Adenylate cyclase permanently uncoupled from hormone receptors in a novel variant of S49 mouse lymphoma cells.

Haga, Tatsuya; Ross, Elliott M.; Anderson, Hannah; Gilman, Alfred G.

doi:10.1073/pnas.74.5.2016

Cited by 160 publications

(23 citation statements)

References 17 publications

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“…The SCG1 Pr°a67 defects were somewhat more severe in a cells than in a cells (Table 2). This phenotype is similar to the effect of the analogous mutation in G~xs (Haga et al 1977;Rail and Harris 1987;Sullivan et al 1987), that is, both result in reduced activation of the respective pathways. The SCG1 e~°a6~ mutant showed similar effects as the SCG1Pr°a67 mutant in a cells {Table 2; Figs.…”

Section: Mutations Resulting M Reduced Mating Are Consistent With Defsupporting

confidence: 51%

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The carboxyl terminus of Scg1, the G alpha subunit involved in yeast mating, is implicated in interactions with the pheromone receptors.

Hirsch

Dietzel²,

Kurjan³

1991

Genes Dev.

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The carboxyl termini of ¢x subunits of mammalian G proteins have been implicated in receptor interactions. We have used a genetic analysis to test such a role for the carboxyl terminus of Scgl, the ot subunit involved in the yeast pheromone response pathway. A 22-amino-acid truncation (scgl A~t'4sl) resulted in defects in growth and cellular morphology. This phenotype is similar to the null phenotype and represents constitutive activation of the pheromone response pathway; it could result from various effects, including protein instability or constitutive guanine nucleotide exchange, as reported for some altered mammalian Gas constructs. A 5-amino-acid truncation (SCG1 °ch't68) resulted in pheromone response and mating defects in both a and a cells, which is consistent with defects in interactions with the pheromone receptors.Lysine-to-proline mutations near the carboxyl terminus (SCG1 pr°467 and SCG1 J'r°46s) resulted in pheromone response and mating defects, the severity of which differed in a and o~ cells. This differential effect in the two mating types suggests that the specificity for the interactions with the two pheromone receptors may involve different residues of the Scgl carboxyl terminus. Mutations leading to constitutive activation of the pathway were recessive, whereas mutations that result in decreased pheromone response and mating were partially dominant. These relationships are consistent with the model for the mechanism of action of the G protein subunits in the pheromone response pathway and indicate the importance of the stoichiometry of components of this system. Signal transduction mediated by regulatory G proteins in mammalian systems involves the interaction of a ligand-receptor complex with an intracellular G protein composed of o~, 13, and ~/subunits (for review, see Gilman 1987). Before activation, the a subunit has GDP bound and is complexed with 13~/. After receptor activation, GDP is replaced by GTP, and the a subunit dissociates from the 13~ complex. The intrinsic GTPase activity of the a subunit hydrolyzes the bound GTP, regenerating the GDP-bound form of the a subunit, which reassociates with 13~/to complete the cycle.A similar system in yeast is involved in the response of cells to peptide pheromones and is required for mating (for review, see Cross et al. 1988}. Cells of a and a mating type secrete a-factor and o~-factor, respectively, and respond to the pheromone secreted by the opposite mating type. Response to pheromone causes arrest of cells in the Present addresses: tRockefeller University,

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Section: Mutations Resulting M Reduced Mating Are Consistent With Defsupporting

confidence: 51%

“…An arginine-to-proline change 6 amino acids from the carboxyl terminus of Gas uncouples Gs from the [3-adrenergic receptor (Haga et al 1977;Rall and Harris 1987;Sullivan et al 1987). This position is highly conserved in Ga subunits, consisting of an arginine or lysine residue.…”

Section: Mutations Resulting M Reduced Mating Are Consistent With Defmentioning

confidence: 99%

The carboxyl terminus of Scg1, the G alpha subunit involved in yeast mating, is implicated in interactions with the pheromone receptors.

Hirsch

Dietzel²,

Kurjan³

1991

Genes Dev.

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“…The GTP analog stimulated the TD-1 adenylate cyclase to nearly the same proportion and extent as that seen in PC12 cells. This is similar to the uncoupled phenotype (UNC) reported in the S49 lymphoma system, in which variants occurred that were resistant to cell killing by agonists and that possessed a dysfunctional Ns protein (18). The Ns protein of that mutant still mediates adenylate cyclase activation if treated with GppNHp, but is uncoupled from plasma membrane hormone receptors.…”

Section: Methodssupporting

confidence: 59%

“…This protein, which is variously designated as coupling factor, G/F, or Ns, binds GTP and the GTPase-resistant analog, GppNHp. GppNHp alone will activate adenylate cyclase in crude membrane preparations (9,18). The ability to be stimulated by GppNHp is therefore indicative of the presence of both a functional adenylate cyclase and an Ns protein.…”

Section: Methodsmentioning

confidence: 99%

Mutants of PC12 cells with altered cyclic AMP responses.

Block¹,

Kon²,

Breckenridge³

1984

Mol. Cell. Biol.

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PC12 cells, derived from a rat pheochromocytoma, were mutagenized and selected in media containing agents known to elevate intracellular concentrations of cyclic AMP (cAMP). More than 40 clones were isolated by selection with cholera toxin or 2-chloroadenosine or both. The variants that were deficient in accumulating cAMP were obtained by using a protocol in which 1 ,IM 8-bromo-cAMP was included in addition to the agonist. Certain of these variants were partially characterized with respect to the site of altered cAMP metabolism. The profiles of adenylate cyclase activity responsiveness of certain variants to guanosine-5'-(0,-y- The use of variants of cloned cells clearly has advanced the understanding of the regulation of cyclic AMP (cAMP) concentration and illuminated the means by which it exerts its effects (13, 23). Phenomena involving cAMP in a particular cell type may not occur generally, however, as exemplified by the diverse relationships of cAMP concentration to proliferation of cultured cells (4).The PC12 cell line, derived from a rat pheochromocytoma, has been studied extensively as a model for growth and differentiation of cells derived from the neural crest. In response to nerve growth factor, the tumor cells undergo striking morphological and functional changes and develop into cells resembling neurons (34). The cells synthesize cAMP in response to the activation of an adenosine receptor coupled to adenylate cyclase (17), but the effect of nerve growth factor on cAMP content is inconsistent and of small magnitude (34). The cyclic nucleotide has been proposed to serve as a permissive or synergistic factor rather than to function as an obligatory intermediate (16), but other evidence supports dissociation of effects of cAMP and of nerve growth factor (14,20,30).Variants of PC12 cells deficient in cAMP accumulation or refractory to its actions should be useful in defining more precisely the role of cAMP in these processes, and additionally they might provide clues linking the cyclic nucleotide to other functions of cells derived from the neural crest. We have applied the rationale initially used in studies of the S49 lymphoma system to the PC12 line, and we have employed procedures closely similar to those of Bothwell et al. (3), who obtained PC12 variants with altered response to nerve growth factor. High intracellular concentrations of cAMP can be obtained in these cells by treating with 2-chloroadenosine, which acts on adenosine receptors in the plasma membrane, or by treating with cholera toxin, which irreversibly activates cyclase. In either case, the treated cells stop dividing, extend processes, and exhibit cytotoxicity with time. In this paper we describe the procedures that have been used to obtain more than 40 different clones and the * Corresponding author. cAMP phenotypes, which appear separable into at least two general categories with respect to forskolin stimulation. Revertants to a wild-type cyclic nucleotide phenotype have been isolated and are also described briefly. Cell culture and ...

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“…1) (22, 44, 46). Most G ␣ mutations that result in receptor coupling defects cluster in G ␣ C termini (19,20,42,43,45,49,55).In the budding yeast Saccharomyces cerevisiae, a G proteinreceptor system is involved in the response of haploid cells to mating pheromones (reviewed in reference 30). The two haploid cell types, MATa and MAT␣, secrete the peptide pheromones a-factor and ␣-factor, respectively, which bind to mating type-specific receptors; the a-factor receptor Ste3p is expressed by MAT␣ cells, and the ␣-factor receptor Ste2p is expressed by MATa cells.…”

mentioning

confidence: 99%

Analysis of the Receptor Binding Domain of Gpa1p, the G_α Subunit Involved in the Yeast Pheromone Response Pathway

Kallal

Kurjan

1997

Molecular and Cellular Biology

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The Saccharomyces cerevisiae G protein ␣ subunit Gpa1p is involved in the response of both MATa and MAT␣ cells to pheromone. We mutagenized the GPA1 C terminus to characterize the receptor-interacting domain and to investigate the specificity of the interactions with the a-and ␣-factor receptors. The results are discussed with respect to a structural model of the Gpa1p C terminus that was based on the crystal structure of bovine transducin. Some mutants showed phenotypes different than the pheromone response and mating defects expected for mutations that affect receptor interactions, and therefore the mutations may affect other aspects of Gpa1p function. Most of the mutations that resulted in pheromone response and mating defects had similar effects in MATa and MAT␣ cells, suggesting that they affect the interactions with both receptors. Overexpression of the pheromone receptors increased the mating of some of the mutants tested but not the wild-type strain, consistent with defects in mutant Gpa1p-receptor interactions. The regions identified by the matingdefective mutants correlated well with the regions of mammalian G ␣ subunits implicated in receptor interactions. The strongest mating type-specific effects were seen for mutations to proline and a mutation of a glycine residue predicted to form a C-terminal ␤ turn. The analogous ␤ turn in mammalian G ␣ subunits undergoes a conformational change upon receptor interaction. We propose that the conformation of this region of Gpa1p differs during the interactions with the a-and ␣-factor receptors and that these mating type-specific mutations preclude the orientation necessary for interaction with one of the two receptors.G protein-mediated signaling involves heterotrimeric proteins that consist of ␣, ␤, and ␥ subunits. G proteins couple to membrane receptors of the rhodopsin/␤-adrenergic family of seven transmembrane-helix receptors (reviewed in reference 9). In the absence of a stimulus, the G protein ␣ subunit is bound to GDP and is complexed with the ␤␥ dimer. When a G protein-linked receptor is stimulated, the G ␣ subunit exchanges GTP for GDP and dissociates from G ␤␥ . After dissociation, G ␣ and/or G ␤␥ interact with targets that are effectors of cellular response pathways.Biochemical and genetic studies in mammalian systems have indicated that G ␣ C termini are involved in receptor interactions. Pertussis toxin modification of a cysteine residue 4 amino acids from the C terminus of G ␣t (transducin) results in reduced affinity for rhodopsin (58). Antibodies against C-terminal peptides of G ␣t and G ␣i2 block the interactions with rhodopsin and the ␣2-adrenergic receptor, respectively (6, 23, 51), and peptides corresponding to the C termini of G ␣t and G ␣s compete for binding to rhodopsin and the ␤-adrenergic receptor, respectively ( Fig. 1) (22, 44, 46). Most G ␣ mutations that result in receptor coupling defects cluster in G ␣ C termini (19,20,42,43,45,49,55).In the budding yeast Saccharomyces cerevisiae, a G proteinreceptor system is involved in the resp...

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Adenylate cyclase permanently uncoupled from hormone receptors in a novel variant of S49 mouse lymphoma cells.

Cited by 160 publications

References 17 publications

The carboxyl terminus of Scg1, the G alpha subunit involved in yeast mating, is implicated in interactions with the pheromone receptors.

The carboxyl terminus of Scg1, the G alpha subunit involved in yeast mating, is implicated in interactions with the pheromone receptors.

Mutants of PC12 cells with altered cyclic AMP responses.

Analysis of the Receptor Binding Domain of Gpa1p, the G_α Subunit Involved in the Yeast Pheromone Response Pathway

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Adenylate cyclase permanently uncoupled from hormone receptors in a novel variant of S49 mouse lymphoma cells.

Cited by 160 publications

References 17 publications

The carboxyl terminus of Scg1, the G alpha subunit involved in yeast mating, is implicated in interactions with the pheromone receptors.

The carboxyl terminus of Scg1, the G alpha subunit involved in yeast mating, is implicated in interactions with the pheromone receptors.

Mutants of PC12 cells with altered cyclic AMP responses.

Analysis of the Receptor Binding Domain of Gpa1p, the Gα Subunit Involved in the Yeast Pheromone Response Pathway

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Analysis of the Receptor Binding Domain of Gpa1p, the G_α Subunit Involved in the Yeast Pheromone Response Pathway