McCune-Albright syndrome (MAS) is characterized by polyostotic fibrous dysplasia, cqaf-au-it lesions, and a variety of endocrine disorders, including precocious puberty, hyperthyroidism, hypercortisolism, growth hormone excess, and hyperprolactinemla. The diverse metabolic abnormalities seen in MAS share the involvement of cells that respond to extracellular signals through activation of the hormone-sensitive adenylyl cyclase system (EC 4.6. (4). The few patients with cortisol excess have adrenal hyperplasia and undetectable levels of adrenocorticotropin (5). Finally, excessive secretion of growth hormone (GH) by subjects with MAS is indistinguishable biochemically from that which occurs in patients who have autonomous GH-secreting pituitary tumors (6). These observations have led to the speculation that MAS is caused by a lesion that results in constitutive activation of adenylyl cyclase.Activity of adenylyl cyclase is regulated by at least two guanine nucleotide-binding (G) proteins; Gs is responsible for stimulation of catalytic activity, whereas another group of G subunits, represented by at least three forms ofGi (7), mediate inhibition of the enzyme (8). Recent studies have demonstrated that mutations in the gene encoding the a subunit of G.

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Ethanol and other drugs of abuse modulate cAMP-PKA signaling within the mesolimbic reward pathway. To understand the role of the cAMP-PKA signal transduction in mediating the effects of ethanol, we have studied ethanol consumption and the sedative effects of ethanol in three lines of genetically modified mice. We report that mice with the targeted disruption of one Gs␣ allele as well as mice with reduced neuronal PKA activity have decreased alcohol consumption compared with their wild-type littermates. Genetic reduction of cAMP-PKA signaling also makes mice more sensitive to the sedative effects of ethanol, although plasma ethanol concentrations are unaffected. In contrast, mice with increased adenylyl cyclase activity resulting from the transgenic expression of a constitutively active form of Gs␣ in neurons within the forebrain are less sensitive to the sedative effects of ethanol. Thus, the cAMP-PKA signal transduction pathway is critical in modulating sensitivity to the sedative effects of ethanol as well as influencing alcohol consumption.Key words: alcohol; sedation; adenylyl cyclase; protein kinase A; cAMP; alcoholismThe cAMP-PKA signal transduction pathway is a ubiquitous cascade that modulates numerous cellular events within neurons Self et al., 1998). The stimulatory G-protein Gs couples and amplifies ligand-induced signals transmitted from receptors to multiple isoforms of adenylyl cyclase. We have previously shown that alcohol-preferring rats have increased adenylyl cyclase activity and increased expression of the ␣ subunit of Gs (Gs␣) in mesolimbic regions of the brain (e.g., nucleus accumbens and ventral tegmental area) compared with alcohol-nonpreferring rats (Froehlich and Wand, 1997). Levels of Gs␣ are similarly increased in blood cell membranes from humans at increased risk for alcoholism (Wand et al., 1994). In addition, a series of studies have identified biochemical abnormalities in this pathway in erythrocyte, lymphocyte, and platelet membranes derived from alcoholic persons (Tabakoff et al., 1988;Gordon et al., 1991;Waltman et al., 1993;Parsian et al., 1996;Menninger et al., 1998). Ethanol and other drugs of abuse modulate cAMP-PKA signaling within the mesolimbic reward pathway (Hoffman and Tabakoff, 1990;Self et al., 1998;Spanagel and Weiss, 1999). Because the magnitude of adenylyl cyclase activity as well as vulnerability to alcoholism are both influenced by strong genetic determinants (Devor et al., 1991;Foroud and Li, 1999), it is conceivable that genes controlling cAMP-PKA signaling in the mesolimbic reward pathway play an important role in determining genetic vulnerability for alcoholism.Compared with the offspring of nonalcohol-dependent parents, the offspring of alcoholics have a 4-to 10-fold increased probability of developing alcoholism during their teenage years and adulthood (Schuckit, 1994(Schuckit, , 2000. Human studies of "at risk" individuals have established a premorbid phenotype characterized by increased drug liking and decreased sensitivity to sedative effects of alcohol...

Albright hereditary osteodystrophy is caused by heterozygous inactivating mutations in GNAS, a gene that encodes not only the alpha-chain of Gs (Galphas), but also NESP55 and XLalphas through use of alternative first exons. Patients with GNAS mutations on maternally inherited alleles are resistant to multiple hormones such as PTH, TSH, LH/FSH, GHRH, and glucagon, whose receptors are coupled to Gs. This variant of Albright hereditary osteodystrophy is termed pseudohypoparathyroidism type 1a and is due to presumed tissue-specific paternal imprinting of Galphas. Previous studies have shown that mice heterozygous for a targeted disruption of exon 2 of Gnas, the murine homolog of GNAS, showed unique phenotypes dependent on the parent of origin of the mutated allele. However, hormone resistance occurred only when the disrupted gene was maternally inherited. Because disruption of exon 2 is predicted to inactivate Galphas as well as NESP55 and XLalphas, we created transgenic mice with disruption of exon 1 to investigate the effects of isolated loss of Galphas. Heterozygous mice that inherited the disruption maternally (-m/+) exhibited PTH and TSH resistance, whereas those with paternal inheritance (+/-p) had normal hormone responsiveness. Heterozygous mice were shorter and, when the disrupted allele was inherited maternally, weighed more than wild-type littermates. Galphas protein and mRNA expression was consistent with paternal imprinting in the renal cortex and thyroid, but there was no imprinting in renal medulla, heart, or adipose. These findings confirm the tissue-specific paternal imprinting of GNAS and demonstrate that Galphas deficiency alone is sufficient to account for the hormone resistance of pseudohypoparathyroidism type 1a.

The G protein ␤␥-dimer is required for receptor interaction and effector regulation. However, previous approaches have not identified the physiologic roles of individual subtypes in these processes. We used a gene knockout approach to demonstrate a unique role for the G protein ␥ 7 -subunit in mice. Notably, deletion of Gng7 caused behavioral changes that were associated with reductions in the ␣ olf -subunit content and adenylyl cyclase activity of the striatum. These data demonstrate that an individual ␥-subunit contributes to the specificity of a given signaling pathway and controls the formation or stability of a particular G protein heterotrimer.The heterotrimeric G proteins control diverse biological processes by conveying signals from cell-surface receptors to intracellular effectors. Although function was originally ascribed to the GTP-bound ␣-subunit, it is now well established that the ␤␥-dimer plays active roles in the signaling process through upstream recognition of receptors and downstream regulation of effectors (1). Molecular cloning has identified at least 5 ␤-and 12 ␥-subunit genes in the mouse and human genomes. Structurally, ␥-subunits are the most diverse, with four subgroups that show less than 50% identity to each other (2). Moreover, ␥-subunits exhibit very different temporal (3, 4) and spatial (5) patterns of expression. These characteristics suggest that ␥-subunits have heterogeneous functions. However, comparison of their biochemical properties has revealed only modest differences (6 -8), perhaps because of the inherent limitations of transfection and reconstitution approaches. Gene ablation in mice has proven to be a powerful approach to identifying the functional roles of several G protein ␣-subunits (9). We report the first use of a gene targeting strategy to identify a unique function for a member of the ␥-subunit family.The G protein ␥ 7 -subunit (G␥ 7 ) was originally cloned from bovine brain (10). In situ hybridization of rat brain sections revealed that mRNA for G␥ 7 is most highly expressed in the striatum (5), where it is found in 40 -50% of medium sized neurons in the caudate putamen (11). The regional expression of mRNA for G␥ 7 in the brain mirrors that of the striatumenriched D 1 dopamine receptor (D1R), 1 G␣ olf , and adenylyl cyclase Type V (12), suggesting involvement of G␥ 7 in the G␣ olf -mediated stimulation of adenylyl cyclase by dopamine. Single cell RT-PCR analysis confirms that D1R and G␥ 7 are expressed in the same subset of rat neurons (13). Ribozyme suppression studies support a role for G␥ 7 in the endogenous ␤-adrenergic receptor pathway (14) and the heterologously expressed D1R pathway in human embryonic kidney cells (13).

Inactivating and activating mutations in the gene encoding G alpha s (GNAS1) are known to be the basis for 2 well-described contrasting clinical disorders, Albright hereditary osteodystrophy (AHO) and McCune-Albright syndrome (MAS). AHO is an autosomal dominant disorder due to germline mutations in GNAS1 that decrease expression or function of G alpha s protein. Loss of G alpha s function leads to tissue resistance to multiple hormones whose receptors couple to G alpha s. By contrast, MAS results from postzygotic somatic mutations in GNAS1 that lead to enhanced function of G alpha s protein. Acquisition of the activating mutation early in life leads to a more generalized distribution of the mosaicism and is associated with the classic clinical triad of polyostotic fibrous dysplasia, endocrine hyperfunction, and café au lait skin lesions described in MAS. Acquisition of a similar activating mutation in GNAS1 later in life presumably accounts for the restricted distribution of the gsp oncogene, and is associated with the development of isolated lesions (for example, fibrous dysplasia, pituitary or thyroid tumors) without other manifestations of MAS. Tissues that are affected by loss of G alpha s function in AHO are also affected by gain of G alpha s function in MAS, thus identifying specific tissues in which the second messenger cAMP plays a dominant role in cell growth, proliferation, or function. Further investigations of the functions of G alpha s and other members of the GTPase binding protein family will provide more insight into the pathogenesis and clinical manifestations of human disease.

Mutations in the yeast gene CYH2 can lead to resistance to cycloheximide, an inhibitor of eukaryotic protein synthesis. The gene product of CYH2 is ribosomal protein L29, a component of the 60S ribosomal subunit. We have cloned the wild-type and resistance alleles of CYH2 and determined their nucleotide sequence. Transcription of CYH2 appears to initiate and terminate at multiple sites, as judged by S1 nuclease analysis. The gene is transcribed into an RNA molecule of about 1082 nucleotides, containing an intervening sequence of 510 nucleotides. The splice junction of the intron resides within a codon near the 5' end of the gene. In confirmation of peptide analysis by Stocklein et al. (1) we find that resistance to cycloheximide is due to a transversion mutation resulting in the replacement of a glutamine by glutamic acid in position 37 of L29.

Emerging evidence suggests that the gamma subunit composition of an individual G protein contributes to the specificity of the hundreds of known receptor signaling pathways. Among the twelve gamma subtypes, gamma3 is abundantly and widely expressed in the brain. To identify specific functions and associations for gamma3, a gene-targeting approach was used to produce mice lacking the Gng3 gene (Gng3-/-). Confirming the efficacy and specificity of gene targeting, Gng3-/- mice show no detectable expression of the Gng3 gene, but expression of the divergently transcribed Bscl2 gene is not affected. Suggesting unique roles for gamma3 in the brain, Gng3-/- mice display increased susceptibility to seizures, reduced body weights, and decreased adiposity compared to their wild-type littermates. Predicting possible associations for gamma3, these phenotypic changes are associated with significant reductions in beta2 and alphai3 subunit levels in certain regions of the brain. The finding that the Gng3-/- mice and the previously reported Gng7-/- mice display distinct phenotypes and different alphabetagamma subunit associations supports the notion that even closely related gamma subtypes, such as gamma3 and gamma7, perform unique functions in the context of the organism.