age, for which she received medications for many years. Menarche was normal and occurred at 12 years. The proband exhibited a round face and brachydactyly, a feature of Albright hereditary osteodystrophy (AHO), with shortened fourth and fifth metacarpals and foreshortened terminal first digits noted during evaluation for short stature at 5 years and 3 months of age (Fig. 2).

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Figure 1. Identification of a new GNAS mutation in PHP. (A) Pedigree of the family. Clinical status is indicated by open symbols (unaffected) and solid symbols (affected). Proband is indicated by the arrow. The presence (+) or absence (−) of the mutation in tested family members is shown. (B) Nucleotide sequence of part of GNAS exon 1 after PCR amplification of genomic DNA either by direct sequence analysis of the amplicon to show sequence of both alleles [(i) and (ii)] or via subcloned DNA [(iii), (iv), (v), and (vi)] to show sequence of one allele only. (i) Unrelated normal individual, both alleles. (ii) Mother, II-5, both alleles. (iii) Father, II-6, allele with (GCC)6 VNTR. (iv) Father, II-6, allele with (GCC)7 VNTR. (v) Proband, III-1, allele with (GCC)6 VNTR. (vi) Proband, III-1, allele with (GCC)7 VNTR. Initiation codon: ATG > ATA mutation. Therefore, the mutation arose de novo on the paternal allele. (C) The mutation removes an NcoI restriction site present in the wild-type sequence. (D) Confirmation of the mutation by restriction enzyme digestion with NcoI of PCR-amplified genomic DNA. The wild-type amplicon (288 bp) is completely cut into two fragments of similar size (140 and 148 bp). The proband's amplicon is incompletely cleaved owing to the presence of one mutant allele.

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Figure 2. Features of Albright hereditary osteodystrophy in proband III-1. Brachydactyly of the hand. Radiographs of the hand showing the shortened fourth and fifth metacarpals and the greatly foreshortened terminal first digit. CA = chronological age; BA = bone age.

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Laboratory data at 5 years and 3 months of age were as follows: serum calcium, 9.5 mg/dL (normal range 8.5 to 10.5 mg/dL); phosphate, 5.3 mg/dL (normal range 4.5 to 5.5 mg/dL); magnesium, 1.6 mg/dL (normal range 1.4 to 2.0 mg/dL); PTH, 42 pg/mL (normal range 10 to 60 pg/mL); T4, 8.6 µg/dL (normal range 4.5 to 10.9 µg/dL); and TSH, 5.14 mIU/L (normal range 0.5 to 5.0 mIU/L). The karyotype was normal 46,XX. Repeat laboratory studies at 5 years and 11 months of age were as follows: serum calcium, 9.5 mg/dL (normal range 8.4 to 10.5 mg/dL); phosphate, 5.3 mg/dL (normal range 3.2 to 6.3 mg/dL); magnesium, 1.9 mg/dL (normal range 1.5 to 2.4 mg/dL); alkaline phosphatase, 206 U/L (normal range 80 to 270 U/L); PTH, 17 to 42 pg/mL (normal range 10 to 65 pg/mL); T4, 10.4 µg/dL (normal range 4.5 to 11.2 µg/dL); TSH, 5.6 mIU/L (normal range 0.5 to 4.5 mIU/L; on 25 µg of levothyroxine); insulin-like growth factor 1 (IGF-1), 170 ng/mL (normal range 117 to 770 ng/mL); and insulin-like growth factor binding protein 3 (IGFBP3), 3.1 mg/L (normal range 2.0 to 4.8 mg/L). Calcium and PTH levels thus were normal, suggestive of PPHP rather than PHP-1a, and persisted normal at subsequent evaluation.

The child's bone age, assessed by the Greulich and Pyle23 method, was advanced relative to her chronologic age at 5 years and 3 months of age by 1.5 years, particularly for the phalanges. The bone age matched the chronologic age at 7 years and 11 months and 8 years and 11 months of age for the carpal bones but appeared advanced for the phalanges subsequent to early fusion of middle phalangeal growth plates (Fig. 2). Premature epiphyseal fusion occurs selectively in the hands and feet of PHP-1a/PPHP patients.24 The phalanges of such patients either lack epiphyses or have epiphyses that are partially fused when they first develop, making accurate measurements of bone age difficult. An anteroposterior view of her hands revealed broadening of the metacarpals and phalanges and shortening of the fourth and fifth metacarpals. Anteroposterior views of the feet showed some broadening of the distal phalanges, particularly of the first digits bilaterally, and mild shortening of the third and fourth metatarsals of the left foot. Anteroposterior and lateral views of the skull showed mild calvarial thickening. There was no evidence of soft tissue calcification or calcification at the level of the basal ganglia.

Levothyroxine replacement was initiated at 25 µg daily at 6 years of age subsequent to a trend of rising TSH levels and a gradual decrease in T4 levels and has been increased gradually over the years. She is currently 18 years old and receives 62.5 µg of levothyroxine daily with normal TSH and free T4 levels. Antibody testing revealed negative anti–thyroid peroxidase and positive anti-thyroglobulin antibodies, suggestive of unrelated autoimmune hypothyroidism. The proband continues to show no evidence of calcium, phosphorus, or PTH abnormalities. She is short with an adult height of 140.3 cm (height SDS is −3.5). This falls significantly short of her genetic potential for height (161.3 cm ± 5 cm). Thus far there is no evidence of soft tissue calcification.

Genomic DNA was isolated from blood leukocytes of the patient and family members (none of whom had clinical abnormalities), and exons 1 to 13 (encoding Gαs) were PCR amplified and sequenced directly by modification of previously described techniques.25, 26 Primer sequence and PCR conditions are available on request. This analysis revealed a heterozygous nucleotide substitution, c.3G > A, that led to a missense mutation at residue 1, Gαs M1I (Fig. 1B). This sequence change led to the loss of an NcoI restriction enzyme site (Fig. 1C). Restriction enzyme digestion of the patient-derived PCR product showed the presence of both the wild-type and mutant alleles (Fig. 1D). The sequence of exons 2 to 13 was normal.

The mutation was not present in genomic DNA from the siblings of the patient, her parents (and the siblings of her mother), and her grandparents (Fig. 1A), all of whom were normal clinically. The DNA encoding the 5' UTR of the Gαs mRNA has a (GCC)n variable number of tandem-repeat polymorphism (Fig. 1B). The repeat numbers (n) of both wild-type alleles of an unrelated normal subject (Fig. 1B, i) and of the patient's mother were 6 (Fig. 1B, ii), whereas the repeat numbers for the wild-type alleles of her father were 6 for one allele (Fig. 1B, iii) and 7 for the other (Fig. 1B, iv). The repeat numbers of the wild-type and mutant alleles of the patient were 6 and 7, respectively (Fig. 1B, v and vi). Hence the M1I mutation had arisen de novo on the paternal allele. Inheritance of the mutation on the paternal allele would be consistent with PPHP.

The mutation at the initiation (+1) AUG codon would be predicted to result in an NH2-terminally truncated Gαs protein lacking the first 59 amino acids and beginning at the methionine at codon 60 in frame with the methionine at +1.

All subjects (or their guardians) gave informed consent for the study, which was approved by the institutional review board of the Massachusetts General Hospital and the ethics committee of the Royal Victoria Hospital.

Immunoprecipitation/Western blot of GNAS proteins in human fetal kidney and liver tissues

Human fetal kidneys and liver samples were collected at the time of therapeutic abortion and frozen at −80 °C. Fetal age was determined by foot length. The study was approved by the Ethics Committee of the Royal Victoria Hospital, and written informed consent was obtained from individual patients.

Homogenized human fetal kidney (n = 3, 18.5 to 20 weeks FA) and liver (n = 1, 17 weeks FA) tissue fragments were solubilized in ice-cold lysis buffer (50 mM Tris [pH 7.5], 0.1% Triton X-100, 150 mM NaCl, and 2 mM EDTA) containing protease and phosphatase inhibitors (Complete Cocktail Tablets; Roche Diagnostics, Laval, Qubec, Canada) for 15 minutes on ice. To remove insoluble material, lysates were centrifuged at 13,000g for 10 minutes at 4 °C. The supernatants were collected, and protein concentrations were measured with the Bradford kit (Bio-Rad Laboratories, Inc., Mississauga, Ontario, Canada) using bovine serum albumin (BSA) as a standard.

Kidney and liver lysate proteins (0.5 to 1 mg) were incubated by rotation in the lysis buffer described earlier with a rabbit antibody to the terminal decapeptide (385 to 394) of human Gαs (06-237, 2 µL; Upstate Biotechnology, Lake Placid, NY, USA) overnight at 4 °C, followed by the addition of 50 µL of protein G–agarose beads (Amersham Biosciences, Baie D'Urfé, Quebec, Canada) for 1 hour at 4 °C. Antibody complexes were washed three times in lysis buffer, followed by a single wash in 50 mM Tris (pH 8.0). Immunoprecipitates were boiled for 5 minutes in Laemmli loading buffer (50 mM Tris [pH 6.8], 2% SDS, 0.1% bromophenol blue, 10% glycerol, and 100 mM DTT), and proteins were resolved by SDS-PAGE (8% to 15%) and transferred (Mini Trans-Blot Cell; BioRad, Hercules, CA, USA) onto polyvinylidene fluoride (PVDF) Immobilon-P transfer membranes (Millipore Corporation, Mississauga, Ontario, Canada).

Membranes were blocked overnight at 4 °C with 5% nonfat dry milk in 1× PBS–Tween 20 (PBST) buffer and then incubated with the same Gαs antibody (1:1000) for 3 hours at room temperature. After washing three times in PBST, membranes were incubated with goat anti-rabbit (1:1000) IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 hour at room temperature. Protein bands were visualized by an enhanced chemiluminescence (ECL) detection system (Perkin Elmer Life Sciences, Inc., Boston, MA, USA). Markers included Benchmark Prestained Protein Ladder and SDS-PAGE Unstained Broad Range Standards (BioRad). Densitometric analyses were carried out using the BioRad GelDoc system.

Reagents for transfection assays

A full-length Gαs cDNA in pcDNA3.1(−) was from the Guthrie cDNA Resource Center (Sayre, PA, USA). The human PTHR1 and TSHR expression vectors were as described previously,27, 28 and the human V2R was kindly provided by Dr Stephane Laporte, McGill University (Montreal, Quebec, Canada).29 Bovine PTH(1–34), bovine TSH, (−)-isoproterenol-(+)-bitartrate (isoproterenol), [Arg8]-vasopressin acetate salt (AVP), and IBMX were from Sigma-Aldrich (Oakville, Ontario, Canada). Polyfect transfection reagent was from Qiagen (Mississauga, Ontario, Canada). Primers were from Alpha DNA (Montreal, Quebec, Canada).

Construction of expression vectors

The truncated (Tr) αs cDNA was made from the Gαs FL cDNA in the pcDNA3.1(−) expression vector by removing exon 1 that contains the Gαs ATG start site; the next in-frame ATG that could function as a translational start site is within exon 2, at position +60, and is predicted to produce a 38-kDa protein product.

To create the A/Bαs cDNA, 5 µg of HEK293 cell total RNA was reverse transcribed with Superscript II (Invitrogen, Burlington, Ontario, Canada) using random primers (Invitrogen). Using the A/B sense primer 5'-CGC TCC CCT GCT CTC TGG CT-3' with the exon 6 antisense primer 5'-AGC CTT GGC ATG CTC ATA GAA TTC G-3', a 440-bp fragment containing 123 bp of exon A/B as well as Gαs exons 2 to 6 was PCR amplified. This amplicon was cloned into pCR2.1-TOPO (Invitrogen). Subsequently, the exon A/B/2–6 sequence was released with EcoRV and BsgI (the site for the latter is within the Gαs cDNA sequence) and subcloned into blunt-ended HindIII/BsgI-cut pcDNA3.1(−) Gαs lacking exons 1 to 6. Thus the resulting expression vector has the exon 2 ATG translation start site and is predicted to produce a 38-kDa protein. The M1I mutant was made from Gαs FL cDNA by replacing the first methionine in exon 1 with isoleucine using the QuikChange II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA), according to the manufacturer's instructions. The correctness of all constructs was confirmed by sequencing.

Transient transfection assays

HEK293 cells were cultured in DMEM with 10% fetal bovine serum/newborn calf serum (FBS/NCS) (50:50) at 37 °C and 5% CO2, plated in 12-well culture plates, and allowed to grow to approximately 60% confluence for 24 hours. The next day, cells were transiently transfected with vectors expressing cDNAs encoding PTHR1, TSHR, or V2R and the full-length Gαs (FL Gαs) with or without truncated Gαs proteins (Tr αs, M1I αs) or empty expression vector (pcDNA3.1). To assess dose-response effects, cells were transfected with FL Gαs and Tr αs cDNAs to obtain 1:1 or 1:2 ratios, taking into account the endogenous Gαs levels. A total of 1 µg of DNA/well was transfected using the Polyfect transfection reagent. After 48 hours, the cells were stimulated for 15 minutes at 25 °C with either vehicle or receptor agonist in increasing concentrations (PTH, 10−7 to 10−9 M; TSH, 1 to 100 mU; isoproterenol, 10−6 to 10−8 M; or AVP, 10−6 to 10−8 M) in the presence of 2 mM phosphodiesterase inhibitor (IBMX), as described previously.30 Following the stimulation, the culture plates were chilled on ice, and the cells were lysed in 0.1 M HCl. Cyclic AMP activity in each well was quantified by EIA (Direct cAMP Immunoassay Kit; Sigma-Aldrich). Four to eight independent experiments were performed for each study. The level of cAMP is expressed as a ratio relative to maximal agonist-induced accumulation by cells expressing FL Gαs (100%).

In a further series of experiments, proximal tubule opossum kidney (OK) cells (CRL-1840, ATCC) or HEK293 cells were seeded at 3 × 105 cells in 2 mL of complete medium (DMEM + 10% FBS) in each well of 6-well tissue culture dishes. After overnight incubation, the cells were transfected (Polyfect, Qiagen) with the following plasmid DNAs from the PathDetect Trans-Reporting System (Catalogue No. 219010; Agilent Technologies, Santa Clara, CA, USA): pFR-Luc reporter plasmid (1.0 µg) and pFA2-CREB fusion trans-activator plasmid (50 ng). Cells were cotransfected with empty vector, pcDNA3.1, or Tr αs, A/Bαs, M1I, or FL Gαs expression vectors as required. HEK293 cells also were cotransfected with the PTHR1 expression vector (100 ng). After overnight incubation, cells were serum-starved (0.5% FBS) for 8 hours, after which the cells were stimulated or not with PTH or isoproterenol for 12 hours in the presence of 2 mM IBMX, and then the cells were lysed for luciferase assay. The results are expressed as luciferase activity as a measure of cAMP production.

Western blot analysis

For each of the preceding experiments, parallel wells were transfected for Western blot analysis of protein expression. Forty-eight hours after transfection, the cells were washed twice with ice-cold PBS, and protein was extracted with a lysis buffer containing 50 mM Tris pH 7.4, 15 mM NaCl, 0.1% SDS, 1% NP40, 0.5% sodium deoxycholate, and protease inhibitors. Then 25 µg of each protein sample was run on 10% SDS-PAGE and then transferred to PVDF membranes. For immunodetection of both FL Gαs and Tr αs proteins, the rabbit COOH-terminal Gαs antibody was used (1:2000). The signal was revealed using goat anti-rabbit IgG conjugated with horseradish peroxidase (1:1000) followed by ECL detection. Protein expression ratios were determined by densitometric analyses using Quantity One software (BioRad).

Statistical analysis

Data are means ± SEM. Statistical significance was assessed initially by ANOVA, followed by repeated-measurements comparison using the Tukey test.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Multiple GNAS protein products are present in human tissues

The relative expression of the various GNAS protein products was examined by immunoprecipitation and Western blot, using a Gαs COOH-terminal antibody, of human fetal kidney and liver samples (Fig. 3A). Besides the 45- and 52-kDa Gαs species, additional approximately 38-kDa and multiple higher-molecular-weight (≥77 kDa) species were observed, predicted to be derived from the mRNAs for A/Bαs and XLαs, respectively. Even on overexposure of the blots, the molecular-weight bands higher and lower than those for the Gαs 45- and 52-kDa species were not present in the rabbit IgG control lane, supporting the Gαs specificity of all the products (data not shown). Hence proteins derived from mRNAs for A/Bαs and XLαs, as well as Gαs, are present in human tissues.

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Figure 3. (A) Gαs-related proteins are present in human tissues. Immunoprecipitation and Western blot analysis of Gαs-related proteins in human fetal kidney (n = 3) and fetal liver (n = 1). The Gαs antibody, raised against the COOH-terminus, recognizes Gαs itself (45- and 52-kDa forms) and the A/Bαs- (38 kDa) and XLαs- (≥77 kDa) derived proteins. Sizes of molecular weight standards are on the left. On longer exposure, the 52-, 45-, and 38-kDa species were apparent in the fetal liver lane but not the rabbit IgG lane (data not shown). (BD) Endogenous and exogenous Gαs protein expression levels in transfected cells. HEK293 cells were transiently transfected with either pcDNA3.1 or vectors expressing full-length Gαs protein (FL Gαs) or the NH2-terminally truncated proteins, Tr αs, or A/Bαs, either individually (BD) or in combination (FL Gαs + Tr αs) (C, D). Forty-eight hours after transfection, cell extracts were made, and Western blot analysis was conducted using the Gαs antibody. Molecular weights of size markers are shown on the left and the positions of Gαs + Tr αs on the right.

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GNAS protein products of HEK293 cells transfected with FL Gas, Tr αs, and A/Bαs

By transient transfection, FL Gαs, Tr αs, or A/Bαs was overexpressed in HEK293 cells, and cell extracts were analyzed by Western blot using a Gαs COOH-terminal antibody (Fig. 3B). HEK293 cells transfected with the empty vector (pcDNA3) expressed basal levels of the two forms of Gαs derived from both exon 3–included and exon 3–excluded mRNAs. Cells transfected with the FL Gαs vector expressed additional amounts of the approximately 52-kDa species consistent with the construct encoding the exon 3–included form, and cells transfected with the Tr αs vector as well as those transfected with the A/Bαs vector expressed a smaller, approximately 38-kDa protein (Fig. 3B).

For the experiments listed below, the amounts of either exogenous FL Gαs or Tr αs proteins were manipulated to achieve either 1:1 or 2:1 ratios relative to total Gαs (endogenous plus exogenous) by transfecting different concentrations of the expression vectors. For example, equal amounts of FL Gαs and Tr αs expression (Fig. 3C) or double the amount of Tr αs expression (Fig. 3D) was demonstrated by Western blot analysis under the different conditions used.

Tr αs modulates PTHR1- and TSHR-stimulated cAMP levels in transfected HEK293 cells

Activation of PTHR1 and TSHR initiates intracellular events resulting in increased cAMP production. HEK293 cells endogenously express Gαs, and when transfected with the PTHR1 expression vector alone, they demonstrated a dose-related increase in cAMP levels in response to PTH (Fig. 4A, B, PTHR1). Cells cotransfected with the PTHR1 and the Gαs vectors exhibited increased responsiveness to PTH with respect to cAMP production at all concentrations tested (Fig. 4A, B, PTHR1 + FL Gαs). Cells cotransfected with the PTHR1 and Tr αs vector exhibited significantly lower cAMP levels in response to PTH relative to those transfected with PTHR1 alone (Figs. 4A, B, PTHR1 + Tr αs), consistent with the truncated protein inhibiting endogenous Gαs function. Furthermore, cells cotransfected with PTHR1 and FL Gαs and Tr αs showed markedly decreased cAMP levels in response to PTH compared with cells transfected with PTHR1 and FL Gαs alone. The inhibition was more marked in the cells transfected with FL Gαs and Tr αs at a 1:2 ratio than in those transfected at a 1:1 ratio (e.g., ∼54% at 1:2 versus ∼35% at 1:1 with 10−8 M PTH; Fig. 4A, B, PTHR1 + FL Gαs + Tr αs).

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Figure 4. Exogenous NH2-terminally truncated Gαs proteins inhibit PTH- and TSH-stimulated cAMP levels in transfected cells. (A, B) HEK293 cells were transiently transfected with expression vectors, either PTHR1 alone, or with FL Gαs, or with Tr αs, or with both FL Gαs and Tr αs. Expression ratio for FL Gαs:Tr αs proteins was 1:1 (A) or 1:2 (B). After 48 hours, cells were challenged with bovine PTH(1–34) (10−9 to 10−7 M) for 15 minutes at 25 °C in the presence of 2 mM IBMX. Levels of cAMP are expressed relative to those induced by 10−8 M PTH in cells expressing PTHR1 + FL Gαs (100%). Data are means ± SEM (n = 4 to 8 independent experiments). *p < 0.05; **p < 0.01; ***p < 0.001 compared with PTHR1 + FL Gαs–transfected cells; +p < 0.05; ++p < 0.01 PTHR1 + Tr αs–transfected versus PTHR1 alone–transfected cells. (C, D) HEK293 cells were transiently transfected with expression vectors, either TSHR alone or with FL Gαs, Tr αs, or with both FL Gαs + Tr αs. The expression ratio for FL Gαs:Tr αs proteins was 1:1 (C) or 1:2 (D). After 48 hours, cells were challenged with 1 to 100 mU of bovine TSH for 15 minutes at 25 °C in the presence of 2 mM IBMX. Levels of cAMP are expressed relative to those induced by 10 mU TSH in cells expressing TSHR + FL Gαs (100%). Data are means ± SEM (n = 5 to 8 independent experiments). **p < 0.01; ***p < 0.001 compared with TSHR + FL Gαs–transfected cells.

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Next, we evaluated the ability of Tr αs to modulate the cAMP responsiveness of HEK293 cells to TSH. HEK293 cells transiently transfected with the TSHR expression vector alone exhibited dose-related increases in cAMP levels to TSH (Fig. 4C, D, TSHR). Cells cotransfected with TSHR and Tr αs exhibited similar TSH-stimulated cAMP levels to those of cells transfected with TSHR alone (Fig. 4C, D, TSHR + Tr αs). However, cells cotransfected with TSHR and FL Gαs and Tr αs showed markedly decreased cAMP levels in response to TSH compared with cells transfected with TSHR and FL Gαs alone. The inhibition was more marked in cells transfected with FL Gαs and Tr αs at a 1:2 ratio relative to cells transfected at a 1:1 ratio (e.g., ∼42% at 1:2 versus ∼25% at 1:1 with 10 or 100 mU TSH; Fig. 4C, D, TSHR + FL Gαs + Tr αs).

Effects of Tr αs on β2AR- and V2R-stimulated cAMP levels in HEK293 cells

Activation of β2AR and V2R, as for PTHR1 and TSHR, initiates intracellular events resulting in increased cAMP production. Therefore, we also tested the influence of the Tr αs protein on FL Gαs activity following β2AR or V2R stimulation. Since HEK293 cells already endogenously express the β2AR, for these experiments, the cells were transfected with the empty pcDNA3.1 vector. HEK293 cells, when transfected with pcDNA3.1 alone, demonstrated a dose-related increase in cAMP levels in response to isoproterenol (Fig. 5A, B, pcDNA3.1). Cells cotransfected with pcDNA3.1 and the Gαs vector exhibited increased responsiveness to isoproterenol with respect to cAMP production (Fig. 5A, B, pcDNA3.1 + FL Gαs). However, cells cotransfected with pcDNA3.1 and Tr αs vector exhibited similar cAMP levels in response to isoproterenol relative to those transfected with pcDNA3.1 alone (Fig. 5A, B, pcDNA3.1 + Tr αs), consistent with a lack of effect of the truncated protein on endogenous Gαs function. Furthermore, although cells cotransfected with pcDNA3.1 and FL Gαs and Tr αs showed a trend toward decreased cAMP levels in response to isoproterenol compared with cells transfected with pcDNA3.1 and FL Gαs alone, the effect was not significant. The inhibition was more evident in the cells transfected with FL Gαs and Tr αs at a 1:2 ratio than those transfected at a 1:1 ratio. (Fig. 5A, B, pcDNA3.1 + FL Gαs + Tr αs), and the cAMP values were significantly different (∼30%, p < 0.001) at 10−7 M isoproterenol (Fig. 5B).

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Figure 5. Exogenous NH2-terminally truncated Gαs proteins exert little or no effect on isoproterenol- or AVP-stimulated cAMP levels in transfected cells. (A, B) HEK293 cells (which endogenously express the β2AR) were transiently transfected with expression vectors, either pcDNA3.1 alone or with FL Gαs, Tr αs, or with both FL Gαs and Tr αs. Expression ratio for FL Gαs:Tr αs proteins was 1:1 (A) or 1:2 (B). After 48 hours, cells were challenged with isoproterenol (10−8 to 10−6 M) for 15 minutes at 25 °C in the presence of 2 mM IBMX. Levels of cAMP are expressed relative to those induced by 10−7 M isoproterenol in cells expressing β2AR + FL Gαs (100%). Data are means ± SEM (n = 4 to 8 independent experiments). ***p < 0.001 compared with pcDNA3.1 + FL Gαs–transfected cells. (C, D) HEK293 cells were transiently transfected with expression vectors, either V2R alone or with FL Gαs, Tr αs, or with both FL Gαs and Tr αs. Expression ratio for FL Gαs:Tr αs proteins was 1:1 (C) or 1:2 (D). After 48 hours, cells were challenged with AVP (10−8 to 10−6 M) for 15 minutes at 25 °C in the presence of 2 mM IBMX. Levels of cAMP are expressed relative to those induced by 10−7 M AVP in cells expressing V2R + FL Gαs (100%). Data are means ± SEM (n = 4 to 5 independent experiments); there were no significant differences between groups.

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HEK293 cells, when transfected with the V2R vector alone, demonstrated a dose-related increase in cAMP levels in response to AVP (Fig. 5C, D, V2R). Cells cotransfected with V2R and the Gαs vector exhibited increased responsiveness to AVP with respect to cAMP production (Fig. 5C, D, V2R + FL Gαs). However, cells cotransfected with pcDNA3.1 and Tr αs vector exhibited similar cAMP levels in response to AVP relative to those transfected with V2R alone (Fig. 5C, D, V2R + Tr αs), consistent with a lack of effect of the truncated protein on endogenous Gαs function. Furthermore, cells cotransfected with V2R and FL Gαs and Tr αs demonstrated no difference at all in the increases in cAMP levels in response to AVP compared with cells transfected with V2R and FL Gαs alone. This lack of effect extended to cells transfected with FL Gαs and Tr αs at both the 1:1 and the 1:2 ratios (Fig. 5C, D, V2R + FL Gαs + Tr αs). This is in contrast to the marked differences noted in the PTHR1 and TSHR experiments described earlier.

Effects of Tr αs on PTHR1- and β2AR-stimulated cAMP levels in OK cells

Proximal tubule OK cells that endogenously express the PTHR1 and β2AR demonstrated dose-related increases in response to PTH (Fig. 6A) or isoproterenol (Fig. 6B). Cotransfection with increasing amounts of the Tr αs vector (25 to 150 ng) led to a dose-related decrease in the cAMP levels stimulated by PTH (Fig. 6A, inset), but no such inhibitory effect was observed with the cAMP levels stimulated by isoproterenol (Fig. 6B and data not shown) even with the highest amount of Tr αs vector transfected. Hence the inhibitory effect exerted by Tr αs was specific for the endogenous PTHR1 in these proximal tubule kidney cells.

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Figure 6. Exogenous NH2-terminally truncated Gαs protein Tr αs inhibits PTH- but not isoproterenol-stimulated cAMP levels in proximal tubule OK cells that endogenously express the PTHR1 and β2AR. OK cells were transiently transfected with the pFR-Luc reporter and pFA2-CREB fusion trans-activator plasmids and either without or with the Tr αs expression vector (100 ng/well of a 6-well culture dish) and stimulated with either bovine PTH(1–34) (10−9 to 10−7 M) (A) or isoproterenol (10−8 to 10−6 M) (B) in the presence of 2 mM IBMX. (Inset) OK cells were transfected either without or with the Tr αs expression vector (25, 50, 100, or 150 ng/well in a 6-well culture dish) and stimulated with bovine PTH(1–34) (10−9 to 10−7 M). Cell extracts were made and luciferase activity determined. Values are reported as luciferase activity as a measure of cAMP production. *p < 0.05; **p < 0.01; ***p < 0.001.

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M1I αs mutant is a truncated Gαs and inhibits FL Gαs activity

We describe a patient heterozygous for a de novo M1I mutation encoded by Gαs exon 1. Inheritance of the mutation on the paternal allele was consistent with the presence of brachydactyly without hormone resistance, that is, PPHP. The mutation at the initiation AUG codon (+1) is predicted to result in an NH2-terminally truncated Gαs protein lacking the first 59 amino acids and beginning at the methionine at codon 60 in frame with the methionine at codon 1. The M1I αs mutant was transfected into HEK293 cells, and the expression of the exogenous Gαs-related proteins was evaluated by Western blot of cell extracts with the COOH-terminal Gαs antibody. The cells expressed a 38-kDa species similar to that seen in cells transfected with the Tr αs construct (Fig. 7A). In addition, a molecular species similar in size to that produced by transfection with the FL Gαs vector was noted, but to a much lesser extent (∼5% to 10%). We hypothesize that translation of the M1I αs mutant mRNA while generating a majority of the NH2-terminally truncated Gαs also yields a small amount of ∼FL Gαs protein, using non-consensus translational start sites in exon 1 [e.g., the leucine codon (CTG) at +4]. In the experiments discussed below, HEK293 cells co-transfected with FL Gαs and M1I αs expressed the full-length and truncated Gαs proteins at close to the 1:1 ratio achieved when FL αs and Tr αs were coexpressed (Fig. 7A).

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Figure 7. The NH2-terminally truncated PPHP M1I αs mutant inhibits PTH-stimulated cAMP levels in transfected cells. (A) HEK293 cells were transiently transfected with either pcDNA3.1 or vectors expressing the NH2-terminally truncated proteins Tr αs or M1I αs, either individually or in a 1:1 combination, FL Gαs + Tr αs or FL Gαs + M1I αs. Forty-eight hours after transfection, cell extracts were made, and Western blot analysis was conducted using the Gαs antibody. Molecular weights of size markers are shown on the left and the positions of Gαs + Tr αs on the right. M1I αs lane: Arrow indicates a 52-kDa Gαs species. (B) HEK293 cells were transiently transfected as described under “Methods” with the pFR-Luc reporter and pFA2-CREB fusion trans-activator plasmids and expression vectors, all with PTHR1, with pcDNA3.1, Tr αs, A/Bαs, M1I αs, or all with the PTHR1 and FL Gαs combination and with pcDNA3.1 or Tr αs or A/Bαs or M1I αs. Cells were challenged with bovine PTH(1–34) (10−8 M) in the presence of 2 mM IBMX, and the luciferase activity of cell extracts was determined. Values are reported as luciferase activity as a measure of cAMP production. **p < 0.01; ***p < 0.001. (C, D) The NH2-terminally truncated forms of Gαs, Tr αs, A/Bαs, and M1I αs inhibit PTH-stimulated cAMP levels in transfected proximal tubule OK cells that endogenously express the PTHR1. (C) OK cells were transiently transfected with either pcDNA3.1 or vectors expressing FL Gαs or the NH2-terminally truncated proteins Tr αs, A/Bαs, or M1I αs. Forty-eight hours after transfection, cell extracts were made and Western blot analysis conducted using the Gαs antibody. A representative result is shown for pcDNA3.1- and Tr αs–transfected cells. Molecular weights of size markers are shown on the left, as well as the positions of Gαs + Tr αs on the right. (D) OK cells were transiently transfected as described under “Methods” with the pFR-Luc reporter and pFA2-CREB fusion trans-activator plasmids and expression vectors and with pcDNA3.1 or Tr αs or A/Bαs or M1I αs or all with FL Gαs and with pcDNA3.1 or Tr αs or A/Bαs or M1I αs. Cells were challenged with bovine PTH(1–34) (10−8 M) in the presence of 2 mM IBMX, and luciferase activity of cell extracts was determined. Values are reported as luciferase activity as a measure of cAMP production. **p < 0.01; ***p < 0.001.

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The M1I αs mutant was tested for its ability to act like Tr αs and A/Bαs and inhibit the PTH (10−8 M) stimulation of cAMP levels. HEK293 cells cotransfected with the PTHR1 and Tr αs or A/Bαs or M1I αs vectors exhibited lower cAMP levels in response to PTH relative to those transfected with PTHR1 only (Fig. 7B), consistent with all the truncated proteins inhibiting endogenous Gαs function. In cells transfected with PTHR1 and FL Gαs, the stimulation with PTH was increased approximately three-fold over that with the endogenous Gαs only, and lower cAMP levels were seen in cells cotransfected with Tr αs or A/Bαs or M1I αs vectors (Fig. 7B). The M1I αs was slightly (but significantly) less effective than Tr αs in inhibiting the PTH-stimulated cAMP levels (Fig. 7B) in cells either transfected with the FL Gαs vector or not. The difference between M1I and Tr αs may well relate to the presence of the approximately 5% to 10% FL Gαs product observed by Western blot (Fig. 7A), which would promote (rather than inhibit) the PTH-stimulated cAMP levels. Thus the M1I αs mutant has a similar but less potent inhibitory effect on FL Gαs compared with Tr αs.

Similar experiments were performed in OK cells but without transfection of the PTHR1 vector. Cotransfection of 100 ng of the truncated Gαs vectors provided a 1:1 ratio of their expression relative to endogenous FL Gαs (Fig. 7C, and data not shown). OK cells transfected with the Tr αs or A/Bαs or M1I αs vectors exhibited lower cAMP levels in response to PTH relative to those not transfected (Fig. 7D), consistent with all these truncated proteins inhibiting endogenous Gαs function. In cells cotransfected with FL Gαs, the stimulation with PTH was increased approximately 2.5-fold over that with the endogenous Gαs only, and lower cAMP levels were seen in cells cotransfected with Tr αs or A/Bαs or M1I αs vectors (Fig. 7D). Thus the cAMP responses in the OK cells and the PTHR1-transfected HEK293 cells were very similar, with the relative levels being slightly less in the OK cells.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

We identified a novel de novo c.3G > A M1I mutation on the paternal allele of a PPHP proband who exhibited brachydactyly as a feature of AHO. There are three previous reports of mutations at the Gαs initiation codon. A proband with PHP-1a and his mother with PPHP were found to have a heterozygous c.1A > G mutation in Gαs exon 1 encoding an M1V mutation.31 The identical mutation was reported in a separate case of PHP.26 A heterozygous c.2T > G M1R mutation was reported in a boy with PPHP.32 In all these cases it would be predicted that translation initiation at the in-frame codon 60 ATG in exon 2 would result in an NH2-terminally truncated Gαs protein. However, no previous functional analysis of such a protein has been conducted. This prompted us to consider how the Gαs mutants, with a truncated NH2-terminus but intact COOH-terminus, identified in the few cases of PHP-1a and PPHP, might have an impact on signaling by PTH and other hormones by inhibiting Gαs. In addition, the loss of methylation of exon A/B leading to abnormal biallelic expression of A/Bαs in PHP-1b patients would predict enhanced levels of NH2-truncated forms of Gαs in PHP-1b in which mutations within GNAS exons 2 to 13 are not found.33 Hence, in this form of PHP, the enhanced levels of the truncated Gαs could negatively influence the signaling of PTH.

Abnormal imprinting patterns of GNAS-derived mRNA have been found in peripheral blood cells of PHP-1b patients. Biallelic expression of A/B αs transcripts (in most cases) and XLαs transcripts (in a few cases) that are normally only paternally expressed were noted.7, 8, 34, 35 The proteins translated from these mRNAs have common COOH-terminal sequence with Gαs itself. There is no consensus translational start site within exon A/B and the next ATG start codon that is in frame with the Gαs sequence is at position 60 in exon 2 of GNAS. In vitro translated exon A/B mRNA yielded an approximately 38-kDa protein (recognized by a COOH-terminal Gαs antibody) consistent with a 335-amino-acid truncated Gαs protein lacking the NH2-terminal βγ-binding and one of the receptor-interacting domains.21, 22 In this study, transfection of either an A/B αs construct or one encompassing only exons 2 to 13 of Gαs (Tr αs) resulted in production of an approximately 38-kDa Gαs-immunoreactive protein. We also identified the endogenous protein in human fetal kidney and liver. It should be noted that a similar protein was present in erythrocyte membranes from the PHP-1a patient with the M1V mutation referred to earlier.31

Inhibitory effects of COOH-terminal peptides on their full-length G protein counterparts have been demonstrated, and even very short peptides blocked GPCR signaling in a Gα-specific manner.36 Coexpression of Gαq(305–359) with full-length Gαq resulted in reduction of α1-AR-mediated inositol phosphate production, whereas coexpression of Gαq(1–54) had no effect.37 Gαs COOH-terminal peptides of 10 to 83 amino acids inhibited the ability of β2AR to activate Gαs and adenylate cyclase.38, 39 These data support the hypothesis considered here that the NH2-terminally truncated Gαs proteins could modify the responsiveness of specific GPCR signaling.

In this study, we show that exogenous expression of the approximately 38-kDa Tr αs protein that is translated from the A/B αs transcript does have a marked impact on Gαs signaling via the PTHR1, less so via the TSHR, even less via the β2AR, and no effect at all via the V2R. With the PTHR1, the inhibitory effect of the Tr αs protein was robust enough to be observed on PTH stimulation of endogenous adenylate cyclase activity. When the FL Gαs was cotransfected, Tr αs exerted a dose-dependent inhibitory effect. While the Tr αs protein did not inhibit the endogenous TSHR–stimulated Gαs activity, the cotransfected Tr αs exerted a dose-response inhibitory effect in the presence of exogenous Gαs. With β2AR, an effect was observed only with cotransfection of the highest dose of Tr αs and at the highest concentration of isoproterenol. The Tr αs protein was without effect on AVP stimulation of cAMP via the V2R under any of the tested conditions. These results are in line with the selective resistance to PTH and TSH observed in PHP-1a and PHP-1b patients. Additional evidence for a specific effect of the Tr αs (and the other truncated Gαs proteins) on PTH versus isoproterenol responsiveness was obtained in the proximal tubule OK cells that endogenously express the PTHR1 and β2AR.

With respect to the Gαs M1I mutation identified in our PPHP patient, we show here that the predominant species encoded by the mutant transcript has an electrophoretic mobility identical to that of the Tr αs protein. This is consistent with translation of the mRNA initiating predominantly at codon 60 to generate the NH2-terminally truncated protein. In addition, a small amount of a molecular species with an electrophoretic mobility close to that of full-length Gαs was noted. Thus it is likely that, infrequently, translation initiates at an in-frame nonconsensus start codon near to the normal start site; the CTG leucine at codon 4 would be a candidate for this. The M1I mutant significantly inhibited the stimulation of cAMP via the PTHR1 when cotransfected with FL Gαs, although it was slightly less effective than the Tr αs, likely owing to the presence of the small amount of the (almost) full-length Gαs species referred to earlier. Note that the effectiveness of the M1I protein is predicted to rely on its parental origin. In tissues in which the paternal Gαs allele is silenced, including the renal proximal tubule and the thyroid, this mutant would not be produced. This is indeed the case in our patient, who has the mutation on the paternal allele.

The precise mechanism whereby the NH2-terminally truncated Gαs protein inhibits GPCR signaling (and in a PTHR1/TSHR-selective fashion) remains to be elucidated. The mechanism may involve competition with the full-length protein for binding to the receptor and/or activation of adenylate cyclase. Gαs COOH-terminal peptides as short as 10 amino acids can bind specifically to Gαs-coupled GPCRs.36 Moreover, the sequences required for binding to and activation of adenylate cyclase are within the truncated protein.40, 41 Therefore, if this is all that is required, the Tr αs should be able to bind the PTHR1 and stimulate cAMP production. However, two additional key elements found within the NH2-terminus are missing, namely, a palmitoylation site that stabilizes the Gαs-receptor, Gαs-βγ, and the Gαs–adenylate cyclase complexes by enhancing membrane localization, as well as protein-protein interactions,36, 42 and the βγ-binding domain. βγ subunits not only stabilize the α subunit but also participate in receptor-mediated G protein activation.42 The present data suggest that the truncated A/Bαs and M1I proteins having the COOH-terminal receptor interaction domain but lacking the NH2-terminal receptor and βγ interaction sites can couple with certain GPCRs but less efficiently than FL Gαs.

As with the A/Bαs exon, XLαs is also paternally expressed but additionally is expressed from the maternal allele in a small subset of PHP-1b patients. Gαs and XLαs share an identical COOH-terminal portion encoded by GNAS exons 2 to 13 but differ in their NH2-terminal portions encoded by different exons.14–16 However, unlike the situation with the A/Bαs and Tr αs proteins and loss of Gαs NH2-terminal functional domains, the COOH-terminal part encoded by the XLαs exon is very homologous with the NH2-terminal functional domain of Gαs and may share its functions and be able to compensate for its loss. In vitro transfection studies have shown that, as with Gαs, XLαs binds βγ subunits and couples agonist stimulation of GPCRs with activation of adenylate cyclase.17–19, 43, 44 However, studies in mice in vivo have been interpreted as suggesting that under some circumstances XLαs might antagonize certain Gαs-dependent pathways.45, 46 Overall, the data are not compelling for XLαs to play similar roles to NH2-terminally truncated Gαs proteins in the situations being considered in this study.

In summary, our results demonstrate that NH2-terminally truncated forms of human Gαs can selectively inhibit the stimulation of adenylate cyclase by the PTHR1 and TSHR. These truncated forms of Gαs may arise by naturally occurring mutations in Gαs itself in PHP-1a and PPHP, or their expression may be enhanced by epigenetic modifications occurring in PHP-1b.

Disclosures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

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

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

We thank all the family members for their participation and their physicians for the clinical evaluation of patients. We thank Dr Stephane Laporte for the human V2R expression vector and the staff at the Royal Victoria Hospital Day Surgery Unit for their help in obtaining human tissue samples.

This work was supported by operating grants from the Canadian Institutes of Health Research (to CGG and GNH) and by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (R37 DK46718 to HJ and RO1 DK073911 to MB). L.C. was supported by Fellowships from the Kidney Foundation of Canada and the Research Institute of the McGill University Health Centre.

Authors' roles: SP and CGG contributed equally to this work; SP, CGG, and GNH were involved in conception and design; SP, CGG, MAK, LC, MM, HJ, and MB did data acquisition; and SP, CGG, LC, MM, HJ, MB, and GNH drafted and/or revised the manuscript.

References

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  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
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