Receptor-Mediated Adenylyl Cyclase Activation Through XL{alpha}s, the Extra-Large Variant of the Stimulatory G Protein {alpha}-Subunit

Murat Bastepe, Yasemin Gunes, Beatriz Perez-Villamil, Joy Hunzelman, Lee S. Weinstein and Harald Jüppner

Endocrine Unit (M.B., Y.G., J.H., H.J.) and Cancer Center (B.P.-V.), Massachusetts General Hospital and MassGeneral Hospital for Children (H.J.), and Harvard Medical School, Boston, Massachusetts 02114; Metabolic Diseases Branch (L.S.W.), National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Harald Jüppner, M.D., Endocrine Unit, Massachusetts General Hospital, 50 Blossom Street, WEL 5, Boston, Massachusetts 02114. E-mail: jueppner{at}helix.mgh.harvard.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
XL{alpha}s, the large variant of the stimulatory G protein {alpha} subunit (Gs{alpha}), is derived from GNAS1 through the use of an alternative first exon and promoter. Gs{alpha} and XL{alpha}s have distinct amino-terminal domains, but are identical over the carboxyl-terminal portion encoded by exons 2–13. XL{alpha}s can mimic some functions of Gs{alpha}, including ß{gamma} interaction and adenylyl cyclase stimulation. However, previous attempts to demonstrate coupling of XL{alpha}s to typically Gs-coupled receptors have not been successful. We now report the generation of murine cell lines that carry homozygous disruption of Gnas exon 2, and are therefore null for endogenous XL{alpha}s and Gs{alpha} (GnasE2-/E2-). GnasE2-/E2- cells transfected with plasmids encoding XL{alpha}s and different heptahelical receptors, including the ß2-adrenergic receptor and receptors for PTH, TSH, and CRF, showed agonist-mediated cAMP accumulation that was indistinguishable from that observed with cells transiently coexpressing Gs{alpha} and these receptors. Our findings thus indicate that XL{alpha}s is capable of functionally coupling to receptors that normally act via Gs{alpha}.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
HETEROTRIMERIC G PROTEINS are essential for cell signaling throughout the body. The stimulatory G protein, Gs, couples activation of a host of different transmembrane receptors to adenylyl cyclase stimulation, leading to intracellular generation of cAMP (1, 2). The structural features of Gs necessary for receptor coupling, nucleotide binding, and adenylyl cyclase stimulation are primarily contained within the {alpha}-subunit (Gs{alpha}), which also possesses an intrinsic GTP phosphohydrolase activity that serves to limit the duration of activation (1, 2).

Gs{alpha} is encoded by GNAS1, an imprinted gene, which gives rise to multiple additional gene products with parent-of-origin specific expression. Encoded by exons 1–13, the almost ubiquitously expressed Gs{alpha} is biallelically transcribed in most tissues (3, 4), with the exception of proximal renal tubular cells and some pituitary cells, where yet-unknown mechanisms lead to paternal silencing of Gs{alpha} expression (5, 6). A large variant of Gs{alpha}, termed XL{alpha}s (for extra-large), is derived from GNAS1 through the use of an alternative promoter and first exon that splices onto the common exon 2 (4, 7). Unlike the widely expressed Gs{alpha}, XL{alpha}s has a more limited tissue distribution. It is primarily found in neuroendocrine tissues with particularly high levels in the pituitary (8, 9), and in all investigated tissues its expression occurs from the paternal GNAS1 allele (4, 7).

Consistent with its broad biological importance, numerous different GNAS1 mutations have been identified in a large number of unrelated patients affected by different disorders such as pseudohypoparathyroidism type Ia (PHP-Ia), progressive osseous heteroplasia, McCune-Albright syndrome, and testotoxicosis associated with PHP-Ia (2, 10, 11, 12, 13, 14). Most of these GNAS1 mutations are located within exons 2–13, thus affecting the amino acid sequence common to Gs{alpha} and XL{alpha}s, and may therefore have broader functional implications than currently appreciated.

XL{alpha}s contains all structural domains of Gs{alpha} except for the amino acid residues encoded by exon 1, which have been implicated in ß{gamma} interaction and in receptor coupling (15, 16). This functionally important domain of Gs{alpha} is replaced in XL{alpha}s by the amino acid sequence encoded by exon XL, which shares significant homology at its carboxyl-terminal end with the region encoded by the Gs{alpha}-specific exon 1 (8, 17). Because of the significant degree of overall sequence homology, XL{alpha}s was predicted to display Gs-like properties in vitro and in vivo. In support of this notion, rat XL{alpha}s has been shown to localize to the plasma membrane in both neuroendocrine and nonneuroendocrine cells (9), and recombinant XL{alpha}s and Gs{alpha} behaved similarly in various functional assays. For example, XL{alpha}s, like Gs{alpha}, was shown to undergo conformational changes upon GTP{gamma}S binding and to bind to ß{gamma} dimers, thus forming a stable heterotrimeric complex (17). Furthermore, when bound to GTP{gamma}S, or when rendered constitutively active through the introduction of a point mutation that confers GTP phosphohydrolase deficiency, XL{alpha}s was shown to stimulate adenylyl cyclase, indicating that it may function as a signaling protein (17).

Nevertheless, several different methods have thus far failed to demonstrate efficient coupling of XL{alpha}s to different G protein-coupled receptors. These studies involved reconstitution of Gs-deficient S49 cyc- cell membranes with in vitro translated XL{alpha}s, the transfection of PC12 cells (that express Gs{alpha} and XL{alpha}s endogenously) with plasmid DNA encoding rat XL{alpha}s, and azidoanilide-GTP photoaffinity labeling of endogenous XL{alpha}s in pituitary membranes. The receptors that failed to show functional coupling of XL{alpha}s in these assays included the ß2-adrenergic receptor (ß2AR) and the receptors for pituitary adenylyl cyclase activating peptide and CRF (17). Thus, the biological role(s) of XL{alpha}s within the cell has remained obscure, which led to the hypothesis that this GNAS1-derived variant of Gs{alpha} transduces primarily the signals of an as-yet-undefined class of cell surface receptors.

However, coupling of XL{alpha}s to typical Gs-coupled receptors could have been missed because of technical limitations, i.e. due to the presence of endogenous XL{alpha}s or Gs{alpha}, or due to insufficient posttranslational modifications of the in vitro translated XL{alpha}s. In an effort to assess the role of XL{alpha}s less ambiguously, we developed cell lines that are homozygous for disruption of Gnas exon 2 (GnasE2-/E2- cells), one of the exons common to Gs{alpha} and XL{alpha}s. Transfections of these null cells revealed that XL{alpha}s functionally couples to several different Gs-coupled receptors.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To be able to analyze the possible function of XL{alpha}s as a signaling molecule, we established clonal murine cell lines that do not express any endogenous XL{alpha}s or Gs{alpha}. Functional ablation of Gnas, the mouse ortholog of GNAS1, has been previously achieved by the targeted disruption of exon 2 (5), which is used by several different transcripts from this locus, including those that encode XL{alpha}s and Gs{alpha} (Fig. 1AGo). Because homozygous inheritance of the disrupted gene leads to lethality on or before embryonic d 10.5 (E10.5) (5), we isolated E9.5 embryos from matings between mice with heterozygous disruption of Gnas exon 2. Some of the embryos were markedly misshapen and significantly smaller, whereas the shape and size of others appeared appropriate for that stage of development. From individual embryos we established primary cultures, which were subsequently immortalized by transformation with mutant SV40 encoding a temperature-sensitive large T antigen. By limiting dilution of the immortalized cultures, we then obtained clonal cell lines for which genotyping by Southern analysis revealed the homozygous disruption of Gnas exon 2 (GnasE2-/E2-; Fig. 1BGo). The GnasE2-/E2- cells grow as a monolayer in F12-DMEM supplemented with fetal bovine serum, and they are maintained at 33 C for stability of the mutant SV40 large T antigen.



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Figure 1. Generation of Murine Cell Lines Lacking Gs{alpha} and XL{alpha}s

A, The Gnas locus. Boxes and connecting lines indicate exons and introns, respectively. Broken lines depict splicing pattern; arrows show direction of transcription; asterisks indicate termination codons. Nesp55, the murine homolog of the neuroendocrine secretory protein 55 (NESP55) in humans, is encoded by two exons in the mouse genome (7 ), as opposed to a single exon in the human genome (35 ). Exon 1A is the alternative first exon giving rise to 1A transcripts, which, similar to Nesp55 and XL{alpha}s transcripts, splice onto exons 2–13 (36 37 38 ). Exons encoding an antisense transcript (39 40 ) are not shown. m, Maternal; p, paternal; bi, bi-allelic expression; horizontal bar, hybridization probe; X, XmnI site. B, Southern blot analysis. The XmnI fragment representing the targeted allele is 9 kb due to an additional XmnI site (in parentheses) within the neomycin-resistance cassette (neoR).

 
To examine whether XL{alpha}s can exert receptor-mediated adenylyl cyclase stimulation, we first chose to study the PTHR1 (PTH receptor 1), a heptahelical receptor for PTH and PTH-related peptide (PTHrP), which typically couples to Gs and Gq (18). We used one of the clonal GnasE2-/E2- cell lines, designated 2B2 (see Fig. 1BGo), to examine coupling of XL{alpha}s to the PTHR1. Using vector-transfected cells, or cells transfected only with the cDNA encoding the PTHR1, 10-8M PTH failed to induce a significant increase in cAMP accumulation over baseline (0.31 ± 0.12 vs. 0.42 ± 0.13 pmol/well, n = 4, before and after PTH treatment, respectively, for cells transfected with PTHR1 cDNA alone; Fig. 2AGo). Although a similar result was obtained in cells transfected with plasmids encoding XL{alpha}s or Gs{alpha} in the absence of the PTHR1, challenge with PTH resulted in a marked elevation of intracellular cAMP in cells cotransfected with cDNAs encoding the PTHR1 and XL{alpha}s (1.88 ± 0.55 vs. 12.39 ± 3.63 pmol/well, n = 9, before and after PTH treatment, respectively). Control experiments with cells transiently expressing both the PTHR1 and Gs{alpha} revealed the expected increase in the level of cAMP after PTH challenge (0.72 ± 0.16 vs. 5.76 ± 1.06 pmol/well, n = 9, before and after PTH treatment, respectively). Western blot analysis using an affinity-purified antibody directed to an epitope within the region encoded by exon 13 (RM antibody, kindly provided by Dr. Paul K. Goldsmith), and thus recognizing both XL{alpha}s and Gs{alpha}, showed comparable expression of the two proteins in GnasE2-/E2- cells transiently transfected with their respective cDNAs. In contrast, no XL{alpha}s or Gs{alpha} immunoreactivity was detected in cells transfected either with the empty vector or with vector encoding the receptor alone (see Fig. 2AGo). cAMP accumulation in cells transiently coexpressing the PTHR1 and XL{alpha}s was dependent on the concentration of PTH (EC50 = 1.76 x 10-10, 95% confidence interval: 1.33 x 10-10 to 2.33 x 10-10M; Fig. 2BGo), and under these conditions, the dose-response relationship was comparable to that obtained in cells coexpressing the PTHR1 and Gs{alpha} (EC50 = 1.25 x 10-10, 95% confidence interval: 9.43 x 10-11 to 1.65 x 10-10M). Upon challenge with PTHrP, a similar dose-response pattern was obtained in 2B2 cells transiently expressing the PTHR1 and XL{alpha}s, and those expressing the PTHR1 and Gs{alpha} (data not shown). These results indicated that XL{alpha}s was capable of coupling the PTHR1 to adenylyl cyclase stimulation.



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Figure 2. Functional Coupling of XL{alpha}s to the PTHR1

A, XL{alpha}s-mediated cAMP accumulation upon agonist stimulation of the PTHR1 (top); absence (open bars) or presence (solid bars) of 10-8 M PTH. Values are mean ± SD of duplicate cAMP determinations; *, P < 0.01 for difference between means of basal and stimulated values (Student’s t test). Western blot analysis showing XL{alpha}s and Gs{alpha} proteins (94 kDa and 52 kDa, respectively) in transfected GnasE2-/E2- cells (bottom) was performed as in Materials and Methods. Data are representative of at least three independent experiments with similar results. B, PTH-induced cAMP formation in cells coexpressing PTHR1 and XL{alpha}s (squares) or PTHR1 and Gs{alpha} (triangles). Values were normalized to the maximal level of cAMP attained in each transfection. Data represent mean ± SEM of four independent experiments.

 
We next tested the TSH receptor (TSHR) for coupling to XL{alpha}s. TSH treatment failed to elicit a cAMP response in 2B2 cells transfected only with cDNA encoding the TSHR, whereas an agonist-dependent cAMP increase was obtained in cells cotransfected with cDNAs encoding the TSHR and XL{alpha}s (Fig. 3AGo), indicating functional coupling of these two proteins. Control experiments with cells coexpressing the TSHR and Gs{alpha} also demonstrated a TSH concentrationdependent cAMP accumulation.



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Figure 3. Functional Coupling of XL{alpha}s to the TSHR (A), CRFR1 (B), and ß2AR (C)

Transfected GnasE2-/E2- cells were challenged with varying concentrations of agonists in the presence of 2 mM isobutylmethylxanthine. Values are mean ± SD of duplicate cAMP determinations. Data are representative of at least three separate experiments.

 
The PTHR1 and the TSHR, both of which demonstrated XL{alpha}s coupling in our experiments, had not been examined previously with regard to this signaling protein. However, previous findings suggested that certain other Gs-coupled receptors, such as the receptor for CRF and ß2AR, couple poorly, if at all, to XL{alpha}s (17). It thus appeared plausible that XL{alpha}s shows selective coupling to only a few receptors. To investigate this possibility, we transfected the 2B2 cells with plasmids encoding the CRF receptor 1 (CRFR1) or the ß2AR. Upon challenge with CRF, cells coexpressing both the CRFR1 and XL{alpha}s showed a concentration-dependent elevation in cAMP accumulation, whereas CRF treatment of cells transiently expressing the CRFR1 alone yielded no significant increase of cAMP (Fig. 3BGo). A similar agonist-dependent cAMP response was obtained in cells expressing the CRFR1 and Gs{alpha}. Isoproterenol challenge of 2B2 cells transfected with cDNAs encoding the ß2AR and XL{alpha}s (or Gs{alpha}) also resulted in a significant, dose-dependent rise in cAMP accumulation, whereas cells transfected with cDNA encoding the ß2AR alone failed to elicit an agonist-induced cAMP response (Fig. 3CGo). An isoproterenol-induced increase in cAMP accumulation was also observed in 2B2 cells transfected only with cDNA encoding XL{alpha}s or Gs{alpha}, suggesting endogenous expression of the ß-adrenergic receptor(s) in these cells (see below). These data thus indicated that XL{alpha}s can also couple to the CRFR1 and the ß2AR.

Two additional clonal cell lines, designated 1B2 and 2D12, which also lacked endogenous XL{alpha}s and Gs{alpha} due to homozygous disruption of Gnas exon 2 (see Fig. 1BGo), were also used for determining receptor-coupling properties of XL{alpha}s. The results obtained with these cells were similar to those described above and thus provided further confirmation that XL{alpha}s can exhibit Gs-like activity (data not shown).

We also evaluated receptor-coupling efficiency of XL{alpha}s at different expression levels by examining agonist-induced cAMP accumulation in 2B2 cells transiently transfected with varying concentrations of cDNA encoding XL{alpha}s. In an attempt to maintain equal transfection efficiency, and thereby obtain varying expression levels of XL{alpha}s in individual transfected cells, the total amount of plasmid DNA used in each transfection was kept constant by adding appropriate amounts of empty vector. Because our initial experiments with 2B2 cells had indicated endogenous expression of the ß-adrenergic receptor(s), we used isoproterenol as an agonist, thus eliminating the possibility of experimental variation in receptor levels. Cells expressing XL{alpha}s, including those transfected with the lowest amount of XL{alpha}s cDNA (0.02 µg/well), responded to 10-5 M isoproterenol with significant increases in cAMP levels over basal, and the response was proportional to the amount of cDNA encoding XL{alpha}s used for transfection (Fig. 4AGo). When 2B2 cells were transfected with the same amounts of cDNA encoding Gs{alpha}, a similar pattern of isoproterenol-induced cAMP response was obtained, although the levels were about 2-fold lower than those obtained for XL{alpha}s cDNA (Fig. 4AGo). To determine whether these quantitative differences in agonist-induced cAMP levels between cells expressing XL{alpha}s and those expressing Gs{alpha} reflected differences in agonist-independent activities of these proteins in transfected cells, we examined cholera toxin (CTX)-induced cAMP accumulation. CTX ADP ribosylates both of these proteins (8, 19), and it has been well established for Gs{alpha}, although never been demonstrated for XL{alpha}s, that this posttranslational modification leads to stimulation of adenylyl cyclase in the absence of receptor activation. Treatment of XL{alpha}s- or Gs{alpha}-transfected cells with 1 µg/ml CTX resulted in elevations of cAMP levels that were proportional to the amount of cDNA used for transfection, and furthermore, the level of CTX-induced cAMP accumulation for each transfection was lower in cells expressing Gs{alpha} than those expressing XL{alpha}s (Fig. 4BGo). Western blot analysis of lysates from cells transfected with varying cDNA amounts indicated lower expression of Gs{alpha} compared with XL{alpha}s for each transfection (data not shown), thus providing a reasonable explanation for the observed differences between agonist-independent activities of these proteins. When data from the experiments with Gs{alpha}-expressing cells that had been stimulated with isoproterenol (Fig. 4AGo) were normalized according to the differences in CTX-induced cAMP levels for each transfection (Fig. 4BGo), the patterns of cAMP accumulation in cells expressing different levels of XL{alpha}s or Gs{alpha} were virtually identical (Fig. 4CGo). These findings thus indicated that the ability of XL{alpha}s for coupling to the ß-adrenergic receptor is comparable to that of Gs{alpha}.



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Figure 4. Comparison of the Effectiveness of XL{alpha}s and Gs{alpha} for Coupling to the Endogenous ß-Adrenergic Receptor(s) Expressed in 2B2 Cells

A, cAMP accumulation stimulated by isoproterenol (10-5 M) in 2B2 cells transfected with varying amounts of cDNA encoding either XL{alpha}s (open squares) or Gs{alpha} (solid squares). Transfected GnasE2-/E2- cells were challenged with agonist for 1 h at room temperature in the presence of 2 mM isobutylmethylxanthine. Basal cAMP levels ranged from 0.42 ± 0.08 to 1.25 ± 0.22 pmol/well for transfections with XL{alpha}s (open circles), and from 0.36 ± 0.05 to 0.73 ± 0.10 pmol/well for transfections with Gs{alpha} (solid circles). B, CTX-induced accumulation of cAMP in cells transfected with varying amounts of cDNA encoding either XL{alpha}s (open squares) or Gs{alpha} (solid squares). Transfected GnasE2-/E2- cells were incubated with 1 µg/ml CTX at 37 C for 2 h, and cAMP measurements were performed as described in Materials and Methods. C, Agonist-induced cAMP accumulation in cells expressing XL{alpha}s (open squares) or Gs{alpha} (solid squares) after normalization according to the differences between the CTX-induced cAMP levels. To normalize agonist-dependent activity for each transfection, the ratio of CTX-XL{alpha}s/CTX-Gs{alpha} (see Materials and Methods for explanation) was multiplied by the cAMP level induced by isoproterenol in Gs{alpha}-expressing cells. All data points represent mean ± SEM of three independent experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To examine whether XL{alpha}s can interact with typically Gs-coupled transmembrane receptors, we established immortalized cell lines that lack endogenous Gs{alpha} and XL{alpha}s. These GnasE2-/E2- cells grow as a monolayer, are easily maintained, and are readily transfected using lipid-based methods. Transformation of these cells with the temperature-sensitive SV40 mutant was performed several weeks after initiation of the primary cultures, during which time an overgrowth of fibroblast-like cells appeared to have taken place. Thus, most of the cloned immortalized cells are likely to be derived from the fibroblastic lineage, and showed, consistent with this hypothesis, a pavement-like morphology (20).

Transient transfection of GnasE2-/E2- cells revealed that XL{alpha}s can mediate signaling of cell surface receptors that are typically coupled to Gs{alpha}. In these cells, receptor-mediated cAMP accumulation through XL{alpha}s appeared comparable to that of Gs{alpha}. Particularly, the abilities of both G protein variants to stimulate adenylyl cyclase in response to the ß-adrenergic receptor activation were similar over a wide range of expression levels, strongly suggesting that XL{alpha}s and Gs{alpha} have similar receptor-coupling efficiencies. In response to CTX treatment, however, these proteins appeared to activate adenylyl cyclase with divergent efficacies (see Fig. 4BGo). Although minor differences between agonist-independent activities of XL{alpha}s and Gs{alpha} might be present, our Western blot data suggest that different expression levels of these signaling proteins in transfected cells were responsible, at least in part, for the observed differences.

Similar to these findings with the endogenous ßadrenergic receptor(s) (class A receptor), no apparent differences were revealed between the abilities of XL{alpha}s and Gs{alpha} for coupling to two other classes of G protein-coupled receptors (class B, PTHR1 and CRFR1; class C, TSHR), or to the transiently expressed ß2AR. Although subtle differences in receptor specificity could be present between XL{alpha}s and Gs{alpha}, elucidation of such differences is likely to require more detailed investigations, e.g. with stably transfected cells. Such cell lines will also be useful for investigating whether both signaling proteins interact selectively with different ß{gamma}-subunits, and/or with proteins that modulate their activity, such as RGS-PX1, a recently reported GTP phosphohydrolase activating protein that attenuates Gs-mediated signaling (21). Furthermore, because Gs{alpha} not only stimulates adenylyl cyclase, but can also interact with other downstream effectors, such as L-type Ca2+ channels (22) and Src-tyrosine kinases (23), it will also be of interest to explore whether XL{alpha}s can mimic these properties of Gs{alpha}.

Our results, particularly those regarding XL{alpha}s coupling to the CRFR1 and ß2AR, are not concordant with the results of a previous study, in which XL{alpha}s largely failed to mediate the actions of these two receptors (17). However, the GnasE2-/E2- cells used in the present study are likely to yield a more sensitive assay for studying the cellular roles of XL{alpha}s (as well as of Gs{alpha} and variants thereof) than transfection assays using cells that express Gs{alpha} (and possibly XL{alpha}s) already endogenously; the absence or presence of endogenous signaling proteins may thus provide a reasonable explanation for the observed discrepancies. On the other hand, assays involving the reconstitution of membranes from S49 cyc- cells that do not contain XL{alpha}s or Gs{alpha} (17) should be as sensitive as those provided by GnasE2-/E2- cells. The lack of receptor-dependent signaling through XL{alpha}s in the S49 cyc- system could therefore indicate that additional confounding factors contribute to signaling in these cells. For instance, improper folding of in vitro translated XL{alpha}s and/or insufficient posttranslational modifications necessary for receptor interaction could account for the observed impairment in receptor coupling of XL{alpha}s. It is also conceivable that XL{alpha}s requires cell-specific cofactors for efficient receptor coupling, and that S49 cyc- lymphoma cells lack these hitherto undefined molecules. Conversely, it appears also possible that ALEX, which is derived through the use of a second open reading frame within exon XL (24), acts as an inhibitor of XL{alpha}s function in S49 cyc- cells, but not in GnasE2-/E2- cells used in the present study. Of note, no agonist-mediated cAMP production was obtained in GnasE2-/E2- cells transfected with receptor cDNA alone (see Figs. 2AGo and 3Go). If ALEX, which does not require an intact Gnas exon 2, is indeed endogenously expressed in these cells, it does not appear to have Gs-like activity. The lack of amino acid homology between this protein and either XL{alpha}s or Gs{alpha} is consistent with this interpretation (24).

Previously, XL{alpha}s was localized to the trans-Golgi network within the cell (8, 25). However, a more recent report has refuted these previous findings by demonstrating that this protein is primarily localized to the plasma membrane (9). Our finding that XL{alpha}s, like Gs{alpha}, can mediate a productive interaction between agonist activated cell-surface receptors and adenylyl cyclases indicates association of XL{alpha}s with the plasma membrane at least in response to agonist challenge. Furthermore, GnasE2-/E2- cells described in our study exhibit elevated cAMP levels in response to CTX treatment when they are transfected with cDNA encoding XL{alpha}s, providing evidence for plasma membrane localization even in the absence of receptor activation.

Although its in vivo efficacy remains to be documented, the ability of XL{alpha}s to couple in vitro to several different agonist-occupied receptors suggests that this splice variant of Gs{alpha} may play an important role in intracellular signaling events. Unlike the widely expressed Gs{alpha}, XL{alpha}s shows high-level expression only in certain parts of the pituitary gland (9); low level expression, however, is detectable by RT-PCR in a broad range of tissues (4, 26). Expression of XL{alpha}s was confirmed in the pituitary and other sections of the developing rat brain using immune sera raised against the repetitive EPAA epitope located within the unique amino-terminal domain encoded by exon XL (27). It is therefore conceivable that XL{alpha}s and its rodent ortholog have an important role in mediating the actions of hypothalamic factors such as GHRH and CRF.

XL{alpha}s could also have a role in proximal renal tubular cells, where Gs{alpha} appears to be derived only from the maternal allele, at least later in development (2, 5, 28). Consequently, GNAS1 mutations that impair Gs{alpha} function, i.e. those identified in patients affected by PHP-Ia or related disorders, lead to renal PTH resistance only when inherited maternally (2, 29). However, hormone resistance is not present at birth, but rather develops over the first years of life (11). This could indicate that XL{alpha}s derived from the nonmutated paternal allele in patients with PHP-Ia mediates the PTH-dependent actions at the PTHR1 in renal proximal tubules. Although XL{alpha}s appears to be expressed only at low levels in the adult kidney (Ref. 26 and Weinstein, L.S., unpublished data), it is conceivable that expression of this signaling protein is higher during fetal and early postnatal life, thus providing a plausible explanation of the delayed onset of PTH resistance in PHP-Ia (2, 11). Assessment of the spatial and temporal expression profiles of XL{alpha}s and Gs{alpha} in the different cells of the renal cortex may thus be required to further clarify some of the potential roles of XL{alpha}s in regulating mineral ion homeostasis.

Findings in mice with heterozygous disruption of Gnas exon 2 suggests that XL{alpha}s may have additional biological roles, although some of these may be specific to rodents (5). About three quarters of mice with a paternally disrupted Gnas, which lack XL{alpha}s entirely due to its exclusive paternal expression, die within 24 h of birth. In contrast, all mice with a maternally disrupted Gnas survive into the first week, although most develop various neurological abnormalities between 6–21 d after birth and die shortly thereafter. These divergent findings in animals with a disrupted exon 2 on either the maternal or the paternal Gnas allele indicate that XL{alpha}s is important during fetal and/or neonatal development.

In addition to these developmental defects, mice with Gnas exon 2 disruption display phenotypic differences in energy metabolism that are dependent on the parental origin of the mutant Gnas allele (30). For example, animals inheriting the disrupted Gnas allele from a male appear to have a markedly higher metabolic rate and are significantly leaner than the wild-type animals or mice carrying the disrupted Gnas exon 2 on the maternal allele, and these metabolic changes appear to be associated with increased neural sympathetic activity in adipose tissue. Furthermore, in addition to a proposed role of XL{alpha}s in the central regulation of energy metabolism, this splice variant of Gs{alpha} may have a role in blood clotting, because a 36-bp insertion in GNAS1 exon XL is thought to be associated with increased agonist-induced cAMP accumulation in platelets and increased trauma-induced bleeding tendency (31). Several lines of evidence thus indicate that XL{alpha}s has unique biological properties, and it will be important to further explore some of these roles in vivo, and in vitro through systems that take advantage of the presented GnasE2-/E2- cells.

In summary, we established cell lines that lack endogenous XL{alpha}s and Gs{alpha} and used these cells to demonstrate that XL{alpha}s can couple efficiently to several different Gs-coupled receptors, thus providing definitive evidence for the conclusion that it can function as a signaling protein. In addition to being expressed only from the paternal allele and besides having a different expression pattern than Gs{alpha}, XL{alpha}s has unique structural elements, some of which could be investigated through our GnasE2-/E2- cells. In addition, because GnasE2-/E2- cells are easily transfectable, they appear to be ideal for evaluating the impact of different GNAS1 mutations on Gs{alpha}- and XL{alpha}s-mediated signaling events at different G protein-coupled receptors.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Experimental Animals
All animal experimentation described in this article was conducted in accordance with accepted standards of humane animal care, and the studies were approved by Massachusetts General Hospital Subcommittee on Research Animal Care (no. 97-4218).

Materials
[Y34]hPTH(1–34)amide (PTH), [Y36]hPTHrP (1–36)amide (PTHrP), and hCRF were synthesized as described previously (32). [32P]dCTP and chemiluminescence immunodetection reagents were from NEN Life Science Products (Boston, MA). Restriction endonucleases were from New England Biolabs, Inc. (Beverly, MA). F12-DMEM was from Life Technologies, Inc. (Gaithersburg, MD). All other chemicals were from Sigma (St. Louis, MO).

Generation of GnasE2-/E2- Cells
The generation of mice with disruption of Gnas exon 2 was previously described (5). To isolate GnasE2-/E2- cells, male and female heterozygotes were mated, and cesarean section was performed to isolate individual E9.5 embryos. Each embryo was minced under sterile conditions in F12-DMEM, containing 10% fetal bovine serum and an antibiotic-antimycotic solution, and placed in a 37 C, 5% CO2 incubator to allow for the propagation of fibroblasts. After seven to ten passages, cells were immortalized through infection with mutant SV40 encoding a temperature-sensitive large T antigen (provided by Dr. Shiow-Shih Tang, Boston, MA) and subsequently maintained at 33 C. Immortalized cells from individual embryos were genotyped using Southern blot analysis; the probe for detection of the wild-type and targeted alleles, and hybridization conditions were described previously (5). Limiting dilution was performed to isolate clonal cells.

Transfections, Western Blot Analysis, and cAMP Measurements
Plasmids containing cDNAs for rat XL{alpha}s (rXL/CDM8) and hemagglutinin (HA)-tagged rat Gs{alpha} (rGsHA/pcDNA1) were from Dr. Wieland B. Huttner (Heidelberg, Germany) and Dr. Melvin I. Simon (Pasadena, CA), respectively. rXL/CDM8 encodes a proline at position 519 (8); by replacing this residue with the wild-type leucine (17), we generated rXLwt/CDM8. This plasmid starts at nucleotide 380 (8) and encodes the entire XL{alpha}s protein sequence (17). Plasmid encoding the rat PTHR1 was described previously (32). Plasmids encoding human CRFR1 (hCRFR1/pcDNA1), human TSH receptor (hTSHR/pSVL), and ß2AR cDNA (hß2AR/CDM8) were from Drs. Imman Assil (Boston, MA), Peter Kopp (Chicago, IL), and Robert J. Lefkowitz (Durham, NC), respectively.

Cells (2 x 105) were seeded into each well of 24-well plates and transfected with 0.4 µg plasmid DNA/well using Effectene (QIAGEN, Valencia, CA). Protein and cAMP measurements were carried out 72 h after transfection as described previously (33), except that, for protein analyses, cells were lysed in 50 mM Tris-HCl (pH 7.8) containing 1% Triton X-100, 175 µg/ml phenylmethylsulfonyl fluoride, 50 µg/ml bacitracin, and 140 mM NaCl. Supernatant was collected after centrifugation at 750 x g. cAMP levels were determined after 1 h of agonist stimulation at room temperature in the presence of 2 mM isobutylmethylxanthine (34). For determination of CTX-induced cAMP formation, cells were treated at 37 C for 2 h with 1 µg/ml CTX diluted in F12-DMEM containing 10% fetal bovine serum. The treatment was followed by a 1-h incubation at room temperature with DMEM containing 35 mM HEPES-NaOH (pH 7.4), 2 mM isobutylmethylxanthine, and 1 mg/ml BSA. The amount of cAMP in each well was determined by a RIA, as described previously (34). For determination of coupling efficiency, cells were transfected with varying amounts of cDNA encoding either XL{alpha}s or Gs{alpha}, whereas the total amount of plasmid DNA in each transfection (0.54 µg/well) was kept constant by addition of empty vector. To normalize agonist-dependent activity for each transfection, CTX-induced cAMP level in XL{alpha}s-expressing cells (CTX-XL{alpha}s) was divided by CTX-induced cAMP level in Gs{alpha}expressing cells (CTX-Gs{alpha}) to yield the ratio CTX-XL{alpha}s/CTX-Gs{alpha}; for each cDNA concentration used for transfection, this ratio was multiplied by the level of cAMP induced by isoproterenol in Gs{alpha}-expressing cells.


    ACKNOWLEDGMENTS
 
We thank Drs. Robert C. Gensure and Ung-Il Chung for insightful discussions, and Dr. Henry M. Kronenberg for critical review of the manuscript.


    FOOTNOTES
 
This work was supported by NIH Grant RO1 46718-06 (to H.J.) from the National Institute of Diabetes and Digestive and Kidney Diseases and by a Research Fellowship (to M.B.) from the National Kidney Foundation.

Abbreviations: ß2AR, ß2-Adrenergic receptor; CRFR1, CRF receptor; CTX, cholera toxin; E9.5 and E10.5, embryonic d 9.5 and 10.5; Gs, stimulatory G protein; Gs{alpha}, {alpha}-subunit of the stimulatory G protein; PHP, pseudohypoparathyroidism; PTHR1, PTH/PTH-related peptide receptor; PTHrP, PTHrelated peptide; TSHR, TSH receptor; XL{alpha}s, extra-large splice variant of Gs{alpha}.

Received for publication January 31, 2002. Accepted for publication April 30, 2002.


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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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