The subunits of the stimulatory regulatory component of adenylate cyclase. Resolution of the activated 45,000-dalton (alpha) subunit.

GTP-binding proteins which participate in signal transduction share a common heterotrimeric structure of the alpha beta gamma-type. In the activated state, the alpha subunit dissociates from the beta gamma complex but remains anchored in the membrane. The alpha subunits of several GTP-binding proteins, such as Go and Gi, are myristoylated at the amino terminus (Buss, J. E., S. M. Mumby, P. J. Casey, A. G. Gilman, and B. M. Sefton. 1987. Proc. Natl. Acad. Sci. USA. 84:7493-7497). This hydrophobic modification is crucial for their membrane attachment. The absence of fatty acid on the alpha subunit of Gs (Gs alpha), the protein involved in adenylate cyclase activation, suggests a different mode of anchorage. To characterize the anchoring domain of Gs alpha, we used a reconstitution model in which posttranslational addition of in vitro-translated Gs alpha to cyc- membranes (obtained from a mutant of S49 cell line which does not express Gs alpha) restores the coupling between the beta-adrenergic receptor and adenylate cyclase. The consequence of deletions generated by proteolytic removal of amino acid sequences or introduced by genetic removal of coding sequences was determined by analyzing membrane association of the proteolyzed or mutated alpha chains. Proteolytic removal of a 9-kD amino-terminal domain or genetic deletion of 28 amino-terminal amino acids did not modify the anchorage of Gs alpha whereas proteolytic removal of a 1-kD carboxyterminal domain abolished membrane interaction. Thus, in contrast to the myristoylated alpha subunits which are tethered through their amino terminus, the carboxy-terminal residues of Gs alpha are required for association of this protein with the membrane.

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Dual regulation of PTH-stimulated adenylate cyclase activity by GTP

AJP Renal Physiology ◽

10.1152/ajprenal.1986.251.5.f858 ◽

1986 ◽

Vol 251 (5) ◽

pp. F858-F864 ◽

Cited By ~ 1

Author(s):

A. P. Teitelbaum ◽

R. A. Nissenson ◽

L. A. Zitzner ◽

K. Simon

Keyword(s):

Adenylate Cyclase ◽

Pertussis Toxin ◽

Regulatory Protein ◽

Inhibitory Effect ◽

Alpha Subunit ◽

Kidney Cells ◽

Intact Cells ◽

Opossum Kidney ◽

Opossum Kidney Cells ◽

Nucleotide Regulation

Guanyl nucleotide regulation of parathyroid hormone (PTH)-activated adenylate cyclase was studied in membrane preparations of cultured opossum kidney cells. Guanosine triphosphate (GTP) (100 microM) decreased PTH-stimulated activity by 70%. Pertussis toxin enhanced PTH stimulation in intact cells and membranes, completely blocked the inhibitory effect of GTP, and catalyzed the [32P]ADP-ribosylation of a 38,000-dalton protein migrating in the position of the alpha-subunit of the inhibitory GTP-regulatory protein Ni. Cholera toxin was used to identify the alpha-subunit of the stimulatory GTP-binding protein Ns, a 42,000-dalton protein. We tested the idea that Ni may be involved in mediating the reduced response of opossum kidney cells to PTH after pretreatment with the hormone (desensitization). GTP inhibited PTH-stimulated activity to approximately the same degree in membranes from PTH-pretreated cells and control cells whether or not the cells had also received pertussis toxin. We conclude that GTP inhibits PTH action in opossum kidney cells through Ni but that PTH-induced desensitization is not mediated by Ni.

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Presence of somatostatin receptors negatively coupled to adenylate cyclase in ectopic growth hormone-releasing hormone- and alpha-subunit- secreting tumors from acromegalic patients responsive to octreotide

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.79.5.1457 ◽

1994 ◽

Vol 79 (5) ◽

pp. 1457-1464 ◽

Cited By ~ 6

Author(s):

J. Bertherat

Keyword(s):

Growth Hormone ◽

Adenylate Cyclase ◽

Somatostatin Receptors ◽

Alpha Subunit ◽

Growth Hormone Releasing Hormone ◽

Releasing Hormone

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G-proteins of fat-cells Role in hormonal regulation of intracellular inositol 1,4,5-trisphosphate

Biochemical Journal ◽

10.1042/bj2400035 ◽

1986 ◽

Vol 240 (1) ◽

pp. 35-40 ◽

Cited By ~ 39

Author(s):

P J Rapiejko ◽

J K Northup ◽

T Evans ◽

J E Brown ◽

C C Malbon

Keyword(s):

Adenylate Cyclase ◽

G Proteins ◽

Pertussis Toxin ◽

Hormonal Regulation ◽

Alpha Subunit ◽

Fat Cells ◽

Fat Cell ◽

Alpha Subunits ◽

Inositol 1,4,5 Trisphosphate ◽

Rat Fat Cells

Pertussis toxin abolishes hormonal inhibition of adenylate cyclase, hormonal stimulation of inositol 1,4,5-trisphosphate accumulation in rat fat-cells, and catalyses the ADP-ribosylation of two peptides, of Mr 39,000 and 41,000 [Malbon, Rapiejko & Mangano (1985) J. Biol. Chem. 260, 2558-2564]. The 41,000-Mr peptide is the alpha-subunit of the G-protein, referred to as Gi, that is believed to mediate inhibitory control of adenylate cyclase by hormones. The nature of the 39,000-Mr substrate for pertussis toxin was investigated. The fat-cell 39,000-Mr peptide was compared structurally and immunologically with the alpha-subunits of two other G-proteins, Gt isolated from the rod outer segments of bovine retina and Go isolated from bovine brain. After radiolabelling in the presence of pertussis toxin and [32P]NAD+, the electrophoretic mobilities of the fat-cell 39,000-Mr peptide and the alpha-subunits of Go and Gt were nearly identical. Partial proteolysis of these ADP-ribosylated proteins generates peptide patterns that suggest the existence of a high degree of homology between the fat-cell 39,000-Mr peptide and the alpha-subunit of Go. Antisera raised against purified G-proteins and their subunits were used to probe immunoblots of purified Gt, Gi, Go, and fat-cell membrane proteins. Although recognizing the 36,000-Mr beta-subunit band of Gt, Gi, Go and a 36,000-Mr fat-cell peptide, antisera raised against Gt failed to recognize either the 39,000- or the 41,000-Mr peptides of fat-cells or the alpha-subunits of Go and Gi. Antisera raised against the alpha-subunit of Go, in contrast, recognized the 39,000-Mr peptide of rat fat-cells, but not the alpha-subunit of either Gi or Gt. These data establish the identity of Go, in addition to Gi, in fat-cell membranes and suggest the possibility that either Go or Gi alone, or both, may mediate hormonal regulation of adenylate cyclase and phospholipase C.

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The effect of iloprost on the ADP-ribosylation of Gsα (the α-subunit of Gs)

Biochemical Journal ◽

10.1042/bj2610841 ◽

1989 ◽

Vol 261 (3) ◽

pp. 841-845 ◽

Cited By ~ 29

Author(s):

L Molina y Vedia ◽

R D Nolan ◽

E G Lapetina

Keyword(s):

Adenylate Cyclase ◽

Cholera Toxin ◽

Alpha Subunit ◽

Α Subunit ◽

Present Evidence ◽

Adp Ribosylation ◽

Specific Antisera ◽

Membrane Bound ◽

Gs Alpha ◽

Adp Ribosyltransferase

Treatment of platelets with a prostacyclin analogue, iloprost, decreased the cholera-toxin-induced ADP-ribosylation of membrane-bound Gs alpha (alpha-subunit of G-protein that stimulates adenylate cyclase; 42 kDa protein) and a cytosolic substrate (44 kDa protein) [Molina y Vedia, Reep & Lapetina (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5899-5902]. This decrease is apparently not correlated with a significant change in the quantity of membrane Gs alpha, as detected by two Gs alpha-specific antisera. This finding contrasts with the suggestion in a previous report [Edwards, MacDermot & Wilkins (1987) Br. J. Pharmacol. 90, 501-510], indicating that iloprost caused a loss of Gs alpha from the membrane. Our evidence points to a modification in the ability of the 42 kDa protein to be ADP-ribosylated by cholera toxin. This modification of Gs alpha might be related to its ADP-ribosylation by endogenous ADP-ribosyltransferase activity. Here we present evidence showing that Gs alpha was ADP-ribosylated in platelets that had been electropermeabilized and incubated with [alpha-32P]NAD+. This endogenous ADP-ribosylation of Gs alpha is inhibited by nicotinamide and stimulated by iloprost.

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Adenylate Cyclase Stimulatory Protein, Alpha Subunit

Encyclopedia of Signaling Molecules ◽

10.1007/978-3-319-67199-4_100134 ◽

2018 ◽

pp. 186-186

Keyword(s):

Adenylate Cyclase ◽

Alpha Subunit ◽

Protein Alpha Subunit

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Absence of pituitary adenylate cyclase-activating polypeptide-stimulated transcription of the human glycoprotein alpha-subunit gene in LbetaT2 gonadotrophs reveals disrupted cAMP-mediated gene transcription

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0310263 ◽

2003 ◽

Vol 31 (2) ◽

pp. 263-278 ◽

Cited By ~ 17

Author(s):

RC Fowkes ◽

KK Sidhu ◽

JK Sosabowski ◽

P King ◽

JM Burrin

Keyword(s):

Adenylate Cyclase ◽

Adenylyl Cyclase ◽

Radioligand Binding ◽

Alpha Subunit ◽

Regulatory Subunit ◽

Pituitary Cells ◽

Hormone Regulation ◽

Glycoprotein Hormone ◽

Altered Expression ◽

Pituitary Adenylate Cyclase

Hormone regulation of anterior pituitary expression of the common glycoprotein hormone alpha-subunit (alphaGSU) is mediated by multiple response elements residing in the first -435 bp of the human promoter. In rat pituitary cells and mouse alphaT3-1 precursor gonadotrophs, the human alphaGSU promoter is strongly responsive to activators of the adenylyl cyclase/cAMP pathway, such as the hypothalamic releasing hormone, pituitary adenylate cyclase-activating polypeptide (PACAP) and forskolin (an adenylyl cyclase activator). However, the role of PACAP and cAMP in regulating alphaGSU transcription in the more differentiated LbetaT2 gonadotroph is unclear. Here, we investigate the regulation of the human alphaGSU promoter by PACAP and forskolin in LbetaT2 and alphaT3-1 gonadotrophs. PACAP failed to stimulate alphaGSU promoter activity or cAMP production in LbetaT2 cells, in marked contrast to alphaT3-1 cells. LbetaT2 gonadotrophs expressed extremely low levels of any PACAP type 1 receptors (PAC(1)-R) isoform by RT-PCR and lacked PAC(1)-R by radioligand binding. Forskolin stimulated the alphaGSU promoter in LbetaT2 cells, but by less than 30% of the response seen in alphaT3-1 gonadotrophs. This blunted cAMP transcriptional effect was not due to different levels of cAMP generation, or altered expression of the cAMP target proteins CREB, Akt, CBP or ICER. However, only LbetaT2 cells showed detectable expression of the protein kinase A type IIalpha regulatory subunit. Binding of activating transcription factor-2 and phosphorylated CREB to the consensus CRE was observed in both LbetaT2 and alphaT3-1 gonadotrophs, yet forskolin failed to stimulate either CRE- or CREB-mediated transcription in LbetaT2 cells. Collectively, these data demonstrate the lack of functional PACAP receptors in LbetaT2 gonadotrophs, and a pronounced attenuation in the responsiveness of this differentiated gonadotroph cell line to cAMP stimulus.

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Glucose repression of fbp1 transcription of Schizosaccharomyces pombe is partially regulated by adenylate cyclase activation by a G protein alpha subunit encoded by gpa2 (git8).

Genetics ◽

10.1093/genetics/138.1.39 ◽

1994 ◽

Vol 138 (1) ◽

pp. 39-45 ◽

Cited By ~ 1

Author(s):

M Nocero ◽

T Isshiki ◽

M Yamamoto ◽

C S Hoffman

Keyword(s):

Adenylate Cyclase ◽

Enzymatic Activity ◽

Schizosaccharomyces Pombe ◽

G Protein ◽

Glucose Repression ◽

Nucleotide Binding ◽

Alpha Subunit ◽

Guanine Nucleotide ◽

Adenylate Cyclase Activation ◽

Guanine Nucleotide Binding

Abstract In the fission yeast Schizosaccharomyces pombe, genetic studies have identified genes that are required for glucose repression of fbp1 transcription. The git2 gene, also known as cyr1, encodes adenylate cyclase. Adenylate cyclase converts ATP into the second messenger cAMP as part of many eukaryotic signal transduction pathways. The git1, git3, git5, git7, git8 and git10 genes act upstream of adenylate cyclase, presumably encoding an adenylate cyclase activation pathway. In mammalian cells, adenylate cyclase enzymatic activity is regulated by heterotrimeric guanine nucleotide-binding proteins (G proteins). In the budding yeast Saccharomyces cerevisiae, adenylate cyclase enzymatic activity is regulated by monomeric, guanine nucleotide-binding Ras proteins. We show here that git8 is identical to the gpa2 gene that encodes a protein homologous to the alpha subunit of a G protein. Mutations in two additional genes, git3 and git5 are suppressed by gpa2+ in high copy number. Furthermore, a mutation in either git3 or git5 has an additive effect in strains deleted for gpa2 (git8), as it significantly increases expression of an fbp1-lacZ reporter gene. Therefore, git3 and git5 appear to act either in concert with or independently from gpa2 (git8) to regulate adenylate cyclase activity.

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