scholarly journals High level expression of transfected G protein αi3subunit is required for plasma membrane targeting and adenylyl cyclase inhibition in NIH 3T3 fibroblasts

FEBS Letters ◽  
1992 ◽  
Vol 312 (2-3) ◽  
pp. 223-228 ◽  
Author(s):  
Sylvie Hermouet ◽  
Philippe de Mazancourt ◽  
Allen M. Spiegel ◽  
Marilyn Gist Farquhar ◽  
Bridget S. Wilson
Keyword(s):  
Plasma Membrane ◽  
Adenylyl Cyclase ◽  
G Protein ◽  
Membrane Targeting ◽  
High Level Expression ◽  
3T3 Fibroblasts ◽  
High Level ◽  
Nih 3T3

scholarly journals G protein-regulated endocytic trafficking of adenylyl cyclase type 9

2020 ◽  
Author(s):  
André M. Lazar ◽  
Roshanak Irannejad ◽  
Tanya A. Baldwin ◽  
Aparna A. Sundaram ◽  
J. Silvio Gutkind ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Adenylyl Cyclase ◽  
G Protein ◽  
Subcellular Location ◽  
Endocytic Pathway ◽  
Heterotrimeric G Proteins ◽  
Camp Production ◽  
Stimulation Of

SummaryGPCRs are increasingly recognized to initiate signaling via heterotrimeric G proteins as they move through the endocytic network, but little is known about how relevant G protein effectors are localized. Here we report dynamic trafficking of adenylyl cyclase type 9 (AC9) from the plasma membrane to endosomes, while adenylyl cyclase type 1 (AC1) remains in the plasma membrane, and stimulation of AC9 trafficking by ligand-induced activation of Gs-coupled GPCRs or Gs. AC9 transits a similar dynamin-dependent early endocytic pathway as activated GPCRs but, in contrast to GPCR trafficking which is regulated by β-arrestin but not Gs, AC9 trafficking is regulated by Gs but not β-arrestin. We also show that AC9, but not AC1, contributes to cAMP production from endosomes. These results reveal dynamic and isoform-specific trafficking of adenylyl cyclase in the endocytic network, and a discrete role of a heterotrimeric G protein in controlling subcellular location of a relevant effector.

scholarly journals Organization of G Proteins and Adenylyl Cyclase at the Plasma Membrane

1997 ◽  
Vol 8 (12) ◽  
pp. 2365-2378 ◽  
Author(s):  
Chunfa Huang ◽  
John R. Hepler ◽  
Linda T. Chen ◽  
Alfred G. Gilman ◽  
Richard G.W. Anderson ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Adenylyl Cyclase ◽  
G Protein ◽  
Buoyant Density ◽  
Cyclase Activity ◽  
Immunogold Electron Microscopy ◽  
Adenylyl Cyclase Activity ◽  
A Cell ◽  
Hormone Sensitive ◽  
Punctate Pattern

There is mounting evidence for the organization and compartmentation of signaling molecules at the plasma membrane. We find that hormone-sensitive adenylyl cyclase activity is enriched in a subset of regulatory G protein-containing fractions of the plasma membrane. These subfractions resemble, in low buoyant density, structures of the plasma membrane termed caveolae. Immunofluorescence experiments revealed a punctate pattern of G protein α and β subunits, consistent with concentration of these proteins at distinct sites on the plasma membrane. Partial coincidence of localization of G protein α subunits with caveolin (a marker for caveolae) was observed by double immunofluorescence. Results of immunogold electron microscopy suggest that some G protein is associated with invaginated caveolae, but most of the protein resides in irregular structures of the plasma membrane that could not be identified morphologically. Because regulated adenylyl cyclase activity is present in low-density subfractions of plasma membrane from a cell type (S49 lymphoma) that does not express caveolin, this protein is not required for organization of the adenylyl cyclase system. The data suggest that hormone-sensitive adenylyl cyclase systems are localized in a specialized subdomain of the plasma membrane that may optimize the efficiency and fidelity of signal transduction.

scholarly journals Activation of 5-HT1A receptors expressed in NIH-3T3 cells induces focus formation and potentiates EGF effect on DNA synthesis.

1992 ◽  
Vol 3 (9) ◽  
pp. 961-969 ◽  
Author(s):  
A Varrault ◽  
J Bockaert ◽  
C Waeber
Keyword(s):  
Dna Synthesis ◽  
Adenylyl Cyclase ◽  
Pertussis Toxin ◽  
Receptor Activation ◽  
Intracellular Camp ◽  
3T3 Cells ◽  
Focus Formation ◽  
3T3 Fibroblasts ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

NIH-3T3 fibroblasts have been transfected with human serotonin 5-HT1A receptors. Clonal cell lines expressed between 40 and 500 fmol receptor/mg. 5-HT1A agonists strongly inhibited nonstimulated- as well as forskolin- or isoproterenol-stimulated adenylyl cyclase. The effects of 5-HT1A receptor activation on cell growth were investigated. 5-HT1A agonists accelerated cell division, generated foci, and increased DNA synthesis. The stimulation of [3H]thymidine incorporation was much stronger when tyrosine kinase receptors were activated concomitantly. Cyclic AMP (cAMP) elevating agents inhibited DNA synthesis induced by all mitogens tested. The mitogenic activity of 5-HT1A agonists did not seem to be linked to adenylyl cyclase inhibition because 1) we were not able to measure any decrease in intracellular cAMP levels under the conditions of DNA synthesis assay and 2) 2',5'-dideoxyadenosine, which strongly inhibited adenylyl cyclase, was not mitogenic and did not modify the mitogenic effects of 5-HT1A agonists. Pertussis toxin completely blocked potentiation of epidermal growth factor effect induced by 8-hydroxy-di-(n-propyl)aminotetralin, a 5-HT1A agonist, but only partially blocked the one induced by insulin. In conclusion, in transfected NIH-3T3 cells, transforming and mitogenic effects of 5-HT1A agonists involve a pertussis toxin-sensitive G protein but do not seem to be linked to adenylyl cyclase inhibition.

scholarly journals Role of soluble adenylyl cyclase in the heart

2012 ◽  
Vol 302 (3) ◽  
pp. H538-H543 ◽  
Author(s):  
Jonathan Chen ◽  
Lonny R. Levin ◽  
Jochen Buck
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Adenylyl Cyclase ◽  
G Protein ◽  
Nutrient Utilization ◽  
Soluble Adenylyl Cyclase ◽  
Atp Production ◽  
Downstream Effector ◽  
Subsequent Activation ◽  
Unique Source

This review discusses the potential place of soluble adenylyl cyclase (sAC) in the framework of signaling in the cardiovascular system. cAMP has been studied as a critical and pleiotropic second messenger in cardiomyocytes, endothelial cells, and smooth muscle vascular cells for many years. It is involved in the transduction of signaling by catecholamines, prostaglandins, adenosine, and glucagon, just to name a few. These hormones can act via cAMP by binding to a G protein-coupled receptor on the plasma membrane with subsequent activation of a heterotrimeric G protein and its downstream effector, transmembrane adenylyl cyclase. This has long been the canonical standard for cAMP production in a cell. However, the relatively recent discovery of a unique source of cAMP, sAC, creates the potential for a shift in this signaling paradigm. In fact, sAC has been shown to play a role in apoptosis in coronary endothelial cells and cardiomyocytes. Additionally, it links nutrient utilization with ATP production in the liver and brain, which suggests one of many potential roles for sAC in cardiac function. The possibility of producing cAMP from a source distal to the plasma membrane provides a critical new building block for reconstructing the cellular signaling infrastructure.

scholarly journals Spatial resolution of cAMP signaling by soluble adenylyl cyclase

2016 ◽  
Vol 214 (2) ◽  
pp. 125-127 ◽  
Author(s):  
Giusi Caldieri ◽  
Sara Sigismund
Keyword(s):  
Plasma Membrane ◽  
Adenylyl Cyclase ◽  
G Protein ◽  
Spatial Resolution ◽  
Camp Signaling ◽  
Receptor Signaling ◽  
Soluble Adenylyl Cyclase ◽  
Spatial Encoding ◽  
Cell Biol ◽  
G Protein Coupled

G protein–coupled receptor signaling starts at the plasma membrane and continues at endosomal stations. In this issue, Inda et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201512075) show that different forms of adenylyl cyclase are activated at the plasma membrane versus endosomes, providing a rationale for the spatial encoding of cAMP signaling.

N-terminal domain of Gpa1 (G protein alpha) subunit) is sufficient for plasma membrane targeting in yeast Saccharomyces cerevisiae

1998 ◽  
Vol 111 (21) ◽  
pp. 3235-3244
Author(s):  
K.M. Gillen ◽  
M. Pausch ◽  
H.G. Dohlman
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
G Protein ◽  
Alpha Subunit ◽  
Membrane Targeting ◽  
Yeast Saccharomyces Cerevisiae ◽  
N Terminus ◽  
Membrane Attachment ◽  
G Protein Alpha Subunit ◽  
Protein Alpha Subunit

G proteins play a central role in transmitting signals from cell surface receptors to effector proteins inside the cell. Signaling can only occur, however, if all these protein components are properly assembled and localized at the plasma membrane. Past studies have shown that certain segments within the N-terminal region of the G protein alpha subunit are necessary for membrane attachment. Here we identify a region within the yeast G alpha (Gpa1) that is sufficient for membrane attachment, as well as for specific targeting to the plasma membrane. Initially, we constructed chimeric proteins that replace the N terminus of mammalian Gsalpha with the corresponding sequence from Gpa1. Gsalpha is inefficiently targeted to the yeast plasma membrane and therefore cannot fully complement the loss of Gpa1. Gpa1-Gsaplha chimeras were assayed for proper membrane localization by functional complementation of a gpa1Delta;) mutant, and by sucrose density gradient fractionation of cell membranes. Most of the chimeras tested, including one with only the N-terminal 7 amino acids from Gpa1, exhibited normal membrane targeting and complementing activity. We also fused various lengths of N-terminal Gpa1 sequence to glutathione-S-transferase (GST), a heterologous protein normally expressed in the cytoplasm. The first 67- 36- or 9-amino acids of Gpa1 were all sufficient to direct GST specifically to the plasma membrane in yeast. This analysis defines the extreme N terminus of Gpa1 as the primary determinant of proper membrane targeting, and represents an essential step towards isolating and identifying G protein-targeting proteins within the plasma membrane.

scholarly journals Nonmyristoylated Abl proteins transform a factor-dependent hematopoietic cell line.

1992 ◽  
Vol 12 (4) ◽  
pp. 1864-1871 ◽  
Author(s):  
G Q Daley ◽  
R A Van Etten ◽  
P K Jackson ◽  
A Bernards ◽  
D Baltimore
Keyword(s):  
Plasma Membrane ◽  
Cell Line ◽  
Hematopoietic Cells ◽  
Cell Types ◽  
Hematopoietic Cell ◽  
Myelogenous Leukemia ◽  
3T3 Fibroblasts ◽  
Nih 3T3

N-terminal myristoylation can promote the association of proteins with the plasma membrane, a property that is required for oncogenic variants of Src and Abl to transform fibroblastic cell types. The P210bcr/abl protein of chronic myelogenous leukemia cells is not myristoylated and does not stably transform NIH 3T3 fibroblasts; however, it will transform lymphoid and myeloid cell types in vitro and in vivo, suggesting that myristoylation is not required for Abl variants to transform hematopoietic cells. To test this hypothesis, we introduced point mutations that disrupt myristoylation into two activated Abl proteins, v-Abl and a deletion mutant of c-Abl (delta XB), and examined their ability to transform an interleukin-3-dependent lymphoblastoid cell line, Ba/F3. Neither of the nonmyristoylated Abl proteins transformed NIH 3T3 fibroblasts, but like P210bcr/abl, both were capable of transforming the Ba/F3 cells to factor independence and tumorigenicity. Nonmyristoylated Abl variants did not associate with the plasma membrane in the transformed Ba/F3 cells. These results demonstrate that Abl proteins can transform hematopoietic cells in the absence of membrane association and suggest that distinct functions of Abl are required for transformation of fibroblast and hematopoietic cell types.

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