scholarly journals Glucose Monitoring in Fission Yeast via the gpa2 Gα, the git5 Gβ and the git3 Putative Glucose Receptor

Genetics ◽  
2000 ◽  
Vol 156 (2) ◽  
pp. 513-521
Author(s):  
Robert M Welton ◽  
Charles S Hoffman
Keyword(s):  
Adenylate Cyclase ◽  
Fission Yeast ◽  
G Protein ◽  
Glucose Repression ◽  
Glucose Monitoring ◽  
Coding Region ◽  
Guanine Nucleotide Binding Protein ◽  
Cyclase Activation ◽  
Adenylate Cyclase Activation ◽  
G Protein Coupled

Abstract The fission yeast Schizosaccharomyces pombe responds to environmental glucose by activating adenylate cyclase. The resulting cAMP signal activates protein kinase A (PKA). PKA inhibits glucose starvation-induced processes, such as conjugation and meiosis, and the transcription of the fbp1 gene that encodes the gluconeogenic enzyme fructose-1,6-bisphosphatase. We previously identified a collection of git genes required for glucose repression of fbp1 transcription, including pka1/git6, encoding the PKA catalytic subunit, git2/cyr1, encoding adenylate cyclase, and six “upstream” genes required for adenylate cyclase activation. The git8 gene, identical to gpa2, encodes the alpha subunit of a heterotrimeric guanine-nucleotide binding protein (Gα) while git5 encodes a Gβ subunit. Multicopy suppression studies with gpa2+ previously indicated that S. pombe adenylate cyclase activation may resemble that of the mammalian type II enzyme with sequential activation by Gα followed by βγ. We show here that an activated allele of gpa2 (gpa2R176H, carrying a mutation in the coding region for the GTPase domain) fully suppresses mutations in git3 and git5, leading to a refinement in our model. We describe the cloning of git3 and show that it encodes a putative seven-transmembrane G protein-coupled receptor. A git3 deletion confers the same phenotypes as deletions of other components of the PKA pathway, including a germination delay, constitutive fbp1 transcription, and starvation-independent conjugation. Since the git3 deletion is fully suppressed by the gpa2R176H allele with respect to fbp1 transcription, git3 appears to encode a G protein-coupled glucose receptor responsible for adenylate cyclase activation in S. pombe.

scholarly journals The Fission Yeast git5 Gene Encodes a Gβ Subunit Required for Glucose-Triggered Adenylate Cyclase Activation

Genetics ◽  
2000 ◽  
Vol 154 (4) ◽  
pp. 1463-1471 ◽  
Author(s):  
Sheila Landry ◽  
Maria T Pettit ◽  
Ethel Apolinario ◽  
Charles S Hoffman
Keyword(s):  
Adenylate Cyclase ◽  
Fission Yeast ◽  
Coiled Coil ◽  
Negative Regulator ◽  
Guanine Nucleotide Binding Protein ◽  
Amino Terminal ◽  
Cyclase Activation ◽  
Adenylate Cyclase Activation ◽  
Guanine Nucleotide Binding ◽  
Camp Levels

Abstract Fission yeast adenylate cyclase is activated by the gpa2 Gα subunit of a heterotrimeric guanine-nucleotide binding protein (G protein). We show that the git5 gene, also required for this activation, encodes a Gβ subunit. In contrast to another study, we show that git5 is not a negative regulator of the gpa1 Gα involved in the pheromone response pathway. While 43% identical to mammalian Gβ's, the git5 protein lacks the amino-terminal coiled-coil found in other Gβ subunits, yet the gene possesses some of the coding capacity for this structure 5′ to its ORF. Although both gpa2 (Gα) and git5 (Gβ) are required for adenylate cyclase activation, only gpa2 is needed to maintain basal cAMP levels. Strains bearing a git5 disruption are derepressed for fbp1 transcription and sexual development even while growing in a glucose-rich environment, although fbp1 derepression is half that observed in gpa2 deletion strains. Multicopy gpa2 partially suppresses the loss of git5, while the converse is not true. These data suggest that Gβ is required for activation of adenylate cyclase either by promoting the activation of Gα or by independently activating adenylate cyclase subsequent to Gα stimulation as seen in type II mammalian adenylate cyclase activation.

Six git genes encode a glucose-induced adenylate cyclase activation pathway in the fission yeast Schizosaccharomyces pombe

1993 ◽  
Vol 105 (4) ◽  
pp. 1095-1100 ◽  
Author(s):  
S.M. Byrne ◽  
C.S. Hoffman
Keyword(s):  
Adenylate Cyclase ◽  
Schizosaccharomyces Pombe ◽  
Glucose Repression ◽  
Signal Transduction Pathway ◽  
Activation Pathway ◽  
Cyclase Activation ◽  
Adenylate Cyclase Activation ◽  
Before And After ◽  
Dependent Protein ◽  
Camp Levels

An important eukaryotic signal transduction pathway involves the regulation of the effector enzyme adenylate cyclase, which produces the second messenger, cAMP. Previous genetic analyses demonstrated that glucose repression of transcription of the Schizosaccharomyces pombe fbp1 gene requires the function of adenylate cyclase, encoded by the git2 gene. As mutations in git2 and in six additional git genes are suppressed by exogenous cAMP, these ‘upstream’ git genes were proposed to act to produce a glucose-induced cAMP signal. We report here that assays of cAMP levels in wild-type and various mutant S. pombe cells, before and after exposure to glucose, show that this is the case. The data suggest that the cAMP signal results from the activation of adenylate cyclase. Therefore these ‘upstream’ git genes appear to encode a glucose-induced adenylate cyclase activation pathway. Assays of cAMP on a strain carrying a mutation in the git6 gene, which acts downstream of adenylate cyclase, indicate that git6 may function to feedback regulate adenylate cyclase activity. Thus git6 may encode a cAMP-dependent protein kinase.

scholarly journals Effect of inactivating mutations on phosphorylation and internalization of the human VPAC2 receptor

2005 ◽  
Vol 34 (2) ◽  
pp. 405-414 ◽  
Author(s):  
Ingrid Langer ◽  
Christelle Langlet ◽  
Patrick Robberecht
Keyword(s):  
Adenylate Cyclase ◽  
Chinese Hamster ◽  
Amino Acid Sequences ◽  
Carboxyl Terminus ◽  
Amino Terminus ◽  
Receptor Phosphorylation ◽  
Conserved Motifs ◽  
Cyclase Activation ◽  
Adenylate Cyclase Activation ◽  
G Protein Coupled

The VPAC2 receptor, as all members of the G-protein-coupled receptor (GPCR)-B family, has two highly conserved motifs in the third intracellular (IC3) loop: a lysine and a leucine located at the amino-terminus and two basic residues separated by a leucine and an alanine at the carboxyl-terminus. This study evaluates the involvement of those conserved amino acid sequences in VPAC2 signal transduction and regulation. The residues were mutated into alanine and mutants were expressed in Chinese hamster ovary (CHO) cells stably transfected with Gα16 and aequorin. Mutation of L310 reduced efficacy of vasoactive intestinal polypeptide (VIP) to stimulate adenylate cyclase activity through Gαs coupling by 75%, without affecting VIP capability to stimulate an increase in [Ca2+]i through Gα16 coupling. Mutation of R325 and, to a lesser extend, K328 reduced VIP efficacy to stimulate [Ca2+]i increase and VIP potency to stimulate adenylate cyclase. The combination of mutations of both amino- and carboxyl-terminus located conserved motifs of the IC3 loop generates an inactive receptor with respect to [Ca2+]i increase and adenylate cyclase activation, but also with respect to receptor phosphorylation and internalization that were indeed directly correlated with the potency of inactivation of the receptors. The amino-terminus of the VPAC2 receptor IC3 loop is thus involved in adenylate cyclase activation and the carboxyl-terminus of the IC3 loop participates in both Gαs and Gα16 coupling. The mutations studied also reduced both receptor phosphorylation and internalization in a manner that appeared directly linked to the alteration of Gαs and Gα16 coupling.

Mechanisms involved in alpha-adrenergic phenomena

1985 ◽  
Vol 248 (6) ◽  
pp. E633-E647 ◽  
Author(s):  
J. H. Exton
Keyword(s):  
Protein Kinase ◽  
Adenylate Cyclase ◽  
Adrenergic Receptors ◽  
Regulatory Protein ◽  
Smooth Muscles ◽  
Target Cells ◽  
Guanine Nucleotide Binding Protein ◽  
Cyclase Activation ◽  
Adenylate Cyclase Activation ◽  
Dependent Protein

Epinephrine and norepinephrine exert many important actions by interacting with alpha 1- and alpha 2-adrenergic receptors in their target cells. Activation of alpha 2-adrenergic receptors causes platelet aggregation and other inhibitory cellular responses. Some of these responses are attributable to a decrease in cAMP due to inhibition of adenylate cyclase. Activation of alpha 2-adrenergic receptors promotes their coupling to an inhibitory guanine nucleotide binding protein (Ni). This coupling promotes the binding of GTP to Ni, causing it to dissociate into subunits. This results in inhibition of the catalytic component of adenylate cyclase. Activation of alpha 1-adrenergic receptors stimulates the contraction of most smooth muscles and alters secretion and metabolism in several tissues. The primary event is a breakdown of phosphatidylinositol-4,5-bisphosphate in the plasma membrane to produce two intracellular "messengers": myo-inositol-1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG). IP3 causes the release of Ca2+ from endoplasmic reticulum, producing a rapid rise in cytosolic Ca2+. Ca2+ binds to the regulatory protein calmodulin, and the resulting complex interacts with specific or multifunctional calmodulin-dependent protein kinases and other calmodulin-responsive proteins, altering their activities and thereby producing a variety of physiological responses. DAG also produces effects by activating a Ca2+-phospholipid-dependent protein kinase (protein kinase C) that phosphorylates and alters the activity of certain cellular proteins. Frequently there is synergism between the IP3 and DAG mechanisms.

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 ◽  
1994 ◽  
Vol 138 (1) ◽  
pp. 39-45 ◽  
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.

scholarly journals Effects of Topical Adenosine Analogs and Forskolin on Rat Pial Arterioles in vivo

1991 ◽  
Vol 11 (1) ◽  
pp. 72-76 ◽  
Author(s):  
Setsuro Ibayashi ◽  
Al C. Ngai ◽  
Joseph R. Meno ◽  
H. Richard Winn
Keyword(s):  
Adenylate Cyclase ◽  
Adenosine Analogs ◽  
Cyclase Activation ◽  
Window Technique ◽  
Adenylate Cyclase Activation ◽  
Pial Arterioles ◽  
Adenosine Agonists ◽  
Cerebral Vasodilation

We utilized the closed window technique to study the in vivo responses of rat pial arterioles to superfused adenosine agonists. Adenosine and its analogs dilated pial arterioles and exhibited the following order of potency: 5′ N-ethylcarboxamide adenosine (NECA) > 2-chloroadenosine (2-CADO) > adenosine = R-N6-phenylisopropyladenosine ( R-PIA) = S-PIA > N6-cyclohexyladenosine (CHA). This potency profile suggests that cerebral vasodilation is mediated through the A2 receptor. Forskolin (10−9 M) potentiated the vasodilation caused by 10−6 M NECA, thus implicating adenylate cyclase activation during NECA-induced vasodilation and providing further support for involvement of the A2 receptor.

CCK receptor phosphorylation exposes regulatory domains affecting phosphorylation and receptor trafficking

2000 ◽  
Vol 279 (6) ◽  
pp. C1986-C1992 ◽  
Author(s):  
Rammohan V. Rao ◽  
Eileen L. Holicky ◽  
Susan M. Kuntz ◽  
Laurence J. Miller
Keyword(s):  
G Protein ◽  
Chinese Hamster ◽  
G Protein Coupled Receptors ◽  
Receptor Internalization ◽  
Ovary Cell ◽  
Guanine Nucleotide Binding Protein ◽  
Receptor Phosphorylation ◽  
Regulatory Domains ◽  
Ovary Cell Line ◽  
G Protein Coupled

Agonist-stimulated phosphorylation of guanine nucleotide-binding protein (G protein)-coupled receptors has been recognized as an important mechanism for desensitization by interfering with coupling of the activated receptor with its G protein. We recently described a mutant of the CCK receptor that modified two of five key sites of phosphorylation (S260,264A) and eliminated agonist-stimulated receptor phosphorylation, despite normal ligand binding and signaling (20). As expected, this nonphosphorylated mutant had impaired rapid desensitization but was ultimately able to be desensitized by normal receptor internalization. Here we demonstrate that this mutant receptor is also defective in resensitization, with abnormal recycling to the cell surface. To explore this, another receptor mutant was prepared, replacing the same serines with aspartates to mimic the charge of serine-phosphate (S260,264D). This mutant was expressed in a Chinese hamster ovary cell line and shown to bind CCK normally. It had accelerated kinetics of signaling and desensitization and was phosphorylated in response to agonist occupation, with all other normal sites of phosphorylation modified. It was internalized like wild-type receptors and was resensitized and trafficked normally. This provides evidence for an additional important function for phosphorylation of G protein-coupled receptors. Phosphorylation may induce a conformational change in the receptor to expose other potential sites of phosphorylation and to expose domains involved in the targeting and trafficking of endosomes. The hierarchical phosphorylation of these sites may play a key role in receptor regulation.

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