scholarly journals Chemical inhibition of myristoylation of the G-protein Gi1α by 2-hydroxymyristate does not interfere with its palmitoylation or membrane association. Evidence that palmitoylation, but not myristoylation, regulates membrane attachment

1996 ◽  
Vol 313 (3) ◽  
pp. 717-720 ◽  
Author(s):  
F GALBIATI ◽  
F GUZZI ◽  
A I. MAGEE ◽  
G MILLIGAN ◽  
M PARENTI
Keyword(s):  
G Protein ◽  
Saturated Fatty Acids ◽  
Membrane Association ◽  
Cytoplasmic Fraction ◽  
Α Subunit ◽  
Cos Cells ◽  
Absolute Requirement ◽  
Membrane Attachment ◽  
Chemical Inhibition ◽  
Protein Myristoylation

The α-subunit of the G-protein Gi1α is normally dually acylated at its N-terminus with the saturated fatty acids myristate and palmitate. Inhibition of protein myristoylation by treatment with 2-hydroxymyristate prevented neither the incorporation of [3H]palmitate nor the membrane association of this protein when expressed in COS cells. Construction of a mutant of Gi1α in which serine-6 was replaced by aspartic acid prevented both myristoylation and palmitoylation, and the expressed protein was found primarily in the cytoplasmic fraction. These data indicate that myristoylation is not an absolute requirement for palmitoylation of Gi1α and that palmitoylation, but not myristoylation, plays a key role in membrane association of this G-protein α-subunit.

scholarly journals N-terminal fatty acylation of the α-subunit of the G-protein Gi1: only the myristoylated protein is a substrate for palmitoylation

1994 ◽  
Vol 303 (3) ◽  
pp. 697-700 ◽  
Author(s):  
F Galbiati ◽  
F Guzzi ◽  
A I Magee ◽  
G Milligan ◽  
M Parenti
Keyword(s):  
Palmitic Acid ◽  
G Protein ◽  
Fatty Acyl ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Α Subunit ◽  
Metabolic Labelling ◽  
Fatty Acylation ◽  
Absolute Requirement ◽  
Cdna Construct

The alpha-subunit of the G-protein Gi1 carries two fatty acyl moieties covalently bound to its N-terminal region: myristic acid is linked to glycine-2 and palmitic acid is linked to cysteine-3. Using site-directed mutagenesis on a cDNA construct of alpha i1 we have generated an alpha i1-G2A mutant, carrying alanine instead of glycine at position 2, and alpha i1-C3S mutant, in which serine replaced cysteine-3 and a double mutant with both substitutions (alpha i1-G2A/C3S). These constructs were individually expressed by transfection in Cos-7 cells, and incorporation of fatty acids into the various mutants was compared with wild-type alpha i1 monitoring metabolic labelling with [3H]palmitate or [3H]myristate. The disruption of the palmitoylation site in alpha i1-C3S did not influence myristoylation, whereas prevention of myristoylation in alpha i1-G2A also abolished palmitoylation. Co-translational myristoylation is thus an absolute requirement for alpha i1 to be post-translationally palmitoylated. The non-palmitoylated alpha i1-C3S showed reduced membrane binding to the same extent as the non-myristoylated/non-palmitoylated alpha i1-G2A and alpha i1-G2A/C3S mutants, indicating that the attachment of palmitic acid is necessary for proper interaction with the membrane.

scholarly journals Plasma Membrane Localization of Gαz Requires Two Signals

1998 ◽  
Vol 9 (1) ◽  
pp. 1-14 ◽  
Author(s):  
Janine Morales ◽  
C. Simone Fishburn ◽  
Paul T. Wilson ◽  
Henry R. Bourne
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Protein Localization ◽  
Receptor Activation ◽  
Α Subunit ◽  
Surface Receptors ◽  
Defective Mutant ◽  
Specific Localization ◽  
Membrane Attachment ◽  
Stable Association

Three covalent attachments anchor heterotrimeric G proteins to cellular membranes: the α subunits are myristoylated and/or palmitoylated, whereas the γ chain is prenylated. Despite the essential role of these modifications in membrane attachment, it is not clear how they cooperate to specify G protein localization at the plasma membrane, where the G protein relays signals from cell surface receptors to intracellular effector molecules. To explore this question, we studied the effects of mutations that prevent myristoylation and/or palmitoylation of an epitope-labeled α subunit, αz. Wild-type αz (αz-WT) localizes specifically at the plasma membrane. A mutant that incorporates only myristate is mistargeted to intracellular membranes, in addition to the plasma membrane, but transduces hormonal signals as well as does αz-WT. Removal of the myristoylation site produced a mutant αz that is located in the cytosol, is not efficiently palmitoylated, and does not relay the hormonal signal. Coexpression of βγ with this myristoylation defective mutant transfers it to the plasma membrane, promotes its palmitoylation, and enables it to transmit hormonal signals. Pulse-chase experiments show that the palmitate attached to this myristoylation-defective mutant turns over much more rapidly than does palmitate on αz-WT, and that the rate of turnover is further accelerated by receptor activation. In contrast, receptor activation does not increase the slow rate of palmitate turnover on αz-WT. Together these results suggest that myristate and βγ promote stable association with membranes not only by providing hydrophobicity, but also by stabilizing attachment of palmitate. Moreover, palmitoylation confers on αz specific localization at the plasma membrane.

scholarly journals Genetic Involvement of a cAMP-Dependent Protein Kinase in a G Protein Signaling Pathway Regulating Morphological and Chemical Transitions in Aspergillus nidulans

Genetics ◽  
2001 ◽  
Vol 157 (2) ◽  
pp. 591-600
Author(s):  
Kiminori Shimizu ◽  
Nancy P Keller
Keyword(s):  
Protein Kinase ◽  
Aspergillus Nidulans ◽  
Signaling Pathway ◽  
G Protein ◽  
Radial Growth ◽  
Morphological Development ◽  
Dependent Protein Kinase ◽  
Α Subunit ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

Abstract In the filamentous fungus Aspergillus nidulans, a heterotrimeric G protein α-subunit and an RGS domain protein, encoded by fadA and flbA, respectively, regulate production of the carcinogenic metabolite sterigmatocystin (ST) and asexual spores (i.e., conidia). We investigated the genetic involvement of the cAMP-dependent protein kinase catalytic subunit (PkaA), a potential downstream target of FadA activity, in ST production and conidiation. Relative to wild type, sporulation was decreased in the pkaA overexpression strain but was not totally absent, as occurs in ΔflbA or fadAG42R (fadA-dominant active) strains. Deletion of pkaA resulted in a hyper-conidiating strain with limited radial growth. This phenotype was epistatic to mutation in flbA or fadA; the double mutants ΔpkaA; ΔflbA and ΔpkaA; fadAG42R recovered sporulation and their radial growth was severely restricted. PkaA overexpression also negatively regulated AflR, the ST biosynthesis-specific transcription factor, both transcriptionally and post-transcriptionally. Deletion of pkaA restored ST production in the ΔflbA background but not in the fadAG42R background. These data provide genetic evidence that the FlbA/FadA signaling pathway regulating ST production and morphological development is partially mediated through PkaA.

scholarly journals Every Detail Matters. That Is, How the Interaction between Gα Proteins and Membrane Affects Their Function

Membranes ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 222
Author(s):  
Agnieszka Polit ◽  
Paweł Mystek ◽  
Ewa Błasiak
Keyword(s):  
Signal Transduction ◽  
G Protein ◽  
G Proteins ◽  
Lipid Membranes ◽  
Signaling Proteins ◽  
Heterotrimeric G Proteins ◽  
Membrane Attachment ◽  
The Individual ◽  
Multicellular Organisms ◽  
G Protein Coupled

In highly organized multicellular organisms such as humans, the functions of an individual cell are dependent on signal transduction through G protein-coupled receptors (GPCRs) and subsequently heterotrimeric G proteins. As most of the elements belonging to the signal transduction system are bound to lipid membranes, researchers are showing increasing interest in studying the accompanying protein–lipid interactions, which have been demonstrated to not only provide the environment but also regulate proper and efficient signal transduction. The mode of interaction between the cell membrane and G proteins is well known. Despite this, the recognition mechanisms at the molecular level and how the individual G protein-membrane attachment signals are interrelated in the process of the complex control of membrane targeting of G proteins remain unelucidated. This review focuses on the mechanisms by which mammalian Gα subunits of G proteins interact with lipids and the factors responsible for the specificity of membrane association. We summarize recent data on how these signaling proteins are precisely targeted to a specific site in the membrane region by introducing well-defined modifications as well as through the presence of polybasic regions within these proteins and interactions with other components of the heterocomplex.

scholarly journals RAGE ligands stimulate angiotensin II type I receptor (AT1) via RAGE/AT1 complex on the cell membrane

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Serina Yokoyama ◽  
Tatsuo Kawai ◽  
Koichi Yamamoto ◽  
Huang Yibin ◽  
Hiroko Yamamoto ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Cardiovascular Diseases ◽  
Epithelial Cells ◽  
G Protein ◽  
Rat Kidney ◽  
Chinese Hamster ◽  
Cellular Responses ◽  
Type I ◽  
Α Subunit ◽  
Mesenchymal Transition

AbstractThe receptor for advanced glycation end-products (RAGE) and the G protein-coupled angiotensin II (AngII) type I receptor (AT1) play a central role in cardiovascular diseases. It was recently reported that RAGE modifies AngII-mediated AT1 activation via the membrane oligomeric complex of the two receptors. In this study, we investigated the presence of the different directional crosstalk in this phenomenon, that is, the RAGE/AT1 complex plays a role in the signal transduction pathway of RAGE ligands. We generated Chinese hamster ovary (CHO) cells stably expressing RAGE and AT1, mutated AT1, or AT2 receptor. The activation of two types of G protein α-subunit, Gq and Gi, was estimated through the accumulation of inositol monophosphate and the inhibition of forskolin-induced cAMP production, respectively. Rat kidney epithelial cells were used to assess RAGE ligand-induced cellular responses. We determined that RAGE ligands activated Gi, but not Gq, only in cells expressing RAGE and wildtype AT1. The activation was inhibited by an AT1 blocker (ARB) as well as a RAGE inhibitor. ARBs inhibited RAGE ligand-induced ERK phosphorylation, NF-κB activation, and epithelial–mesenchymal transition of rat renal epithelial cells. Our findings suggest that the activation of AT1 plays a central role in RAGE-mediated cellular responses and elucidate the role of a novel molecular mechanism in the development of cardiovascular diseases.

scholarly journals The G-Protein α-Subunit GasC Plays a Major Role in Germination in the Dimorphic FungusPenicillium marneffei

Genetics ◽  
2003 ◽  
Vol 164 (2) ◽  
pp. 487-499 ◽  
Author(s):  
Sophie Zuber ◽  
Michael J Hynes ◽  
Alex Andrianopoulos
Keyword(s):  
G Protein ◽  
Germination Rate ◽  
Yeast Cells ◽  
Class Iii ◽  
Temperature Dependent ◽  
Gene Encoding ◽  
Α Subunit ◽  
G Protein Α Subunit ◽  
Opportunistic Human Pathogen

AbstractThe opportunistic human pathogen Penicillium marneffei exhibits a temperature-dependent dimorphic switch. At 25°, multinucleate, septate hyphae that can undergo differentiation to produce asexual spores (conidia) are produced. At 37° hyphae undergo arthroconidiation to produce uninucleate yeast cells that divide by fission. This work describes the cloning of the P. marneffei gasC gene encoding a G-protein α-subunit that shows high homology to members of the class III fungal Gα-subunits. Characterization of a ΔgasC mutant and strains carrying a dominant-activating gasCG45R or a dominant-interfering gasCG207R allele show that GasC is a crucial regulator of germination. A ΔgasC mutant is severely delayed in germination, whereas strains carrying a dominant-activating gasCG45R allele show a significantly accelerated germination rate. Additionally, GasC signaling positively affects the production of the red pigment by P. marneffei at 25° and negatively affects the onset of conidiation and the conidial yield, showing that GasC function overlaps with functions of the previously described Gα-subunit GasA. In contrast to the S. cerevisiae ortholog Gpa2, our data indicate that GasC is not involved in carbon or nitrogen source sensing and plays no major role in either hyphal or yeast growth or in the switch between these two forms.

scholarly journals Central role of G protein Gαi2 and Gαi2+ vomeronasal neurons in balancing territorial and infant-directed aggression of male mice

2019 ◽  
Vol 116 (11) ◽  
pp. 5135-5143 ◽  
Author(s):  
Anne-Charlotte Trouillet ◽  
Matthieu Keller ◽  
Jan Weiss ◽  
Trese Leinders-Zufall ◽  
Lutz Birnbaumer ◽  
...  
Keyword(s):  
G Protein ◽  
Sensory Neurons ◽  
Neural Activity ◽  
Parental Behavior ◽  
Vomeronasal Organ ◽  
Male Infant ◽  
Lateral Septum ◽  
Male Mice ◽  
Protein Subunit ◽  
Α Subunit

Aggression is controlled by the olfactory system in many animal species. In male mice, territorial and infant-directed aggression are tightly regulated by the vomeronasal organ (VNO), but how diverse subsets of sensory neurons convey pheromonal information to limbic centers is not yet known. Here, we employ genetic strategies to show that mouse vomeronasal sensory neurons expressing the G protein subunit Gαi2 regulate male–male and infant-directed aggression through distinct circuit mechanisms. Conditional ablation of Gαi2 enhances male–male aggression and increases neural activity in the medial amygdala (MeA), bed nucleus of the stria terminalis, and lateral septum. By contrast, conditional Gαi2 ablation causes reduced infant-directed aggression and decreased activity in MeA neurons during male–infant interactions. Strikingly, these mice also display enhanced parental behavior and elevated neural activity in the medial preoptic area, whereas sexual behavior remains normal. These results identify Gαi2 as the primary G protein α-subunit mediating the detection of volatile chemosignals in the apical layer of the VNO, and they show that Gαi2+ VSNs and the brain circuits activated by these neurons play a central role in orchestrating and balancing territorial and infant-directed aggression of male mice through bidirectional activation and inhibition of different targets in the limbic system.

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