Coupling of muscarinic receptors to GTP proteins in postmortem human brain — alterations in Alzheimer's disease (original) (raw)
Related papers
Neurodegeneration, 1996
THE ACETYLCHOLINE MUSCARINIC receptor family consists of five different gene products that activate different signal transduction pathways via intermediate GTP binding 'G'-proteins. The m2 and m4 receptors are coupled via pertussis toxin-sensitive G-proteins to the inhibition of adenylyl cyclase activity (Peralta et al., 1988), whereas the m1, m3 and m5 receptors act via pertussis toxin-insensitive G-proteins to stimulate phospholipases C, D and A2 (Conklin et al., 1988; Peralta et al., 1988; Sandman et al., 1991). The m1, m2 and m3 gene products correspond, respectively, to the pharmacologically defined M1, M2 and M3 receptor types. The idea that disrupted acetylcholine muscarinic receptor-mediated signal transduction could limit the success of cholinergic replacement therapies for Alzheimer's disease dementia (Fowler et al., 1990; Flynn et al., 1991) has provided the impetus for a number of studies to determine the functional integrity of muscarinic receptors in this disorder. When investigating the effects of guanylylimidodiphosphate (Gpp[NH]p) on carbachol agonist displacement of [ 3 H]pirenzepine antagonist binding, Smith et al. and Flynn and colleagues found that the ability of putative muscarinic M1 receptors to form high affinity agonistreceptor complexes with G-proteins was impaired in Alzheimer's disease parietal (Smith et al., 1987) and pre-frontal (Flynn et al., 1991) cortices. Muscarinic receptor-G-protein interactions have also been suggested to be disrupted in Alzheimer's disease thalamus (Warpman et al., 1993), whereas others have
Neuroscience …, 1995
The efficacy of acetylcholine muscarinic M2 receptor-G-protein coupling was investigated in Alzheimer's disease and control neocortical membranes by measuring the effects of MgC12 and 5'-guanylylimidodiphosphate (Gpp[NH]p) on high-affinity [3H]oxotremorine-M ([3H]OXO-M) binding. MgC12 gave similar enhancements of [3H]OXO-M binding in Alzheimer's disease and control occipital cortex. In contrast, MgC12 enhanced [3H]OXO-M binding was significantly higher in Alzheimer's disease superior temporal cortex, compared to controls. MgCl* enhanced [3H]OXO-M binding in both the occipital and temporal cortices of the Alzheimer's disease cases was reversed to control levels by Gpp[NH]p. It is concluded that the number of high-affinity muscarinic M2 sites is increased in Alzheimer's disease superior temporal, but not occipital, cortex and that M2 sites in both regions maintain an efficient Gprotein coupling.
Neuroscience Letters, 1995
The efficacy of acetylcholine muscarinic M2 receptor-G-protein coupling was investigated in Alzheimer's disease and control neocortical membranes by measuring the effects of MgC12 and 5'-guanylylimidodiphosphate (Gpp[NH]p) on high-affinity [3H]oxotremorine-M ([3H]OXO-M) binding. MgC12 gave similar enhancements of [3H]OXO-M binding in Alzheimer's disease and control occipital cortex. In contrast, MgC12 enhanced [3H]OXO-M binding was significantly higher in Alzheimer's disease superior temporal cortex, compared to controls. MgCl* enhanced [3H]OXO-M binding in both the occipital and temporal cortices of the Alzheimer's disease cases was reversed to control levels by Gpp[NH]p. It is concluded that the number of high-affinity muscarinic M2 sites is increased in Alzheimer's disease superior temporal, but not occipital, cortex and that M2 sites in both regions maintain an efficient Gprotein coupling.
Regulation of muscarinic receptor binding by guanine nucleotides and N-ethylmaleimide
Journal of supramolecular structure, 1980
The regulation of muscarinic receptor binding by guanine nucleotides and N-ethylmaleimide (NEM) was investigated using the agonist ligand, [3H] cis methyldioxolane ([3H]CD). Characterization studies on rat forebrain homogenates show that [3H]CD binding was linear with tissue concentration and was unaffected by a change in pH from 5.5 to 8.0. The regional variation in [3H]CD binding in the rat brain correlated generally wit [3H](-)3-quinuclidinyl benzilate ([3H](-)QNB) binding, although the absolute variation in binding was somewhat less. At a concentration of 100 microM, the GTP analogue, guanyl-5'-yl imidodiphosphate [Gpp(NH)p], caused a 43-77% inhibition of [3H]CD binding in the corpus striatum, ileum, and heart. The results of binding studies using several Gpp(NH)p concentrations demonstrated that the potency of this guanine nucleotide for inhibition of [3H]CD binding, Gpp(NH)p caused an increase in (3H](-)QNB binding in the heart heart and ileum and no change in [3H](-)QNB b...
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.
Preservation of acetylcholine muscarinic M2 receptor G-protein interactions 1995
The efficacy of acetylcholine muscarinic M2 receptor-G-protein coupling was investigated in Alzheimer's disease and control neocortical membranes by measuring the effects of MgC12 and 5'-guanylylimidodiphosphate Gpp[NH]p) on high-affinity [3H]oxotremorine-M ([3H]OXO-M) binding. MgC12 gave similar enhancements of [3H]OXO-M binding in Alzheimer's disease and control occipital cortex. In contrast, MgC12 enhanced [3H]OXO-M binding was significantly higher in Alzheimer's disease superior temporal cortex, compared to controls. MgCl* enhanced [3H]OXO-M binding in both the occipital and temporal cortices of the Alzheimer's disease cases was reversed to control levels by Gpp[NH]p. It is concluded that the number of high-affinity muscarinic M2 sites is increased in Alzheimer's disease superior temporal, but not occipital, cortex and that M2 sites in both regions maintain an efficient Gprotein coupling. Despite convincing evidence that the early degenera-0304-3940/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(95)11281-W
Brain Research, 1991
The coupling of aa-adrenoceptors to guanine nucleotide binding G-proteins was investigated in cerebral cortical membranes from control and Alzheimer's disease brain by characterizing the effects of MnCI 2 and Gpp[NH]p on [3H]clonidine binding. The manganese induced elevation of [3H]clonidine binding was apparent in both control and Alzheimer's disease samples and the effect showed no significant difference between the two groups in the frontal cortex. However, the MnC12 concentration dependence curves for control and Alzheimer's disease samples were significantly different from one another in the temporal cortex, whereas the pattern of stimulation by MnCI 2 remained the same. The guanine nucleotide analogue, Gpp[NH]p inhibited [3H]clonidine binding in a concentration-dependent manner, the profiles of inhibition showing no significant differences between control and Alzheimer's disease samples. Analysis of the effect of Gpp[NH]p on [3H]clonidine saturation binding curves showed no significant differences between control and Alzheimer's disease samples in either frontal (K d = 9.68-+ 1.38, 9.1-+ 2.6 nM; Bma x = 40.23 _+ 4.33, 44.3 • 9.4 fmol/mg, control and Alzheimer's disease values, respectively), or temporal (Ka = 11.61 +-4.04, 5.38-+ 2.5 nM; Bma x = 52.0-+ 14.0, 31.07-8.00 fmol/mg control and Alzheimer's disease values, respectively) cortices. [3H]Clonidine binding parameters, measured in the presence of manganese were significantly different in the temporal cortex in Alzheimer's disease compared to control samples, (K d = 0.53-0.06, 1.02 _+ 0.16 nM; Bm~x = 46.22 _+ 2.58, 82.41-+ 5.48 fmol/mg, control and Alzheimer's disease values, respectively), whereas in the frontal cortex binding parameters, in the presence of manganese, remained the same between groups (K a = 0.61 4-0.05, 0.64 _+ 0.11 nM; Bma x = 52.35-+ 2.0, 50.50-+ 4.27 fmol/mg, control and Alzheimer's disease values, respectively). It is concluded that there are no gross disturbances in the a 2-adrenoceptor-G-protein complex in Alzheimer's disease brain.
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
Archives of Gerontology and Geriatrics, 1993
Receptor binding assays and in vitro macroautoradiography were used to analyze muscarinic cholinergic receptors (MCR) in the cerebral frontal cortex of Alzheimer's disease (AD), senile dementia of Alzheimer type (SDAT), and age-matched control brains at autopsy. Total MCR binding, detected by [3H]quiniclinidyl benzilate binding, did not differ significantly between the 3 groups. The concentrations of MI subtype (MI-R), detected by [3H]pirenzepine binding, and high affinity state MCRs, however, were significantly lower in AD than in control and SDAT frontal cortices. No differences were detected in the affinity of these receptors for their ligands. The MCRs in AD frontal cortex were more sensitive to the agonist carbachol than were control MCRs. Autoradiography revealed a complete destruction of the laminar distribution of MCR and MI-R in AD and SDAT frontal cortices. Forskolin and phorbol ester binding sites, used to analyze second messenger systems, were significantly and markedly reduced in AD frontal cortex. In addition, coupling between MCR and second messenger systems was supersensitive in AD frontal cortex. Our findings that there are alterations in the structural distribution of MCR as well as reductions and abnormalities in second messenger systems in AD cerebral frontal cortex, suggest that drug therapy with acetylcholine precursors, choline esterase inhibitors and muscarinic agonists cannot eliminate symptoms in dementia patients. Furthermore, they point out the need for techniques to diagnose the disease prior to disintegration of the neuronal network, and the need for therapies to delay or prevent the progression of structural changes.
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.