Characterization of M 2 muscarinic receptor activation of different G protein subtypes (original) (raw)
Related papers
The EMBO Journal, 1990
Communicated by P.Seeburg Relatively little is understood concerning the mechanisms by which subtypes of receptors, G proteins and effector enzymes interact to transduce specific signals. Through expression of normal, hybrid and deletion mutant receptors in Xenopus oocytes, we determined the G protein coupling characteristics of the functionally distinct m2 and m3 muscarinic acetylcholine receptor (mAChR) subtypes and identified the critical receptor sequences responsible for G protein specificity. Activation of a pertussis toxin insensitive G protein pathway, leading to a rapid and transient release of intracellular Ca2+ characteristic of the m3 receptor, could be specified by the transfer of as few as nine amino acids from the m3 to the m2 receptor. In a reciprocal manner, transfer of no more than 21 residues from the m2 to the m3 receptor was sufficient to specify activation of a pertussis toxin sensitive G protein coupled to a slow and oscillatory Ca2' release pathway typical of the m2 subtype. Notably, these critical residues occur within the same region of the third cytoplasmic domain of functionally distinct mAChR subtypes.
FEBS Letters, 1990
Muscarinic acetylcholine receptors purified from porcine atrium were phosphorylated, depending on the presence of agonists, by a protein kinase partially purified from porcine brain, which had similar properties to the)%adrenergic receptor kinase. GTP-binding regulatory proteins (Go) had dual effects on the phosphorylation of muscarinic receptors, i.e. stimulation at lower concentrations and inhibition at higher concentrations. The stimulatory effect was reproduced with the By subunit of Go and the inhibitory effect with the combination of the a and By subunits.
Journal of Biological Chemistry
Muscarinic acetylcholine receptors purified from porcine brain were reconstituted with two kinds of GTP-binding proteins (Gi and Go). The binding of agonists was affected by guanine nucleotides when the receptor was reconstituted with either Gi or Go, but not in the absence of one of the GTP-binding proteins. The displacement curves with agonists for the [ 3 H ] q~i n~clidinyl benzylate ([3H]QNB) binding were explained by assuming there are two sites with different affinities for a given agonist. The proportion of the high affinity site increased with increasing concentrations of the GTP-binding proteins, and the maximum value represented 50-70% of the total [3H]QNB-binding sites. Reconstitution of the receptor with both Gi and Go did not increase the proportion any further. These results indicate that Gi and Go interact with the same site, which rules out the possibility that there are two kinds of muscarinic receptors, one interacting with Gi and the other with Go. GDP as well as GTP decreased the affinity for the agonists of the muscarinic receptors reconstituted with Gi or Go. The conversion of GDP to GTP during the incubation was less than 1%, indicating that the effect of GDP is not due to its conversion to GTP, and that the binding of either GTP or GDP with the GTP-binding proteins suppresses their interaction with the receptor. Muscarinic agonists are known to induce different responses including (a) increases in the cGMP level, (b) decreases in the CAMP level, (c) breakdown of polyphosphoinositides, and (d) changes in ion permeability. Recently, several lines of evidence have accumulated that indicate that the activation of these reactions by various receptors, including muscarinic receptors, is mediated through activation of GTP-binding proteins (G-proteins'). Adenylate cyclase is known to be inhibited by an inhibitory G-protein (Gi or Ni) (see review in Ref. 1). The breakdown of polyphosphoinosi
Molecular Pharmacology, 2011
We have used alanine-scanning mutagenesis followed by functional expression and molecular modeling to analyze the roles of the 14 residues, Asn422 to Cys435, C-terminal to transmembrane (TM) helix 7 of the M 1 muscarinic acetylcholine receptor. The results suggest that they form an eighth (H8) helix, associated with the cytoplasmic surface of the cell membrane in the active state of the receptor. We suggest that the amide side chain of Asn422 may act as a cap to the C terminus of TM7, stabilizing its junction with H8, whereas the side chain of Phe429 may restrict the relative movements of H8 and the C terminus of TM7 in the inactive ground state of the receptor. We have identified four residues, Phe425, Arg426, Thr428, and Leu432, which are important for G protein binding and signaling. These may form a docking site for the C-terminal helix of the G protein ␣ subunit, and collaborate with G protein recognition residues elsewhere in the cytoplasmic domain of the receptor to form a coherent surface for G protein binding in the activated state of the receptor.
Neuroscience Research Supplements, 1988
Musca~inic receptors were purified from porcine cerebrum an@ atrium (a specific [ H]QNB binding activity, 2-4 nmol/ mg of protein). A new G protein (tentatively termed Gn) was separated from other G proteins, Gi and Go, by DEAE-Toyopearl column chromatography and biochemical analysis indicated that Gn is a novel protein distinct from Gi or Go, but not a proteolytic fragment of them. Muscarinic receptors reconstituted with G proteins showed GTP-sensitive high affinity for acetylcholine, irrespective of the species of G proteins and origin of receptors. The proportion of the high affinity sites increased with the concentration of G proteins up to 80% of the total sites, the order of potency being Go, Gi and Gn. The GTP-sensitive high affinity binding was not observed when each G protein was pretreated with IAP. These results indicate that both cerebral and atrial receptors, which are probably composed of a mixture of four subtypes and predominantly a single subtype respectively, are capable of interacting with three different G proteins and activating them in an IAP-sensitive manner. The results also suggest that if a single receptor subtype interacts with a single kind of G protein in situ, there must be some mechanism(s) or contribution of other component(s) that allows only the specific interaction. Muscarinic receptors purified from porcine brain were phosphorylated by protein kinase C purified from the same tissue. Alpha subunits of Gi, Go and Gn and gamma subunits of Gi and Go but not Gn were also phosphorylated by the same enzyme. These phosphorylations were independent of the presence of acetylcholine or guanine nucleotides. The GTPsensitive high affinity binding of receptors reconstituted with G proteins were observed irrespective of phosphorylation, indicating that the interaction between the receptor and G protein is not affected by their phosphorylation as far as the interaction between purified receptors and purified G proteins is concerned.
Muscarinic receptors modulating acetylcholine release from insect synaptosomes
Comparative biochemistry and physiology. C, Comparative pharmacology and toxicology, 1989
1. Cholinergic synapses in the central nervous system of insects contain inhibitory muscarinic receptors whose stimulation by agonists leads to a diminished output of acetylcholine; antagonists, like atropine, facilitate acetylcholine release. 2. The receptors involved appear to be of the M2-subtype. Upon activation of presynaptic receptors a significant reduction of the intrasynaptosomal cyclic AMP level as well as a significantly increased membrane potential was observed. 3. The observed membrane hyperpolarization is apparently not a consequence of a lower cyclic AMP level, thus both effects may offer alternative or synergistical mechanisms for modulating transmitter release.
Journal of biochemistry, 2014
We expressed the fusion proteins of the muscarinic acetylcholine receptor M2 subtype (M2 receptor) with a maltose-binding protein (MBP) and various G protein α subunits (Gα(i1-i3/o)) at its N- and C-terminals, respectively (MBP-M2-Gα(i/o)), in Escherichia coli, and examined the effect of G protein βγ subunits (Gβγ) on the receptor-Gα interaction as assessed by agonist- and GDP-dependent [(35)S]GTPγS binding of the fusion proteins. We found that (i) Gβγ promoted both the agonist-dependent and -independent [(35)S]GTPγS binding with little effect on the guanine nucleotide-sensitive high-affinity agonist binding, (ii) the specific [(35)S]GTPγS binding activity was much greater for MBP-M2-Gα(oA) than for MBP-M2-Gα(i1-i3) in the absence of Gβγ, whereas Gβγ preferentially promoted the agonist-dependent decrease in the affinity for GDP of MBP-M2-Gα(i1-i3) rather than of MBP-M2-Gα(oA), and (iii) the proportion of agonist-dependent [(35)S]GTPγS binding was roughly 50% irrespective of species ...
Two types of muscarinic acetylcholine receptors in Drosophila and other arthropods
Cellular and Molecular Life Sciences, 2013
482: 547-551) published the crystal structure of the human m2 mAChR, revealing 14 amino acid residues forming the binding pocket for QNB. These residues are identical between the human m2 and the D. melanogaster and T. castaneum A-type mAChRs, while many of them are different between the human m2 and the B-type receptors. Using bioinformatics, one orthologue of the A-type and one of the B-type mAChRs could also be found in all other arthropods with a sequenced genome. Protostomes, such as arthropods, and deuterostomes, such as mammals and other vertebrates, belong to two evolutionarily distinct lineages of animal evolution that split about 700 million years ago. We found that animals that originated before this split, such as cnidarians (Hydra), had two A-type mAChRs. From these data we propose a model for the evolution of mAChRs.
Naunyn-Schmiedeberg's Archives of Pharmacology, 1992
The non-selective labelled antagonist [3H]Nmethyl-scopolamine ([3H]NMS) was used to identify muscarinic acetylcholine receptors in rat duodenum smooth muscle membranes. Saturation and kinetic experiments revealed a binding site with a Ko-value of 0.2-0.3nmol/1 and a receptor concentration (Bmax) of 100 fmol/mg protein. The affinities of eight selective muscarinic antagonists were determined and compared with those at M1 (rat cerebral cortex), M2 (rat heart), M3 (rat submandibular gland) and M4 (data from D6rje et al. 1991) receptors. The "M2-selective" agent AF-DX 116, the group of "M2/M4-selective" compounds himbacine, AF-DX 384, AQ-RA 741 and methoctramine but also the "M3-selective" HHSiD showed affinities corresponding to M2 and/or M~ sites. The intermediate affinity of 4-DAMP favours a mixed Mz/M¢ receptor population mainly containing M2 receptors. Two compounds, pirenzepine and AQ-RA 741, displayed biphasic displacement curves indicating the presence of a small population of putative M 1 receptors. The rat duodenum antagonist binding profile, however, is not consistent with the presence of M3 receptors. We further demonstrate a concentration-dependent stimulation of [35S]GTP[S] binding to duodenal G proteins by the muscarinic agonist oxotremorine. Estimation of the binding parameters of GTP[S] in absence and presence of oxotremorine provided evidence for a catalytic activation of G proteins by agonist-activated muscarinic receptors in rat duodenal membranes and a strong signal amplification on the G protein level.
Phosphorylation of muscarinic receptors: regulation by G proteins
Life Sciences, 1993
Effects of G proteins on the phosphorylation of muscarinic receptors (mAChRs) have been examined. Cerebral but not atrial mAChRs were phosphorylated by any one of three types of protein kinase C and 4-6 mol of phosphate were incorporated per mol of mAChR, mostly in the 12-14 kDa from the carboxyterminus. Atrial mAChRs were better substrates of cAMP-dependent protein kinase than cerebral mAChRs. Phosphorylation of mAChRs by protein kinase C or cAMP-dependent protein kinase was not dependent on the presence of agonists and G proteins except that a slight inhibition by G proteins was observed probably because G proteins were also substrates of the two kinases. Agonistdependent phosphorylation of atrial mAChRs or recombinant human mAChRs (m2 subtype) by a kinase (mAChR kinase), which is the same or very similar to 15 adrenergic receptor kinase (15ARK), was found to be regulated by the G proteins in a dual manner; stimulation by G protein ~y subunits and inhibition by G protein cc15y trimer. The inhibition by the G protein trimer is restored by addition of guanine nucleotides and is considered to be due to the formation of a ternary complex of agonist, mAChR and guanine nucleotide free G proteins. The stimulation by G protein 15Y subunits was also observed for the light-or agonist-dependent phosphorylation of rhodopsin and 15AR by the mAChR kinase but not for the lightdependent phosphorylation of rhodopsin by rhodopsin kinase. The phosphorylation by 15ARK 1 was also found to be stimulated by G protein 15v subunits. The 15v subunit is considered to interact with the extra 130 amino acid residue carboxyterminal tail of 15ARK, which does not exist in rhodopsin kinase, and the interaction results in the activation of the kinase. We may assume that the G protein coupled receptor kinase is an effector of G protein 15v subunits and that one of the functions of 15y subunits is to stimulate the phosphorylation of G protein coupled receptors thereby facilitating their desensitization.