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REVIEW www.nature.com/clinicalpractice/endmet Mechanisms of Disease: mutations of G proteins and G-protein-coupled receptors in endocrine diseases Andrea G Lania, Giovanna Mantovani and Anna Spada* INTRODUCTION S U M M A RY G proteins and G-protein-coupled receptors (GPCRs) mediate the effects of a number of hormones. Genes that encode these molecules are subject to loss-of function or gain-of-function mutations that result in endocrine disorders. Loss-of-function mutations prevent signaling in response to the corresponding agonist and cause resistance to hormone actions, which mimics hormone deficiency. Gain-of-function mutations lead to constitutive, agonist-independent activation of signaling, which mimics hormone excess. Disease-causing mutations of GPCRs have been identified in patients with various disorders of the pituitary–thyroid, pituitary– gonadal and pituitary–adrenal axes, and in those with abnormalities in food intake, growth, water balance and mineral-ion turnover. The only mutational changes in G proteins unequivocally associated with endocrine disorders occur in GNAS (guanine nucleotide-binding protein G-stimulatory subunit α, or Gsα). Heterozygous loss-of-function mutations of GNAS in the active, maternal allele cause resistance to hormones that act through Gsα-coupled GPCRs, whereas somatic gain-of-function mutations cause proliferation of endocrine cells that recognize cyclic AMP as a mitogen. The study of mutations in G proteins and GPCRs has already had major implications for understanding the molecular basis of rare endocrine diseases, as well as susceptibility to multifactorial disorders that are associated with polymorphisms in these genes. KEYWORDS cyclic AMP, endocrine diseases, G protein, G-protein-coupled receptor, GSP oncogene REVIEW CRITERIA We searched PubMed for publications using different combinations of the following search terms: “endocrine disease”, “GPCR”, “G protein”, “GNAS”, “gene mutation”, “pituitary–thyroid axis”, “thyrotropin-releasing hormone receptor”, “thyroidstimulating hormone receptor”, “pituitary–gonadal axis”, “G-protein-coupled receptor 54”, “gonadotropin-releasing hormone receptor”, “gonadotropin hormone receptor”, “pituitary–adrenal axis”, “adrenocorticotropic hormone receptor”, “melanocortin 4 receptor”, “growth-hormone-releasing hormone receptor”, “vasopressin V2 receptor”, “diabetes insipidus”, “SIADH”, “calcium-sensing receptor”, and “parathyroid hormone receptor”. All selected papers were English-language, fulltext articles. A number of references were not included because of space restrictions. AG Lania is a Research Associate, G Mantovani is a Postdoctoral Fellow and A Spada is a Professor of Endocrinology at the Endocrine Unit, Department of Medical Sciences, University of Milan, Fondazione IRCCS Ospedale Maggiore, Policlinico, Mangiagalli, Regina Elena, Milan, Italy. Advances in understanding the molecular biology of signal transduction have had a considerable impact on several aspects of basic research and clinical medicine. G-protein-coupled receptors (GPCRs) are one of the major classes of signaling proteins; they mediate numerous physiologic processes of relevance to endocrinology. In the past few years, studies have identified genes that code for G proteins and GPCRs as loci for lossof-function and gain-of-function mutations that result in human diseases. This article will briefly describe genetic alterations of G proteins and GPCRs that lead to endocrine diseases (Tables 1 and 2). G-PROTEIN-COUPLED RECEPTORS GPCRs constitute one of the largest classes of proteins (encoded by more than 800 genes in the human genome). GPCRs are responsible for signal transduction1 and mediate the effects of endogenous ligands, such as neurotransmitters, neuropeptides, hormones and ions, as well as sensory stimuli, such as light, odorants and gustatory compounds. There are, moreover, many GPCRs (so-called orphan GPCRs) for which the endogenous agonist is still unknown. GPCRs share significant structural homology, and are predicted to exhibit a common, ‘serpentine’ structure: seven hydrophobic regions that form transmembrane α-helices, connected by alternating extracellular and intracellular loops2 (Figure 1). A general model for GPCR activity has postulated that GPCRs are in equilibrium between an active and an inactive state, and that interaction with a GPCR agonist elicits or stabilizes a conformational change in the GPCR, which favors binding to a G protein (or to multiple G proteins) and signal generation inside the cell.3 Correspondence *Endocrine Unit, Department of Medical Sciences, University of Milan, Fondazione IRCCS Ospedale Maggiore, Policlinico, Mangiagalli, Regina Elena, Via Francesco Sforza 35, 20122 Milan, Italy [email protected] Mutations in genes that encode GPCRs can cause losses or gains of function.4 Loss-of-function (inactivating) mutations prevent signaling in Received 27 February 2006 Accepted 10 July 2006 www.nature.com/clinicalpractice doi:10.1038/ncpendmet0324 DECEMBER 2006 VOL 2 NO 12 MAIN FEATURES OF ENDOCRINE DISEASES CAUSED BY GPCR MUTATIONS NATURE CLINICAL PRACTICE ENDOCRINOLOGY & METABOLISM 681 ©2006 Nature Publishing Group REVIEW www.nature.com/clinicalpractice/endmet Table 1 Mutations in genes for GPCRs and G proteins that lead to disorders in pituitary axes. Gene Mutation Disease OMIM# Pituitary–thyroid axis TRHR Inactivating, germline Isolated central hypothyroidism TSHa 188545 TSHR Inactivating, germline Activating, somatic Activating, germline Resistance to Toxic thyroid adenomab Hereditary toxic thyroid hyperplasiab Familial gestational hyperthyroiditisb 275200 603372 609152 603373 GNAS Inactivating, germline Activating, somatic Resistance to TSH in PHP1aa Toxic thyroid adenomab Thyroid adenoma in MASb Thyroid carcinoma 103580 603372 174800 NA Pituitary–gonadal axis KISS1R Inactivating, germline Hypogonadotropic hypogonadism 146110 GNRHR Inactivating, germline Familial idiopathic hypogonadotropic hypogonadism 146110 LHCGR Inactivating, germline Activating, germline Male pseudohermaphroditisma Hypergonadotropic hypogonadisma Male-limited pituitary-independent precocious pubertyb 152790 152790 176410 FSHR Inactivating, germline Activating, germline Hypergonadotropic hypogonadism with amenorrheaa Ovarian hyperstimulation syndrome 238320 136435 GNAS Inactivating, germline Activating, somatic Resistance to LH and FSH in PHP1aa Precocious puberty in MASb Leydig cell tumor Ovarian granulosa cell tumors 103580 174800 NA NA Pituitary–adrenal axis MC2R Inactivating, germline Familial glucocorticoid deficiencya 202200 GIPR, LHCGR, or β-adrenergic receptor genes Aberrant expression Cushing’s syndrome 219080 GNAS Activating, somatic Adrenal adenoma, bilateral adrenal hyperplasia NA aPhenotype of hormone deficiency associated with normal or high serum levels of biologically active hormone and absence of autoimmunity. bPhenotype of hormone excess associated with low or undetectable serum levels of corresponding hormone and absence of autoimmunity. Abbreviations: FSH, follicle-stimulating hormone; FSHR, follicle-stimulating hormone receptor; GIPR, gastric inhibitory polypeptide receptor; GNAS, guanine nucleotide-binding protein G-stimulatory subunit α; GNRHR, gonodatropin-releasing hormone receptor; GPCR, G-protein-coupled receptor; KISS1R, kisspeptin 1 receptor (previously termed G-protein-coupled receptor 54); LH, luteinizing hormone; LHCGR, luteinizing hormone and choriogonadotropin receptor; MAS, McCune–Albright syndrome; MC2R, melanocortin 2 receptor; NA, not available; OMIM#, Online Mendelian Inheritance in Man accession number; PHP1a, pseudohypoparathyroidism type 1a; TRHR, TSH-releasing hormone receptor; TSHR, TSH receptor. response to the corresponding agonist, and cause resistance to hormone action, which mimics hormone deficiency.4 Clinically significant impairment of signal transduction generally requires loss of function of both alleles and, therefore, most endocrine diseases caused by inactivating GPCR mutations are recessive, though several exceptions have been reported. These mutations can be missense, nonsense or frameshift mutations that result in absent or functionally defective proteins, which exhibit abnormal targeting to the cell membrane, trafficking or degradation.5 They may involve any portion of the receptor, particularly the membrane-spanning helices. 682 NATURE CLINICAL PRACTICE ENDOCRINOLOGY & METABOLISM ©2006 Nature Publishing Group Gain-of-function mutations in GPCRs lead to constitutive, agonist-independent activation of signaling, which mimics states of hormone excess. These activating mutations are almost always missense mutations that are able to disrupt the normal inhibitory constraints that maintain the receptor in its inactive conformation. Most endocrine diseases caused by activating GPCR mutations are inherited in a dominant manner. Interestingly, activating mutations in GPCRs that are coupled to the cyclic AMP (cAMP)-dependent pathway (e.g. the TSH receptor, TSH-R) are also associated with proliferative effects. The phenotype caused by inactivating or activating GPCR mutations depends on the LANIA ET AL. DECEMBER 2006 VOL 2 NO 12 REVIEW www.nature.com/clinicalpractice/endmet Table 2 Mutations in genes for GPCRs and G proteins that lead to disorders in weight, growth, water balance and mineral-ion turnover. Disorder Gene Mutation Disease OMIM# Obesity MC4R Inactivating, germline Obesity 155541 GH and growth GHRHR GHSR GNAS Inactivating, germline Inactivating, germline Inactivating, germline Activating, somatic Isolated GH deficiency Short stature Resistance to GHRH in PHP1aa GH-secreting pituitary adenoma GH-secreting pituitary adenoma in MAS 139191 601898 103580 139320 174800 Water balance AVPR2 Inactivating, germline Activating, germline Nephrogenic diabetes insipidus type 1a Nephrogenic syndrome of inappropriate antidiuresisb 304800 300539 Mineral-ion turnover CASR Inactivating, germline Familial hypocalciuric hypercalcemia (heterozygous) Neonatal severe primary hyperparathyroidism (homozygous) Familial hypercalciuric hypocalcemia Blomstrand chondrodysplasia Jansen-type metaphyseal chondrodysplasiab Resistance to PTH in PHP1aa 145980 PTHR1 GNAS Activating, germline Inactivating, germline Activating, germline Inactivating, germline 239200 146200 215045 156400 103580 aPhenotype of hormone deficiency associated with normal-to-high serum levels of biologically active hormone and absence of autoimmunity. bPhenotype of hormone excess associated with low or undetectable serum levels of the corresponding hormone and absence of autoimmunity. Abbreviations: AVPR2, arginine vasopressin receptor 2; CASR, calcium-sensing receptor; GH, growth hormone; GHRH, growth-hormone-releasing hormone; GHRHR, growth-hormone-releasing hormone receptor; GHSR, ghrelin and GH secretagogue receptor; GNAS, guanine nucleotide-binding protein G-stimulatory protein subunit α; GPCR, G-protein-coupled receptor; MAS, McCune–Albright syndrome; MC4R, melanocortin 4 receptor; OMIM#, Online Mendelian Inheritance in Man accession number; PHP1a, pseudohypoparathyroidism type 1a; PTH, parathyroid hormone; PTHR1, parathyroid hormone receptor 1. specific receptor involved, the range of tissues it is expressed in, and whether the mutation occurs in germline (gamete) cells—and, therefore, affects every cell that expresses the gene— or is somatic (present only in a subpopulation of cells that express the gene) and leads to focal manifestations of disease. GPCR MUTATIONS AND THE PITUITARY–THYROID AXIS TSH-releasing hormone receptor Central hypothyroidism is mostly caused by tumors or infiltrative diseases of the hypothalamic–pituitary region. At present, only one patient with a defect in TSH signaling caused by resistance to the actions of TSH-releasing hormone (TRH) has been described.6 In particular, this individual, who showed the typical biochemical parameters of central hypothyroidism (low thyroid hormone and lownormal TSH levels) was a compound heterozygote for two different loss-of-function mutations in the TRH receptor gene (i.e. the patient had inherited a different mutation from each parent). In this individual, the lack of TSH and prolactin response to a TRH-stimulation test predicted the loss-of-function mutations in TRHR. No DECEMBER 2006 VOL 2 NO 12 LANIA ET AL. gain-of-function mutations have been reported so far. TSH receptor The TSH-R has a marked propensity toward overactivity or underactivity, probably because of molecular instability. Indeed, the large number of different, naturally occurring TSHR mutations provided an initial basis for characterization of the protein domains required for TSH-R activation, which were subsequently confirmed by in vitro investigations. Germline, loss-of-function mutations induce either a partial or a complete resistance to TSH. The resultant disease phenotypes range from mild, subclinical hypothyroidism, to congenital, severe hypothyroidism, depending on the type of mutation and the affected individual’s homozygous, compound heterozygous or heterozygous status.4,7–9 Gain-of-function mutations can occur at the somatic or germline level. The two somatic, missense substitutions that were originally found in toxic thyroid adenomas (Ala623Ile and Asp619Gly) were the first naturally occurring activating mutations of a GPCR to be associated with tumorigenesis.10 Since then, several TSHR mutations, mostly located in the NATURE CLINICAL PRACTICE ENDOCRINOLOGY & METABOLISM 683 ©2006 Nature Publishing Group REVIEW www.nature.com/clinicalpractice/endmet N terminus GPCR C terminus γ GDP α γ β β α GTP Second messenger Hormone secretion Gene transcription Figure 1 Schematic representation of GPCRs and G proteins. GPCRs are expressed on the cell surface and share a common, ‘serpentine’ structure characterized by seven hydrophobic regions that form transmembrane α-helices, which are connected by alternating extracellular and intracellular loops. The interaction of an agonist with its binding site (which can be located in the seven transmembrane domains or in the extracellular loops and N terminus) elicits, or stabilizes, a conformational change in the receptor. This conformational change allows a specific G protein to bind, and a signaling cascade to be generated, inside the cell. The cytoplasmic loops are similar among GPCRs. This similarity is consistent with the presence of a common mechanism, by which hundreds of GPCRs activate only a few dozen G proteins. The G proteins are composed of the α subunit, which is specific for each G protein, and the common β and γ subunits. The GDP-bound α subunit binds tightly to the β–γ complex and is inactive, whereas the GTPbound α subunit can dissociate from the β–γ complex and activate effector proteins. Interaction of an agonist with a specific GPCR induces activation of the G protein, by facilitating the exchange of GDP for GTP on the α subunit. The duration of such dissociation is determined by the rate of α-subunit-mediated hydrolysis of GTP. Signaling via second messengers can then lead to gene transcription and hormone secretion. Abbreviations: α, β, γ, G-protein subunits; GPCR, G-protein-coupled receptor. should be screened for TSHR mutations, and should be treated early and aggressively to avoid tumor recurrence. A particular mutation in the extracellular N-terminal domain of the TSH-R (Lys183Arg) confers an abnormally high sensitivity to human chorionic gonadotropin (hCG) on this receptor, which leads to a rare syndrome referred to as familial gestational hyper thyroidism.13 Although some degree of thyroid stimulation by hCG is frequently observed during the first trimester of pregnancy, in a family with the Lys183Arg mutation, a mother and daughter (both heterozygous for this mutation) were severely hyperthyroid only during each of their pregnancies.13 GPCR MUTATIONS AND THE PITUITARY–GONADAL AXIS Kisspeptin 1 receptor Gonadotropin-releasing hormone (GnRH) is critical for normal secretion of folliclestimulating hormone (FSH) and luteinizing hormone (LH), and for pubertal development and reproduction. The evidence indicates that at the onset of puberty, activation of GnRH neurons (and consequently GnRH release from the forebrain) is mediated by kisspeptin 1, the endogenous ligand of the kisspeptin 1 receptor (KiSS-1R, encoded by KISS1R, previously named GPR54), which is expressed in GnRH neurons. Loss-of-function KiSS-1R mutations cause congenital hypogonadotropic hypogonadism in both sexes, characterized by delayed puberty, low sex steroid levels, and low-normal gonadotropin levels that are responsive to GnRH.14 Gonadotropin-releasing hormone receptor third cytoplasmic loop or in the adjacent sixth transmembrane segment, have been identified in about 80% of toxic thyroid adenomas.9,11 These mutations confer constitutive activity on the TSH-R, and result in TSH-independent cAMP accumulation and consequent endocrine hyperfunction, together with clonal expansion of the mutated cell. When these same TSHR substitutions occur in the germline, they cause a rare form of hyperthyroidism characterized by variable age of onset, goiter and absence of autoimmunity, referred to as hereditary toxic thyroid hyperplasia.12 Patients with familial or congenital, nonautoimmune hyperthyroidism 684 NATURE CLINICAL PRACTICE ENDOCRINOLOGY & METABOLISM ©2006 Nature Publishing Group Several loss-of-function mutations of the GnRH receptor, which can be present in either homozygous or compound heterozygous forms, have been identified in patients with familial idiopathic hypogonadotropic hypogonadism.15,16 These mutations frequently cause reduced expression of the GnRH receptor at the plasma membrane, and its increased retention and/or degradation in the endoplasmic reticulum.5 The disease affects males and females, and the resulting phenotypes span a continuum between complete and partial gonadotropic deficiency (also termed ‘fertile eunuch’, in men). No gainof-function mutations of the GnRH receptor have been reported.17 LANIA ET AL. DECEMBER 2006 VOL 2 NO 12 REVIEW www.nature.com/clinicalpractice/endmet Luteinizing hormone receptor Loss-of-function and gain-of-function mutations have been identified in the LH receptor (LHR) gene, LHCGR.18 Homozygous, lossof-function LHR mutations cause Leydig cell hypoplasia or agenesis and a variable phenotype—from complete male pseudohermaphroditism, characterized by XY genotype with female external genitalia, high LH, normal FSH and barely detectable testosterone levels that are unresponsive to hCG treatment, to very mild undervirilization.18,19 Affected females present with a relatively mild phenotype, characterized by amenorrhea with normal development of primary and secondary sexual characteristics, elevated LH, normal or elevated FSH and low estradiol levels.18 Familial, male-limited, pituitary-independent precocious puberty (also known as familial testotoxicosis) was the first-known example of an inherited endocrine disease caused by a constitutively active GPCR (in this case, the LHR).20 The disease is characterized by testicular enlargement before 3–4 years of age, with high testosterone and low gonadotropin levels. Asp578Gly substitution is the most common cause of familial testotoxicosis in the US. Interestingly, Asp578His, which occurs exclusively at the somatic level, causes Leydig cell transformation and adenoma formation.21 In females, activating LHR mutations do not seem to cause a recognizable phenotype, probably because of low LHR expression in prepubertal ovaries. Follicle-stimulating hormone receptor Loss-of-function and gain-of-function mutations have been identified in the FSH receptor (FSH-R) gene, FSHR.18 Inactivating mutations have been reported in females with hypergonadotropic hypogonadism, primary or early-onset secondary amenorrhea, variable development of secondary sex characteristics and premature arrest of follicular maturation.18,22 Affected males have normal androgen production and reduced sperm quality, but remain fertile. Despite the rarity of FSH-R mutations, there is good correlation between genotype and phenotype, and both these factors should be considered when managing these patients. Individuals with mutations that cause mild phenotypes may still respond to stimulation with high doses of FSH.18 To date, one activating mutation in FSHR has been reported, in a hypophysectomized man with persistent spermatogenesis in spite of DECEMBER 2006 VOL 2 NO 12 LANIA ET AL. undetectable gonadotropins.23 More recently, substitutions in the serpentine domain of FSH-R (Thr449Ile and Asp567Asn) that confer sensitivity to hCG have been reported in women with recurrent ovarian hyperstimulation syndrome, a complication that can occur following hCG administration for in vitro fertilization.18,24 GPCR MUTATIONS AND ADRENAL FUNCTION Familial glucocorticoid deficiency is a rare, autosomal recessive disease due to defective functioning or trafficking of melanocortin 2 receptor (MC2-R, formerly known as adrenocorticotropic hormone [ACTH] receptor). Approximately 25% of individuals with familial glucocorticoid deficiency have loss-of-function missense mutations in MC2R and 20% have mutations in MRAP, which encodes melanocortin 2 receptor accessory protein—a small, single-transmembrane-domain protein that is required for MC2-R expression at the cell surface. Familial glucocorticoid deficiency is potentially lethal. Affected individuals are subject to hypoglycemia and infection, in infancy or childhood. Typically, they have low levels of glucocorticoids and extremely high levels of ACTH and α-melanocyte-stimulating hormone, which results in skin hyperpigmentation.25,26 No gain-of-function mutations of MC2-R have been found in cortisol-secreting tumors. In these tumors the cAMP pathway, which is physiologically stimulated by ACTH, is frequently activated by aberrant GPCRs. In particular, abnormal expression of the receptor for gastric inhibitory polypeptide causes ‘food-dependent’ Cushing’s syndrome, in which cortisol levels increase after meals in response to the physiological postprandial rise of gastric inhibitory polypeptide.27 Other GPCRs that are aberrantly expressed in cortisol-secreting adenomas and associated with Cushing’s syndrome are the LHR and β-adrenergic receptors.28 GPCR MUTATIONS AND OBESITY Loss-of-function mutations in melanocortin 4 receptor (MC4-R)—which, together with melanocortin receptor 3, is the predominant anorexigenic receptor involved in hypothalamic control of food intake—is the most common monogenic disorder in obesity, accounting for 1–6% of cases of early-onset or severe adult obesity.29,30 Loss of MC4-R function is generally caused by heterozygous missense mutations, NATURE CLINICAL PRACTICE ENDOCRINOLOGY & METABOLISM 685 ©2006 Nature Publishing Group REVIEW www.nature.com/clinicalpractice/endmet which segregate with obesity and show incomplete penetrance. There is a good correlation between the severity of defective MC4-R function in vitro and the amount of food ingested at a test meal.31 Affected individuals exhibit hyperphagia that often starts in the first year of life, severe obesity, increased bone mineral density, increased linear growth, and severe hyperinsulinemia.30 GPCR MUTATIONS AND GROWTH DISORDERS Growth-hormone releasing hormone receptor Homozygous, loss-of-function mutations of the growth hormone (GH)-releasing-hormone receptor gene (GHRHR) are a rare cause of familial, isolated, GH deficiency.32,33 Affected patients show early and severe growth failure during the first year of life, with low GH and insulin-like growth factor 1 levels that are unresponsive to GH-releasing hormone. They have particular phenotypic characteristics—a high-pitched voice, mild frontal bossing, and abdominal adiposity. No gain-of-function mutations in GHRHR have been described.34 Ghrelin and GH secretagogue receptor In 2006, a missense mutation within the first exon of GHSR (the gene that encodes GHS-R, the receptor for ghrelin and GH secretagogues), was identified in two unrelated families. This mutation is a substitution that has been found in homozygous and heterozygous forms, and has a dominant mode of inheritance and incomplete penetrance. The mutation segregates with short stature and results in decreased cellsurface expression of GHS-R, which selectively impairs the constitutive activity of GHS-R while preserving its ability to respond to ghrelin.35 GPCR MUTATIONS AND WATER BALANCE Loss-of-function mutations in the vasopressin V2 receptor (AVPR V2) are present in approximately 90% of patients with nephrogenic diabetes insipidus (NDI), a rare disorder due to renal distal nephron insensitivity to the action of antidiuretic hormone (ADH, also known as arginine vasopressin).36,37 NDI is an X-linked recessive disease and affected patients are male, although female patients with NDI and heterozygous mutations in the AVPR2 gene have been reported, in whom the condition is probably caused by skewed X inactivation.38,39 The remaining 10% of patients have autosomal NDI, due to mutations in AQP2, the aquaporin-2 renal collecting duct water channel 686 NATURE CLINICAL PRACTICE ENDOCRINOLOGY & METABOLISM ©2006 Nature Publishing Group gene.37 To date, more than 200 distinct AVPRV2 mutations, which are associated with nonspecific symptoms in the first year of life (e.g. anorexia, vomiting, fever, growth and development retardation, and excretion of large volumes of hypotonic urine) and frequently complicated by severe dehydration in adult life, have been identified.37 These mutations are scattered throughout the AVPR V2 protein, with the exception of the C-terminal and N-terminal tails.40 In 2005, hemizygous gain-of-function AVPR2 mutations were identified in two unrelated male infants with signs and symptoms consistent with the syndrome of inappropriate ADH secretion (SIADH), a condition characterized by inappropriately concentrated urine, hyponatremia, hypo-osmolality and natriuresis.41 In contrast to classical SIADH, this particular subtype of the condition (termed ‘nephrogenic syndrome of inappropriate antidiuresis’) is characterized by low ADH levels, consistent with the presence of activating AVPR V2 mutations.41 Taking into account that 10–20% of patients with SIADH have low ADH levels, it is tempting to speculate that the nephrogenic syndrome of inappropriate antidiuresis might not be so rare. GPCR MUTATIONS AND MINERAL ION TURNOVER Calcium-sensing receptor The calcium-sensing receptor (CaSR) allows parathyroid and kidney tubular cells to sense the extracellular calcium concentrations and to regulate parathyroid hormone (PTH) secretion and tubular calcium reabsorption, accordingly. By this mechanism, elevated calcium concentrations inhibit PTH secretion and calcium reabsorption in the kidney thick ascending limb and, therefore, increase urinary calcium excretion. Both loss-of-function and gain-of-function mutations of the CaSR have been identified.42–44 Homozygous loss-of-function missense or nonsense mutations, scattered throughout most of the CaSR, cause neonatal severe primary hyperparathyroidism,42,43 a rare disease that requires early diagnosis and parathyroidectomy to permit affected individuals to survive. The loss of a functional CaSR means that neither PTH secretion nor calcium reabsorption is inhibited even in the presence of high calcium concentrations, which results in hypercalcemia, hypercalciuria and parathyroid tumors. In heterozygous individuals these mutations cause familial hypocalciuric hypercalcemia, a rare, LANIA ET AL. DECEMBER 2006 VOL 2 NO 12 REVIEW www.nature.com/clinicalpractice/endmet autosomal-dominant disorder characterized by hypercalcemia, inappropriately low urinary calcium excretion and (usually) normal PTH levels. This disorder is considered to be benign and is, therefore, often termed familial benign hypocalciuric hyercalcemia, although affected individuals probably have an increased incidence of chondrocalcinosis, pancreatitis and osteomalacia.43 Gain-of-function mutations of CaSR are associated with familial hypercalciuric hypocalcemia syndrome. These mutations are located within the extracellular domain of the protein, and lead to suppression of PTH secretion and to inappropriately increased calcium excretion in the presence of low serum calcium concentrations.43–47 This condition needs to be distinguished from hypoparathyroidism, since patients with CaSR mutations can experience increased hypercalciuria, nephrocalcinosis and renal impairment if treated with calcium and vitamin D supplementation.48 Parathyroid hormone receptor 1 Loss-of-function and gain-of-function mutations in the PTH receptor 1 gene cause two disorders characterized by abnormalities in endochondral bone formation. Loss-of-function mutations in PTH receptor 1 cause Blomstrand chondrodysplasia, a recessive disorder characterized by accelerated skeletal maturation, inappropriate cartilage ossification, craniofacial malformations, coarctation of the aorta, and fetal death.49 Heterozygous gain-of-function mutations are responsible for Jansen-type metaphyseal chondrodysplasia. This disease is a rare form of shortlimbed dwarfism that is usually associated with normal or suppressed levels of serum PTH and severe hypercalcemia and hypophosphatemia, which mimics primary hyperparathyroidism.50 of the reaction is determined by GTP hydrolysis. The α-subunit-triggered effectors are enzymes of second-messenger metabolism, such as cAMPdependent and phosphatidylinositol kinases, and ion channels. They induce short-term effects on hormone secretion, neurotransmission and muscle contraction as well as long-term effects on gene transcription52 (Figure 1). The seventeen α subunits that have been cloned so far are divided into major subfamilies, according to the nature of the generated second messenger. G-PROTEIN MUTATIONS AND DISEASE To date, only guanine nucleotide-binding protein G-stimulating subunit α (Gsα) and guanine nucleotide-binding protein G-transducing activity α1 subunit (Gtα, also known as the transducin α1 chain) have been unequivocally associated with human diseases. Indeed, data that reported somatic gain-of-function mutations of Gi2α protein (which is involved in the inhibition of adenylyl cyclase and activation of mitogenic pathways) in subsets of ovarian sex cord stromal tumors, adrenal cortex tumors and pituitary nonfunctioning tumors have not been subsequently confirmed.51 Loss-of-function mutations of the Gtα gene (GNAT1) cause autosomal-dominant stationary night blindness (Nougaret syndrome).53 Loss-of-function or gain-of-function mutations in the gene that encodes Gsα (GNAS, formerly known as GNAS1) cause endocrine disorders characterized by hormone resistance or hormone excess, respectively.54 Germline, loss-of-function mutations are always heterozygous, and gain-of-function mutations are always somatic, presumably because complete loss or constitutive activation of GNAS in the germline is lethal.54 Loss-of-function mutations of GNAS G PROTEINS G proteins are molecular switches whose activities are determined by their interaction with guanine nucleotides, hence their name (Figure 1). They are composed of three subunits—α, β and γ. The functional specificity of each G protein depends on the α subunit, which differs from one G protein to another.51 The GDP-bound α subunit binds tightly to the β–γ complex and is inactive, whereas the GTP-bound α subunit can dissociate from the β–γ complex and activate effector proteins. The interaction of an agonist with a specific GPCR causes the exchange of GDP for GTP in the α subunit, whereas the duration DECEMBER 2006 VOL 2 NO 12 LANIA ET AL. Heterozygous, loss-of-function mutations of GNAS cause Albright’s hereditary osteodystrophy (AHO), a syndrome characterized by several physical features, including short stature, obesity, subcutaneous ossifications, brachydactyly and mental deficiency.54–56 Consistent with the presence of heterozygous mutations, haploinsufficiency is probably responsible for these multiple defects. In addition to the AHO phenotype, patients who inherit the mutation from their mother develop resistance to the actions of PTH, which is frequently associated with resistance to other hormones that act via Gsα-coupled receptors, such as TSH, GHRH and NATURE CLINICAL PRACTICE ENDOCRINOLOGY & METABOLISM 687 ©2006 Nature Publishing Group REVIEW www.nature.com/clinicalpractice/endmet gonadotropins. This rare condition is referred to as pseudohypoparathyroidism type 1a (PHP1a).54,56 Female patients develop resistance to PTH, TSH, GHRH and gonadotropins, of variable severity and time course.56–58 Patients who inherit the same GNAS mutations from their father have AHO without any resistance to hormone action, a condition defined as pseudopseudohypoparathyroidism (PPHP). The differences between the PHP1a and PPHP phenotypes are caused by genomic GNAS imprinting (imprinting is an epigenetic event that causes expression of one allele, either maternal or paternal, to be suppressed). Indeed, Gsα is biallelically expressed in most tissues, whereas it is primarily expressed from the maternal allele in others, particularly renal proximal tubules, thyroid, gonads and pituitary.57,59,60 In these tissues, mutations in the active, maternal allele lead to Gsα deficiency and hormone resistance (i.e. PHP1a) whereas mutations in the inactive, paternal allele have little or no effect (i.e. PPHP); moreover, silencing of one allele can be total or partial, and may occur either during embryogenesis or in postnatal life, which is consistent with the observed variation in the PHP1a phenotype. Some patients with AHO develop a severe form of ectopic ossification referred to as progressive osseous heteroplasia, in which connective tissues and muscles are invaded by large, ossified plaques. Since the mutations in patients with AHO or progressive osseous heteroplasia are similar, other genetic or environmental factors are presumed to be involved in this process.61 An intriguing missense mutation of GNAS has been identified in two unrelated males who presented with AHO, PTH resistance and testotoxicosis. This substitution (Ala366Ser) causes a rapid GDP release from Gsα, and results in activation of cAMP production at the reduced temperature of the testis and in LH-independent stimulation of Leydig cells; however, the mutant protein is thermolabile at 37 °C, which results in the AHO phenotype.62 Resistance to PTH in the absence of AHO and other endocrine abnormalities is referred to as PHP1b, a usually sporadic but occasionally familial defect with an autosomal-dominant pattern of transmission. Linkage analysis has mapped the genetic locus to a small region of chromosome 20q13.3, where GNAS is located. The proposed mechanism of disease is the presence of specific deletions, which differ in familial 688 NATURE CLINICAL PRACTICE ENDOCRINOLOGY & METABOLISM ©2006 Nature Publishing Group and sporadic PHP1b and disrupt long-range imprinting of the control elements of this locus, with consequent decreased GNAS transcription in renal proximal tubules.63–66 Gain-of-function mutations of GNAS Activating GNAS mutations were first identified in GH-secreting pituitary adenomas.67 The only substitutions so far described occur at either Arg201 or Gln227 and cause constitutive activation of cAMP formation, by inhibition of the intrinsic GTPase activity of the G-protein α subunit. Since pituitary somatotrophs belong to a cell type that recognizes cAMP as a mitogen, GNAS can be considered to be a proto-oncogene that is converted into an oncogene, designated GSP (Gs protein), in selected cell types. Following the identification of GSP mutations in about 30–40% of GH-secreting adenomas, analogous mutations have been detected in a small subset of other pituitary tumors, in 5–10% of toxic thyroid adenomas and thyroid cancers, in some cortisol-secreting adenomas and in a significant proportion of ovarian and testicular stromal Leydig cell tumors.68 In the past 3 years, these same mutations have been described in girls with premature thelarche, and in juvenile ovarian granulosa cell tumors.69,70 With the exception of juvenile ovarian granulosa cell tumors, neoplastic lesions associated with the GSP oncogene are benign, well-differentiated, hormone-hypersecreting adenomas, without significant clinical and biochemical differences from lesions that express wild-type Gsα protein;71 this phenomenon is probably caused by the activation of counter-regulatory mechanisms.72,73A variety of Gsα mutations at Arg201 lead to McCune–Albright syndrome (MAS), a rare disorder characterized by polyostotic fibrous dysplasia, café-au-lait skin hyperpigmentation, and autonomous hyperfunction of several endocrine glands.74 These tissue-specific effects are caused by mosaicism for the Gsα mutation, consistent with a somatic GNAS mutation that occurs as an early postzygotic event. Interestingly, in patients with MAS who have acromegaly, as well as in isolated GH-secreting adenomas, these GNAS mutations are in the maternal allele, presumably because in somatotroph cells Gsα is almost exclusively expressed from this allele.75 Finally, substitutions at Arg201 are also found in isolated fibrous dysplasia and in intramuscular myxomas, which occur outside of the context of typical MAS.76 LANIA ET AL. DECEMBER 2006 VOL 2 NO 12 REVIEW www.nature.com/clinicalpractice/endmet Table 3 Clinical and biochemical features of ‘nonsyndromic’ diseases, which might be caused by GPCR mutations that lead to endocrine disorders. Clinical features Biochemical feature Possible GPCR affected (type of mutation) Normal or low TSH, low FT4, absent TSH and prolactin responses to TRH test Moderately high TSH, normal FT4, no goiter, no antibodies to thyroglobulin or thyroperoxidase High TSH, low FT4, no goiter, no antibodies to thyroglobulin or thyroperoxidase TRH receptor (inactivating) Low TSH, high FT4, no antibodies to thyroglobulin, or TSH receptor Low TSH, high FT4 TSH receptor (activating) Low sex steroids and low-normal LH and FSH, responsive to GnRH test Low sex steroids and low-normal LH and FSH, poorly responsive to GnRH test Kisspeptin 1 receptor (inactivating) Androgenic insufficiency with ‘normal’ spermatogenesis, eunuchoidal features Low LH and testosterone levels, normal FSH GnRH receptor (inactivating) Complete male pseudohermaphroditism, to very mild undervirilization with hypoplasia and/or agenesis of Sertoli cells High LH, normal FSH, barely detectable testosterone, unresponsive to hCG LH receptor (inactivating) Primary amenorrhea with normal development of primary and secondary sexual characteristics High LH, normal or high FSH, and low estradiol and progesterone, unresponsive to hCG LH receptor (inactivating) Male precocious puberty with normal pituitary MRI High testosterone and low LH and FSH with prepubertal response to GnRH test LH receptor (activating) Primary or early-onset secondary amenorrhea, variable development of secondary sex characteristics and premature arrest of follicular maturation High LH, high FSH FSH receptor (inactivating) Ovarian hyperstimulation syndrome during in vitro fertilizationa None FSH receptor (activating) Low cortisol, high ACTH, no response to ACTH stimulation test, normal renin–angiotensin system Melanocortin 2 receptor (inactivating) Thyroid disorders Isolated central hypothyroidism with normal pituitary MRI Partial hypothyroidism Complete congenital hypothyroidism Juvenile hyperthyroidism with goiter Gestational hyperthyroidism with spontaneous remission after delivery TRH receptor (inactivating) TRH receptor (inactivating) Gonadal disorders Delayed puberty GnRH receptor (inactivating) Adrenal disorders Isolated central hypoadrenalism aSymptoms range from discomfort to potentially life-threatening, massive ovarian enlargement and capillary leak with fluid sequestration. Abbreviations: ACTH, adrenocorticotropic hormone; FSH, follicle-stimulating hormone; FT4, free T4; GnRH, gonadotropin-releasing hormone; GPCR, G-protein-coupled receptor; hCG, human chorionic gonadotropin; LH, luteinizing hormone; TRH, TSH-releasing hormone. Polymorphic variants of GPCRs and G proteins The association of polymorphic variants of GPCRs and G proteins with disorders of multifactorial etiology has been widely investigated. In this respect, susceptibility to obesity and type 2 diabetes has been found to be associated with polymorphisms of DRD2 (which encodes the dopamine D2 receptor) and HTR2C, which encodes the 5-hydroxytryptamine (serotonin) 2C receptor. Susceptibility to type 1 diabetes seems to be associated with CC chemokine receptor 2 variants. Previous studies also reported an increased risk of hypertension associated with DECEMBER 2006 VOL 2 NO 12 LANIA ET AL. genetic variants of β2 adrenergic and angiotensin II type 1 receptors.77 On the basis of the notion that genetic polymorphisms could influence a patient’s response to drugs, GPCRs have been investigated in pharmacogenetic studies. In this respect, two known polymorphisms in FSHR seem to be of clinical relevance, since they are associated with an altered ovarian response to pharmacologic doses of FSH and should, therefore, be considered in women who are undergoing controlled ovarian hyperstimulation.78 As for G proteins, studies have reported associations between polymorphic variants of GNAS and hypertension, alcohol consumption, and NATURE CLINICAL PRACTICE ENDOCRINOLOGY & METABOLISM 689 ©2006 Nature Publishing Group REVIEW www.nature.com/clinicalpractice/endmet Table 4 Clinical and biochemical features of ‘nonsyndromic’ endocrine diseases, which might be caused by GPCR mutations that lead to disorders in weight, growth, water balance and mineral-ion turnover. Clinical features Biochemical feature Possible GPCR affected (type of mutation) Hyperinsulinemia Melanocortin 4 receptor (inactivating) Low GH and insulin-like growth factor 1, unresponsive to GHRH test GHRH receptor (inactivating) Nephrogenic diabetes insipidus Hypernatremia, low urine osmolality, normal or high ADH, no response of urinary cyclic AMP to vasopressin AVPR V2 (inactivating) Nephrogenic syndrome of inappropriate antidiuresis Hyponatremia, low serum osmolality, inappropriately high urine osmolality, undetectable ADH levels AVPR V2 (activating) Severe neonatal hyperparathyroidism High PTH, hypercalcemia, hypophosphatemia, hypercalciuria CaSR (inactivating, homozygous) Familial hypocalciuric hypercalcemia Normal PTH, hypercalcemia, hypermagnesemia, hypocalciuria CaSR (inactivating, heterozygous) Familial hypercalciuric hypocalcemia Low PTH, hypocalcemia, hyperphosphatemia, hypomagnesemia, hypercalciuria CaSR (activating) Obesity Early-onset or severe adult obesity, associated with hyperphagia, increased linear growth, increased bone mass Growth disorders Dwarfism associated with highpitched voice, mild frontal bossing and abdominal adiposity Water balance disorders Mineral-ion turnover disorders Abbreviations: ADH, antidiuretic hormone; AVPR V2, arginine vasopressin receptor 2; CaSR, calcium-sensing receptor; GH, growth hormone; GHRH, GH-releasing hormone; GPCR, G-protein-coupled receptor; PTH, parathyroid hormone. cigarette smoking,79,80 whereas the 825C>T poly morphism in GNB3, which encodes guanine nucleotide-binding protein subunit β3 (transducin beta chain 3) has been associated with hypertension, atherosclerosis, and type 2 diabetes.80,81 More clinical studies are warranted to confirm these associations, and to investigate the possible usefulness of genotyping for GPCR and G-protein variants. CONCLUSIONS GPCRs and G proteins are molecules that are required for the transmission of a wide series of endogenous and exogenous signals. The broad range of physiologic functions that are associated with GPCRs explains why at least half of the currently available medications target this receptor family. Naturally occurring loss-of-function or gain-of-function mutations in genes that encode GPCRs and G proteins are associated with human endocrine-related diseases, which mimic 690 NATURE CLINICAL PRACTICE ENDOCRINOLOGY & METABOLISM ©2006 Nature Publishing Group hormone deficiency or excess, respectively. The study of the phenotypic consequences of these mutations, in naturally occurring cases as well as in knockout and transgenic mouse models, has already had major implications for understanding structure–function relationships in GPCRs and G proteins, as well as tissue expression specificity, and function redundancy of these molecules. The diseases caused by these genetic defects are rare, and diagnosis requires careful clinical and biochemical work-up, great awareness of the potential variation in phenotypes, and close collaboration between clinical and molecular endocrinologists. A schematic summary that outlines the appropriate diagnostic tests that can lead clinicians to suspect the various mutations described in this article is given in Tables 3 and 4. There are significant numbers of patients in whom no mutations of candidate genes have been found, which suggests that further research into potentially disease-causing mutations, informed by careful analysis of their relevant LANIA ET AL. DECEMBER 2006 VOL 2 NO 12 REVIEW www.nature.com/clinicalpractice/endmet phenotypes, is likely to be fruitful. Moreover, polymorphic variants that result in subtle functional differences might contribute to multifactorial disease susceptibility. Unfortunately, once GPCR and G-protein mutations are identified in a patient, the implications for their treatment are, as yet, rather limited. It is predictable, however, that novel approaches to treatment— such as the development of inverse agonists that can block constitutively activated GPCRs, or methods to rescue the function of inactive GPCRs—will be available in the future. 6 7 8 9 10 11 KEY POINTS ■ Genes that encode G-protein-coupled receptors (GPCRs) and G proteins can have loss-of-function or gain-of-function mutations, which result in endocrine disorders ■ Loss-of-function mutations in GPCRs and G proteins prevent signaling in response to the corresponding agonist, and cause resistance to hormone action, which mimics hormone deficiency ■ ■ Gain-of-function mutations in GPCRs and G proteins lead to constitutive, agonistindependent activation of signaling, which mimics hormone excess The diseases caused by genetic defects in GPCRs and G proteins are rare, and diagnosis requires careful clinical and biochemical work-up as well as close collaboration between clinical and molecular endocrinologists 12 13 14 15 16 17 18 ■ The study of the phenotypic consequences of mutations in GPCRs and G proteins has already had major implications for understanding structure–function relationships of these molecules, even if the implications for treatment of patients who carry such mutations are limited, at present 19 20 21 References 1 Fredriksson R and Schioth HB (2005) The repertoire of G-protein-coupled receptors in fully sequenced genomes. Mol Pharmacol 67: 1414–1425 2 Hollmann MW et al. (2005) Receptors, G proteins and their interactions. Anesthesiology 103: 1066–1078 3 Perez DM and Karnik SS (2005) Multiple signaling states of G-protein-coupled receptors. Pharmacol Rev 57: 147–161 4 Spiegel AM and Weinstein LS (2004) Inherited diseases involving G proteins and G protein-coupled receptors. Annu Rev Med 55: 27–39 5 Brothers SP et al. (2004) Human loss-of-function gonadotropin-releasing hormone receptor mutants retain wild-type receptors in the endoplasmic reticulum: molecular basis of the dominant-negative effect. Mol Endocrinol 18: 1787–1797 DECEMBER 2006 VOL 2 NO 12 LANIA ET AL. 22 23 24 25 26 Collu R et al. (1997) A novel mechanism for isolated central hypothyroidism: inactivating mutations in the thyrotropin-releasing hormone receptor gene. J Clin Endocrinol Metab 82: 1561–1565 Abramowicz MJ et al. (1997) Familial congenital hypothyroidism due to inactivating mutation of the thyrotropin receptor causing profound hypoplasia of the thyroid gland. J Clin Invest 99: 3018–3024 Sunthornthepvarakul T et al. (1995) Resistance to thyrotropin caused by mutations in the thyrotropinreceptor gene. N Engl J Med 332: 155–160 Davies TF et al. (2005) Thyrotropin receptor-associated diseases: from adenomas to Graves disease. J Clin Invest 115: 1972–1983 Parma J et al. (1993) Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenomas. Nature 365: 649–651 Krohn K et al. (2005) Molecular pathogenesis of euthyroid and toxic multinodular goiter. Endocr Rev 26: 504–524 Fuhrer et al. (1997) Identification of a new thyrotropin receptor germline mutation (Leu629Phe) in a family with neonatal onset of autosomal dominant nonautoimmune hyperthyroidism. J Clin Endocrinol Metab 82: 4234–4238 Rodien P et al. (1998) Familial gestational hyperthyroidism caused by a mutant thyrotropin receptor hypersensitive to human chorionic gonadotropin. N Engl J Med 339: 1823–1826 de Roux N et al. (2003) Hypogonadotropic hypogonadism due to loss of function of the KiSS1derived peptide receptor GPR54. Proc Natl Acad Sci USA 100: 10972–10976 de Roux N et al. (1997) A family with hypogonadotropic hypogonadism and mutations in the gonadotropinreleasing hormone receptor. N Engl J Med 337: 1597–1602 Karges B et al. (2003) Clinical and molecular genetics of the human GnRH receptor. Hum Reprod Update 9: 523–530 Chanson P et al. (1998) Absence of activating mutations in the GnRH receptor gene in human pituitary gonadotroph adenomas. Eur J Endocrinol 139: 157–160 Themmen APN (2005) An update of the pathophysiology of human gonadotropin subunit and receptor gene mutations and polymorphisms. Reproduction 130: 263–274 Berthezene F et al. (1976) Leydig-cell agenesis: a cause of male pseudohermaphroditism. N Engl J Med 295: 969–972 Shenker A et al. (1993) A constitutively activating mutation of the luteinizing hormone receptor in familial male precocious puberty. Nature 365: 652–654 Liu G et al. (1999) Leydig-cell tumors caused by an activating mutation of the gene encoding the luteinizing hormone receptor. N Engl J Med 341: 1731–1736 Aittomaki K et al. (1995) Mutation in the folliclestimulating hormone receptor gene causes hereditary hypergonadotropic ovarian failure. Cell 82: 959–968 Gromoll J et al. (1996) An activating mutation of the follicle-stimulating hormone receptor autonomously sustains spermatogenesis in a hypophysectomized man. J Clin Endocrinol Metab 81: 1367–1370 Kaiser UB (2003) The pathogenesis of the ovarian hyperstimulation syndrome. N Engl J Med 349: 729–732 Clark AJ et al. (1993) Familial glucocorticoid deficiency associated with a point mutation in the adrenocorticotropin receptor. Lancet 341: 461–462 Clark AJ et al. (2005) Inherited ACTH insensitivity illuminates the mechanisms of ACTH action. Trends Endocrinol Metab 16: 451–457 NATURE CLINICAL PRACTICE ENDOCRINOLOGY & METABOLISM 691 ©2006 Nature Publishing Group REVIEW www.nature.com/clinicalpractice/endmet 27 Lacroix A et al. (1992) Gastric inhibitory polypeptidedependent cortisol hypersecretion: a new cause of Cushing’s syndrome. N Engl J Med 327: 974–980 28 Lacroix A et al. (2001) Ectopic and abnormal hormone receptors in adrenal Cushing’s syndrome. Endocr Rev 22: 75–110 29 Yeo GS et al. (1998) A frameshift mutation in MC4R associated with dominantly inherited human obesity. Nat Genet 20: 111–112 30 Farooqi IS and O’Rahilly S (2005) Monogenic obesity in humans. Annu Rev Med 56: 443–458 31 Govaerts C et al. (2005) Obesity-associated mutations in the melanocortin 4 receptor provide novel insights into its function. Peptides 26: 1909–1919 32 Maheshwari HG et al. (1998) Phenotype and genetic analysis of a syndrome caused by an inactivating mutation in the growth hormone-releasing hormone receptor: dwarfism of Sindh. J Clin Endocrinol Metab 83: 4065–4074 33 Alba M and Salvatori R (2004) Familial growth hormone deficiency and mutations in the GHRH receptor gene. Vitam Horm 69: 209–220 34 Lee EJ et al. (2001) Absence of constitutively activating mutations in the GHRH receptor in GH-producing pituitary tumors. J Clin Endocrinol Metab 86: 3989–3995 35 Pantel J et al. (2006) Loss of constitutive activity of the growth hormone secretagogue receptor in familial short stature. J Clin Invest 116: 760–768 36 Rosenthal W et al. (1992) Molecular identification of the gene responsible for congenital nephrogenic diabetes insipidus. Nature 359: 233–235 37 Knoers NV and Deen PM (2001) Molecular and cellular defects in nephrogenic diabetes insipidus. Pediatr Nephrol 16: 1146–1152 38 van Lieburg AF et al. (1995) Clinical phenotype of nephrogenic diabetes insipidus in females heterozygous for a vasopressin type 2 receptor mutation. Hum Genet 96: 70–78 39 Nomura Y et al. (1997) Detection of skewed X-inactivation in two female carriers of vasopressin type 2 receptor gene mutation. J Clin Endocrinol Metab 82: 3434–3437 40 Ala Y et al. (1998) Functional studies of twelve mutant V2 vasopressin receptors related to nephrogenic diabetes insipidus: molecular basis of a mild clinical phenotype. J Am Soc Nephrol 9: 1861–1872 41 Feldman BJ et al. (2005) Nephrogenic syndrome of inappropriate antidiuresis. N Engl J Med 352: 1884–1889 42 Pollak MR et al. (1993) Mutations in the human Ca2+ sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Cell 75: 1297–1303 43 Thakker RV (2004) Diseases associated with the extracellular calcium-sensing receptor. Cell Calcium 35: 275–282 44 Hendy GN et al. (2000) Mutations of the calciumsensing receptor (CASR) in familial hypocalciuric hypercalcemia, neonatal severe hyperparathyroidism, and autosomal dominant hypocalcemia. Hum Mutat 16: 281–296 45 Baron J et al. (1996) Mutations in the Ca2+-sensing receptor gene cause autosomal dominant and sporadic hypoparathyroidism. Hum Mol Genet 5: 601–606 46 Okazaki R et al. (1999) A novel activating mutation in calcium-sensing receptor gene associated with a family of autosomal dominant hypocalcemia. J Clin Endocrinol Metab 84: 363–366 47 Lovlie K et al. (1996) The Ca2+ sensing receptor gene (PCAR1) mutation T151M in isolated autosomal dominant hypoparathyroidism. Hum Genet 98: 129–133 692 NATURE CLINICAL PRACTICE ENDOCRINOLOGY & METABOLISM ©2006 Nature Publishing Group 48 Lienhardt A et al. (2001) Activating mutations of the calcium-sensing receptor: management of hypocalcemia. J Clin Endocrinol Metab 86: 5313–5323 49 Jobert A-S et al. (1998) Absence of functional receptors for parathyroid hormone and parathyroid hormone-related peptide in Blomstrand chondrodysplasia. J Clin Invest 102: 34–40 50 Schipani E et al. (1996) Constitutively active receptors for parathyroid hormone and parathyroid hormone-related peptide in Jansen’s metaphyseal chondrodysplasia. N Engl J Med 335: 708–714 51 Lyons et al. (1990) Two G protein oncogenes in human endocrine tumors. Science 249: 655–659 52 Wettschureck N and Offermanns S (2005) Mammalian G proteins and their cell type specific functions. Physiol Rev 85: 1159–1204 53 Dryja TP et al. (1996) Missense mutation in the gene encoding the α subunit of rod transducin in the Nougaret form of congenital stationary night blindness. Nat Genet 13: 358–360 54 Weinstein LS et al. (2004) Minireview: GNAS: normal and abnormal functions. Endocrinology 145: 5459–5464 55 Weinstein LS et al. (1990) Mutations of the Gsαsubunit gene in Albright hereditary osteodystrophy detected by denaturing gradient gel electrophoresis. Proc Natl Acad Sci USA 87: 8287–8290 56 Weinstein LS et al. (2001) Endocrine manifestations of stimulatory G protein α-subunit mutations and the role of genomic imprinting. Endocr Rev 22: 675–705 57 Mantovani G et al. (2003) Growth hormone-releasing hormone resistance in pseudohypoparathyroidism type Ia: new evidence for imprinting of the Gsα gene. J Clin Endocrinol Metab 88: 4070–4074 58 Germain-Lee EL et al. (2003) Growth hormone deficiency in pseudohypoparathyroidism type 1a: another manifestation of multihormone resistance. J Clin Endocrinol Metab 88: 4059–4069 59 Mantovani G et al. (2002) The Gsα gene: predominant maternal origin of transcription in human thyroid gland and gonads. J Clin Endocrinol Metab 87: 4736–4740 60 Hayward BE et al. (2001) Imprinting of the Gsα gene GNAS1 in the pathogenesis of acromegaly. J Clin Invest 107: R31–R36 61 Shore EM et al. (2002) Paternally inherited inactivating mutations of the GNAS1 gene in progressive osseous heteroplasia. N Engl J Med 346: 99–106 62 Iiri T et al. (1994) Rapid GDP release from Gsα in patients with gain and loss of endocrine function. Nature 371: 164–168 63 Juppner H et al. (1998) The gene responsible for pseudohypoparathyroidism type Ib is paternally imprinted and maps in four unrelated kindreds to chromosome 20q13.3. Proc Natl Acad Sci USA 95: 11798–11803 64 Linglart A et al. (2005) A novel STX16 deletion in autosomal dominant pseudohypoparathyroidism type Ib redefines the boundaries of a cis-acting imprinting control element of GNAS. Am J Hum Genet 76: 804–814 65 Bastepe M et al. (2005) Deletion of the NESP55 differentially methylated region causes loss of maternal GNAS imprints and pseudohypoparathyroidism type Ib. Nat Genet 37: 25–27 66 Liu J et al. (2005) Distinct patterns of abnormal GNAS imprinting in familial and sporadic pseudohypoparathyroidism type IB. Hum Mol Genet 14: 95–102 LANIA ET AL. DECEMBER 2006 VOL 2 NO 12 REVIEW www.nature.com/clinicalpractice/endmet 67 Landis CA et al. (1989) GTPase inhibiting mutations activate the α chain of Gs and stimulate adenylyl cyclase in human pituitary tumours. Nature 340: 692–696 68 Lania A et al. (2001) G-protein mutations in endocrine diseases. Eur J Endocrinol 145: 543–559 69 Roman R et al. (2004) Activating GNAS1 gene mutations in patients with premature thelarche. J Pediatr 145: 218–222 70 Kalfa N et al. (2006) Activating mutations of the stimulatory G protein in juvenile ovarian granulosa cell tumors: a new prognostic factor? J Clin Endocrinol Metab 91: 1842–1847 71 Spada A et al. (1990) Clinical, biochemical, and morphological correlates in patients bearing growth hormone-secreting pituitary tumors with or without constitutively active adenylyl cyclase. J Clin Endocrinol Metab 71: 1421–1426 72 Lania A et al. (1998) Constitutively active Gsα is associated with an increased phosphodiesterase activity in human growth hormone-secreting adenomas. J Clin Endocrinol Metab 83: 1624–1628 73 Ballare E et al. (1998) Activating mutations of the Gsα gene are associated with low levels of Gsα protein in growth hormone-secreting tumors. J Clin Endocrinol Metab 83: 4386–4390 DECEMBER 2006 VOL 2 NO 12 LANIA ET AL. 74 Weinstein LS et al. (1991) Activating mutations of the stimulatory G protein in the McCune–Albright syndrome. N Engl J Med 325: 1688–1695 75 Mantovani G et al. (2004) A parental origin of Gsα mutations in the McCune–Albright syndrome and in isolated endocrine tumors. J Clin Endocrinol Metab 89: 3007–3009 76 Shenker A et al. (1995) Osteoblastic cells derived from isolated lesions of fibrous dysplasia contain activating somatic mutations of the Gsα gene. Hum Mol Genet 4: 1675–1676 77 Thompson MD et al. (2005) The G protein-coupled receptors: pharmacogenetics and disease. Crit Rev Clin Lab Sci 42: 311–392 78 Gromoll J and Simoni E (2005) Genetic complexity of FSH receptor function. Trends Endocrinol Metab 8: 368–373 79 Jia H et al. (1999) Association of the Gsα gene with essential hypertension and response to β-blockade. Hypertension 34: 8–14 80 Siffert W (2005) G protein polymorphisms in hypertension, atherosclerosis, and diabetes. Annu Rev Med 56: 17–28 81 Siffert W et al. (1998) Association of a human G-protein β3 subunit variant with hypertension. Nat Genet 18: 45–48 Acknowledgments This work was supported by Associazione Italiana per la Ricerca sul Cancro (AIRC), Milan, and Ricerca Corrente Funds of Fondazione Policlinico Istituti di Ricovero e Cura a Carattere Scientifico (IRCCS), Milan, Italy. Competing interests The authors declared they have no competing interests. NATURE CLINICAL PRACTICE ENDOCRINOLOGY & METABOLISM 693 ©2006 Nature Publishing Group