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Endocrinology, doi:10.1210/en.2005-1487
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Endocrinology Vol. 147, No. 5 2253-2262
Copyright © 2006 by The Endocrine Society

Coding GNAS Mutations Leading to Hormone Resistance Impair in Vitro Agonist- and Cholera Toxin-Induced Adenosine Cyclic 3',5'-Monophosphate Formation Mediated by Human XL{alpha}s

Agnès Linglart, Matthew J. Mahon, Mohammad A. Kerachian, David M. Berlach, Geoffrey N. Hendy, Harald Jüppner and Murat Bastepe

Endocrine Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School (A.L., M.J.M., M.B.), Boston, Massachusetts 02114; Pediatric Endocrinology and Institut National de la Santé et de la Recherche Médicale, Unité 561, Saint Vincent de Paul Hospital (A.L.), Paris, France; Departments of Medicine, Physiology, and Human Genetics, McGill University (M.A.K., D.M.B., G.N.H.), and Calcium Research Laboratory, and Hormones and Cancer Research Unit, Royal Victoria Hospital (M.A.K., D.M.B., G.N.H.), Montréal, Québec, Canada; and Pediatric Nephrology Unit, MassGeneral Hospital for Children and Harvard Medical School (H.J.), Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Dr. Murat Bastepe, Endocrine Unit, Massachusetts General Hospital, 50 Blossom Street, Thier 501, Boston, Massachusetts 02114. E-mail: bastepe{at}helix.mgh.harvard.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Most loss of function mutations of GNAS identified in different forms of pseudohypoparathyroidism disrupt not only the stimulatory G protein {alpha}-subunit (Gs{alpha}), but also its paternally expressed variant, XL{alpha}s. However, the possibility that XL{alpha}s deficiency contributes to disease pathogenesis has remained unexplored. We therefore examined the signaling property of human XL{alpha}s and the effects of one novel (XL{alpha}sH704P or Gs{alpha}H362P) and two previously described (XL{alpha}sDelI724 and XL{alpha}sY733X or Gs{alpha}DelI382 and Gs{alpha}Y391X, respectively) GNAS mutations on either XL{alpha}s or Gs{alpha} activity. Confocal immunofluorescence microscopy detected human XL{alpha}s immunoreactivity at the plasma membrane of transduced mouse embryonic fibroblasts null for endogenous Gs{alpha} and XL{alpha}s (GnasE2–/E2– cells). Cholera toxin- and isoproterenol-induced cAMP accumulation in GnasE2–/E2– cells transiently expressing wild-type human XL{alpha}s was similar to that in cells transiently expressing wild-type Gs{alpha}. Human XL{alpha}s, like Gs{alpha}, mediated PTH-induced cAMP accumulation in GnasE2–/E2– cells coexpressing PTH receptor type 1 and either of these proteins. Moreover, overexpression of human XL{alpha}s or Gs{alpha} markedly enhanced the PTH-induced cAMP accumulation in opossum kidney cells that endogenously express PTH receptor type 1. In contrast, each XL{alpha}s mutant failed to mediate isoproterenol- and PTH-induced cAMP accumulation in transduced GnasE2–/E2– cells. XL{alpha}sDelI724 showed a reduced cholera toxin response over the basal level compared with wild-type XL{alpha}s, and XL{alpha}sH704P completely failed to respond to cholera toxin. These findings were comparable to those observed with each corresponding Gs{alpha} mutant transiently expressed in GnasE2–/E2– cells. Thus, mutations that typically inactivate Gs{alpha} also impair XL{alpha}s activity, consistent with a possible role for XL{alpha}s deficiency in diseases caused by paternal GNAS mutations.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE {alpha}-SUBUNIT OF the stimulatory G protein (Gs{alpha}), which mediates the actions of numerous hormones, paracrine/autocrine factors, and neurotransmitters, is encoded by GNAS, a complex locus with multiple imprinted gene products (1). Heterozygous mutations within GNAS exons encoding Gs{alpha} are found in patients with pseudohypoparathyroidism type Ia (PHP-Ia), who show end-organ resistance to PTH and some additional hormones that act through Gs{alpha}-coupled receptors (2, 3). These patients also display characteristic, but variable, physical features, collectively termed Albright’s hereditary osteodystrophy (AHO), including obesity, short stature, brachydactyly, ectopic ossification, and/or mental retardation (4). Although Gs{alpha} mutations are inherited maternally in these patients, the same mutations are inherited paternally in patients who lack hormone resistance but display AHO, a disorder termed pseudo-pseudohypoparathyroidism (PPHP) (5, 6, 7). Thus, PHP-Ia and PPHP can be found in the same kindreds, and hormone resistance develops only after maternal inheritance of Gs{alpha} mutations. Heterozygous inactivating Gs{alpha} mutations are also associated with progressive osseous heteroplasia (POH), which is characterized by severe ectopic ossification involving skeletal muscles and deep connective tissue (8). POH may represent an extreme manifestation of AHO, because several of the Gs{alpha} mutations are identical with those found in patients with PHP-Ia or PPHP (8), and some patients with POH also exhibit some of the typical AHO features and/or hormone resistance (9, 10).

In most tissues, Gs{alpha} expression is biallelic (11, 12, 13). However, in some tissues, such as renal proximal tubules, thyroid, pituitary, and ovaries, Gs{alpha} is derived predominantly from the maternal allele, i.e. paternal Gs{alpha} transcription is silenced (14, 15, 16, 17), thus explaining the parental origin-specific inheritance of hormone resistance in PHP-Ia/PPHP kindreds. In contrast, AHO features may be the result of Gs{alpha} haploinsufficiency, which has been demonstrated in growth plate chondrocytes through analysis of mice chimeric for wild-type cells and cells that carry heterozygous disruption of Gnas exon 2 (18). Nonetheless, the severity and expression of each AHO feature vary markedly among patients, and therefore, it remains possible that certain AHO features also follow an imprinted mode of inheritance. In fact, this may be true for POH, which develops upon paternal inheritance in most cases (19).

Among the imprinted gene products of GNAS, XL{alpha}s is the only one that shares protein sequence identity with Gs{alpha} (12, 20). XL{alpha}s transcripts use a distinct first exon encoding the unique N terminus of XL{alpha}s (the XL domain) but share exons 2–13 that also encode Gs{alpha}. Furthermore, the C-terminal end of the XL domain shows high homology to the region of Gs{alpha} encoded by exon 1. The promoter driving the expression of XL{alpha}s is active only on the paternal allele. Unlike the ubiquitously expressed Gs{alpha}, XL{alpha}s is expressed more abundantly in pituitary, adrenal gland, and nervous system, although its mRNA has been detected in other tissues, such as kidney, pancreas, growth plate, and adipose tissue (21, 22, 23). In vivo knockout experiments indicated that XL{alpha}s is required for postnatal adaptation to feeding and for glucose and energy metabolism, possibly functioning through mechanisms that do not involve Gs{alpha}-like cell signaling (14, 21, 24). However, consistent with the structural similarities between Gs{alpha} and XL{alpha}s, it has been shown that rat XL{alpha}s can mimic Gs{alpha} functionally in transfected cells (25, 26). A similar signaling role has remained unexplored for human XL{alpha}s, which diverges markedly from its rat ortholog in the XL domain.

In this study we first established that human XL{alpha}s can act similarly to Gs{alpha} in cultured cells. Because Gs{alpha} and XL{alpha}s share the amino acid sequences encoded by exons 2–13, all disease-causing GNAS mutations, with the exception of those located in exon 1, affect not only the transcript encoding Gs{alpha}, but also the transcript encoding XL{alpha}s. Therefore, we introduced some of the GNAS mutations that cause different forms of PHP into the Gs{alpha} and the XL{alpha}s backbone and examined the abilities of these mutant proteins to mediate adenylyl cyclase stimulation. Our results showed that these mutations disrupt cAMP accumulation mediated not only through Gs{alpha}, but also through XL{alpha}s.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients
At presentation, patient 1 was a 23-month-old short, obese girl [weight, 15.5 kg (>95th percentile); height, 81 cm (10th–25th percentile)] with a large tongue, developmental delay, chronic constipation, and frequent episodes of choking. She was noted to have coarse facial features with mild hypotelorism and markedly small hands. Biochemical investigations showed a normal T4 level (6.8 µg/dl; normal range, 5–12) with elevated TSH (18.5 µU/ml; normal range, <10). One month after initial presentation, the patient was admitted to the hospital due to a seizure presumed to be secondary to hypocalcemia; her serum calcium level was 5.9 mg/dl. Her serum phosphorous (11.3 mg/dl; age-matched normal range, 4.5–5.5) and PTH (28.2 µlEq/ml; normal range, 2.4–5.9) levels were elevated. PHP was thus suspected as the cause of her physical and biochemical abnormalities. Genomic DNA was isolated from blood leukocytes, and exons 1–13 (encoding Gs{alpha}) were PCR amplified and directly sequenced. This analysis revealed a heterozygous nucleotide substitution, c.1085 A->C, which led to a missense mutation at residue 362, Gs{alpha}H362P (Fig. 1AGo). This mutation did not introduce or remove a restriction site by itself. However, for analysis purposes, PCR was performed to amplify the region across this mutation, using a reverse primer designed to introduce a BseMI site into the product amplified from the wild-type allele only (Fig. 1BGo). Thus, restriction enzymatic digestion of the patient-derived PCR product with BseMI showed the presence of both the wild-type (109 bp) and the mutant (136 bp) allele; note that the 27-bp fragment generated from the wild-type product was not visualized (Fig. 1CGo). Based on this genetic defect, patient 1 was diagnosed with PHP-Ia.


Figure 1
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  		FIG. 1. Identification of a new GNAS mutation in PHP-Ia. A, Direct nucleotide sequence analysis of GNAS exon 13 after PCR amplification from genomic DNA of patient 1 (right) and a normal control (left). Superimposition of the sequence traces shows the heterozygous c.1085 A->C mutation, which predicts a missense amino acid substitution at residue 362 with respect to Gs{alpha}. B, The mutation removes a BseMI restriction site, which was introduced in the PCR product through the use of a reverse primer that contained a T->G substitution 3 bp downstream of the mutation. C, Confirmation of the mutation by restriction enzymatic digestion with BseMI of PCR-amplified genomic DNA. In the presence of the restriction endonuclease, patient 1 shows an additional band of 136 bp corresponding to the nondigested mutant allele.

 
The mutation was not present in genomic DNA from the mother (father is deceased) or the siblings, who all lacked the distinctive clinical features of the proband. Analysis of polymorphic markers in the region of chromosome 20q13 encompassing GNAS indicated haplotype sharing between the patient and several of her healthy siblings (data not shown). This finding indicated that the identified mutation was de novo and based on the presence of PTH and TSH resistance in the patient, which typically develops only if a GNAS mutation is inherited from a female obligate carrier (5, 6), it appears likely that the mutation occurred on the maternal GNAS allele.

Patients 2 and 3 with Gs{alpha}DelI382 and Gs{alpha}Y391X mutations have been previously described (27, 28). Briefly, Gs{alpha}DelI382 was identified in three boys with isolated PTH resistance, and their unaffected mother and maternal grandfather. This mode of inheritance was consistent with the paternal imprinting described for the autosomal dominant form of PHP-Ib (29), and the in vitro findings were suggestive of resistance toward PTH, but not TSH, LH, and isoproterenol. Consequently, Gs{alpha}DelI382 was thought to cause a form of PHP-Ib (27). Gs{alpha}Y391X was found in a patient who was originally diagnosed with PHP-Ic according to the presence of AHO and multihormone resistance despite apparently normal Gs{alpha} bioactivity; the assay used to determine Gs{alpha} bioactivity examined the ability of patient-derived erythrocyte Gs{alpha} to stimulate adenylyl cyclase in the presence of guanosine 5'-0-(3-thio)triphosphate (28).

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

Materials
[Y34]Human PTH-(1–34) amide (PTH) was synthesized at the Massachusetts General Hospital Biopolymer Core Facility. cDNA encoding the yellow fluorescent protein (YFP) was derived from the plasmid pEYFP-N1 (BD Clontech, Palo Alto, CA). Chemiluminescence immunodetection reagents were purchased from PerkinElmer Life Science (Norwalk, CT), restriction endonucleases were obtained from New England Biolabs (Beverley, CA), and isoproterenol, cholera toxin (CTX), isobutylmethylxanthine (IBMX), and other chemicals were purchased from Sigma-Aldrich Corp. (St. Louis, MO).

Adenoviral constructs
cDNAs encoding hemagglutinin-tagged rat Gs{alpha} and the human PTH receptor type 1 (PTHR1) were previously described (30, 31). Note that rat and human Gs{alpha} are virtually identical and differ from each other only at residue 139, which is an asparagine in rat and an aspartic acid in human. To generate cDNA encoding human XL{alpha}s, the XL exon-encoded portion was amplified by PCR using the PAC clone 806M20 as a template and primers with XbaI and AvrII linkers. This fragment (nucleotide 120,694–121,953 of GenBank AL132655) was used to replace the exon 1-encoded portion of the cDNA encoding Gs{alpha} (32), which was previously modified through silent mutagenesis to contain an AvrII site at the junction of exons 1 and 2. The constructed human XL{alpha}s cDNA was predicted to encode a protein of 736 amino acids based on Q5JWF2 (UniProtKB/TrEMBL). Three mutations were introduced independently into both Gs{alpha} and XL{alpha}s using QuikChange (Stratagene, La Jolla, CA).

Adenoviral vectors were generated according to the manufacturer’s recommendations (ViraPower Adenoviral Expression System, Invitrogen Life Technologies, Inc., Carlsbad, CA). Briefly, each cDNA was cloned into a shuttle vector (pENTR1A) and subsequently transferred into the empty adenoviral vector (pAd/CMV/V5-DEST) by enzymatic recombination. The correctness of the recombinant constructs was verified by restriction analysis and nucleotide sequencing (Massachusetts General Hospital DNA Core Facility). Viral particles were packaged and amplified in HEK293a cells, which were transfected with each linearized plasmid through the use of Effectene (QIAGEN, Valencia, CA). The concentration of the viral lysates ranged between 5 x 107 and 109 particle-forming units/ml.

Generation of the XL-specific antibody
A peptide corresponding to residues 260–276 of human XL{alpha}s (RRVYYDEGVASSDDDSS) was synthesized at the Massachusetts General Hospital Biopolymer Core Facility with a cysteine residue at the amidated C terminus for subsequent conjugation to keyhole limpet hemocyanin. Immunization of rabbits and collection and screening of antisera were performed at Cocalico Biologicals (Reamstown, PA).

Transduction of cells, Western blot analyses, and cAMP measurements
Fibroblastic GnasE2–/E2– cells were generated from murine embryos homozygous for disruption of Gnas exon 2 and therefore lack both Gs{alpha} and XL{alpha}s (25). GnasE2–/E2– cells were plated in 24-well plates at a density of 105 cells/well, and OK cells were plated in 48-well plates at a density of 4 x 104 cells/well the day before transduction, which was performed by adding the appropriate volume of the virus solution to the growth medium. For analysis of protein expression on Western blots and determination of cAMP accumulation in GnasE2–/E2– cells and OK cells, a multiplicity of infection (number of viral particles divided by the number of cells) of 10 or 300 was used, respectively. Western blot analysis was carried out as described previously (25), using equal amounts of protein per lane (30–50 µg) and antiexon 13 antibody for detection of Gs{alpha} and its mutants (Santa Cruz Biotechnologies, Inc., Santa Cruz, CA) and XL-specific antibody for detection of XL{alpha}s and its mutants. For detection of both XL{alpha}s and Gs{alpha} in transduced OK cells, antiexon 13 antibody was used. Signal was detected using goat antirabbit IgG conjugated with horseradish peroxidase (Santa Cruz Biotechnologies, Inc.), followed by chemiluminescence. cAMP measurements before and after CTX treatment or after treatment with PTH or isoproterenol were completed 48 h after transduction. Cells were incubated with agonists in the presence of the phosphodiesterase inhibitor IBMX, and the amount of cAMP in each well was determined by RIA, as previously described (25). Nonlinear regression analysis was performed with DeltaPad PRISM software (GraphPad, Inc., San Diego, CA) for analysis of data from PTH concentration-cAMP response experiments.

Immunocytochemistry and confocal microscopy
GnasE2–/E2– cells were grown and transduced (multiplicity of infection, 500) in collagen-coated, four-well chamber slides. Cells were fixed by 4% paraformaldehyde/PBS and permeabilized with 0.1% Triton X-100. After blocking with 0.5% BSA/PBS, cells were incubated for 90 min with either the antiexon 13 Gs{alpha} antibody (1:500) or the XL-specific antibody (1:200). Cells were washed four times with 0.5% BSA/PBS and incubated for 90 min with a Cy3-labeled goat antirabbit antibody (1:1000; Amersham Biosciences, Arlington Heights, IL). After washing with 0.5% BSA/PBS, slides were mounted with Vectashield mounting medium (Vector Laboratories, Inc., Burlingame, CA) and analyzed using a Zeiss Axioplan confocal fluorescent microscope (Carl Zeiss, Inc., New York, NY).

Statistical analyses
One-way ANOVA was used to determine the significance of observed differences among CTX-induced cAMP levels, before and after normalization to basal levels, in cells expressing wild-type Gs{alpha} or wild-type XL{alpha}s and their mutants. This was followed, for each analysis yielding a significant F ratio, by Tukey’s multiple pairwise comparison test. Student’s t test was used to determine significance of observed differences between the Gs{alpha}- and XL{alpha}s-mediated cAMP levels in GnasE2–/E2– or OK cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Human XL{alpha}s is localized to the plasma membrane in GnasE2–/E2– cells
Although previous studies located rat XL{alpha}s in the trans-Golgi network (20, 33), it has been subsequently shown that this protein is predominantly expressed at the plasma membrane of PC12 cells and transfected HeLa and COS-7 cells (23). Of the six cysteine residues in the rat XL domain identified as targets for palmitoylation and as being critical for membrane targeting (33), only two are conserved in human XL{alpha}s. Moreover, the sequence of the XL domain is poorly conserved between rat and human, except for two short segments (Fig. 2Go). Thus, to determine whether human XL{alpha}s also localizes to the plasma membrane, we examined Gs{alpha}- and XL{alpha}s-deficient mouse embryonic fibroblasts (GnasE2–/E2– cells) (25) transduced with an adenoviral vector containing cDNA encoding either human XL{alpha}s or Gs{alpha}. This adenoviral expression system delivered cDNA into more than 95% of GnasE2–/E2– cells, as judged by transduction of cDNA encoding YFP, used here as a control (data not shown). A polyclonal antihuman XL{alpha}s antibody recognizing an epitope within the unique XL domain (XL-specific antibody) was used to detect human XL{alpha}s, and a polyclonal antibody raised against the C-terminal region of both Gs{alpha} and XL{alpha}s (antiexon 13 antibody) was used to detect Gs{alpha}. Western blot analysis showed the absence of both proteins in lysates of GnasE2–/E2– cells transduced only with adenovirus containing cDNA for YFP (Fig. 3AGo). In contrast, each signaling protein was detected in lysates of GnasE2–/E2– cells transduced with adenovirus containing its cDNAs (Fig. 3AGo). Of note, similar to the findings reported for rat XL{alpha}s (20), the apparent electrophoretic mobility of human XL{alpha}s corresponded to a larger molecular mass (~100 kDa) than that predicted from the amino acid sequence (80 kDa). Indirect confocal immunofluorescence microscopy detected both human XL{alpha}s and Gs{alpha} at the plasma membrane, although there was also punctate perinuclear and intracellular staining for human XL{alpha}s and some cytoplasmic staining for Gs{alpha} (Fig. 3BGo). These findings indicated that human XL{alpha}s localizes to the plasma membrane, consistent with its acting in a manner similar to Gs{alpha}.


Figure 2
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  		FIG. 2. Schematic representation of Gs{alpha} and XL{alpha}s. Exons encoding human XL{alpha}s and Gs{alpha} are given above and below, respectively, each rectangle depicting the main structural features. Amino acid numbering of human XL{alpha}s is based on Q5JWF2 (UniProtKB/TrEMBL). Note that alternatively spliced additional small exons between exons XL and 2 (12 ) are not shown. Figure 2, Regions encoded by different first exons; Figure 2, the region of high homology between the N-terminal domains of XL{alpha}s and Gs{alpha}; {blacksquare}, the proline-rich region (PRR) and the highly charged domain (HCD) conserved between human and rat XL{alpha}s. Roman numerals indicate the switch regions. Figure 2, {alpha}3ß5 and {alpha}4ß6 loops. The positions of the investigated mutations are indicated with arrowheads.

 

Figure 3
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  		FIG. 3. Expression and subcellular localization of Gs{alpha} and XL{alpha}s in GnasE2–/E2– cells. A, Proteins from lysates of cells transduced with adenoviral vectors encoding YFP, Gs{alpha}, or human XL{alpha}s were separated by 9% SDS-PAGE. Blots were probed with either antiexon 13 antibody or XL-specific antibody. B, GnasE2–/E2– cells transduced with adenoviral vectors encoding Gs{alpha} or human XL{alpha}s or nontransduced cells were analyzed by confocal indirect immunofluorescence microscopy using Cy3-labeled antirabbit IgG recognizing the antiexon 13 antibody (left) or the XL-specific antibody (right). Photographs represent four experiments that showed similar results.

 
Human XL{alpha}s functions as a signaling protein
We have previously shown that in transfected GnasE2–/E2– cells, rat XL{alpha}s can stimulate adenylyl cyclase in response to receptor activation (25). Although both rat XL{alpha}s and human XL{alpha}s contain most of the functionally important domains of Gs{alpha}, each has distinct XL domains that are much less conserved. Therefore, we first examined the ability of human XL{alpha}s to stimulate adenylyl cyclase. Adenoviral expression of human XL{alpha}s in GnasE2–/E2– cells resulted in a basal cAMP level more than 10-fold higher than that in nontransduced cells (7.6 ± 1.6 vs. 0.5 ± 0.2 pmol/well). Treatment of the XL{alpha}s-transduced cells with 0.1 µg/ml CTX, which directly activates Gs{alpha} by ADP-ribosylating its guanosine triphosphatase domain, also led to a marked increase in cAMP levels (186.8 ± 12.5 pmol/well), which was even higher than that reached in Gs{alpha}-expressing cells under the same conditions (109.6 ± 18.8 pmol/well; Fig. 4AGo). To assess agonist-induced cAMP accumulation through human XL{alpha}s, we treated transduced GnasE2–/E2– cells with 10–5 M isoproterenol, an agonist of the endogenous ß-adrenergic receptor (25). This treatment elicited pronounced cAMP accumulation in both XL{alpha}s-expressing (57.6 ± 3.6 pmol/well) and Gs{alpha}-expressing (65.2 ± 7.3 pmol/well) cells. In GnasE2–/E2– cells cotransduced with cDNA encoding PTHR1 and either Gs{alpha} or human XL{alpha}s, PTH stimulation also raised cAMP levels markedly, although the level in cells expressing XL{alpha}s (138.1 ± 9.8 pmol/well) was lower than that in cells expressing Gs{alpha} (204.5 ± 28.7 pmol/well). cAMP accumulation in XL{alpha}s-expressing cells was dependent on the agonist concentration (EC50, 1.78 x 10–9 M; 95% confidence interval, 5.69 x 10–10 to 5.54 x 10–9), and the potency of activation was similar to that observed in GnasE2–/E2– cells coexpressing Gs{alpha} and PTHR1 (EC50, 1.38 x 10–9 M; 95% confidence interval, 5.89 x 10–10 to 3.26 x 10–9; Fig. 4BGo). To determine whether human XL{alpha}s can mediate agonist-induced signaling in a different cell type, we used an opossum kidney cell line (OK cells) that expresses PTHR1 (as well as Gs{alpha}) endogenously (34, 35). As shown in Fig. 4CGo, adenoviral overexpression of either Gs{alpha} or human XL{alpha}s in OK cells also resulted in marked increases in basal cAMP accumulation compared with nontransduced OK cells, and the basal cAMP levels in OK cells transiently overexpressing human XL{alpha}s appeared to be higher than the basal cAMP levels in cells overexpressing Gs{alpha}. PTH stimulation led to marked increases over the basal cAMP levels in both cells overexpressing Gs{alpha} and those overexpressing human XL{alpha}s. Western blot analysis using the antiexon 13 antibody showed comparable levels of human XL{alpha}s and Gs{alpha} expression in OK cells transduced individually with adenovirus containing cDNA encoding human XL{alpha}s or Gs{alpha} (Fig. 4DGo). These results indicated that human XL{alpha}s, like Gs{alpha}, was able to mediate both receptor-activated and receptor-independent stimulation of adenylyl cyclase in GnasE2–/E2– and OK cells.


Figure 4
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  		FIG. 4. Human XL{alpha}s mediates adenylyl cyclase stimulation. A, cAMP was measured in transduced GnasE2–/E2– cells without stimulation ( Figure 4), after 0.1 µg/ml CTX treatment (Figure 4), or agonist stimulation ({square}, 10–5 M isoproterenol; {blacksquare}, 10–8 M PTH). The PTH response was determined in cells cotransduced with cDNA for PTHR1 and either human XL{alpha}s or Gs{alpha}. Agonist treatment was performed in the presence of 2 mM IBMX. Data are the mean ± SEM of three independent experiments with duplicate determinations. B, Concentration-response relationship of PTH-induced cAMP formation in GnasE2–/E2– cells cotransduced with PTHR1 cDNA and either Gs{alpha} ({blacksquare}) or XL{alpha}s ({square}). Data represent the mean ± SEM of three independent experiments after normalization to the maximal response in each experiment. The basal cAMP levels were 12.6, 71.7, and 38.3 pmol/well in Gs{alpha}-transduced cells and 90.1, 48.4, and 57.8 pmol/well in XL{alpha}s-transduced cells. The maximal cAMP levels were 908, 327, and 295 pmol/well in Gs{alpha}-transduced cells and 1598, 335, and 285 pmol/well in XL{alpha}s-transduced cells. C, Basal ({square}) and 10–8 M PTH-induced ({blacksquare}) cAMP accumulation in nontransduced (NT) OK cells and OK cells transduced with adenovirus encoding either human XL{alpha}s or Gs{alpha}. The inset shows an enlarged version of data obtained from nontransduced cells. Data are the mean ± SEM of two or more independent experiments with duplicate determinations. D, Western blot analysis showing adenoviral expression of XL{alpha}s and Gs{alpha} in transduced OK cells, detected by antiexon 13 antibody. Note the endogenous expression of the long (52 kDa) and short (45 kDa) Gs{alpha} splice variants, which differ from each other by the presence or absence of sequences derived from exon 3. *, Significantly different from the levels obtained in Gs{alpha}-expressing cells (P < 0.05); {ddagger}, different from the basal cAMP level obtained in Gs{alpha}-expressing cells (P = 0.087); #, different from the basal cAMP level obtained in XL{alpha}s-expressing cells (P = 0.066).

 
Naturally occurring GNAS mutations impair signaling through both Gs{alpha} and human XL{alpha}s
Most mutations identified in patients with PHP-Ia impair Gs{alpha} activity (2). Therefore, we investigated the effects of such mutations on the activity of human XL{alpha}s. We first tested a novel mutation located in GNAS exon 13, which was identified in a patient with PHP-Ia (see Materials and Methods). Neither CTX nor isoproterenol treatment elicited any significant elevation of cAMP in GnasE2–/E2– cells transduced with cDNA encoding this previously unreported Gs{alpha} mutant (Gs{alpha}H362P; Fig. 5AGo). Moreover, PTH failed to induce an elevation of intracellular cAMP in GnasE2–/E2– cells coexpressing PTHR1 and Gs{alpha}H362P (Fig. 5BGo). These results were consistent with a loss of function mutation with respect to Gs{alpha} activity. Under similar conditions, we tested a human XL{alpha}s mutant carrying the same amino acid change at the analogous location (XL{alpha}sH704P). Similar to the findings obtained in cells expressing Gs{alpha}H362P, isoproterenol, PTH, and CTX failed to induce cAMP accumulation in GnasE2–/E2– cells expressing XL{alpha}sH704P (Fig. 5Go, A and B). Thus, the effect of this mutation on the signaling activity of XL{alpha}s appeared to be similar to its effect on the signaling activity of Gs{alpha}.


Figure 5
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  		FIG. 5. The novel GNAS mutation impairs both Gs{alpha}- and XL{alpha}s-mediated cAMP accumulation. A, cAMP was measured in GnasE2–/E2– cells transduced with Gs{alpha}, Gs{alpha}H362P, XL{alpha}s, or XL{alpha}sH704P after CTX or isoproterenol treatment. B, PTH-induced cAMP response of GnasE2–/E2– cells coexpressing PTHR1 and each of the wild-type and mutant proteins. Data, expressed as the fold increase in the cAMP level over the basal level (depicted as a dashed line), represent the mean ± SEM of two independent experiments. The expression of each wild-type and mutant protein in transduced GnasE2–/E2– cells was determined by Western blot (C) and confocal indirect immunofluorescence microscopy (D). Gs{alpha} and Gs{alpha}H362P were detected by the antiexon 13 antibody, and XL{alpha}s and XL{alpha}sH704P were detected by the XL-specific antibody.

 
The {alpha}4ß6 loop of Gs{alpha}, where His362 is located, is implicated in both effector activation and receptor interaction (36, 37). It is therefore possible that the unresponsiveness of Gs{alpha}H362P or human XL{alpha}sH704P reflects deficient effector activation and/or deficient receptor interaction. Alternatively, poor cellular expression of each mutant protein could account for the observed functional defect. Western blot analysis using either the antiexon 13 antibody or the XL-specific antibody showed that the expression of each mutant in transduced GnasE2–/E2– cells was lower than that of its wild-type counterpart, with the reduction being more pronounced for Gs{alpha}H362P (Fig. 5CGo). Confocal immunofluorescence microscopy also revealed reduced fluorescence intensity in GnasE2–/E2– cells expressing either Gs{alpha}H362P or human XL{alpha}sH704P as well as a lack of plasma membrane staining (Fig. 5DGo), indicating that this mutation affected both protein expression levels and intracellular targeting, which could account, at least in part, for the poor responsiveness of these mutants to agonist and CTX stimulation.

Loss of function Gs{alpha} mutations have also been identified in patients diagnosed with PHP-Ib or PHP-Ic: 1) residue Ile382 was deleted in three brothers with apparently selective resistance to PTH, who were therefore diagnosed as having PHP-Ib; and 2) residue Tyr391 was mutated to a termination codon in a patient with manifestations of AHO and multihormone resistance despite apparently normal Gs{alpha} bioactivity, who was hence diagnosed as having PHP-Ic (28). Note that the identification of a coding Gs{alpha} mutation in the latter case may suggest that this patient actually had PHP-Ia. To determine whether the same amino acid changes also impair signaling through XL{alpha}s, we tested the ability of the homologous XL{alpha}s mutants, XL{alpha}sDelI724 and XL{alpha}sY733X, to mediate adenylyl cyclase stimulation. Basal cAMP accumulation appeared comparable among GnasE2–/E2– cells transiently expressing individual Gs{alpha} and XL{alpha}s mutants or their wild-type counterparts, except that the basal cAMP level in cells expressing XL{alpha}sDelI724 was significantly higher than that in cells expressing wild-type XL{alpha}s (Fig. 6AGo). All four mutants seemed to respond to CTX; however, normalization of the data according to basal values revealed that the CTX-induced cAMP response through Gs{alpha}DelI382 and XL{alpha}sDelI724 was significantly reduced compared with that in cells expressing either wild-type Gs{alpha} or wild-type XL{alpha}s (Fig. 6AGo). Isoproterenol completely failed to induce intracellular cAMP accumulation in GnasE2–/E2– cells expressing each of the mutant Gs{alpha} and XL{alpha}s proteins; similarly, there was no PTH-induced cAMP accumulation in cells coexpressing PTHR1 and any of the mutant proteins (Fig. 6AGo). Western blot analysis using the antiexon 13 antibody or the XL-specific antibody showed that Gs{alpha}DelI382 and XL{alpha}sDelI724 were expressed at slightly higher levels than their wild-type counterparts (Fig. 6BGo). The XL{alpha}sY733X mutant was also expressed at levels comparable to wild-type XL{alpha}s. The expression of Gs{alpha}Y391X in GnasE2–/E2– cells was not detected by Western blot analysis. This was most likely due to the presence of the mutation within the epitope recognized by the antiexon 13 antibody, because Western blots using the same antibody also failed to detect XL{alpha}sY733X (data not shown). Nevertheless, a pronounced response to CTX treatment in GnasE2–/E2– cells expressing Gs{alpha}Y391X (see Fig. 6AGo) indicated that the recombinant mutant protein was indeed expressed in these cells. Overall, these results indicated that the effect of each of these GNAS mutations on Gs{alpha} activity was similar to its effect on XL{alpha}s activity, which is consistent with the hypothesis that the patients carrying these mutations on the paternal allele have impaired signaling through both Gs{alpha} and XL{alpha}s.


Figure 6
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  		FIG. 6. Effects of the previously identified Gs{alpha}DelI382 and Gs{alpha}Y391X mutations on XL{alpha}s-mediated adenylyl cyclase stimulation. A, cAMP was measured in GnasE2–/E2– cells transduced with Gs{alpha}, Gs{alpha}DelI382, Gs{alpha}Y391X, XL{alpha}s, XL{alpha}sDelI724, or XL{alpha}sY733X after CTX or agonist treatment (isoproterenol or PTH). Results are either expressed as the cAMP level (picomoles per well; top) or the fold increase in the cAMP level over the basal level (bottom). Data represent the mean ± SEM of two independent experiments. B, Western blot was used to determine the expression of each wild-type and mutant protein in transduced GnasE2–/E2– cells. Wild-type Gs{alpha} and the two Gs{alpha} mutants were detected by the antiexon 13 antibody, and wild-type XL{alpha}s and the two XL{alpha}s mutants were detected by the XL-specific antibody. Note that the Gs{alpha}Y391X mutant was not detected on Western blot due to disruption of the epitope recognized by the antiexon 13 antibody. *, Significantly different from the corresponding cAMP level in cells expressing wild-type XL{alpha}s or Gs{alpha} (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We showed that human XL{alpha}s localizes to the plasma membrane as well as other intracellular sites; consistent with this finding, it is capable of mediating basal and receptor-activated adenylyl cyclase stimulation. Furthermore, we demonstrated that naturally occurring, loss of function GNAS mutations impair not only the activity of Gs{alpha}, but also the activity of XL{alpha}s. Although these findings were obtained in vitro, and thus, their direct relevance to biology needs to be verified, our results are consistent with the hypothesis that XL{alpha}s deficiency, in addition to Gs{alpha} deficiency, may play a role in disorders associated with paternal GNAS mutations.

Reports of PHP-Ia and PPHP cases in the literature have indicated that inactivating GNAS mutations lead to AHO regardless of parental origin, and that mutations in GNAS exon 1, which are not predicted to disrupt XL{alpha}s, also cause AHO (with or without hormone resistance) (2, 3). These findings argue against a role for XL{alpha}s in the pathogenesis of PPHP or PHP-Ia. However, there is remarkable patient to patient variation in the manifestation of AHO, and currently available data are not sufficient to rule out a possible correlation between the parental origin of GNAS mutations and the variety and severity of typical AHO features. In fact, POH, which appears to be an extreme manifestation of AHO in some patients, is strongly associated with inactivating GNAS mutations that are inherited paternally (19), suggesting that there may be parent of origin specific effects on the development or severity of certain AHO features. Thus, it is possible that XL{alpha}s deficiency occurring after paternal, but not maternal, inheritance of the GNAS coding mutations adds to or modifies the phenotypic presentation of AHO features in PPHP patients. Providing support for this hypothesis, we have previously shown that growth plate chondrocytes heterozygous for paternal disruption of Gnas exon 2 (lacking XL{alpha}s as well as one copy of Gs{alpha}) undergo hypertrophic differentiation more prematurely than chondrocytes heterozygous for maternal disruption of Gnas exon 2 (with normal XL{alpha}s expression despite the lack of one copy of Gs{alpha}) (18). If applicable to humans with inactivating GNAS mutations, this finding would predict that the average height of patients with PPHP is less than that of patients with PHP-Ia. However, such a difference in stature has not been established, and it is plausible that systemic endocrinopathies that occur after maternal inheritance of inactivating GNAS mutations, such as GH deficiency (38, 39), abolish the predicted difference in the degree of short stature between patients with PPHP and those with PHP-Ia. Careful comparisons between phenotypes associated with maternal and paternal GNAS mutations and those associated with paternal mutations of GNAS exon 1 and paternal mutations of other GNAS exons are necessary to determine whether XL{alpha}s deficiency could play a role in the development of certain AHO features.

It is important to note that because of the limited tissue distribution of XL{alpha}s, its deficiency, unlike Gs{alpha} deficiency, would be expected to influence the function of a relatively small number of tissues. Therefore, even if XL{alpha}s simply mimics Gs{alpha}, which is hardly the case based on data from in vivo models (14, 21, 24), the phenotype caused by XL{alpha}s deficiency after paternal inheritance is not predicted to be the same as that caused by Gs{alpha} deficiency after maternal inheritance. Hence, the hypothesis that XL{alpha}s deficiency could contribute to the disorders caused by paternally inherited GNAS mutations is not incompatible with the finding that the clinical features of patients with PPHP and those with PHP-Ia differ from each other.

XL{alpha}s is expressed paternally and, therefore, is not affected by maternal GNAS mutations. It is therefore plausible that in patients with PHP-Ia, paternally expressed, intact XL{alpha}s mediates some responses that typically depend on Gs{alpha}. For example, predominantly maternal expression of Gs{alpha} has been demonstrated in the whole pituitary (40), a tissue in which XL{alpha}s is expressed abundantly (20). Although the types of pituitary cells that show Gs{alpha} imprinting and abundant XL{alpha}s expression are not precisely known, it is possible that XL{alpha}s-mediated signaling could account for the lack of resistance to corticotropin-releasing factor in PHP-Ia patients (41, 42). A similar mechanism could explain why renal PTH resistance does not develop until after the first year of life in patients with PHP-Ia (43) as well as in patients with PHP-Ib who carry maternal imprinting mutations of GNAS (44, 45, 46). Northern blot analysis revealed early postnatal expression of mouse XL{alpha}s mRNA in the kidney (21), and we detected a decline in both XL{alpha}s mRNA and protein levels in whole kidneys of mice within the first week after birth (Linglart, A., and M. Bastepe, unpublished observations). It is therefore possible that XL{alpha}s mediates the renal actions of PTH in patients with PHP-Ia and PHP-Ib during the first year(s) of postnatal development. Our finding that XL{alpha}s can mediate PTH-induced cAMP accumulation in OK cells, which have some characteristics of proximal tubular cells (47, 48), is consistent with this hypothesis. Future investigations will distinguish the mechanisms that involve XL{alpha}s from those that involve cell type- and developmental stage-specific Gs{alpha} imprinting in preventing hormone resistance in these settings.

Data from OK cells transiently overexpressing comparable levels of either Gs{alpha} or XL{alpha}s suggest that XL{alpha}s may have a higher basal activity than Gs{alpha}. Although such an enhanced, agonist-independent activity for XL{alpha}s is also evident from elevated levels of CTX-induced cAMP accumulation in GnasE2–/E2– cells expressing XL{alpha}s compared with Gs{alpha}, the response to PTH does not appear to be mediated as effectively by XL{alpha}s as by Gs{alpha}. Because the data were obtained in transduced cells that express comparable Gs{alpha} and XL{alpha}s levels, differences in expression levels are not sufficient to explain these findings. Additional investigations are necessary to verify these results and, subsequently, to investigate the mechanisms underlying the functional differences between XL{alpha}s and Gs{alpha}.

Despite a complete loss of agonist-induced response, the Gs{alpha}Y391X and XL{alpha}sY733X mutants showed CTX-induced cAMP generation that appeared to be only slightly reduced. These results suggest a predominant impairment of receptor interaction, rather than adenylyl cyclase activation, which is consistent with the importance of the C terminus of Gs{alpha} in receptor coupling (37). These findings also explain the normal guanosine 5'-0-(3-thio)triphosphate response of erythrocyte Gs{alpha} derived from the patient with heterozygous Gs{alpha}Y391X mutation (28). In cells expressing the XL{alpha}sDelI724 mutant, the basal level of cAMP accumulation was significantly elevated compared with that in cells expressing either the corresponding Gs{alpha} mutant (Gs{alpha}DelI382) or the wild-type XL{alpha}s. Although the Western blot analysis showing a slightly higher expression level of XL{alpha}sDelI724 mutant than the others could explain this finding, the possibility that the deletion of Ile724 in XL{alpha}s leads to constitutive XL{alpha}s activity remains to be explored. In contrast, both XL{alpha}sDelI724 and Gs{alpha}DelI382 failed to stimulate adenylyl cyclase in response to PTH and isoproterenol. These findings differ from those reported previously by Wu et al. (27), who showed that the DelI382 mutation causes uncoupling of Gs{alpha} from PTHR1, but not from the ß2-adrenergic receptor. This discrepancy could reflect the differences in the cell types used in these analyses. HEK293 cells, a cell line derived from human embryonic kidney, were used by Wu et al. (27), whereas GnasE2–/E2– cells, a cell line derived from mouse embryonic fibroblasts, were used in our study (25). In addition to being from a different lineage, HEK293 cells, unlike GnasE2–/E2– cells, express endogenous wild-type Gs{alpha}, which may render data analysis difficult by providing a high basal and agonist-induced cAMP response that needs to be carefully subtracted from the responses mediated by mutant Gs{alpha} proteins.

In vivo data from mice with targeted disruption of XL{alpha}s (14, 21, 24) and from children with paternal deletions that involve GNAS (49) suggest that XL{alpha}s is important in adaptation to feeding in the newborn and for glucose and energy metabolism. Because mice homozygous for Gs{alpha} ablation die in utero and, therefore, were not investigated (14, 50, 51), and because mice heterozygous for Gs{alpha} ablation alone (50, 51) do not phenocopy mice with XL{alpha}s ablation alone (21), it appears likely that certain cellular roles of XL{alpha}s significantly differ from the roles of Gs{alpha}. These unique roles may be conferred at the molecular level by the XL domain and may involve interaction with other proteins, such as ALEX, an alternative translation product of XL{alpha}s mRNA shown to interact with the XL domain (52, 53). Future investigations are necessary to understand the significance of XL{alpha}s activity in different cellular responses.

In summary, our results demonstrate that human XL{alpha}s can mediate receptor-activated adenylyl cyclase stimulation, and that naturally occurring GNAS mutations impair signaling through XL{alpha}s as well as Gs{alpha}. Thus, XL{alpha}s may contribute to Gs{alpha} signaling in physiological and pathological conditions, and its deficiency might have a role in the pathogenesis of diseases that are caused by paternal GNAS mutations.


   Acknowledgments
 
We thank all family members for their participation, and their physicians (Drs. Lynne Levitsky and Pierre Lecomte) for their clinical evaluation of the patients. We also thank Dr. Hesham Tawfeek and Mr. Robert Tyszkowski for advice and technical help in confocal immunofluorescence microscopy.


   Footnotes
 
This work was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases [RO1-46718-10 (to H.J.) and KO1-DK-062973 (to M.B.)], an operating grant from the Canadian Institutes of Health Research (to G.N.H.), and a Research Fellowship Grant from the European Society of Pediatric Endocrinology (to A.L.).

A.L., M.J.M., M.A.K., D.M.B., G.N.H., H.J., and M.B. have nothing to declare.

First Published Online February 16, 2006

Abbreviations: AHO, Albright’s hereditary osteodystrophy; CTX, cholera toxin; Gs{alpha}, stimulatory G protein; IBMX, isobutylmethylxanthine; PHP, pseudohypoparathyroidism; PPHP, pseudo-pseudohypoparathyroidism; POH, progressive osseous heteroplasia; PTHR1, PTH receptor type 1; YFP, yellow fluorescent protein.

Received November 22, 2005.

Accepted for publication February 3, 2006.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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C. Aydin, N. Aytan, M. J. Mahon, H. A. W. Tawfeek, N. W. Kowall, A. Dedeoglu, and M. Bastepe
Extralarge XL{alpha}s (XXL{alpha}s), a Variant of Stimulatory G Protein {alpha}-Subunit (Gs{alpha}), Is a Distinct, Membrane-Anchored GNAS Product that Can Mimic Gs{alpha}
Endocrinology, August 1, 2009; 150(8): 3567 - 3575.
[Abstract] [Full Text] [PDF]

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 J. Pharmacol. Exp. Ther.Home page
A. I. Kaya, O. Ugur, S. S. Oner, M. Bastepe, and H. O. Onaran
Coupling of {beta}2-Adrenoceptors to XL{alpha}s and G{alpha}s: A New Insight into Ligand-Induced G Protein Activation
J. Pharmacol. Exp. Ther., April 1, 2009; 329(1): 350 - 359.
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 Mol Biol EvolHome page
S. Wadhawan, B. Dickins, and A. Nekrutenko
Wheels within Wheels: Clues to the Evolution of the Gnas and Gnal Loci
Mol. Biol. Evol., December 1, 2008; 25(12): 2745 - 2757.
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 PhysiologyHome page
S. Krechowec and A. Plagge
Physiological Dysfunctions Associated with Mutations of the Imprinted Gnas Locus
Physiology, August 1, 2008; 23(4): 221 - 229.
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 J. Clin. Endocrinol. Metab.Home page
V. Mariot, S. Maupetit-Mehouas, C. Sinding, M.-L. Kottler, and A. Linglart
A Maternal Epimutation of GNAS Leads to Albright Osteodystrophy and Parathyroid Hormone Resistance
J. Clin. Endocrinol. Metab., March 1, 2008; 93(3): 661 - 665.
[Abstract] [Full Text] [PDF]

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 J EndocrinolHome page
A. Plagge, G. Kelsey, and E. L Germain-Lee
Physiological functions of the imprinted Gnas locus and its protein variants G{alpha}s and XL{alpha}s in human and mouse
J. Endocrinol., February 1, 2008; 196(2): 193 - 214.
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 Proc. Natl. Acad. Sci. USAHome page
N. Makita, J. Sato, P. Rondard, H. Fukamachi, Y. Yuasa, M. A. Aldred, M. Hashimoto, T. Fujita, and T. Iiri
Human Gs{alpha} mutant causes pseudohypoparathyroidism type Ia/neonatal diarrhea, a potential cell-specific role of the palmitoylation cycle
PNAS, October 30, 2007; 104(44): 17424 - 17429.
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 Hum Mol GenetHome page
S. Michienzi, N. Cherman, K. Holmbeck, A. Funari, M. T. Collins, P. Bianco, P. G. Robey, and M. Riminucci
GNAS transcripts in skeletal progenitors: evidence for random asymmetric allelic expression of Gs{alpha}
Hum. Mol. Genet., August 15, 2007; 16(16): 1921 - 1930.
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