Andrew Shenker

Activating mutations of the stimulatory G protein in the McCune-Albright syndrome.

BACKGROUND The McCune-Albright syndrome is a sporadic disease characterized by polyostotic fibrous dysplasia, café au lait spots, sexual precocity, and hyperfunction of multiple endocrine glands. These manifestations may be explained by a somatic mutation in affected tissues that results in activation of the signal-transduction pathway generating cyclic AMP (cAMP). We analyzed DNA from tissues of patients with the McCune-Albright syndrome for the presence of activating mutations of the gene for the alpha subunit of the G protein (Gs alpha) that stimulates cAMP formation. METHODS Genomic DNA fragments encompassing regions (exons 8 and 9) previously found to contain activating missense mutations of the Gs alpha gene (gsp mutations) in sporadically occurring pituitary tumors were amplified in tissues from four patients with the McCune-Albright syndrome by the polymerase chain reaction. The amplified DNA was analyzed for mutations by denaturing gradient gel electrophoresis and allele-specific oligonucleotide hybridization. RESULTS We detected one of two activating mutations within exon 8 of the Gs alpha gene in tissues from all four patients, including affected endocrine organs (gonads, adrenal glands, thyroid, and pituitary) and tissues not classically involved in the McCune-Albright syndrome. In two of the patients, histidine was substituted for arginine at position 201 of Gs alpha, and in the other two patients cysteine was substituted for the same arginine residue. In each patient the proportion of cells affected varied from tissue to tissue. In two endocrine organs, the highest proportion of mutant alleles was found in regions of abnormal cell proliferation. CONCLUSIONS Mutations within exon 8 of the Gs alpha gene that result in increased activity of the Gs protein and increased cAMP formation are present in various tissues of patients with the McCune-Albright syndrome. Somatic mutation of this gene early in embryogenesis could result in the mosaic population of normal and mutant-bearing tissues that may underlie the clinical manifestations of this disease.

Severe endocrine and nonendocrine manifestations of the McCune-Albright syndrome associated with activating mutations of stimulatory G protein Gs**

McCune-Albright syndrome (MCAS) is a sporadic disease classically including polyostotic fibrous dysplasia, café au lait spots, sexual precocity, and other hyperfunctional endocrinopathies. An activating missense mutation in the gene for the alpha subunit of GS, the G protein that stimulates cyclic adenosine monophosphate formation, has been reported to be present in these patients. The mutation is found in variable abundance in different affected endocrine and nonendocrine tissues, consistent with the mosaic distribution of abnormal cells generated by a somatic cell mutation early in embryogenesis. We describe three patients with MCAS who had profound endocrine and nonendocrine disease and who died in childhood. Two of the patients were severely ill neonates whose complex symptoms did not immediately suggest MCAS. A mutation of residue Arg201 of GS alpha was found in affected tissues from all three children. A review of the literature and unpublished case histories emphasizes the existence of other patients with severe and unusual clinical manifestations. We conclude that the manifestations of MCAS are more extensive than is generally appreciated, and may include hepatobiliary disease, cardiac disease, other nonendocrine abnormalities, and sudden or premature death.

The histopathology of fibrous dysplasia of bone in patients with activating mutations of the Gsα gene : Site-specific patterns and recurrent histological hallmarks

Gsα mutations and histopathology have been analysed in a series of 13 patients with fibrous dysplasia (FD) of bone, including 12 patients with the McCune–Albright syndrome (MAS) and one patient with monostotic FD. Activating mutations (either R201C or R201H) of the gene encoding the α subunit of the stimulatory G protein, Gs, were detected in all cases, including the case of monostotic FD, using a variety of techniques [reverse transcription‐polymerase chain reaction (RT‐PCR) with allele‐specific primers, allele‐specific oligonucleotide hybridization, and DNA sequencing]. A spectrum of bone lesions associated with such mutations was identified and it was possible to recognize three primary, but distinct, histological patterns, defined here as Chinese writing type, sclerotic/Pagetoid type, and sclerotic/hypercellular type, which are characteristically associated with the axial/appendicular skeleton, cranial bones, or gnathic bones, respectively. Features of FD histopathology were characterized by confocal fluorescence microscopy, which allowed the definition of osteogenic cell shape changes and ‘Sharpey fibre bone’ as common denominators of all histological subtypes. Defining characteristics of the different subtypes, two of which diverge from standard descriptions of FD and have never been characterized before, were dependent on the amount and structure of bone tissue within the FD lesion. These data emphasize the non‐random (site‐specific) variability of FD histopathology in patients carrying activating mutations of the Gsα gene and provide additional evidence for the occurrence of Gsα mutations in cases of FD other than typical MAS. Copyright

The Role of Asp578 in Maintaining the Inactive Conformation of the Human Lutropin/Choriogonadotropin Receptor

A constitutively activating mutation encoding Asp578→Gly in transmembrane helix 6 of the lutropin/choriogonadotropin receptor (LHR) is the most common cause of gonadotropin-independent, male-limited precocious puberty. This mutant LHR produces a 4.5-fold increase in basal cAMP when expressed in COS-7 cells. To better understand the normal role of Asp578 in the LHR we studied the effect of seven other amino acid substitutions at this position. No agonist binding or response was detected with the Asp578→Pro mutant. Agonist binding affinity was unaffected by the other substitutions and estimated receptor concentrations ranged from 11 to 184% of wild type. Substitution of Asp578 with Asn, a similarly sized, uncharged residue, did not produce agonist-independent activation. In contrast, replacement with Glu, Ser, or Leu caused 4.9-5.6-fold stimulation of basal cAMP. Substitution with Tyr (8.5-fold) or Phe (7.5-fold) had a greater activating effect. Only the Tyr, Phe, and Leu mutants showed constitutive activation of the inositol phosphate pathway. Our data suggest that it is the ability of the Asp578 side chain to serve as a properly positioned hydrogen bond acceptor, rather than its negative charge, that is important for stabilizing the inactive state of the LHR. A bulky aromatic side chain at position 578 may further destabilize the inactive receptor conformation.

Constitutive activation of cyclic AMP but not phosphatidylinositol signaling caused by four mutations in the 6th transmembrane helix of the human thyrotropin receptor

Four different somatic mutations (F631C, T632I, D633E, and D633Y) in the putative 6th transmembrane helix of the human thyrotropin receptor (TSHR) were recently described in hyperfunctioning thyroid adenomas [Porcellini et al. (1994) J. Clin. Endocrinol. Metab. 79, 657‐661]. We transiently expressed these mutant receptors in Cos‐7 cells and measured [125I]TSH binding, basal and TSH‐stimulated cAMP production, and phosphatidylinositol hydrolysis. The concentration of receptors expressed at the cell surface was lower for the mutants than for the wild type (WT) TSHR. Compared to the WT, all four mutant receptors caused a marked increase in basal cAMP levels, but did not increase basal production of inositol phosphates. This suggests that autonomous thyroid function and adenoma formation may be related to constitutive activation of the cAMP pathway alone. A cluster of conserved residues at the base of the 6th transmembrane helix of the TSHR and other glycoprotein hormone receptors appears important for maintaining an inactive receptor conformation.

G protein mutations in human disease

The heterotrimeric G proteins couple cell-surface receptors for extracellular signals to intracellular effectors that generate second messengers. Abnormal G protein signalling, resulting from posttranslational modifications by bacterial toxins, altered gene expression, or gene mutations, may lead to diverse biological consequences. Mutations within G protein alpha subunit genes that lead to either constitutive activation or loss of function have been identified. Such G protein mutations play a role in the pathogenesis of several human diseases, including sporadic endocrine tumors, McCune-Albright syndrome, and Albright hereditary osteodystrophy.

Activating Mutations of the Luteinizing Hormone Receptor Gene in Familial Testotoxicosis

Heptahelical, membrane-spanning cell-surface receptors utilize he-terotrimeric guanine nucleotide binding proteins (G proteins) to transmit extracellular signals to cellular effectors (1–4). Diverse stimuli, including photons, hormones, neurotransmitters, odorants, proteases, and ions, trigger conformational changes in the receptor transmembrane helices that are relayed to the cytoplasmic surface, leading to G protein activation (Fig. 20.1A).