Subtype Differences in Pre-Coupling of Muscarinic Acetylcholine Receptors (original) (raw)
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Proceedings of the National Academy of Sciences, 1993
We have tested the hypothesis that guaninenucleotide-binding-protein-coupled receptors may be able to interact with each other at a molecular level. To address this question, we have initially created two chimeric receptors, a2/m3 and m3/a2, in which the C-terminal receptor portions (containing transmembrane domains VI and VII) were exchanged between the a2c-adrenergic and the m3 muscarinic receptor. Transfection of COS-7 cells with either of the two chimeric constructs alone did not result in any detectable binding activity for the muscarinic ligand N-[3H]methylscopolamine or the adrenergic ligand [3H]rauwolscine. However, cotransfection with a2/m3 and m3/a2 resulted in the appear-
Characterization of M 2 muscarinic receptor activation of different G protein subtypes
Neurochemistry International, 2004
The M2 muscarinic acetylcholine receptor (mAChR) expressed in insect cells (Spodoptera frugiperda, Sf9) using the baculovirus system formed active functional complexes with coexpressed Gi as well as with Go proteins, while no complexes could be detected with internal G proteins. Comparison of the abilities of different muscarinic agonists and partial agonists to increase [35S]GTPγS binding revealed no significant differences between
Agonist binding to M1 muscarinic receptors is sensitive to guanine nucleotides
European Journal of Pharmacology: Molecular Pharmacology, 1989
Putative M 1 (high-affinity pirenzepine) muscarinic receptors in rabbit hippocampal membranes, treated with 0.1 mM N-ethylmaleimide (NEM), were selectively labeled with [3H]pirenzepine. A single class of binding sites was labeled with a K d of 3.4 nM, consistent with the pharmacologically-defined M 1 subtype of muscarinic receptors. While full muscarinic agonists bound to high-and low-affinity states of [3H]pirenzepine-labeled M1 sites with a KL/K H ratio of approximately 100, the ratio for partial muscarinic agonists was approximately 10. The high-affinity binding of all agonists tested required divalent cations, and was interconverted to low-affinity binding in the presence of the non-hydrolyzable GTP analogue, guanylyl imidodiphosphate (GppNHp). Direct labeling of the high-affinity agonist state of M 1 receptors was achieved with 5 nM [3H]oxotremorine-M by selectively uncoupling the high-affinity agonist state of M 2 (low-affinity pirenzepine) receptors with NEM. The rate of dissociation of [3H]Pxotremorine-M from M 1 receptors was accelerated 6-fold by GppNHp. These results provide further evidence which suggests that putative M 1 muscarinic receptors activate second messenger systems by coupling to NEM-insensitive guanine nucleotide-binding proteins.
Muscarinic receptor family interacting proteins: role in receptor function
Journal of neuroscience methods, 2011
a b s t r a c t G protein-coupled receptors constitute one of the most important families of membrane receptors through which cells respond to extracellular stimuli. Receptors of this superfamily likely function as signal transduction complexes. The identification and analysis of their components provide new insights into a better understanding of these receptors' function and regulation. We used tandem-affinity purification and mass spectrometry as a systematic approach to characterize multiprotein complexes in the acetylcholine muscarinic receptor subfamily. To overcome the limitations associated with membrane protein receptor solubilization with detergents, we developed a strategy in which receptors are co-expressed with a cytoplasmic minigene construct, encoding the third intracellular loop and the C-terminal tail tagged to the tandem-affinity-cassette of each receptor subtype. Numerous protein complexes were identified, including many new interactions in various signalling pathways. Systematic identification data set together with protein interactions reported in the literature revealed a high degree of connectivity. These allow the proposal, for the first time, of an outline of the muscarinic interactome as a network of protein complexes and a context for a more reasoned and informed approach to drug discovery and muscarinic receptor subtype specificities.
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.
α-Helical distorting substitutions disrupt coupling between m3 muscarinic receptor and G proteins
FEBS Letters, 1993
Acetylcholme stimulation of the m3 or m2 muscarinic receptor expressed in Xenopus lurris oocytes Induces either a fast transient or slowly oscillating calcmm-sensitive chloride current. The speed of these currents reflects the efficiency of receptor coupling to guanine nucleotide-bmding proteins and phosphatidylinositol (PI) turnover. Point mutations of the m3 receptor were made in a region of the third cytoplasmic loop to test whether receptor function relied on an a-helical structure of the G protein-coupling domain. Prolme substitution for glutamate at position 257 disrupted the m3 response. Also, smgle alanine msertions between residues 259 and 260 disrupted the m3 receptor-stimulated response while double alanme insertions at this site had no effect. Based on these results, we suggest that a region of the third cytoplasmic loop of the m3 receptor possesses an amphipathic a-helical conformation. Xenopus oocyte; G protem-coupling. Amphipathic a-helix; Seven transmembrane-spannmg receptors, Mastoparan *Subtypes of the mAChR are designated as ml, m2, m3. m4 and m5 as defined by the Fourth International Symposium on Subtypes of Muscarimc Receptors.
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.
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.
Evidence for a Tandem Two-site Model of Ligand Binding to Muscarinic Acetylcholine Receptors
Journal of Biological Chemistry, 2000
After short preincubations with N-[ 3 H]methylscopolamine ([ 3 H]NMS) or R(؊)-[ 3 H]quinuclidinyl benzilate ([ 3 H]QNB), radioligand dissociation from muscarinic M 1 receptors in Chinese hamster ovary cell membranes was fast, monoexponential, and independent of the concentration of unlabeled NMS or QNB added to reveal dissociation. After long preincubations, the dissociation was slow, not monoexponential, and inversely related to the concentration of the unlabeled ligand. Apparently, the unlabeled ligand becomes able to associate with the receptor simultaneously with the already bound radioligand if the preincubation lasts for a long period, and to hinder radioligand dissociation. When the membranes were preincubated with [ 3 H]NMS and then exposed to benzilylcholine mustard (covalently binding specific ligand), [ 3 H]NMS dissociation was blocked in wild-type receptors, but not in mutated (D99N) M 1 receptors. Covalently binding [ 3 H]propylbenzilylcholine mustard detected substantially more binding sites than [ 3 H]NMS. The observations support a model in which the receptor binding domain has two tandemly arranged subsites for classical ligands, a peripheral one and a central one. Ligands bind to the peripheral subsite first (binding with lower affinity) and translocate to the central subsite (binding with higher affinity). The peripheral subsite of M 1 receptors may include Asp-99. Experimental data on [ 3 H]NMS and [ 3 H]QNB association and dissociation perfectly agree with the predictions of the tandem two-site model.