Molecular Cloning of Complementary DNA for the Alpha Subunit of the G Protein that Stimulates Adenylate Cyclase

Harris, Bruce A.; Robishaw, Janet D.; Mumby, Susanne M.; Gilman, Alfred G.

doi:10.1126/science.3839937

Cited by 203 publications

(74 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…5), as previously reported [20]. This result is in accordance with data obtained from cerebral cortex [21], neuroblastoma-glioma hybrids [22] and astroglial cells [7].…”

Section: Cellssupporting

confidence: 82%

Cyclic AMP regulation of messenger RNA level of the stimulatory GTP‐binding protein Gsα

Dib¹,

Jamali²,

Correze³

1994

European Journal of Biochemistry

View full text Add to dashboard Cite

The existence of a CAMP-dependent regulation of the expression of the a-subunit of the stimulatory guanine nucleotide-binding protein (Gs) in a well characterized astroglial cells culture was established. The culture of astroglial cells for 3-6 h with isoproterenol (10 pM) or forskolin (10 pM) (a CAMP-inducing agent) increased (200-400%) the response of adenylylcyclase to agents which bypass the receptor; GTP, GTP [S] or forskolin. For prolonged exposure times (15 h or more) to isoproterenol or forskolin, the adenylylcyclase activity decreased to the value observed in control cells. The same biphasic response of adenylylcyclase to isoproterenol (10 pM) plus GTP (10 pM) occurred in membrane fractions from cells cultured with forskolin, whereas a diminished response to isoproterenol was observed in isoproterenol-treated cells, indicating that the P-adrenergic receptor was desensitized. To understand the molecular mechanism of these phenomena, we measured the levels of the a subunits of the guanine-nucleotide binding protein (Gs and Gi) by Western-blot analysis. The culture of astroglial cells with isoproterenol or forskolin (3 -24 h) resulted in a transient increase of both the Gsa and the Gia protein levels, while the level of Gp subunits was unaffected.We also identified Gsa protein (about 40% of the total cellular protein) in the supernatant fraction of astroglial cells but its level was not modified by the stimulation of cells by forskolin.The level of Gsa mRNA measured by Northern-blot analysis was transiently increased (200%) after stimulation of astroglial cells with isoproterenol or forskolin for an incubation period of 6-9 h, then returned to that of control cells for longer period of time. In addition, the Gsa mRNA level was threefold increased when cells were cultured for 2-6 h with 8-bromoadenosine 3':5'-cyclic monophosphate (10 pM), a permeant analogue of CAMP. These results indicate that CAMP induces a time-dependent increase of Gsa mRNA.The half-life of Gsa protein and Gsa mRNA were determined. Pulse-chase studies revealed that the decay of Gsa protein was clearly biphasic with an early phase (5-6 h) and a slower second phase (20-25 h) but the treatment of cells with forskolin did not accelerate or slow down the turnover of Gsa protein.The use of actinomycin D to estimate the stability of Gsa mRNA showed that the half-life of Gsa mRNA was approximately 7 -8 h and was not different in cells treated with 8-bromoadenosine 3' 5'-cyclic monophosphate and in non-treated cells.In contrast, treatment of cells with cycloheximide led to a significant increase of Gsa mRNA but did not abolish the effect of forskolin.These studies suggest that the expression of Gsa mRNA is regulated at the transcriptional level and does not require protein synthesis.In a previous study, we have demonstrated that thyrotropin, via a CAMP-dependent mechanism, regulates the expression of the stirnulatory guanine-nucleotide-binding regulatory component of adenylylcyclase (Gs)

show abstract

“…5), as previously reported [20]. This result is in accordance with data obtained from cerebral cortex [21], neuroblastoma-glioma hybrids [22] and astroglial cells [7].…”

Section: Cellssupporting

confidence: 82%

Cyclic AMP regulation of messenger RNA level of the stimulatory GTP‐binding protein Gsα

Dib¹,

Jamali²,

Correze³

1994

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…This reactivity could be blocked by coincubation with low concentrations of free peptide (not shown). In contrast, no reactivity was seen in membranes from cyc-$49 cells [30], which lack mRNA corresponding to Gs-te [31] ( fig.l, lane 3). These findings demonstrate the specific reactivity of the anti-peptide antibodies with both of the major size classes of as identified by protein purification [32] and cDNA cloning [22][23][24][25][26][27].…”

Section: Resultsmentioning

confidence: 81%

Receptor and effector interactions of G_s Functional studies with antibodies to the α_s carboxyl‐terminal decapeptide

et al. 1989

View full text Add to dashboard Cite

Antibodies generated to a synthetic decapeptide, RMHLRQYELL, representing the carboxyl-terminus of Gs-ct have been characterized in immunoblots and functional studies. This antibody, designated RM, reacts exclusively with a doublet of proteins of 52 and 45 kDa in immunoblots of bovine brain and wild-type $49 murine lymphoma cell membranes. No such reactivity is seen in membranes from cyc-$49 cells, which lack Gs. RM blocks receptor-mediated activation of Gs and adenylyl cyclase in membranes from wild-type $49 cells. RM could also immunoprecipitate adenylyl cyclase activity in detergent extracts from GTP[),]S-or fluoride-preactivated bovine brain membranes; thus binding of cts to effector and carboxyl-terminal antibody was mutually compatible. Such experiments provide an approach for the elucidation of functionally relevant interactions of G-proteins with receptors and effectors in the membrane.

show abstract

“…The absence of a detectable hybridization signal [8] suggests that the gene encoding Gso~ is not transcribed in these cells or that the mRNA is unable to accumulate to levels detectable in hybridization to total RNA. Like the uncoupled cell, G~o~ mRNA does not accumulate in After the DNAs were resolved through a 0.8°70 agarose gel and blotted, they were hybridized to a Gso~ cDNA probe from bovine brain [8]. Molecular mass markers (kilobase pairs) are a HindlIl restriction digest of bacteriophage A DNA.…”

Section: Expression Of G~ Message In $49 Cellsmentioning

confidence: 98%

“…For Northern analysis, 10/zg of total RNA was denatured with glyoxal and resolved through a 1.5% agarose gel prior to transfer to a nitrocellulose membrane [10]. The blot was hybridized to a labeled singlestranded cDNA probe [11] corresponding to an internal EcoRI-BamHI fragment of the bovine brain cDNA encoding G~c~ [8]. Hybridization was performed in the presence of 50% formamide, 5 x SSC, 1 x Denhardt's solution, 20 mM sodium phosphate, 100/~g/ml salmon sperm DNA and 1% SDS.…”

Section: Rna Analysismentioning

confidence: 99%

“…This charge difference may be the result of a single amino acid change or of a covalent post-translational modification. The latter possibility was eliminated [6] by fusing $49 uncoupled cells to $49 cyc-cells which do not produce the cr-subunit of G~ [7,8]. The resulting heterokaryon behaved phenotypically as the parent $49 uncoupled cell.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Identification of the lesion in the stimulatory GTP‐binding protein of the uncoupled S49 lymphoma

Rall¹,

Harris²

1987

FEBS Letters

Self Cite

View full text Add to dashboard Cite

The stimulatory GTP-binding protein (GO of the uncoupled mutant of $49 lymphoma cells is deficient in its ability to transduce hormonal signals from ligand-bound fl-adrenergic receptors to the catalytic component of adenylate cyclase. In order to define the genetic defect in the G~ of uncoupled $49 cells, a complementary DNA clone encoding the ~-subunit of Gs was analyzed and the deduced primary structure of the defective subunit compared to that of the wild-type subunit. A single nucleotide transversion was found that coded for a proline rather than an arginine at residue 389. The results indicate a domain of the ~-subunit of Gs that specifically interacts with hormone receptors.G-protein; Adenylate cyclase; Uncoupled mutant; ($49 lymphoma)

show abstract

Molecular Cloning of Complementary DNA for the Alpha Subunit of the G Protein that Stimulates Adenylate Cyclase

Cited by 203 publications

References 37 publications

Cyclic AMP regulation of messenger RNA level of the stimulatory GTP‐binding protein Gsα

Cyclic AMP regulation of messenger RNA level of the stimulatory GTP‐binding protein Gsα

Receptor and effector interactions of G_s Functional studies with antibodies to the α_s carboxyl‐terminal decapeptide

Identification of the lesion in the stimulatory GTP‐binding protein of the uncoupled S49 lymphoma

Contact Info

Product

Resources

About

Molecular Cloning of Complementary DNA for the Alpha Subunit of the G Protein that Stimulates Adenylate Cyclase

Cited by 203 publications

References 37 publications

Cyclic AMP regulation of messenger RNA level of the stimulatory GTP‐binding protein Gsα

Cyclic AMP regulation of messenger RNA level of the stimulatory GTP‐binding protein Gsα

Receptor and effector interactions of Gs Functional studies with antibodies to the αs carboxyl‐terminal decapeptide

Identification of the lesion in the stimulatory GTP‐binding protein of the uncoupled S49 lymphoma

Contact Info

Product

Resources

About

Receptor and effector interactions of G_s Functional studies with antibodies to the α_s carboxyl‐terminal decapeptide