Physiological functions of the imprinted Gnas locus and its protein variants Gas and XLas in human and mouse

The stimulatory a-subunit of trimeric G-proteins Gas, which upon ligand binding to seven-transmembrane receptors activates adenylyl cyclases to produce the second messenger cAMP, constitutes one of the archetypal signal transduction molecules that have been studied in much detail. Over the past few years, however, genetic as well as biochemical approaches have led to a range of novel insights into the Gas encoding guanine nucleotide binding protein, a-stimulating (Gnas) locus, its alternative protein products and its regulation by genomic imprinting, which leads to monoallelic, parental origin-dependent expression of the various transcripts. Here, we summarise the major characteristics of this complex gene Journal of Endocrinology (2008) 196, 193–214 0022–0795/08/0196–193 q 2008 Society for Endocrinology Printed in Great locus and describe the physiological roles of Gas and its ‘extra large’ variant XLas at post-natal and adult stages as defined by genetic mutations. Opposite and potentially antagonistic functions of the two proteins in the regulation of energy homeostasis and metabolism have been identified in Gnasand Gnasxl (XLas)-deficient mice, which are characterised by obesity and leanness respectively. A comparison of findings in mice with symptoms of the corresponding human genetic disease ‘Albright’s hereditary osteodystrophy’/‘pseudohypoparathyroidism’ indicates highly conserved functions as well as unresolved phenotypic differences. Journal of Endocrinology (2008) 196, 193–214 The stimulatory G-protein signalling cycle Heterotrimeric G-proteins that are composed of a, b and g-subunits, mediate signal transduction from a large number of activated seven-transmembrane receptors to diverse intracellular effector pathways. Many general aspects of G-protein signalling have been covered in recent excellent reviews (Cabrera-Vera et al. 2003, Wettschureck & Offermanns 2005). The Gs class of a-subunits is characterised by its ability to stimulate adenylyl cyclases (ACs) to produce the second messenger molecule cAMP. It comprises two genes, Gnas (GNAS in human) and guanine nucleotide binding protein, a stimulating, olfactory type (Gnal), which encode Gas and Gaolf respectively. While Gnas is generally regarded as a ubiquitouslyexpressed gene,Gnal expression is limited to the olfactory epithelium and a few brain regions, in which it largely replaces Gnas expression with very little overlap of the twoa-subunits (Belluscio et al. 1998, Zhuang et al. 2000, Herve et al. 2001). We will focus here on novel findings related to the Gas-subunit, its gene locus, variant protein isoforms and physiological functions. G-proteins undergo a cycle of active and inactive states during the signal transduction process as summarised for Gas in Fig. 1. The inactive formof theG-protein consists of a trimercomprising Gas in association with bandg-subunit complexes at the plasma membrane, whereby Gas occupies the GDP nucleotide-bound conformation. Membrane anchorage of the aand g-subunits is achieved via lipid modifications, in the case of Gas palmitoylation of the NH2-terminus (Kleuss & Krause 2003). band g-subunits form a very tight and stable complex (Wettschureck & Offermanns 2005). A ligand-bound G-protein coupled receptor (GPCR) activates the Gs-protein through promoting the exchange of GDP for GTP on the a-subunit, which results in its dissociation from the receptor and the band g-complexes. The free Gas subunit can now interact with and stimulate its effectorACuntil the intrinsic GTPase activity (hydrolysis ofGTP) of the a-subunit returns it into the inactive GDP-bound form, which reassociates with the band g-complexes, to enter a new cycle (Sunahara et al. 1997, Cabrera-Vera et al. 2003). Very little is known about specificities in the interactions between Gas and the 5 differentb-subunits and 12g-subunits that have been identified, nor whether specific combinations of these subunits preferentially interact with certain GPCRs. The Gas effector AC comprises a family of proteins encoded by nine different genes in mammalian genomes, termed type DOI: 10.1677/JOE-07-0544 Britain Online version via http://www.endocrinology-journals.org Figure 1 Scheme of the signalling cycle of the trimeric Gs-protein. (I) The inactive, trimeric Gs-protein, consisting of a-, band g-subunits, is associated with the plasma membrane via lipid modifications. The as-subunit, e.g. Gas or XLas, is in its GDP-bound conformation. (II) Agonist binding to a Gs-coupled seven-transmembrane receptor (GPCR) causes a conformational switch in the a-subunit, which also involves an exchange of GDP for GTP, leading to its activation and dissociation from band g-subunits. (III) The active, GTP-bound form of Gas/XLas interacts with and activates transmembrane adenylyl cyclases type I–IX, resulting in increased formation of the second messenger cAMP. (IV) The intrinsic GTP hydrolysis activity of Gas/XLas, which can be stimulated by GTPaseactivating enzymes (GAPs), results in its inactivation and reassociation with band g-subunits. A PLAGGE and others . Gnas imprinting and functions 194 I–IX, all of which are large transmembrane proteins with a bipartite catalytic domain (Kamenetsky et al. 2006, Willoughby & Cooper 2007). Although all transmembrane ACs can be stimulated by Gas, they vary in their responsiveness to additional regulators, e.g. Gai, G band g-subunits, Ca 2C and protein kinases (Kamenetsky et al. 2006, Willoughby & Cooper 2007). Most cell types express several AC genes, but certain isoforms dominate in specific tissues (Hanoune & Defer 2001, Krumins & Gilman 2006, Willoughby & Cooper 2007). In the context of some of the physiological functions of Gas discussed below, it is noteworthy, for example, that AC III exerts a specific role in brown adipose tissue (BAT). In rodents, AC III expression and AC activity in BAT is transiently increased during the neonatal period, when offspring are especially sensitive to environmental conditions and maintenance of body temperature (Chaudhry et al. 1996). Stimulation of this signalling pathway results in increased lipolysis and heat production in mitochondria. AC III is strongly upregulated upon stimulation by the sympathetic nervous system, e.g. adrenergic receptor stimulation (Granneman 1995). The last step of the G-protein cycle (Fig. 1), the inactivation of the Gas subunit and re-association with band g-subunits into the trimeric complex, is triggered by the intrinsic Journal of Endocrinology (2008) 196, 193–214 GTPase activity of Gas (Cabrera-Vera et al. 2003). Generally, the hydrolysis of GTP by a-subunits is stimulated in vivo by GTPase-activating proteins (GAPs). In the case of Gas, several proteins have been demonstrated to exert a GAP function, including regulator of G-protein signalling 2 (RGS2; Abramow-Newerly et al. 2006, Roy et al. 2006), AC V itself (Scholich et al. 1999), RGS-PX1 (Zheng et al. 2001) and cysteine string protein (Natochin et al. 2005). Their importance in Gas signalling in vivo remains to be confirmed. The Gas variant XLas also stimulates cAMP signalling from activated receptors The identification in PC12 cells of an alternative ‘extra large’ form of the as subunit, XLas, brought novel aspects to this signalling pathway (Kehlenbach et al. 1994). The XLas protein was found to be mostly identical in sequence to Gas, apart from the NH2-terminal domain, which was replaced by a different(w370aminoacid)sequence.Asdetailedbelow,thetwo variants are transcribed from alternative promoters/first exons of the Gnas gene and spliced onto shared downstream exons from exon 2 onwards. The novel, XL-specific NH2-terminus consists www.endocrinology-journals.org Gnas imprinting and functions . A PLAGGE and others 195 of a repeated, alanine-rich motif, a proline-rich domain, a highly charged and cysteine-containing region and a sequence motif that includes a stretch of leucines and is highly conserved among alla-subunits (Fig. 2A; Kehlenbach et al. 1994). While the repeat motif varies among mammals (Hayward et al. 1998a, Freson et al. 2003), the other XL-specific domains are well conserved. The function of the proline-rich domain is uncertain; however, the cysteine residues serve for lipid anchorage (palmitoylation) to the plasma membrane similar to Gas (Ugur & Jones 2000), while the leucine-containing motif participates in the binding of G-proteinbandg-subunits (Kehlenbach et al. 1994, Lambright et al. 1996, Klemke et al. 2000). The ability of XLas to act as a Figure 2 Scheme of the protein domains of Gas and XLas encoded by protein regions encoded by Gnas (Gas) and Gnasxl (XLas) first exons. Th the binding of band g-subunits. The Gnasxl specific exon contains f region with cysteines and charged amino acids (Cys/charged AA) that and a domain containing an alanine-rich repetitive motif. The C-term human), is identical. (B) The exon–intron structure (coding exons fille imprinted Gnas locus are depicted. The maternally and paternally inh indicate the promoters and transcriptional direction of the individual R by MMM; DMRs at Nespas/Gnasxl and exon 1A represent imprinting encoded proteins are shown above and below the genomic locus. Gn paternal allele in some cell types (hatched blue box). Gnasxl shows exc 2–12 of Gnas (exons 2–13 in human GNAS). The Gnasxl-specific first e a protein termed Alex. Nesp is expressed exclusively from the matern specific exon. Only a single, uninterrupted Nesp-specific exon is foun non-coding, regulatory RNAs; Nespas transcripts exist in multiple splice specific splicing onto exon N1 exclusively in neural tissues leads to p XLN1 protein (existence of a corresponding GasN1 protein is uncerta www.endocrinology-journals.org fully functional Gs-protein, i.e. binding of band g-subunits, activation of AC and coupling of activated receptors, was established in biochemical assays (Klemke et al. 2000) and in transfections of fibroblasts that lack endogenous Gs-proteins (Bastepe et al. 2002, Linglart et al. 2006); the characteristics of cAMP signalling were identical for XLas and Gas (for rat and humanversions) in these transfection studies (Bastepe et al. 2002, Linglart et al. 2006). Neuroendocrine cell lines that express both proteins endogenously have not yet been analysed (see also Klemke et al. 2000). While Gas is regarded as being more or less ubiquitously expressed, XLas shows a much restricted expression pattern, their first exons and of the imprinted Gnas locus. (A) Conserved e first exons encode conserved amino acids (bg) that contribute to urther protein regions that are conserved among mammals, e.g. a mediates lipid membrane anchorage, a proline-rich domain (Pro) inus of the two proteins, encoded by exons 2–12 (exons 2–13 in d), promoter activities and alternative splicing of the murine erited alleles are indicated in red and blue respectively. Arrows NAs. Regions of differential DNA methylation (DMRs) are marked control regions (ICRs). Splicing patterns of the transcripts and as is expressed biallelically in most tissues, but is silenced on the lusive paternal allele-specific expression and is spliced onto exons xon also contains a second potential open reading frame (ORF) for al allele. The Nesp55 ORF is contained within the second Nespd in human. Exon 1A (exon A/B in human) and Nespas produce d and unspliced forms that extend beyond the Nesp exons. Tissueremature transcription termination and expression of a truncated in). Journal of Endocrinology (2008) 196, 193–214 A PLAGGE and others . Gnas imprinting and functions 196 being mostly confined to neural and endocrine tissues (Pasolli et al. 2000, Pasolli & Huttner 2001, Plagge et al. 2004). At embryonic stages, XLas is already detectable from mid-gestation onwards in regions of neurogenesis and in early differentiating neurons, mainly in areas of the midbrain, hindbrain and spinal cord, including the sympathetic trunk and ganglia (Pasolli & Huttner 2001). At later embryonic stages expression was also found in the hypothalamus and the pituitary (adenohypophysis and pars intermedia). In the neonatal brain, XLas expression is confined to distinct regions of the midbrain and hindbrain, e.g. the centre of the noradrenergic system of the brain (locus coeruleus), laterodorsal tegmental nucleus, motor nuclei that innervate orofacial muscles (hypoglossal, motortrigeminal and facial nuclei), as well as scattered cells in the medulla oblongata (Plagge et al. 2004). Further, sites of expression include the neuroendocrine pituitary (pars anterior and intermedia), the catecholaminergic adrenal medulla and some peripheral tissues, e.g. white adipose tissue (WAT) and BAT, pancreas, heart, kidney and stomach (Plagge et al. 2004). There are indications that this expression pattern changes towards adulthood, as no XLas was detected in adult adipose tissues, kidney and heart, but expression persists in brain, pancreatic islets, the pituitary and adrenal glands (Pasolli et al. 2000, Xie et al. 2006). The Gnas locus: alternative promoters, splicing and regulation by genomic imprinting Although the location of the Gas encodingGnas gene onmouse distal chromosome 2/human chromosome 20q13.2–q13.3 and its exon–intron structure had been known for some time (Blatt et al. 1988, Kozasa et al. 1988, Gejman et al. 1991, Levine et al. 1991, Rao et al. 1991, Peters et al. 1994), and despite some early indications for alternative upstream promoters (Ishikawa et al. 1990, Swaroop et al. 1991), the full complexityof theGnas locus was only discovered through work in a different field, i.e. genomic imprinting. Imprinting affects a small number of genes in the mammalian genome, currently comprising w90 identified transcription units (see databases: http://igc.otago. ac.nz/home.html and http://www.mgu.har.mrc.ac.uk/ research/imprinting/index.html). It describes a phenomenon of gene regulation in mammals, whereby one of the two chromosomal alleles is silenced depending on its parental origin. Thus, an imprinted gene is expressed from either the paternally or the maternally inherited chromosome, and this monoallelic, parent of origin-dependent transcription is achieved through mechanisms of DNA methylation, as well as chromatin modifications (Reik & Walter 2001, Morison et al. 2005, Edwards & Ferguson-Smith 2007). Separate screens for imprinted genes in human and mouse resulted in the identification of the XLas-specific first exon of the Gnas locus and an additional exon and promoter, which initiates a transcript that also splices onto downstream Gnas exons but encodes an unrelated, previously identified protein termed Nesp55 (Fig. 2B; Hayward et al. 1998a,b, Kelsey et al. Journal of Endocrinology (2008) 196, 193–214 1999, Peters et al. 1999). The Gnas locus is now known to comprise a complex arrangement of three protein-coding and two non-coding transcripts regulated by imprinting mechanisms. We will describe the murine locus here, but most features are conserved in humans. As the mechanisms of regulation of the locus by genomic imprinting are currently under much investigation, we will only focus on the main characteristics here, but see Peters et al. (2006) for a recent review. The protein coding transcripts The three protein transcriptsGnas,Gnasxl andNesp each initiate at separate promoters/first exons, but share most of the downstream exons (Fig. 2B; Plagge & Kelsey 2006, Weinstein et al. 2007). TheGas encodingGnas transcript is composed of 12 exons (13 in human, due to an additional intron interrupting exon 9). Most cell types express two variants of the Gas protein, a small (45 kDa) and a long 52 (kDa) version, which are functionally equivalent (Graziano et al. 1989, Levis & Bourne 1992) and are generated through alternative splicing of the 15 codons comprising exon 3. Both Gas versions can vary further by the inclusion of a single serine residue, added through usage of an alternative splice acceptor site at exon 4 (Bray et al. 1986, Kozasa et al. 1988). The Gnas promoter and exon 1, which encodes amino acids 1–45 of Gas, do not carry primary marks of genomic imprinting (Liu et al. 2000b) and in most tissues transcription occurs equally from both alleles. In a subset of tissues or cell types, however, expression is monoallelic and restricted to the maternal allele, e.g. in proximal renal tubules, anterior pituitary, thyroid gland and ovary (Yu et al. 1998, Hayward et al. 2001, Germain-Lee et al. 2002, 2005, Mantovani et al. 2002, 2004, Liu et al. 2003); this is relevant to human inherited disorders that are associated with hormone resistance symptoms, as discussed below. Imprinting of Gnas in adipose tissue is still contentious, as some studies showed predominant maternal allele-specific expression ( Yu et al. 1998, Williamson et al. 2004), while others found no such preference (Mantovani et al. 2004, Chen et al. 2005, Germain-Lee et al. 2005). It remains to be clarified whether these discrepant data reflect the analysis of different developmental stages, implying a change in the imprinting status of the Gnas transcript in adipose tissue during the lifetime. In general, tissue-specific imprinting of Gnas has been difficult to demonstrate, since a small amount of transcripts derived from the paternal allele is often detected among the majority that stems from the maternally inherited allele. Whether this is due to incomplete silencing of the paternal allele or a mixture of cell types with imprinted and non-imprinted expression in the tissue samples analysed is unresolved. A second promoter and first exon are located w30 kb upstream of Gnas exon 1 and initiates the Gnasxl transcript (Fig. 2B; Hayward et al. 1998a, Kelsey et al. 1999, Peters et al. 1999), which is spliced onto exon 2–12 of Gnas. This splice form retains the Gnas open reading frame (ORF) and translates into the XLas protein as a NH2-terminal variant of www.endocrinology-journals.org Gnas imprinting and functions . A PLAGGE and others 197 Gas (Kehlenbach et al. 1994). In contrast to Gnas, the Gnasxl promoter is silenced on the maternal chromosome and activates transcription exclusively from the paternal allele. Apart from the full-length Gnasxl transcript, a prominent truncated form, encoding the protein XLN1, is found in neuroendocrine tissues only (brain, pituitary, adrenal medulla; Klemke et al. 2000, Plagge et al. 2004). This truncation is due to alternative, neural tissue-specific splicing of exon N1, which is located between exons 3 and 4 and contains a termination codon and polyadenylation signal. Originally, exon N1 was described as causing neural-specific truncation of the Gnas transcript (Crawford et al. 1993) but, in contrast to XLN1 (Klemke et al. 2000), it remains uncertain whether a corresponding GasN1 protein is stably expressed. The neural N1 proteins retain the residues for membrane anchorage and part of the domain interacting with band g-subunits (Klemke et al. 2000), but lack the major functional domains that are encoded by the downstream exons as well as further residues for interaction with band g-complexes (Lambright et al. 1996). The significance of the exon N1 splice forms, if any, remains to be determined. The complexity of the Gnasxl transcript is further increased through the highly unusual feature in mammalian mRNAs of a second potential ORF, which is shifted by C1 nucleotide, begins a short distance downstream of the XLas start codon and terminates at the end of the Gnasxl-specific exon (Klemke et al. 2001). This ORF encodes a protein termed Alex, which is conserved, but unrelated to G-proteins (Klemke et al. 2001, Nekrutenko et al. 2005). Although Alex was detected in PC12 cells and human platelets (Klemke et al. 2001, Freson et al. 2003), its abundance, expression level and significance in vivo remain unclarified. As a third promoter for a protein-coding transcript within the Gnas locus, the Nesp promoter and first exon are located w15 kb upstream of the Gnasxl exon (Hayward et al. 1998b, Kelsey et al. 1999, Peters et al. 1999). Although the single human NESP-specific exon is interrupted by a short intron in the mouse genome, the downstream splicing onto exons 2–12 of Gnas is conserved and occurs similarly to Gnasxl and Gnas itself. Nesp is imprinted in an opposite way to Gnasxl being expressed only from the maternally derived allele (Hayward et al. 1998b, Kelsey et al. 1999, Peters et al. 1999). The ORF, which encodes the neuroendocrine secretory protein of Mr 55 000 (Ischia et al. 1997), is confined to the Nesp-specific exon, and the shared downstream exons function as 3 0-untranslated sequence. The Nesp55 protein has similarities with the chromogranin family, is associated with secretory vesicles in neuroendocrine cells and is regarded as a marker for the constitutive secretory pathway (Fischer-Colbrie et al. 2002). Little is known about its molecular function, but the protein is processed into peptides to variable extent in different cell types (Lovisetti-Scamihorn et al. 1999). In agreement with its predominant expression in the nervous system and endocrine tissues (Bauer et al. 1999a,b), mice deficient for Nesp55 show a behavioural phenotype, specifically an altered response to novel environments (Plagge et al. 2005, Isles et al. www.endocrinology-journals.org manuscript in preparation) but, in contrast to Gasand XLas-deficient mice (see below), they exhibit no major effects on development, growth or metabolism. Non-coding transcripts and imprinting marks The complexity of the Gnas locus is not limited to the protein-coding transcripts, but is increased by the occurrence of non-coding transcripts and differentially methylated regions of DNA (DMRs). As noted above, we will only briefly describe how these features relate to how imprinting in the locus is controlled (see also Peters et al. 2006). Two untranslated transcripts are produced from separate promoters within the locus (Fig. 2B). The paternal allelespecific exon 1A transcript (exon A/B in human) is initiated w2.4 kb upstream of Gnas exon 1 (Ishikawa et al. 1990, Swaroop et al. 1991, Liu et al. 2000b, Peters et al. 2006) within a CpG dinucleotide-rich cis-regulatory region that is methylated on the maternal allele (exon 1A DMR). This transcript also splices onto exon 2 of Gnas. The second noncoding RNA, Nespas, begins w2.1 kb upstream of the Gnasxl-specific exon, but it is transcribed in the opposite direction, i.e. antisense to Nesp (Hayward & Bonthron 2000, Wroe et al. 2000, Williamson et al. 2006), and is transcribed solely from the paternal allele from within a CpG-rich DMR (methylated on the maternal allele; Coombes et al. 2003). An increasing evidence points towards a role for such non-coding RNA in the regulation of the imprinted, monoallelic expression of the coding transcripts (Pauler et al. 2007). The DMRs at exon 1A andNespas have been shown to be of central importance for the imprinting of the locus (Williamson et al. 2004, 2006, Liu et al. 2005). At both sites, differential methylation of the maternal allele is established in oocytes and maintained after fertilisation and into adulthood in all somatic tissues (Liu et al. 2000b, Coombes et al. 2003). Such germline differences in DNA methylation are characteristic of imprinting control regions (ICRs; Spahn & Barlow 2003). A third DMR located at the Nesp promoter is unmethylated in oocytes and sperm, but acquires methylation on the paternal allele during embryonic development (Liu et al. 2000b, Coombes et al. 2003). The roles of the exon 1A and Nespas DMRs have been demonstrated through targeted deletion in mice (Williamson et al. 2004, 2006, Liu et al. 2005). These studies show that the exon 1A region controls the tissue-specific imprinting of Gnas without affecting the upstream transcription units (Williamson et al. 2004, Liu et al. 2005). Deletion of the exon 1A DMR and promoteron the paternal (normally unmethylated) allele leads to upregulation in cis of the usually silenced expression of Gnas in imprinted tissues. The exact nature of the silencing mechanism exerted by the paternal exon 1A region onGnas transcription is unknown at present (Peters et al. 2006). Deletion of the Nespas promoter, in contrast, affects the imprinting status of all transcripts of the locus (Williamson et al. 2006), such that the Nespas DMR can be regarded as the principal ICR for the locus. Thus, when Nespas transcription Journal of Endocrinology (2008) 196, 193–214 A PLAGGE and others . Gnas imprinting and functions 198 is ablated on the paternal allele, Nesp and Gnas become derepressed, while Gnasxl and the exon 1A transcript are downregulated. Furthermore, the Nesp DMR loses and the exon 1A DMR gains methylation on the paternal allele (Williamson et al. 2006). The molecular mechanisms through which this ICR controls the imprinted expression of all transcripts of the Gnas locus remain to be elucidated. Physiological functions of the gene products as revealed by mutations in mice and humans It has been known for some time that inactivating mutations in the human GNAS gene are associated with the inherited disorder ‘Albright’s hereditaryosteodystrophy’ (AHO)/‘pseudohypoparathyroidism’ (PHP; Levine et al. 1980, 1983a, Patten et al. 1990, Weinstein et al. 1990, Davies & Hughes 1993). Fuller Albright and his colleagues originally described a disorder characterised by hypocalcaemia, hyperphosphataemia and end organ resistance (in proximal renal tubules) to the main plasma Ca regulator parathyroid hormone (PTH), and therefore named the disease PHP (Albright et al. 1942). As PTH levels are not reduced, but typically elevated, and since GNAS is biallelicallyexpressed in the calcium-reabsorbing thick ascending limb of the kidney, hypercalciuria does usually not occur in these patients. They also described other specific somatic and developmental abnormalities in these patients and the disorder is now known to include the following additional symptoms: a round face with a ‘short, thickset figure’, early closure of the epiphyses with resultant shortening of one or more metacarpals or metatarsals (brachydactyly), s.c. ectopic ossifications, dental hypoplasia, obesity and cognitive abnormalities of varying degrees from learning disabilities to severe retardation (Albright et al. 1942, 1952, Weinstein et al. 2001, Levine 2002). Albright and colleagues also noticed patients who showed many of the latter physical features, but had normal calcium, phosphate and PTH levels (Albright et al. 1952). They termed this combination of symptoms, which was not associated with hormone resistance, ‘pseudopseudohypoparathyroidism’ (PPHP). Both conditions are also referred to as AHO, and identical mutations in GNAS that affect the protein coding sequence can cause AHO with or without hormone resistance. It was Davies & Hughes (1993) who described for the first time the association of the syndromes with the parental origin of the mutation. Thus, paternal inheritance of a GNAS exon mutation results in (AHO-)PPHP, while maternal inheritance is associated with additional resistance to PTH (and other hormones, see below; Levine et al. 1983a) and is now termed ‘PHP type Ia’ (PHP-Ia; Weinstein et al. 2001). Some of the typical features of AHO are shown in Fig. 3A–F and are summarised in Table 1; however, not all features are present in all patients. The recent analysis of several mouse models with deficiencies of the individual protein products has deepened our understanding of the associated physiological and endocrine functions (Plagge & Kelsey 2006, Weinstein et al. 2007). Not surprisingly, homozygous deficiency of Gas is Journal of Endocrinology (2008) 196, 193–214 incompatible with life as embryos die soon after implantation (Yu et al. 1998, Chen et al. 2005, Germain-Lee et al. 2005). Heterozygous mutations of the different proteins of the Gnas locus cause distinct dysfunctions (Table 1). In the case of Gas some aspects of the phenotype vary with the parental origin of the mutation, reflecting its imprinted expression, while other dysfunctions occur after both maternal and paternal transmission, indicating haploinsufficiency of Gas in some tissues. Heterozygous loss of Gas in mice recapitulates many aspects of the human disorders, but haploinsufficiency effects seem to be more prevalent in human than in mice. Furthermore, the consequences of loss of XLas in mice differ and are in several respects opposite to those of specific loss of Gas, despite their similar capability to activate the cAMP signalling pathway. Before discussing the physiological and endocrine roles of the different proteins and evaluating the (in some aspects limited) extent of functional conservation between the two species (Table 1), it should be noted that activating or gain of function mutations ofGnashave also been identified. These are beyond the scope of this review, but have been summarised elsewhere recently (Hayward et al. 2001, Weinstein et al. 2006, 2007). Furthermore, a separate human disorder associated with the GNAS locus is not due to mutations affecting the proteincoding sequences, but is caused by deregulated imprinting and gene expression control. Originally, it has been characterised by PTH resistance only without clear AHO symptoms and was therefore termed ‘PHP type Ib’ (PHP-Ib; Bastepe & Jüppner 2005). Our current understanding of PHP-Ib is briefly summarised towards the end of this review. Post-natal physiological functions All manipulations in mice that lead to lack of maternal allelespecific Gas or XLas show an impaired neonatal phenotype with reduced survival (Cattanach & Kirk 1985, Yu et al. 1998, Cattanach et al. 2000, Plagge et al. 2004, Chen et al. 2005, Germain-Lee et al. 2005). Heterozygous deficiency of Gas in mice, generated through deletion of Gnas exon 1, results in a neonatal phenotype on maternal transmission (Chen et al. 2005, Germain-Lee et al. 2005). The paternally inherited deletion has few consequences at this developmental stage, although some mortality was observed in an inbred strain background (Germain-Lee et al. 2005). For exon1 mice a survival rate to weaning age of 34–51% was observed, again varying with the genetic background used. Most of the losses occur within 3 days after birth, and may result from a severe s.c. oedema, which has been described in several mouse models lacking maternal allele-specific Gas protein (Cattanach & Kirk 1985, Yu et al. 1998, Cattanach et al. 2000, Chen et al. 2005). The physiological cause of the oedema, which resolves within a few days after birth, is currently unclear, although a placental dysfunction has been suggested (Chen et al. 2005, Weinstein et al. 2007). Another consequence of loss of Gas expression from the maternal allele is the development of www.endocrinology-journals.org Gnas imprinting and functions . A PLAGGE and others 199 profound obesity in adulthood (discussed in detail below). The increase in adiposity arises already during the post-natal stage, as has been documented in mice with maternally inherited mutations of exons 2 and 6 (Cattanach et al. 2000, Yu et al. 2000, Plagge & Kelsey 2006). Despite their increased lipid accumulation and adipose tissue mass, these mice remain underweight until after weaning. Comparatively little information on post-natal symptoms is available from case studies of AHO/PHP-Ia patients who carry mutations in GNAS exons on the maternal chromosome. An s.c. oedema has not been documented. However, a few reports describe an early onset of some symptoms characteristic of PHP-Ia at later juvenile or adult stages (see also below; Levine et al. 1985, Weisman et al. 1985, Yokoro et al. 1990, Scott & Hung 1995, Yu et al. 1999, Riepe et al. 2005, Gelfand et al. 2006). From these studies a pattern seems to emerge in which abnormal thyroid function and resistance to thyroid-stimulating hormone (TSH), due to deficient receptor signalling via Gas, are among the first symptoms detectable: typically, TSH levels are elevated in PHP-Ia at birth (Levine et al. 1985, Weisman et al. 1985, Yokoro et al. 1990, Yu et al. 1999). The s.c. ossifications can also develop from the first few months onwards, while resistance to PTH, hypocalcaemia and hyperphosphataemia are usually detected only at later stages of infancy or juvenile age (Eddy et al. 2000, Riepe et al. 2005, Gelfand et al. 2006, 2007). Progressive osseous heteroplasia (POH), a more severe form of extraskeletal ossification with invasion into deeper tissues, can also begin early on, and has been described in association with paternally inherited as well as spontaneously occurring GNAS mutations (Eddy et al. 2000, Shore et al. 2002, Faust et al. 2003, Gelfand et al. 2007). In general, ossification symptoms are a classical AHO feature, as they can occur upon mutations of the maternal or paternal allele. Loss of paternally expressed XLas (through gene targeting of the Gnasxl-specific exon) causes lethality in inbred mouse strains, but 15–20% of mutants survive into adulthood if maintained on an outbred genetic background (Cattanach et al. 2000, Plagge et al. 2004, Xie et al. 2006). Deficient pups become distinguishable from wild-type littermates within 1 or 2 days after birth, due to a failure to thrive, characterised by severe growth retardation, poor suckling, hypoglycaemia, hypoinsulinaemia, lack of adipose reserves and inertia (Plagge et al. 2004). This phenotype is most likely related to pleiotropic functions of XLas in the central nervous system (CNS, e.g. orofacial motornuclei in the context of suckling activity), as well as peripheral tissues that are involved in the maintenance of energy homeostasis (e.g. adipose tissues, pancreas; Plagge et al. 2004). Impairment in neonatal feeding, growth and maintenance of energy balance is found not only in mice with a specific mutation of the Gnasxl exon but also in other mutants that lack XLas (Plagge & Kelsey 2006, Weinstein et al. 2007). Thus, mice that carry two copies of the maternally inherited gene locus and no paternal copy (MatDp.dist2) show narrow, flat-sided bodies with reduced adiposity in BAT, hypoactivity, failure to suckle and lethality www.endocrinology-journals.org within a day after birth (Cattanach & Kirk 1985, Williamson et al. 1998). Two further mutations, a deletion of exon 2 and a point mutation in exon 6 (termed Oed-Sml), affect both Gas and XLas upon paternal transmission (Yu et al. 1998, 2000, Cattanach et al. 2000, Skinner et al. 2002); however, the phenotypes of exon2 mice and Sml mice are identical in many respects to Gnasxl deficiency (Yu et al. 1998, 2000, Cattanach et al. 2000, Plagge & Kelsey 2006, Weinstein et al. 2007). The similarity of the phenotypes of these latter two mutations to the Gnasxl mutation indicates that in mice the loss of XLas is dominant over the simultaneous loss of paternal allele-derived Gas. Furthermore, as the paternally inherited exon 6 point mutation does not affect the other two proteins expressed from the Gnasxl exon (XLN1 and Alex), this indicates that loss of XLas is the main cause for the lack of paternal function phenotypes (Plagge & Kelsey 2006, Weinstein et al. 2007). The post-natal phenotype of XLas deficiency improves at around weaning age; no further premature mortality occurs fromthis stage onwards, although adults remain lean (seebelow). It is not unlikely that changes in XLas expression underlie these phenotype changes, since it has been shown for adipose tissue that Gnasxl expression becomes downregulated during the second half of the post-natal period (Xie et al. 2006). It is currently uncertain whether XLas has a similar role in human neonatal physiology. The classical descriptions of patients with AHO/PPHP do not include comparable symptoms. As PPHP patients carry paternally inherited mutations in GNAScoding exons, similar to exon2 and Sml mice, XLas function would be expected to be impaired and dominant over loss of paternally expressed Gas. However, these mutations cause the same common AHO features as in maternally inherited PHP-Ia (plus additional hormone resistances). A conclusive human case study, which could distinguish between XLas functions and paternal haploinsufficiency of Gas by analysing paternally inherited GNAS exon 1 mutations, has not yet been published (Patten et al. 1990, Fischer et al. 1998, Aldred & Trembath 2000, Mantovani et al. 2000, Long et al. 2007). However, other rare genetic anomalies that disrupt the GNAS locus and XLas expression, e.g. large chromosomal deletions and maternal uniparental disomies (UPD) of chromosome 20q13.2– q13.3, have been associated with neonatal impairments. Patients with maternal UPD20q13.2–q13.3, who lack a corresponding paternal allele and can be compared with MatDp.dist2 mice described above, show preand post-natal growth retardation (Chudoba et al. 1999, Eggermann et al. 2001, Salafsky et al. 2001, Velissariou et al. 2002). The 20q13.2–q13.3 deletions that include the GNAS locus on the paternal allele, also lead to growth retardation, failure to thrive, feedingdifficulties requiring artificial feeding, hypotonia and adipose tissue abnormalities (Aldred et al. 2002, Genevieve et al. 2005), reminiscent ofGnasxl knockout mice. Although these cases of chromosomal deletions and UPD20s require careful interpretation, as other potentially contributing genes might also be affected, they nevertheless encourage an investigation of PPHP patients for post-natal symptoms, as far as this is feasible and records are available. Journal of Endocrinology (2008) 196, 193–214 A PLAGGE and others . Gnas imprinting and functions 200 Journal of Endocrinology (2008) 196, 193–214 www.endocrinology-journals.org Gnas imprinting and functions . A PLAGGE and others 201 No null mutations for the Gnasxl-specific exon have been reported in humans, but a polymorphism in the XLas domain, which results in varied numbers of a 12 amino acid NH2terminal repeat unit, has been associated with symptoms such as growth retardation, unexplained mental retardation and brachydactyly (Freson et al. 2001, 2003). Further characterisation of the patients as well as the biochemical functionality of the XLas repeat variants is required. Physiological functions in adulthood The roles of the proteins of the Gnas-locus at adult stages have been characterised in more detail, both in human and mouse (Table 1). The symptoms common to PHP-Ia and PPHP, which occur independently of parental origin and are due to haploinsufficiency of Gas in cells with biallelic expression of GNAS, as well as the hormone resistances associated with PHP-Ia upon maternal inheritance of mutations, fully develop towards adulthood. With regard to Gas, many parallels have now been described between the human diseases and corresponding mouse models, although a role of XLas in humans remains uncertain.