The Family of G Proteins (original) (raw)
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The Biochemical journal, 1989
The effect of activation of the alpha-subunit(s) of the stimulatory guanine-nucleotide-binding protein, Gs, on levels of this polypeptide(s) associated with the plasma membrane of L6 skeletal myoblasts was ascertained. Incubation of these cells with cholera toxin led to a time- and concentration-dependent 'down-regulation' of both 44 and 42 kDa forms of Gs alpha as assessed by immunoblotting with an anti-peptide antiserum (CS1) able to identify the extreme C-terminus of Gs. The effect of cholera toxin was specific for Gs; levels of Gi alpha in membranes of cholera toxin-treated cells were not different from untreated cells. Down-regulation of Gs was absolutely dependent upon prior ADP-ribosylation, and hence activation of Gs and was not mimicked by other agents which elevate intracellular levels of cyclic AMP. Pretreatment with pertussis toxin, which catalyses ADP-ribosylation of Gi but not of Gs, did not down-regulate either Gi or Gs, demonstrating that covalent modificatio...
Journal of Biological Chemistry, 2002
A ligand-independent activator of heterotrimeric brain G-protein was partially purified from detergentsolubilized extracts of the neuroblastoma-glioma cell hybrid NG108-15. The G-protein activator (NG108-15 Gprotein activator (NG-GPA)) increased [ 35 S]guanosine 5-O-(thiotriphosphate) ([ 35 S]GTP␥S) to purified brain G-protein in a magnesium-dependent manner and promoted GDP dissociation from G␣ o. The NG-GPA also increased GTP␥S binding to purified, recombinant G␣ i2 , G␣ i3 , and G␣ o , but minimally altered nucleotide binding to purified transducin. The NG-GPA increased GTP␥S binding to membrane-bound G-proteins and inhibited basal, forskolin-and hormone-stimulated adenylyl cyclase activity in DDT 1-MF-2 cell membranes. In contrast to G-protein coupled receptor-mediated activation of heterotrimeric G-proteins in DDT 1-MF-2 cell membrane preparations, the action of the NG-GPA was not altered by treatment of the cells with pertussis toxin. ADP-ribosylation of purified brain G-protein also failed to alter the increase in GTP␥S binding elicited by the NG-GPA. Thus, the NG-GPA acts in a manner distinct from that of a G-protein coupled receptor and other recently described receptor-independent activators of G-protein signaling. These data indicate the presence of unexpected regulatory domains on G i /G o proteins and suggest the existence of pertussis toxin-insensitive modes of signal input to G i /G o signaling systems.
Studies on Nucleotide and Receptor Regulation of GiProteins: Effects of Pertussis Toxin
Molecular Endocrinology, 1989
In intact membranes as well as after reconstitution into phospholipid vesicles, pertussis toxin (PT)-mediated ADP-ribosylation of G proteins causes loss of receptor-mediated regulation of effectors and/or G protein-mediated regulation of receptor binding. Studies were carried out to test which of several discrete steps known to constitute the basal and receptor-stimulated regulatory cycles of G ; proteins are affected by PT. Experiments with the G s-deficient Gi-regulated adenylyl cyclase of eye" S49 cell membranes indicated that PT blocks G, activation by GTP without affecting GDP dissociation or GTP binding to a major extent. This suggested that the block lies in the transition of inactive GTP-G; to active GTP-Gi (G to G* transition). Experiments with purified Gi in solution and after incorporation into phospholipid vesicles showed that PT does not increase or decrease the intrinsic GTPase activity of G,. Experiments in which G, was incorporated into phospholipid vesicles with rhodopsin, a receptor that interacts with Gi to stimulate the rate of guanosine 5'-O-(3-thio)triphosphate binding and GTP hydrolysis, indicated that PT does not affect the basal GTPase activity of G { , but blocks its activation by the photoreceptor. Taken together the results indicate that PT-mediated ADP ribosylation has two separate effects, one to block the interaction of receptor with Gi and another to impede the GTP-induced activation reaction from occurring, or that PT has only one effect, that of blocking interaction with receptors. In this latter case the present results add to a mounting series of data that are consistent with the hypothesis that unoccupied receptors are not inactive, but exhibit a basal agonist-independent activity responsible for the various effects of GTP observed on G protein-coupled effector functions in intact membranes. (Molecular Endocrinology 3: 1115-1124, 1989
Biochimica Et Biophysica Acta-molecular Cell Research, 1997
A cDNA encoding the rat μ-opioid receptor was expressed stably in a Rat-1 fibroblast cell line. Expression of this receptor was demonstrated with specific binding of the μ-opioid selective ligand [][d-Ala2,N-MePhe4,Gly5-ol]-enkephalin ([]DAMGO). In membranes of clone μ11 cells DAMGO produced a robust, concentration-dependent stimulation of basal high affinity GTPase activity. Cholera toxin-catalyzed []ADP-ribosylation in membranes of this clone labelled a 40 kDa Gi family polypeptide(s) that was markedly enhanced by the addition of DAMGO. Antisera against Gi2α and Gi3α were both able to immunoprecipitate a []-radiolabelled 40 kDa polypeptide(s) from DAMGO and cholera-toxin treated membranes of clone μ11, indicating that the μ-opioid receptor was able to interact effectively with both Gi2 and Gi3 in Rat-1 fibroblasts. A series of peptides derived from the δ-opioid receptor sequence were assessed for their ability to modify agonist-stimulated G protein activation and [] agonist binding to the receptor. In membranes from the clone μ11, specific binding of []DAMGO was reduced by peptides corresponding to the NH2-terminal region of the third intracellular loop (i3.1) and the carboxyl-terminal tail (i4) of this receptor. Agonist stimulated GTPase activity and DAMGO dependent cholera toxin-catalyzed []ADP-ribosylation were inhibited by peptides derived from the proximal (i3.1) and the distal portion (i3.3) of the third intracellular loop. Peptide i3.1 also inhibited DAMGO-stimulated []guanosine-5′-O-(3-thio)triphosphate ([]GTPγS) binding in the same membranes. In contrast, peptides derived from the second intracellular loop were without any effect.
European Journal of Pharmacology: Molecular Pharmacology, 1990
Rat glioma C6 BU1 cells were treated in tissue culture with cholera toxin. Incubation of membranes derived from these cells with fresh cholera toxin and [32P]NAD+ failed to promote incorporation of radioactivity into polypeptides corresponding to forms of G~a. This is generally assumed to reflect prior ADP ribosylation of these polypeptides in vivo using endogenous NAD + as substrate. However, immunological studies with anfi-peptide antisera which identify all forms of Gsa demonstrated that concentrations of this polypeptide v'ere now substantially reduced in ihe membranes. This effect was specific for Gsa as neither the a-subunits of the pertussis toxin-sensitive G-proteins G~2 and Gi3, nor the ,8 subunit common to the various G-proteins were lost in parallel. Pertussis toxin-catalysed ADP dbosylation did not cause the downregulation of G~a nor of the a-subunits of G~2 or G,3 although it did cause ADP ribosylation of the entire complement of both Gi2 and GI3 in the membranes. Despite the reduction in levels of immunoreactive Gsa from the membranes of cholera toxin-treated cells, no alterations in levels of mRNA corresponding to this G-protein were noted. G-proteins; Downregulation; Cholera toxin: Pertussis toxin
Molecular Endocrinology, 1997
Although it is well established that activated LH/ human (h) CG receptor stimulates adenylyl cyclase activity (via the heterotrimeric stimulatory guanine nucleotide-binding protein, G s) and in some cells stimulates phospholipase C activity, there is no evidence for a direct physical interaction between the LH/CG receptor and G s or any other G protein(s). We conducted studies using cholera toxin (CTX) and pertussis toxin (PTX) to determine which G␣ proteins were associated with the LH/CG receptor in ovarian follicular membranes. Since hormone-dependent, CTX-catalyzed ADP ribosylation (AR) constitutes evidence that a G␣ protein is specifically associated with a receptor, CTX-catalyzed AR of membrane proteins was examined both in the presence and absence of guanine nucleotides to determine which G proteins exhibit hCG-dependent labeling by [ 32 P]NAD. Results demonstrated the time-and hCG-dependent AR of both a 45-kDa protein and a 48/50-kDa doublet as well as a 40-kDa protein that was also sensitive to AR by PTX in a time-and hCG-dependent manner. Using anti-G protein antisera to specifically immunoprecipitate photoaffinity-labeled G proteins, we were able to identify the 45-and 48/50 kDa proteins as the short and long forms of G s ␣ and the 40-kDa protein as G i ␣. A monoclonal anti-hCG antibody immunoprecipitated the activated LH/CG receptor along with the long and short forms of G s ␣ and G i. These results suggest that a portion of G i along with the long and short forms of G s ␣ are associated physically with the LH/CG receptor in ovarian follicular membranes.
Cellular Signalling, 2002
This review focuses on the coupling specificity of the Ga and Gbg subunits of pertussis toxin (PTX)-sensitive G i/o proteins that mediate diverse signaling pathways, including regulation of ion channels and other effectors. Several lines of evidence indicate that specific combinations of G protein alpha, beta and gamma subunits are required for different receptors or receptor -effector networks, and that a higher degree of specificity for Ga and Gbg is observed in intact systems than reported in vitro. The structural determinants of receptor -G protein specificity remain incompletely understood, and involve receptor -G protein interaction domains, and perhaps other scaffolding processes. By identifying G protein specificity for individual receptor signaling pathways, ligands targeted to disrupt individual pathways of a given receptor could be developed. D
Toxins, 2015
Pathogenesis of cholera diarrhea requires cholera toxin (CT)-mediated adenosine diphosphate (ADP)-ribosylation of stimulatory G protein (Gsα) in enterocytes. CT is an AB5 toxin with an inactive CTA1 domain linked via CTA2 to a pentameric receptor-binding B subunit. Allosterically activated CTA1 fragment in complex with NAD+ and GTP-bound ADP-ribosylation factor 6 (ARF6-GTP) differs conformationally from the CTA1 domain in holotoxin. A surface-exposed knob and a short α-helix (formed, respectively, by rearranging "active-site" and "activation" loops in inactive CTA1) and an ADP ribosylating turn-turn (ARTT) motif, all located near the CTA1 catalytic site, were evaluated for possible roles in recognizing Gsα. CT variants with one, two or three alanine substitutions at surface-exposed residues within these CTA1 motifs were tested for assembly into holotoxin and ADP-ribosylating activity against Gsα and diethylamino-(benzylidineamino)-guanidine (DEABAG), a small subs...
Molecular Endocrinology, 1987
ADP ribosylation of membranes by pertussis toxin (PT) and cholera toxin (CT) was studied as a function of addition of ATP, various guanine nucleotides, Mg 2+ , and inorganic phosphate (Pi). ADP ribosylation of a 40 kilodalton (kDa) band by PT is markedly enhanced by ATP and GTP and is strongly inhibited by Pi or Mg 2+. GTP analogs (GTP7S and GMP-adenyl-5'-yl imidodiphosphate) were less effective. In contrast, ADP ribosylation of two substrates for CT (of 42 and 50 kDa) is stimulated by Pi, Mg 2+ , and GTP or GTP analogs such as GTP7S, but is unaffected by ATP. These stimulatory conditions correlate well with GTP-mediated activation of stimulated nucleotide-binding regulatory component of adenyl cyclase. Optimal conditions for ADP ribosylation by PT do not correlate simply with conditions thought to lead to stabilization of an inactive form of inhibitory nucleotide-binding regulatory component of adenyl cyclase (Gj) or Gi-like protein; rather, the data suggest the involvement of both a stimulatory nucleotide site on PT (positively effected by either ATP or GTP) and a stabilizing site on the PT substrate (affected by GDP, GDP0S, or GTP). Treatment of membranes with Lubrol PX increased ADP ribosylation by PT by as much as 25-to 30-fold, but inhibited the action of CT. Using defined conditions for ADP ribosylation by PT and CT, distinct labeling patterns were observed in thyroid, brain, corpus luteum, liver, heart, and erythrocytes membranes. All membranes were more intensely labeled by PT rather than CT.