Dominant negative mutations in the α-factor receptor, a G protein-coupled receptor encoded by the STE2 gene of the yeast Saccharomyces cerevisiae

1999 ◽  
Vol 261 (6) ◽  
pp. 917-932 ◽  
Author(s):  
L. M. Leavitt ◽  
C. R. Macaluso ◽  
K. S. Kim ◽  
N. P. Martin ◽  
M. E. Dumont
Keyword(s):  
Saccharomyces Cerevisiae ◽  
G Protein ◽  
Dominant Negative ◽  
G Protein Coupled Receptor ◽  
Yeast Saccharomyces Cerevisiae ◽  
Dominant Negative Mutations ◽  
G Protein Coupled

scholarly journals Affinity purification and characterization of a G-protein coupled receptor, Saccharomyces cerevisiae Ste2p

2007 ◽  
Vol 56 (1) ◽  
pp. 62-71 ◽  
Author(s):  
Byung-Kwon Lee ◽  
Kyung-Sik Jung ◽  
Cagdas Son ◽  
Heejung Kim ◽  
Nathan C. VerBerkmoes ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
G Protein ◽  
Affinity Purification ◽  
G Protein Coupled Receptor ◽  
Purification And Characterization ◽  
G Protein Coupled

scholarly journals Functional and Physical Interactions among Saccharomyces cerevisiae α-Factor Receptors

2012 ◽  
Vol 11 (10) ◽  
pp. 1276-1288 ◽  
Author(s):  
Austin U. Gehret ◽  
Sara M. Connelly ◽  
Mark E. Dumont
Keyword(s):  
Saccharomyces Cerevisiae ◽  
G Protein ◽  
Dominant Negative ◽  
Negative Effects ◽  
Content Type ◽  
Functional Interactions ◽  
Wide Range ◽  
Physical Interactions ◽  
Mutant Receptors ◽  
G Protein Coupled

ABSTRACT The α-factor receptor Ste2p is a G protein-coupled receptor (GPCR) expressed on the surface of MAT a haploid cells of the yeast Saccharomyces cerevisiae . Binding of α-factor to Ste2p results in activation of a heterotrimeric G protein and of the pheromone response pathway. Functional interactions between α-factor receptors, such as dominant-negative effects and recessive behavior of constitutive and hypersensitive mutant receptors, have been reported previously. We show here that dominant-negative effects of mutant receptors persist over a wide range of ratios of the abundances of G protein to receptor and that such effects are not blocked by covalent fusion of G protein α subunits to normal receptors. In addition, we detected dominant effects of mutant C-terminally truncated receptors, which had not been previously reported to act in a dominant manner. Furthermore, coexpression of C-terminally truncated receptors with constitutively active mutant receptors results in enhancement of constitutive signaling. Together with previous evidence for oligomerization of Ste2p receptors, these results are consistent with the idea that functional interactions between coexpressed receptors arise from physical interactions between them rather than from competition for limiting downstream components, such as G proteins.

Carbon source induced yeast-to-hypha transition in Candida albicans is dependent on the presence of amino acids and on the G-protein-coupled receptor Gpr1

2005 ◽  
Vol 33 (1) ◽  
pp. 291-293 ◽  
Author(s):  
M.M. Maidan ◽  
J.M. Thevelein ◽  
P. Van Dijck
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Candida Albicans ◽  
Carbon Source ◽  
G Protein ◽  
High Glucose ◽  
Glucose Concentration ◽  
G Protein Coupled Receptor ◽  
Hypha Formation ◽  
G Protein Coupled

Yeast-to-hypha transition in Candida albicans can be induced by a wide variety of factors, including specific nutrients. We have started to investigate the mechanism by which some of these nutrients may be sensed. The G-protein-coupled receptor Gpr1 is required for yeast-to-hypha transition on various solid hypha-inducing media. Recently we have shown induction of Gpr1 internalization by specific amino acids, e.g. methionine. This suggests a possible role for methionine as a ligand of CaGpr1. Here we show that there is a big variation in methionine-induced hypha formation depending on the type of carbon source present in the medium. In addition high glucose concentrations repress hypha formation whereas a concentration of 0.1%, which mimics the glucose concentration present in the bloodstream, results in maximal hypha formation. Hence, it remains unclear whether Gpr1 senses sugars, as in Saccharomyces cerevisiae, or specific amino acids like methionine.

scholarly journals Role of G Protein-Coupled Receptor Kinases on the Agonist-Induced Phosphorylation and Internalization of the Follitropin Receptor

1999 ◽  
Vol 13 (6) ◽  
pp. 866-878 ◽  
Author(s):  
Maria de Fatima M. Lazari ◽  
Xuebo Liu ◽  
Kazuto Nakamura ◽  
Jeffrey L. Benovic ◽  
Mario Ascoli
Keyword(s):  
Cell Line ◽  
G Protein ◽  
Dominant Negative ◽  
Receptor Internalization ◽  
Dominant Negative Mutant ◽  
Deficient Mutant ◽  
G Protein Coupled Receptor ◽  
Western Blots ◽  
293 Cells ◽  
G Protein Coupled

Abstract The experiments presented herein were designed to identify members of the G protein-coupled receptor kinase (GRK) family that participate in the agonist-induced phosphorylation and internalization of the rat FSH receptor (rFSHR). Western blots of human kidney 293 cells (the cell line used in transfection experiments) and MSC-1 cells (a cell line derived from Sertoli cells that displays many of the differentiated functions of their normal counterparts) reveal the presence of GRK2 and GRK6 in both cell lines as well as GRK4 in MSC-1 cells. Cotransfection of 293 cells with the rFSHR and GRK2, GRK4α, or GRK6 resulted in an increase in the agonist-induced phosphorylation of the rFSHR. Cotransfections of the rFSHR with GRKs or arrestin-3 enhanced the agonist-induced internalization of the rFHSR, and combinations of GRKs and arrestin-3 were more effective than the individual components. To characterize the involvement of endogenous GRKs on phosphorylation and internalization, we inhibited endogenous GRK2 by overexpression of a kinase-deficient mutant of GRK2 or Gαt, a scavenger of Gβγ. We also inhibited endogenous GRK6 by overexpression of a kinase-deficient mutant of GKR6. All three constructs were effective inhibitors of phosphorylation, but only the kinase-deficient mutant of GRK2 and Gαt inhibited internalization. The inhibition of internalization induced by these two constructs was less pronounced than that induced by a dominant-negative mutant of the nonvisual arrrestins, however. The finding that inhibitors of GRK2 and GRK6 impair phosphorylation, but only the inhibitors of GRK2 impair internalization, suggests that different GRKs have differential effects on receptor internalization.

scholarly journals Fractalkine stimulates angiogenesis by activating the Raf-1/MEK/ERK- and PI3K/Akt/eNOS-dependent signal pathways

2006 ◽  
Vol 291 (6) ◽  
pp. H2836-H2846 ◽  
Author(s):  
Seon-Jin Lee ◽  
Seung Namkoong ◽  
Young-Mi Kim ◽  
Chun-Ki Kim ◽  
Hansoo Lee ◽  
...  
Keyword(s):  
G Protein ◽  
Umbilical Vein ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Signal Pathways ◽  
No Production ◽  
G Protein Coupled Receptor ◽  
Angiogenic Activity ◽  
Enos Phosphorylation ◽  
G Protein Coupled

Fractalkine (FKN) has been implicated in modulation of angiogenesis and vascular inflammation, but the underlying mechanism has not been elucidated. We have investigated the molecular mechanism by which FKN regulates angiogenesis. We found that recombinant FKN increases in vitro proliferation, migration, and tube formation of human umbilical vein endothelial cells and stimulates in vivo angiogenesis. FKN-induced angiogenesis was accompanied by phosphorylation of ERK, Akt, and endothelial nitric oxide (NO) synthase (eNOS), as well as an increase in NO production. These biochemical events and angiogenesis were completely inhibited by the G protein-coupled receptor inhibitor pertussis toxin. Inhibitors of Raf-1, MEK, phosphatidylinositol 3-kinase (PI3K), and eNOS or transfection with dominant-negative forms of ERK and Akt significantly suppressed the angiogenic activity of FKN. However, inhibitors of Raf-1 and MEK or a dominant-negative ERK mutant blocked FKN-induced ERK, but not Akt and eNOS, phosphorylation. The PI3K inhibitor and a dominant-negative mutant of Akt suppressed Akt and eNOS phosphorylation and NO production. Our results demonstrated that FKN stimulated angiogenesis by activating the Raf-1/MEK/ERK and PI3K/Akt/eNOS/NO signal pathways via the G protein-coupled receptor CX3CR1, indicating that two pathways are required for full angiogenic activity of FKN. This study suggests that FKN may play an important role in the pathophysiological process of inflammatory angiogenesis.

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