Selective interactions of μ-opioid receptors with pertussis toxin-sensitive G proteins: involvement of the third intracellular loop and the c-terminal tail in coupling (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...
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
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.
Journal of Neurochemistry, 1988
Pertussis toxin-catalyzed ADP-ribosylation of the guanine nucleotide-binding proteins Gi and Go is shown to proceed in Mg*+-digitonin extracts from rat brain; the M, 4 1,000 and M, 39,000 peptides are labelled there as in the membranes. The ADP-ribosylation in detergent solution retains the differential sensitivity to guanine nucleotide analogues. This reaction also removes the partial inhibition by the guanine nucleotides of the binding of opioid agonists, as does the same treatment in the membranes. The partial inhibition of agonist binding by Na', however, is left unchanged. The binding of the antagonist naloxone is little affected by Na+ or by guanine nucleotides in the treated membranes, but the treated soluble receptors show an enhanced binding in high-Na+ medium, although still guanine nucleotide insensitive. The data suggest that the toxin reaction in the absence of guanine nucleotides and agonist stabilizes the opioid receptor in a receptor-G-protein coupled state which is no longer sensitive to guanine nucleotides but retains its sensitivity to the Na+ ions. Key Words: Rat brain opioid receptors-Pertussis toxin-Gi/Go proteins. Wong Y. H. et al. ADP-ribosylation with pertussis toxin modulates the GTP-sensitive opioid ligand binding in digitonin-soluble extracts of rat brain membranes. J. Neurochem. 51, 114-121 (1988). Abbreviations used: BSA, bovine serum albumin; DADLE, ~AIa'[tyro~yl-3,5--'H]D-Leu~-enkephalin; ('HIDSLET, [tyrosylacid. 3,5-3H(N)]~Se8-~-Leu5-L-ThP-enkephalin; DTT, dithiothreitol; GDPPS, guanosine-5'-0-(2-thiodiphosphate); Gpp(NH)p, guanosine-5-(j3,y-imido)triphosphate; kDa, kilodalton(s); PMSF, phenylmethylsulphonyl fluoride; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; TES-KOH, potassium salt of N-tris(hydroxymethyl)methyl-2-aminoethanesulphonic
The Biochemical journal, 1988
We investigated the mechanisms of receptor-mediated stimulation of high-affinity GTPase activity in response to opioid peptides and to foetal-calf serum in membranes of the neuroblastoma X glioma hybrid cell line NG108-15. Increases in GTPase activity in response to both of these ligands was abolished by prior exposure of the cells to pertussis toxin. Pertussis toxin in the presence of [32P]NAD+ catalysed incorporation of radioactivity into a broad band of approx. 40 kDa in membranes prepared from untreated, but not from pertussis-toxin-pretreated, cells. Additivity studies indicated that the responses to opioid peptides and to foetal-calf serum were mediated by separate guanine-nucleotide-binding proteins (G-proteins). Whereas opioid peptides produced an inhibition of adenylate cyclase in membranes of untreated cells, foetal-calf serum did not. Affinity-purified antibodies which recognize the C-terminus of the inhibitory G-protein identified a 40 kDa polypeptide in membranes of NG1...
δ-Opioid receptors exhibit high efficiency when activating trimeric G proteins in membrane domains
Journal of Neurochemistry, 2003
Low-density membrane fragments (domains) were separated from the bulk of plasma membranes of human embryonic kidney (HEK)293 cells expressing a d-opioid (DOP) receptor-G i 1a fusion protein by drastic homogenization and flotation on equilibrium sucrose density gradients. The functional activity of trimeric G proteins and capacity of the DOP receptor to stimulate both the fusion protein-linked G i 1a and endogenous pertussis-toxin sensitive G proteins was measured as D-Ala2, D-Leu5-enkephalin stimulated high-affinity GTPase or guanosine-5¢-[c-35 S]triphosphate ([ 35 S]GTPcS) binding. The maximum D-Ala2-D-Leu5 enkephalin (DADLE)-stimulated GTPase was two times higher in low-density membrane fragments than in bulk of plasma membranes; 58 and 27 pmol/mg/min, respectively. The same difference was obtained for [ 35 S]GTPcS binding. Contrarily, the low-density domains contained no more than half the DOP receptor binding sites (B max ¼ 6.6 pmol/mg versus 13.6 pmol/mg). Thus, when corrected for expression levels of the receptor, low-density domains exhibited four times higher agonist-stimulated GTPase and [ 35 S]GTPcS binding than the bulk plasma membranes. The regulator of G protein signaling RGS1, enhanced further the G protein functional activity but did not remove the difference between domain-bound and plasma membrane pools of G protein. The potency of the agonist in functional studies and the affinity of specific [ 3 H]DADLE binding to the receptor were, however, the same in both types of membranes -EC 50 ¼ 4.5 ± 0.1 · 10 )8 and 3.2 ± 1.4 · 10 )8 M for GTPase; K d ¼ 1.2 ± 0.1 and 1.3 ± 0.1 nM for [ 3 H]DADLE radioligand binding assay. Similar results were obtained when sodium bicarbonate was used for alkaline isolation of membrane domains. By contrast, detergentinsensitive membrane domains isolated following treatment of cells with Triton X100 exhibited no DADLE-stimulated GTPase or GTPcS binding. Functional coupling between the DOP receptor and cognate G proteins was also blocked by high-energy ultrasound and repeated freezing-thawing. Our data indicate, for the first time, that membrane domains isolated using 'detergent-free' procedures exhibit higher efficiency of coupling between a G protein-coupled receptor and its corresponding G protein(s) than bulk plasma membranes. Detergent-extraction diminishes these interactions, even when the receptor and G proteins are physically tethered together.
Molecular and Cellular Biochemistry, 1994
ADP-ribosylation factors (ARFs) comprise a family of ~20 kDa guanine nucleotide-binding proteins that were discovered as one of several cofactors required in cholera toxin-catalyzed ADP-ribosylation of G, the guanine nucleotide-binding protein responsible for stimulation of adenylyl cyclase, and was subsequently found to enhance all cholera toxin-catalyzed reactions and to directly interact with, and activate the toxin. ARF is dependent on GTP or its analogues for activity, binds GTP with high affinity in the presence of dimyristoylphosphatidylcholine/cholate and contains consensus sequences for GTP-binding and hydrolysis. Six mammalian family members have been identified which have been classified into three groups (Class I, II, and III) based on size, deduced amino acid sequence identity, phylogenetic analysis and gene structure. ARFs are ubiquitous among eukaryotes, with a deduced amino acid sequence that is highly conserved across diverse species. They have recently been shown to associate with phospholipid and Golgi membranes in a GTP-dependent manner and are involved in regulating vesicular transport.
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
Biochimica et Biophysica Acta (BBA) - General Subjects, 1984
We have found in water-soluble extracts of rat liver (and RL-PR-C cloned rat hepatocytes), prepared in the absence of detergent, a factor that markedly enhances basal, isoproterenol and cholera toxin activation of adenylate cyclase of rigorously washed hepatocyte membranes, in the absence of added GTP. The factor, which has characteristics of a protein with an Mr of approx. 35000, has been fractionated from crude cytosol by gel filtration, and then further purified over 50-fold by sequential ion-exchange chromatography. The site of action of the protein appears to be at the level of the guanine nucleotide regulatory (G) protein of the plasma membrane adenylate cyclase complex, as the factor, cooperatively with GTP, also permitted cholera toxin to ADP-ribosylate (from 32P-labeled NAD) two integral membrane proteins that migrated on SDS-polyacrylamide gel electrophoresis gels with the mobilities (Mr approx. 46 000 and 48 000) generally observed for the guanine nucleotide regulator protein subunits. In this system, isoproterenol did not stimulate ADP-ribosylation, in either the presence or absence of the liver protein factor.