Gs Alpha Protein Research Articles

Role of receptor‐mediated endocytosis, endosomal acidification and cathepsin D in cholera toxin cytotoxicity

Using the in situ liver model system, we have recently shown that, after cholera toxin binding to hepatic cells, cholera toxin accumulates in a low-density endosomal compartment, and then undergoes endosomal proteolysis by the aspartic acid protease cathepsin-D [Merlen C, Fayol-Messaoudi D, Fabrega S, El Hage T, Servin A, Authier F (2005) FEBS J272, 4385-4397]. Here, we have used a subcellular fractionation approach to address the in vivo compartmentalization and cytotoxic action of cholera toxin in rat liver parenchyma. Following administration of a saturating dose of cholera toxin to rats, rapid endocytosis of both cholera toxin subunits was observed, coincident with massive internalization of both the 45 kDa and 47 kDa Gsalpha proteins. These events coincided with the endosomal recruitment of ADP-ribosylation factor proteins, especially ADP-ribosylation factor-6, with a time course identical to that of toxin and the A subunit of the stimulatory G protein (Gsalpha) translocation. After an initial lag phase of 30 min, these constituents were linked to NAD-dependent ADP-ribosylation of endogenous Gsalpha, with maximum accumulation observed at 30-60 min postinjection. Assessment of the subsequent postendosomal fate of internalized Gsalpha revealed sustained endolysosomal transfer of the two Gsalpha isoforms. Concomitantly, cholera toxin increased in vivo endosome acidification rates driven by the ATP-dependent H(+)-ATPase pump and in vitro vacuolar acidification in hepatoma HepG2 cells. The vacuolar H(+)-ATPase inhibitor bafilomycin and the cathepsin D inhibitor pepstatin A partially inhibited, both in vivo and in vitro, the cAMP response to cholera toxin. This cathepsin D-dependent action of cholera toxin under the control of endosomal acidity was confirmed using cellular systems in which modification of the expression levels of cathepsin D, either by transfection of the cathepsin D gene or small interfering RNA, was followed by parallel changes in the cytotoxic response to cholera toxin. Thus, in hepatic cells, a unique endocytic pathway was revealed following cholera toxin administration, with regulation specificity most probably occurring at the locus of the endosome and implicating endosomal proteases, such as cathepsin D, as well as organelle acidification.

Endogenous Expression of Adenosine A1, A2 and A3 Receptors in Rat C6 Glioma Cells

Inhibitory and stimulatory adenosine receptors have been identified and characterized in both membranes and intact rat C6 glioma cells. In membranes, saturation experiment performed with [(3)H]DPCPX, selective A(1)R antagonist, revealed a single binding site with a K (D) = 9.4 +/- 1.4 nM and B (max) = 62.7 +/- 8.6 fmol/mg protein. Binding of [(3)H]DPCPX in intact cell revealed a K (D) = 17.7 +/- 1.3 nM and B (max )= 567.1 +/- 26.5 fmol/mg protein. On the other hand, [(3)H]ZM241385 binding experiments revealed a single binding site population of receptors with K (D) = 16.5 +/- 1.3 nM and B (max) = 358.9 +/- 52.4 fmol/mg protein in intact cells, and K (D) = 4.7 +/- 0.6 nM and B (max) = 74.3 +/- 7.9 fmol/mg protein in plasma membranes, suggesting the presence of A(2A) receptor in C6 cells. A(1), A(2A), A(2B) and A(3 )adenosine receptors were detected by Western-blotting and immunocytochemistry, and their mRNAs quantified by real time PCR assays. Gialpha and Gsalpha proteins were also detected by Western-blotting and RT-PCR assays. Furthermore, selective A(1)R agonists inhibited forskolin- and GTP-stimulated adenylyl cyclase activity and CGS 21680 and NECA stimulated this enzymatic activity in C6 cells. These results suggest that C6 glioma cells endogenously express A(1) and A(2) receptors functionally coupled to adenylyl cyclase inhibition and stimulation, respectively, and suggest these cells as a model to study the role of adenosine receptors in tumoral cells.

Cyclic GMP modulates the expression of Gi protein and adenylyl cyclase signaling in vascular smooth muscle cells

We have recently shown that the nitric oxide (NO) donor, SNAP, decreased the expression of Gialpha proteins and associated functions in vascular smooth muscle cells. Because NO stimulates soluble guanylyl cyclase and increases the levels of guanosine 3\',5\'-cyclic monophosphate (cGMP), the present studies were undertaken to investigate whether cGMP can also modulate the expression of Gi proteins and associated adenylyl cyclase signaling. A10 vascular smooth muscle cells (VSMCs) and primary cultured cells from aorta of Sprague Dawley rats were used for these studies. The cells were treated with 8-bromoguanosine 3\',5\'-cyclic monophosphate (8BrcGMP) for 24 h and the expression of Gialpha proteins was determined by immunobloting techniques. Adenylyl cyclase activity was determined by measuring [32P]cAMP formation for [alpha-32P]ATP. Treatment of cells with 8-BrcGMP (0.5 mM) decreased the expression of Gialpha-2 and Gialpha-3 by about 30-45%, which was restored towards control levels by KT5823, an inhibitor of protein kinase G. On the other hand, the levels of Gsalpha protein were not altered by this treatment. The decreased expression of Gialpha proteins by 8Br-cGMP treatment was reflected in decreased Gi functions. For example, the inhibition of forskolin (FSK)-stimulated adenylyl cyclase activity by low concentrations of GTPgammaS (receptor-independent Gi functions) was significantly decreased by 8Br-cGMP treatment. In addition, exposure of the cells to 8Br-cGMP also resulted in the attenuation of angiotensin (Ang) II- and C-ANP4-23 (a ring-deleted analog of atrial natriuretic peptide [ANP])-mediated inhibition of adenylyl cyclase activity (receptor-dependant functions of Gi). On the other hand, Gsalpha-mediated stimulations of adenylyl cyclase by GTPgammaS, isoproterenol and FSK were significantly augmented in 8Br-cGMP-treated cells. These results indicate that 8Br-cGMP decreased the expression of Gialpha proteins and associated functions in VSMCs. From these studies, it can be suggested that 8Br-cGMP-induced decreased levels of Gi proteins and resultant increased levels of cAMP may be an additional mechanism through which cGMP regulates vascular tone and thereby blood pressure.

Pseudohypoparathyroidism Type 1a with Congenital Hypothyroidism

Pseudohypoparathyroidism type la (PHP-1a) is an uncommon disorder that results from an inactivating mutation in the GNAS gene. It can present with resistance to several hormones, in addition to parathyroid hormone (PTH). Patients may have the classic Albright's hereditary osteodystrophy (AHO) phenotype and can develop resistance to thyroid stimulating hormone (TSH), gonadotropins, growth hormone releasing hormone (GHRH), and other hormones that rely on the Gsalpha protein to regulate signal transmission at their receptors. We report two siblings with PHP-1a and congenital hypothyroidism. The patients were found to have a heterozygous mutation at nucleotide 305 in exon 4 (c305C-->A) of the GNAS gene, which has not been previously linked to congenital hypothyroidism.

Clinical approach to clarifying the mechanism of abnormal bone metabolism in McCune–Albright syndrome

Recent advances in our knowledge of the abnormal bone metabolism associated with McCune-Albright syndrome (MAS) is briefly reviewed. Polyostotic fibrous bone dysplasia and hypophosphatemia are well-known characteristics of MAS. To clarify the mechanism of bone dysplasia, an approach that uses human cells isolated from MAS patients was important. It is now clear that normal skeletal stromal cells without mutation of the Gsalpha protein are necessary for the presence of bone dysplasia and that exaggerated production of interleukin-6 by fibrous bone cells with mutation of the Gsalpha protein is linked to the increased number of osteoclasts in bone tissues. The observation of increased bone resorption by the increased osteoclasts is one of the reasons for using bisphosphonates to treat the bone lesions of MAS. The key observation of the mechanism of hypophosphatemia in MAS was in a clinical report, which suggested that the presence of some humoral factors regulate phosphate metabolism. Recently, the humoral factor that causes hypophosphatemia in MAS was clarified to be fibroblast-growth factor 23 (FGF-23), although the possibility of some other humoral factors was not excluded. This is because a humoral factor inhibiting intestinal phosphate transport is present in culture medium obtained from the cells derived from fibrous bone dysplasia. The abnormal vitamin D mechanism in response to hypophosphatemia in MAS patients also proved recently to be caused by the increased circulating FGF-23 levels. The lines of evidence described suggest that FGF-23 and other factors may coexist, causing hyperphosphaturia and impaired intestinal absorption of phosphate, respectively.

Molecular Mechanisms of Impaired Urinary Concentrating Ability in Glucocorticoid-Deficient Rats

The purpose of this study was to examine urinary concentrating ability and protein expression of renal aquaporins and ion transporters in glucocorticoid-deficient (GD) rats in response to water deprivation as compared with control rats. Rats underwent bilateral adrenalectomies, followed only by aldosterone replacement (GD) or both aldosterone and dexamethasone replacement (control). As compared with control rats, the GD rats demonstrated a decrease in cardiac output and mean arterial pressure. In response to 36-h water deprivation, GD rats demonstrated significantly greater urine flow rate and decreased urine osmolality as compared with control rats at comparable serum osmolality and plasma vasopressin concentrations. The initiator of the countercurrent concentrating mechanism, the sodium-potassium-2 chloride co-transporter, was significantly decreased, as was the medullary osmolality in the GD rats versus control rats. There was also a decrease in inner medulla aquaporin-2 (AQP2) and urea transporter A1 (UT-A1) in GD rats as compared with control rats. There was a decrease in outer medulla Gsalpha protein, an important factor in vasopressin-mediated regulation of AQP2. Immunohistochemistry studies confirmed the decreased expression of AQP2 and UT-A1 in kidneys of GD rats as compared with control. In summary, impairment in the urinary concentrating mechanism was documented in GD rats in association with impaired countercurrent multiplication, diminished osmotic equilibration via AQP2, and diminished urea equilibration via UT-A1. These events occurred primarily in the relatively oxygen-deficient medulla and may have been initiated, at least in part, by the decrease in mean arterial pressure and thus renal perfusion pressure in this area of the kidney.

Proteolytic activation of internalized cholera toxin within hepatic endosomes by cathepsin D

We have defined the in vivo and in vitro metabolic fate of internalized cholera toxin (CT) in the endosomal apparatus of rat liver. In vivo, CT was internalized and accumulated in endosomes where it underwent degradation in a pH-dependent manner. In vitro proteolysis of CT using an endosomal lysate required an acidic pH and was sensitive to pepstatin A, an inhibitor of aspartic acid proteases. By nondenaturating immunoprecipitation, the acidic CT-degrading activity was attributed to the luminal form of endosomal cathepsin D. The rate of toxin hydrolysis using an endosomal lysate or pure cathepsin D was found to be high for native CT and free CT-B subunit, and low for free CT-A subunit. On the basis of IC(50) values, competition studies revealed that CT-A and CT-B subunits share a common binding site on the cathepsin D enzyme, with native CT and free CT-B subunit displaying the highest affinity for the protease. By immunofluorescence, partial colocalization of internalized CT with cathepsin D was confirmed at early times of endocytosis in both hepatoma HepG2 and intestinal Caco-2 cells. Hydrolysates of CT generated at low pH by bovine cathepsin D displayed ADP-ribosyltransferase activity towards exogenous Gsalpha protein suggesting that CT cytotoxicity, at least in part, may be related to proteolytic events within endocytic vesicles. Together, these data identify the endocytic apparatus as a critical subcellular site for the accumulation and proteolytic degradation of endocytosed CT, and define endosomal cathepsin D an enzyme potentially responsible for CT cytotoxic activation.

Association between β-adrenergic receptor polymorphisms and their G-protein-coupled receptors with body mass index and obesity in women: a report from the NHLBI-sponsored WISE study

The beta-adrenergic receptor (betaAR) genes are candidate genes for obesity because of their roles in energy homeostasis and promotion of lipolysis in human adipose tissue. Objective is to determine the association between obesity and polymorphisms in genes of the beta(1)AR (ADRB1), beta(2)AR (ADRB2), beta(3)AR (ADRB3), Gs protein alpha (GNAS1), to which all three beta-receptors couple and the G protein beta3 subunit (GNB3), to which beta(3)ARs couple. A case-control genetic association study. A total of 643 black or white women enrolled in Women's Ischemia Syndrome Evaluation (WISE) study. Genotypes were determined by PCR with single primer extension. Associations between genotype and body mass index (BMI), waist-to-hip ratio (WHR), waist circumference, and obesity were made. Polymorphisms in the three betaAR genes, GNAS1, and GNB3 were not associated with BMI, WHR, waist circumference, or obesity. Linear and logistic regression analyses found no contribution of either genotype or haplotype with anthropometric measurements or obesity. Our study suggests that among American women with suspected coronary heart disease, polymorphisms in the betaARs and their G-protein-coupled receptors do not contribute to increased BMI, WHR, waist circumference, or obesity. Given that 50% of all women die from coronary heart disease, and a higher percentage have heart disease during their lifetime, our results are likely generalizable to many American women.

Genetics and Proteomics of Pituitary Tumors

Genetics and proteomics determine structure and function of normal tissues, and the molecular alterations that underlie tumorigenesis result in changes in these aspects of tissue biology in neoplasms. We review the known genetic alterations in pituitary tumors. These include the oncogenic Gsalpha protein (GSP)-activating mutations, and pituitary tumor-derived fibroblast growth factor receptor-4 (ptd-FGFR4), as well as tumor suppressor gene mutations associated with multiple endocrine neoplasia type 1 (MEN1). Other candidates identified from expression profiling include pituitary tumor-transforming gene (PTTG), GADD45, and bone morphogenic protein (BMP)4. Proteomic changes in pituitary tumors include classical alterations identified by immunohistochemistry as well as epigenetic reductions in p27. The underlying mechanisms for dysregulated cell adhesive molecules including cadherins and FGFRs are reviewed. The combined use of genetic and proteomic approaches will enhance novel drug therapeutic development.

Distinct patterns of abnormal GNAS imprinting in familial and sporadic pseudohypoparathyroidism type IB

Pseudohypoparathyroidism type IB (PHPIB) is associated with abnormal imprinting of GNAS, the gene encoding the heterotrimeric G protein Gsalpha and other alternative products. The gene contains three differentially methylated regions (DMRs) located upstream of the Gsalpha promoter (from upstream to downstream): the paternally methylated NESP55 promoter region, the maternally methylated NESP antisense (NESPAS)/XLalphas promoter region and the maternally methylated exon 1A region located just upstream of the Gsalpha promoter. We have now performed a detailed analysis of the GNAS methylation profile in 20 unrelated PHPIB probands. Consistent with prior results, all have loss of exon 1A imprinting (a paternal epigenotype on both alleles). All five probands with familial disease had a deletion mutation within the closely linked STX16 gene and a GNAS imprinting defect involving only the exon 1A region. In contrast, the STX16 mutation was absent in all sporadic cases. The majority of these patients had abnormal imprinting of the more upstream regions in addition to the exon 1A imprinting defect, with eight of 15 having a paternal epigenotype on both alleles throughout the GNAS locus. In virtually all cases, the imprinting status of the NESP55 and NESPAS/XLalphas promoters is concordant, suggesting that their imprinting is co-regulated, whereas the imprinting of the NESPAS/XLalphas promoter region and XLalphas first exon is not always concordant even though they are closely linked and lie within the same DMR. Familial and sporadic forms of PHPIB have distinct GNAS imprinting patterns that occur through different defects in the imprinting mechanism.

Gonadotropin-releasing hormone initiates multiple signaling pathways in human GH-secreting adenomas.

Abnormal GH responses to GnRH test, observed in about 15% of patients with acromegaly, have been reported exclusively in patients bearing tumors without gsp mutation. The absence of responsiveness to GnRH in gsp+ tumors was not predicted on the basis of the mechanism of GnRH action that mainly involves the activation of calcium and protein kinase C dependent pathways. The aim of the present study was to investigate in detail the transduction of GnRH signaling in these tumors. GH-secreting adenomas removed from patients in vivo responsive to GnRH test were studied. Tumor DNA was screened for Gsalpha and GnRH receptor gene sequences. Intracellular calcium ([Ca2+]i) and cAMP levels were measured in dispersed cells and adenylyl cyclase (AC) activity in membrane preparations. DNA analysis showed wild sequence of both Gsalpha and GnRH receptor genes. GnRH caused a significant increase in intracellular Ca2+ that was associated with a significant stimulation of cAMP accumulation. In these cells neither TRH nor GHRP-6 were effective in causing significant modifications of cAMP levels, despite their ability to increase [Ca2+]i. Finally, GnRH was able to directly stimulate AC from 11.1 +/- 3.3 pmol/mg prot/min to 26.9 +/- 5.4 (p<0.005). We report that GnRH was effective in increasing both [Ca2+]i and AC in GH-secreting adenomas removed from responsive patients. The ability of GnRH to signal through Gsalpha protein may account for the lack of GH responses to GnRH observed in acromegalic patients with tumors carrying gsp mutation.

Tissue-specific imprinting of the G protein Gsalpha is associated with tissue-specific differences in histone methylation.

The G protein Gsalpha is imprinted in a tissue-specific manner, being primarily expressed from the maternal allele in some tissues, such as renal proximal tubules. The Gsalpha promoter is unmethylated, but is downstream of a differentially methylated region [the exon 1A differentially methylated region (DMR)] that is methylated on the maternal allele. Maternal Gsalpha null mutations or loss of maternal-specific exon 1A methylation leads to pseudohypoparathyroidism types 1A or 1B, respectively. We now have examined the chromatin state of each parental allele within the exon 1A-Gsalpha promoter region by chromatin immunoprecipitation of samples derived from mice with heterozygous deletions within the region using antibodies to covalently modified histones. The exon 1A DMR had allele-specific differences in histone acetylation and methylation, with histone acetylation and H3 lysine 4 (H3K4) methylation of the paternal allele, and H3 lysine 9 (H3K9) methylation of the maternal allele. Both parental alleles had similar levels of histone acetylation and H3K4 methylation within the Gsalpha promoter and first exon, with no H3K9 methylation. In liver, where Gsalpha is biallelically expressed, both parental alleles had similar levels of tri- and dimethylated H3K4 within the Gsalpha first exon. In contrast, in renal proximal tubules there was a greater ratio of tri- to dimethylated H3K4 of Gsalpha exon 1 in the more transcriptionally active maternal as compared with the paternal allele. These results show that allele-specific differences in Gsalpha expression correlate in a tissue-specific manner with allele-specific differences in the extent of H3K4 methylation, and are the first demonstration that chronic transcriptional activation in mammals is correlated with trimethylation of H3K4.

Gene expression analysis reveals evidence for inactivation of the TGF-beta signaling cascade in autonomously functioning thyroid nodules.

Molecular events that lead to the development of autonomously functioning thyroid nodules (AFTNs) are somatic mutations of the thyrotropin receptor (TSHR) in approximately 60% of the nodules and less frequently, somatic mutations in the Gsalpha protein. However, AFTNs without known mutations indicate that other causes remain to be identified. Moreover, the impact of constitutively activating TSHR mutations on the signal transduction network of the thyroid epithelial cell is unknown. We therefore investigated gene expression in 15 AFTNs and their surrounding tissue using Affymetrix GeneChips. Most prominently, data analysis revealed a changed pattern of gene expression in the TGF-beta signaling cascade and 25 differentially regulated genes in AFTNs, including thyroid peroxidase, type I iodothyronine deiodinase and sialyltransferase (SIAT) 1. Strikingly coexpression of SIAT 1 and TSHR in COS-7 cells increased TSH binding and cell surface expression of the TSHR. Moreover, differences in gene expression patterns for AFTNs with and without TSHR mutations indicate specific alterations of signal transduction in AFTNs without TSHR mutations. These results suggest that AFTNs with TSHR mutations harbor further mechanisms of forward stimulation. Furthermore, they give important leads to elucidate the molecular etiology of AFTNs without TSHR mutations.

Purification of recombinant G protein alpha subunits from Escherichia coli.

The purification of recombinant G protein a subunits expressed in Escherichia coli (E. coli) is a convenient and inexpensive method to obtain homogeneous preparations of protein for biochemical and biophysical analyses. Wild-type and mutant forms of G alpha are easily produced for analysis of their intrinsic biochemical properties, as well as for reconstitution with receptors, effectors, regulators, and G protein beta gamma subunits. Methods are described for the expression of Gi alpha and Gs alpha proteins in E. coli. Protocols are provided for the purification of untagged G protein a subunits using conventional chromatography and histidine (His)-tagged subunits using metal chelate chromatography. Modification of G alpha with myristate can be recapitulated in E. coli by expressing N-myristoyltransferase (NMT) with its G protein substrate. Protocols for the production and purification of myristoylated G alpha are presented.

Resolution of Gsα and Gqα/G11α proteins in membrane domains by two-dimensional electrophoresis: the effect of long-term agonist stimulation.

Low-density membrane-domain fractions were prepared from S49 lymphoma cells and clone e2m11 of HEK293 cells expressing a large number of thyrotropin-releasing hormone receptor (TRH-R) and G(11)alpha by flotation on sucrose density gradients. The intact cell structure was broken by detergent-extraction, alkaline-treatment or drastic homogenization. Three types of low-density membranes were resolved by two-dimensional electrophoresis and analyzed for G(s)alpha (S49) or G(q)alpha/G11) (e2m11) content. Four individual immunoblot signals of Gsalpha protein were identified in S49 lymphoma cells indicating complete resolution of the long G(s)alpha L+/-ser and short G(s)alpha S+/-ser variants of G(s)alpha. All these were diminished by prolonged agonist (isoprenaline) stimulation. In e2m11-HEK cells, five different immunoblot signals were detected indicating post-translational modification of G proteins of G(q)alpha/G(11)alpha family. The two major spots corresponding to exogenously (over)expressed G(11)alpha and endogenous G(q)alpha were reduced; the minor spots diminished by hormonal stimulation. Parallel analysis by silver staining of the total protein content indicated that no major changes in protein composition occurred under these conditions. Our data thus indicate that agonist-stimulation of target cells results in down-regulation of all different members of G(s) and G(q)/G(11) families. This agonist-specific effect may be demonstrated in crude membrane as well as domain/raft preparations and it is not accompanied by changes in overall protein composition.

Ghrelin mRNA and GH secretagogue receptor mRNA in human GH-producing pituitary adenomas is affected by mutations in the alpha subunit of G protein.

Ghrelin and its receptor, growth hormone secretagogue (GHS) receptor (GHSR), are expressed in the normal pituitary gland and various types of pituitary adenoma. Somatic mutations in the subunit of Gs alpha protein (gsp), which led to a constitutive activation of adenylyl cyclase, are reported in GH-producing pituitary adenomas. We analysed the relationship between ghrelin mRNA and GHSR mRNA expression levels in gsp mutation-positive and -negative GH-producing pituitary adenomas. Pituitary adenoma tissue was obtained at surgery from 20 patients with acromegaly. The expression levels of human ghrelin mRNA and GHSR mRNA were quantified using a competitive RT-PCR method. To detect the gsp mutations, amplified Gs alpha subunit cDNA fragments were sequenced directly using RT-PCR method. There was no significant difference in the expression of ghrelin mRNA between mutation-positive and -negative adenomas. The expression of GHSR mRNA was significantly lower in gsp mutation-positive than -negative adenomas. There was a significant negative correlation between the levels of ghrelin mRNA and GHSR mRNA expression in mutation-negative adenomas; no such correlation was found in mutation-positive adenomas. These results suggest that GHSR mRNA expression is downregulated by ghrelin in gsp mutation-negative GH-producing pituitary adenomas, and that changes in intracellular signalling pathways in gsp mutation-positive GH-producing pituitary adenomas affect the expression of G protein-coupled receptors such as GHSR. The absence of negative correlation between ghrelin and GHSR expression might be induced by lowered GHSR expression in gsp mutation-positive GH-producing adenomas.

Functional assessment of the thyrotropin receptor-beta subunit.

Posttranslational processing of the TSH receptor (TSHR) involves proteolysis of a single chain holoreceptor into TSHR-alpha (or A) and TSHR-beta (or B) subunits, which remain associated via disulfide bonds and which may then form oligomers. As both uncleaved and cleavage-derived forms of this receptor have been reported to bind TSH and transduce signals, reasons for this cleavage into alpha- and beta-subunits have remained enigmatic. Recently we suggested that TSHR cleavage was related to receptor oligomerization and now we have asked if cleavage influenced the binding of G proteins to this receptor. Furthermore, as TSHR-alpha subunits are subject to shedding from the cell surface membrane, we have examined whether the remaining TSHR-beta subunits could mediate signaling themselves, either constitutively and /or ligand-induced. We found that only the cleaved form of the TSHR in transfected Chinese hamster ovary cells was able to bind Gsalpha protein, suggesting that cleavage of the native TSH receptor was associated with receptor activation. We also found that independently expressed TSHR-beta subunits on stable cell lines were unable to mediate either constitutive or TSH-induced signaling, as monitored by their inability to induce cAMP accumulation. These data suggested that receptor cleavage was intimately associated with receptor activation in the wild-type TSH receptor and that the residual TSHR-beta subunits left on the thyroid cell membrane, after TSHR cleavage and subsequent TSHR-alpha shedding, were essentially silent and did not participate in signal transduction.

Functional polymorphisms in the paternally expressed XLalphas and its cofactor ALEX decrease their mutual interaction and enhance receptor-mediated cAMP formation.

The paternally expressed extra-large stimulatory G protein gene (XLalphas) is a splice variant of the stimulatory G-protein gene (Gsalpha) consisting of XL-exon1 and exons 2-13 of Gsalpha. A second open reading frame (ORF) in XL-exon1, that completely overlaps the XL-domain ORF, encodes ALEX, which is translated from the XLalphas mRNA and binds the XL-domain of XLalphas. We previously demonstrated that a paternally inherited functional polymorphism in XL-exon1, consisting of a 36 bp insertion and two nucleotide substitutions, is associated with Gs hyperfunction in platelets, leading to an increased trauma-related bleeding tendency and is accompanied by neurological problems and brachydactyly in two families. Here, we describe eight additional patients with brachydactyly, who inherited the same XLalphas polymorphism paternally and who show Gs hyperfunction in their platelets and fibroblasts. All carriers also have an elongated ALEX protein, as a consequence of the paternally inherited insertion. The in vitro interaction between the two elongated XLalphas and ALEX proteins is markedly reduced. Moreover, XLalphas or ALEX can be co-immunoprecipitated with an antibody against either ALEX or XLalphas in platelets from a control but hardly from patients with the XLalphas/ALEX insertion. In contrast to the strong interaction between the two wild-type proteins, we suggest that this defective association results in unimpeded receptor-stimulated activation of XLalphas. The paternally inherited double XLalphas/ALEX functional polymorphism is also associated with elevated platelet membrane Gsalpha protein levels. Both phenomena contribute to increased Gs signaling in patients with platelet hypersensitivity towards Gs-agonists and may be accompanied by neurological problems or growth deficiency.

Molecular analysis of the GNAS1 gene for the correct diagnosis of Albright hereditary osteodystrophy and pseudohypoparathyroidism.

Pseudohypoparathyroidism (PHP) is a heterogeneous disease characterized by PTH resistance and classified as types Ia, Ib, Ic, and II, according to its different pathogenesis and phenotype. PHP-Ia patients show Gsalpha protein deficiency, PTH resistance, and typical Albright hereditary osteodystrophy (AHO). Heterozygous mutations in the GNAS1 gene encoding the Gsalpha protein have been identified both in PHP-Ia and in pseudopseudohypoparathyroidism (PPHP), a disorder with isolated AHO. A single GNAS1 mutation may be responsible for both PHP-Ia and PPHP in the same family when inherited from the maternal and the paternal allele, respectively, suggesting that GNAS1 is an imprinted gene. To evaluate whether molecular diagnosis is a useful tool to characterize AHO and PHP when testing for Gsalpha activity and PTH resistance is not available, we have performed GNAS1 mutational analysis in 43 patients with PTH resistance and/or AHO. Sequencing of the whole coding region of the GNAS1 gene identified 11 mutations in 18 PHP patients, eight of which have not been reported previously. Inheritance was ascertained in 13 cases, all of whom had PHP-Ia: the mutated alleles were inherited from the mothers, who had AHO (PPHP), consistent with the proposed imprinting mechanism. GNAS1 molecular analysis confirmed the diagnosis of PHP-Ia and PPHP in the mutated patients. Our results stress the usefulness of this approach to obtain a complete diagnosis, expand the GNAS1 mutation spectrum, and illustrate the wide mutation heterogeneity of PHP and PHP-Ia.

Albright's hereditary osteodystrophy and pseudohypoparathyroidism.

Pseudohypoparathyroidism (PHP) and Albright's hereditary osteodystrophy (AHO) are not interchangeable terms. AHO describes a constellation of physical features, including short adult stature, obesity, brachydactyly, and ectopic ossifications. PHP means end-organ resistance to PTH and is subclassified into types Ia, Ib, and Ic and type II. Pseudopseudohypoparathyroidism (PPHP) is a term used for individuals with AHO who have normal end-organ responses to PTH. Both the PHPIa and PPHP forms of AHO result from heterozygous deactivating mutations in the GNAS1 gene associated with a 50% reduction in bioactivity of the Gsalpha protein that it encodes. The GNAS1 gene is subject to tissue-specific genomic imprinting. Patients with mutations on their maternally derived allele are likely to have associated PHPIa, whereas mutations on the paternal allele usually cause PPHP. Isolated PTH resistance (PHPIb) can result from mutations within the GNAS1 gene but is more commonly caused by epigenetic imprinting abnormalities affecting the upstream exon 1A. The causes of PHP type Ic and PHP type II are not yet clear, and the latter is likely to be heterogeneous.