G protein specificity (original) (raw)
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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.
Proceedings of the National Academy of Sciences, 2000
Inwardly rectifying potassium (K ؉) channels gated by G proteins (Kir3.x family) are widely distributed in neuronal, atrial, and endocrine tissues and play key roles in generating late inhibitory postsynaptic potentials, slowing the heart rate and modulating hormone release. They are directly activated by G ␥ subunits released from G protein heterotrimers of the G i/o family upon appropriate receptor stimulation. Here we examine the role of isoforms of pertussis toxin (PTx)-sensitive G protein ␣ subunits (Gi␣1-3 and Go␣A) in mediating coupling between various receptor systems (A 1, ␣2A, D2S, M4, GABA B1a؉2, and GABAB1b؉2) and the cloned counterpart of the neuronal channel (Kir3.1؉3.2A). The expression of mutant PTx-resistant G i/o␣ subunits in PTx-treated HEK293 cells stably expressing Kir3.1؉3.2A allows us to selectively investigate that coupling. We find that, for those receptors (A 1, ␣2A) known to interact with all isoforms, Gi␣1-3 and Go␣A can all support a significant degree of coupling to Kir3.1؉3.2A. The M 4 receptor appears to preferentially couple to G i␣2 while another group of receptors (D2S, GABAB1a؉2, GABA B1b؉2) activates the channel predominantly through G␥ liberated from G oA heterotrimers. Interestingly, we have also found a distinct difference in G protein coupling between the two splice variants of GABAB1. Our data reveal selective pathways of receptor activation through different G i/o␣ isoforms for stimulation of the G protein-gated inwardly rectifying K ؉ channel.
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.
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
The CGRP receptor can couple via pertussis toxin sensitive and insensitive G proteins
FEBS Letters, 1998
Swiss 3T3 cell lines were constructed co-expressing receptor activity modifying protein (RAMP) 1 with the calcitonin receptor-like receptor (CRLR), and showed IPS I-calcitonin-generelated peptide (CGRP) 1 binding indicative of a type I CGRP receptor. Application of CGRP1 led to an increase in cAMP, which in 2/5 cell lines was augmented following pertussis toxin (PTX) pre-treatment. In Xenopus oocytes, expression of RAMP1, which potentiates an endogenous CGRP receptor, led to constitutive activation of co-expressed GIRK potassium channels. This potassium current was increased following CGRP application or co-expression of CRLR, but decreased by PTX or co-expression of transducin. We conclude that the CGRP receptor can signal to both PTX sensitive and insensitive G proteins.
The Journal of biological chemistry, 1991
Molecular cloning of cDNAs encoding alpha-subunits of guanine nucleotide-binding regulatory proteins (G-proteins) has revealed the existence of nine species of alpha-subunits. We have identified two additional G-protein alpha-subunits, which we refer to as GL1 alpha and GL2 alpha, by isolating bovine liver cDNA clones that cross-hybridized at reduced stringency with bovine Gi1 alpha-subunit cDNA. The deduced amino acid sequences of GL1 alpha and GL2 alpha share 83% identity with each other and show 45-55% identity with those of other known G-protein alpha-subunits. Both GL1 alpha and GL2 alpha lack a consensus site for ADP-ribosylation by pertussis toxin. Messenger RNA corresponding to GL2 alpha was detected in all tissues examined, but GL1 alpha mRNA was detected only in liver, lung, and kidney. Antiserum prepared against a synthetic pentadecapeptide corresponding to the deduced carboxyl terminus of GL2 alpha specifically reacted with a 40-kDa protein in mouse liver, brain, lung, h...
Regional effects of pertussis toxin in vivo and in vitro on GABAB receptor binding in rat brain
Neuroscience, 1993
Al~raet--Agonist binding to GABA a receptors modulates the activity of the guanine nucleotide binding proteins, Go and Gi. These G proteins are ADP-ribosylated by pertussis toxin and this prevents them from coupling to the GABA e receptor resulting in a reduction in high-alfmity GABA B binding. GTP, which binds to a different site on the G protein ~t subunit, also reduces the affinity of the receptor for the G protein, and this can be used as a "marker" for G protein-GABA e receptor linkage. We have examined GABA e binding site distribution in rat brain after unilateral intrahippocampal pcrtussis toxin injection in vivo, and after incubating brain slices in pertussis toxin/n vitro, using the technique of receptor autoradiography. The effect of pcrtussis toxin was compared with that of GTPTS on GABAB binding.
FEBS Letters, 2000
We previously demonstrated the functional coupling of the rat neurotensin receptor NTS1 with G‐proteins on transfected CHO cell homogenates by showing modulation of agonist affinity by guanylyl nucleotides and agonist‐mediated stimulation of [35S]GTPγS binding. In the present study, we observed that Gi/o‐type G‐protein inactivation by pertussis toxin (PTx) resulted in a dramatic reduction of the NT‐induced [35S]GTPγS binding whereas the effect of guanylyl nucleotide was almost not affected. As expected, NT‐mediated phosphoinositide hydrolysis and intracellular calcium mobilization were not altered after PTx treatment. This suggests the existence of multiple signaling cascades activated by NT. Accordingly, using PTx and the PLC inhibitor U‐73122, we showed that both signaling pathways contribute to the NT‐mediated production of arachidonic acid. These results support evidence for a dual coupling of the NTS1 with PTx‐sensitive and insensitive G‐proteins.
The Journal of Physiology, 2003
In neuronal cells, the influx of Ca2+ ions through voltage‐dependent L‐type calcium (L) channels couples excitation to multiple cellular functions. In addition to voltage, several neurotransmitters, hormones and cytokines regulate L channel gating via binding to G‐protein‐coupled receptors. Intracellular molecules that modify G‐protein activity – such as regulator of G‐protein‐signalling (RGS) proteins – are therefore potential candidates for regulating Ca2+ influx through L channels. Here we show that a novel RGS2 splice variant from chick dorsal root ganglion (DRG) neurons, RGS2L, reduces bradykinin (BK)‐mediated inhibition of neuronal L channels and accelerates recovery from inhibition. Chick RGS2 reduces the inhibition mediated by both the pertussis toxin (PTX)‐sensitive (Gi/o‐coupled) and the PTX‐insensitive (presumably Gq/11‐coupled) pathways. However, we demonstrate for the first time in a living cell that the extent of coupling to each pathway varies with RGS2L concentration...