GTP analogues promote release of the α subunit of the guanine nucleotide binding protein, Gi2, from membranes of rat glioma C6 BU1 cells

Milligan, Graeme; Mullaney, Ian; Unson, Cecilia G.; Marshall, Lawrence M.; Spiegel, Allen M.; McArdle, Hayley

doi:10.1042/bj2540391

Cited by 39 publications

(23 citation statements)

References 29 publications

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“…The data described in this report are generally consistent with this model. Some investigators have observed a slow, fractional release of ao and ai2 from cultured cell membranes exposed to poorly hydrolyzed analogs of GTP (29,30). We (unpublished results) and others (6,31) have not observed release of native a subunits from membranes by analogs of GTP or by hormones.…”

Section: Resultsmentioning

confidence: 86%

G-protein alpha-subunit expression, myristoylation, and membrane association in COS cells.

Mumby

Heukeroth

Gordon

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

268

179

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Myristoylation of seven different a subunits of guanine nucleotide-binding regulatory proteins (G proteins) was examined by expressing these proteins in monkey kidney COS cells. Metabolic labeling studies of cells transfected with cytomegalovirus-based expression vectors indicated that[3H]myristate was incorporated into ail, a,2, a13, a0, at, and a, but not as subunits. The role of myristoylation in the association of a subunits with membranes was analyzed by sitedirected mutagenesis and by substitution of myristate with a less hydrophobic analog, 10-(propoxy)decanoate (li-oxamyristate). Myristoylation of a0 was blocked when an alanine residue was substituted for its amino-terminal glycine, as was association of the protein with membranes. Substitution of the myristoyl group with l1-oxamyristate affected the cellular distribution of a subset of acylated at subunits. The results are consistent with a model wherein the hydrophobic interaction of myristate with the bilayer permits continued association of the protein with the plasma membrane when G-protein a subunits dissociate from fty.Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) transduce signals across cell membranes by coupling receptors for chemical and sensory stimuli to effectors such as enzymes and ion channels. The best studied systems of this type are hormone-sensitive adenylyl cyclase (stimulated by the G protein GU) and the light-sensitive cyclic GMP phosphodiesterase in photoreceptor cells (stimulated by transducin or Gt) (1, 2). Other G proteins of relevance to this work include a group of four related substrates for pertussis toxin (Gil, GQ2, GU3, and GO), which appear to regulate ion channels, and GU, a newly appreciated G protein whose role in transmembrane signaling remains to be defined (3-5).G proteins are associated with the inner face of the plasma membrane, where they are positioned to interact with membrane-bound receptors and effectors. With the exception of Gt, detergent is required to extract G proteins from membranes. However, the deduced amino acid sequences of G protein a, f3, and y subunits lack obvious hydrophobic domains that could account for G protein-membrane interactions, and the molecular basis for the interaction of G proteins with the plasma membrane is largely unknown. Sternweis (6) demonstrated that the G-protein 13y subunit complex accounted for the detergent-binding properties of heterotrimeric G proteins. Furthermore, he demonstrated that ,fy could be incorporated into phospholipid vesicles and that G-protein a subunits would then interact stoichiometrically with fBy-containing vesicles.

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Section: Resultsmentioning

confidence: 86%

G-protein alpha-subunit expression, myristoylation, and membrane association in COS cells.

Mumby

Heukeroth

Gordon

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

268

179

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“…Surprisingly enough, the present results argue against the involvement of any of the traditional transmembrane signals in this binding process. However, it was recently demonstrated that pertussis toxin did not prevent the dissociation between the o~-and the/33,-subunits, even if the a-subunit was efficiently ribosylated (28). Consequently, any involvement of the dissociation process itself or the/33,-subunit in cellular regulation can not be adequately tested with pertussis toxin.…”

Section: Discussionmentioning

confidence: 99%

Association of ligand-receptor complexes with actin filaments in human neutrophils: a possible regulatory role for a G-protein.

Särndahl

Lindroth

Bengtsson

et al. 1989

The Journal of Cell Biology

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Abstract. Most ligand-receptor interactions result in an immediate generation of various second messengers and a subsequent association of the ligund-receptor complex to the cytoskeleton. Depending on the receptor involved, this linkage to the cytoskeleton has been suggested to play a role in the termination of second messenger generation and/or the endocytic process whereby the ligand-receptor complex is internalized. We have studied how the binding of chemotactic peptide-receptor complexes to the cytoskeleton of human neutrophils is accomplished. As much as 76% of the tritiated formylmethionyl-leucyl-phenylalanine (fMet-Leu-[3H]Phe) specifically bound to intact cells, obtained by a 30-s stimulation with 20 nM fMet-Leu-[3H]Phe, still remained after Triton X-100 extraction. Preincubating intact cells with dihydrocytochalasin B (dhCB) or washing the cytoskeletal preparation with a high concentration of potassium, reduced the binding of ligand-receptor complexes to the cytoskeleton by 46% or more. Inhibition of fMet-Leu-Phe-induced generation of second messengers by ADP-ribosylating the ,-subunit of the receptor-coupled G-protein with pertussis toxin, did not reduce the binding of ligandreceptor complexes to the cytoskeleton. However, using guanosine-5'-O-(2-thiodiphosphate) (GDP/~S) to prevent the dissociation of the fMet-Leu-Phe-associated G-protein within electrically permeabilized cells, led to a pronounced reduction (62%) of the binding between ligand-receptor complexes and the cytoskeleton. In summary, in human neutrophils the rapid association between chemotactic peptide-receptor complexes and the cytoskeleton is dependent on filamentous actin. This association is most likely regulated by the activation and dissociation of the fMetLeu-Phe-associated G-protein.H UMAN neutrophils and macrophages play an important role in the body defense against microorganisms. A fundamental quality of these cells, enabling them to perform these duties, is their ability to move and engulf particles. These motile events are dependent on dynamic alterations and reorganization of their cytoskeleton (36).The motile activity is induced by the specific binding of a ligand, a chemotactic factor or a phagocytic opsonin, to its respective receptor. The formation of ligand-receptor complexes triggers the generation of various transmembrane signals responsible for the activation of different effector systems in these cells (35). The signal transduction system activated by chemotactic factors in human neutrophils is generally agreed to involve a guanine nucleotide binding protein, G-protein (15). Evidence for the involvement of this G-protein is usually tested by using pertussis toxin (1,4,8,25,31), which ADP-ribosylates and thereby inhibits the activity of the oL-subunit of the G-protein (9, 24). In the absence of permssis toxin the chemotactic factor-receptor complex, via the ¢x-subunit of the G-protein, causes an increase in the activity of phospholipase C. This results in an increased breakdown of phosphafidylinositol 4,5-bisphosphat...

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“…Substantial down-regulation of G,a from cell membranes has been observed in L6 skeletal myoblasts treated with cholera toxin to ADP ribosylate the G-proteins [7], although under these conditions the G , a lost from the plasma membrane was not detected intracellularly and is presumed to have been degraded. By contrast, non-hydrolysable guanine nucleotide analogues can displace both G , a [7] and G , a [8] from purified plasma membranes in vitro. However, in concurrence with our observations, translocation of a G-protein from the plasma membrane of intact cells during hormonal stimulation remains to be demonstrated.…”

Section: Biochemical Society Transactionsmentioning

confidence: 94%

Internalization of hepatic glucagon receptors is not accompanied by a significant movement of Gsα

Pollock¹,

Forder²,

Creba³

et al. 1990

Biochemical Society Transactions

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The effects of phosphodiesterase Ill inhibition (by ICI 145730) and phosphodiesterase IV inhibition (by rolipram) on cyclic AMP and the phosphorylase activation ratio are also given in Table 1. As with adenylate cyclase activation, there is some disparity between the concentration of cyclic AMP produced and the phosphorylase activity ratio generated by the two cornpounds. From the Table, it can be seen that ICI 145730 appears to require more cyclic AMP to activate phosphorylase than rolipram. As with forskolin, the effectiveness of cyclic AMP levels altered by 1C1 145730 increases with time, whereas that produced by rolipram does not.Since the measurements of phosphorylase were made on extracts o f the cells, enhancement of the activity of phosphorylase must have resulted from the covalent modification of the enzyme by phosphorylase kinase. None of the compounds used in this study elevates [Caz+], in this cell line (A. Simpson & P. R. Kemp, unpublished observations), therefore, activation of phosphorylase kinase must have resulted from protein kinase A activity. As shown above for a given increase in cyclic AMP, isoprenaline appears to increase phosphorylase activity to a greater extent than forskolin.The results presented here indicate a disparity between the increase in cyclic AMP produced by activation of adenylate cyclase and the stimulation of phosphorylase via protein kinase A. The data therefore suggest that in the A,,, cell line cyclic AMP and the enzymes of cyclic AMP metabolism are compartmented and that cyclic AMP can diffuse between these compartments since the cyclic AMP required to generate a given phosphorylase activity ratio decreases with time in response to forskolin and ICI 145730, but increases with time in response to isoprenaline and rolipram.

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GTP analogues promote release of the α subunit of the guanine nucleotide binding protein, Gi2, from membranes of rat glioma C6 BU1 cells

Cited by 39 publications

References 29 publications

G-protein alpha-subunit expression, myristoylation, and membrane association in COS cells.

G-protein alpha-subunit expression, myristoylation, and membrane association in COS cells.

Association of ligand-receptor complexes with actin filaments in human neutrophils: a possible regulatory role for a G-protein.

Internalization of hepatic glucagon receptors is not accompanied by a significant movement of Gsα

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