scholarly journals Agonist activation of transfected human M1 muscarinic acetylcholine receptors in CHO cells results in down-regulation of both the receptor and the α subunit of the G-protein Gq

1993 ◽  
Vol 289 (1) ◽  
pp. 125-131 ◽  
Author(s):  
I Mullaney ◽  
M W Dodd ◽  
N Buckley ◽  
G Milligan
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Cho Cells ◽  
Inositol Phosphate ◽  
Muscarinic Acetylcholine Receptors ◽  
Down Regulation ◽  
Α Subunit ◽  
Muscarinic Acetylcholine ◽  
Alpha Subunits ◽  
M1 Muscarinic

CHO cells stably transfected with cDNA encoding the human M1 muscarinic acetylcholine (HM1) receptor were treated with the cholinergic agonist carbachol at various concentrations for differing times. Levels of the HM1 receptor and of a range of G-proteins were subsequently measured. Carbachol treatment of the transfected cells caused a substantial down-regulation of cellular levels of the alpha subunit of Gq (Gq alpha), but did not significantly alter cellular levels of the alpha subunits of Gs or Gi2. A small decrease in levels of G-protein beta-subunit was also produced. Parallel assessment of agonist-induced down-regulation of the HM1 receptor demonstrated that it was lost in concert with the G-protein. Similar concentrations of carbachol (5 microM) were required to produce half-maximal stimulation of inositol phosphate generation and loss of each of the HM1 receptor and Gq alpha, and half-maximal losses of both receptor and Gq alpha were produced by 3 h of treatment with 1 mM-carbachol. By contrast, treatment of the non-transfected parental CHO cells, which do not express detectable levels of the receptor, with carbachol had no effect on cellular Gq alpha levels. Concurrent treatment of the HM1-expressing CHO cells with carbachol and cycloheximide indicated that suppression of protein synthesis de novo did not mimic the effect of carbachol, and hence even complete inhibition of transcription of the Gq alpha gene and/or translation of pre-existing Gq alpha mRNA could not account for the agonist-induced effect. We have previously noted that cellular levels of both Gs alpha [McKenzie and Milligan (1990) J. Biol. Chem. 265, 17084-17093] and the alpha subunits of the pertussis-toxin-sensitive G-proteins Gi1, Gi2 and Gi3 [Green, Johnson and Milligan (1990) J. Biol. Chem. 265, 5206-5210] can be regulated in certain cell systems by agonist activation of receptors expected to interact with these G-proteins. These results demonstrate that the same is true of Gq alpha and suggest that agonist-induced co-ordinate loss of receptors and associated G-proteins may be a more common feature than has been appreciated to date.

scholarly journals Kinetics of M1 muscarinic receptor and G protein signaling to phospholipase C in living cells

2010 ◽  
Vol 135 (2) ◽  
pp. 81-97 ◽  
Author(s):  
Björn H. Falkenburger ◽  
Jill B. Jensen ◽  
Bertil Hille
Keyword(s):  
Kinetic Model ◽  
Phospholipase C ◽  
G Protein ◽  
G Proteins ◽  
Quantitative Description ◽  
Muscarinic Acetylcholine Receptors ◽  
Gtpase Activity ◽  
Nucleotide Exchange ◽  
Time Courses ◽  
M1 Muscarinic

G protein–coupled receptors (GPCRs) mediate responses to external stimuli in various cell types. Early events, such as the binding of ligand and G proteins to the receptor, nucleotide exchange (NX), and GTPase activity at the Gα subunit, are common for many different GPCRs. For Gq-coupled M1 muscarinic (acetylcholine) receptors (M1Rs), we recently measured time courses of intermediate steps in the signaling cascade using Förster resonance energy transfer (FRET). The expression of FRET probes changes the density of signaling molecules. To provide a full quantitative description of M1R signaling that includes a simulation of kinetics in native (tsA201) cells, we now determine the density of FRET probes and construct a kinetic model of M1R signaling through Gq to activation of phospholipase C (PLC). Downstream effects on the trace membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) and PIP2-dependent KCNQ2/3 current are considered in our companion paper in this issue (Falkenburger et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.200910345). By calibrating their fluorescence intensity, we found that we selected transfected cells for our experiments with ∼3,000 fluorescently labeled receptors, G proteins, or PLC molecules per µm2 of plasma membrane. Endogenous levels are much lower, 1–40 per µm2. Our kinetic model reproduces the time courses and concentration–response relationships measured by FRET and explains observed delays. It predicts affinities and rate constants that align well with literature values. In native tsA201 cells, much of the delay between ligand binding and PLC activation reflects slow binding of G proteins to receptors. With M1R and Gβ FRET probes overexpressed, 10% of receptors have G proteins bound at rest, rising to 73% in the presence of agonist. In agreement with previous work, the model suggests that binding of PLC to Gαq greatly speeds up NX and GTPase activity, and that PLC is maintained in the active state by cycles of rapid GTP hydrolysis and NX on Gαq subunits bound to PLC.

scholarly journals Understanding G Protein Selectivity of Muscarinic Acetylcholine Receptors Using Computational Methods

2019 ◽  
Vol 20 (21) ◽  
pp. 5290
Author(s):  
Luis Santiago ◽  
Ravinder Abrol
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Protein Interactions ◽  
Intracellular Signaling ◽  
Acetylcholine Receptors ◽  
Experimental Studies ◽  
Muscarinic Acetylcholine Receptors ◽  
Structural Basis ◽  
Receptor Subtypes ◽  
Muscarinic Acetylcholine

The neurotransmitter molecule acetylcholine is capable of activating five muscarinic acetylcholine receptors, M1 through M5, which belong to the superfamily of G-protein-coupled receptors (GPCRs). These five receptors share high sequence and structure homology; however, the M1, M3, and M5 receptor subtypes signal preferentially through the Gαq/11 subset of G proteins, whereas the M2 and M4 receptor subtypes signal through the Gαi/o subset of G proteins, resulting in very different intracellular signaling cascades and physiological effects. The structural basis for this innate ability of the M1/M3/M5 set of receptors and the highly homologous M2/M4 set of receptors to couple to different G proteins is poorly understood. In this study, we used molecular dynamics (MD) simulations coupled with thermodynamic analyses of M1 and M2 receptors coupled to both Gαi and Gαq to understand the structural basis of the M1 receptor’s preference for the Gαq protein and the M2 receptor’s preference for the Gαi protein. The MD studies showed that the M1 and M2 receptors can couple to both Gα proteins such that the M1 receptor engages with the two Gα proteins in slightly different orientations and the M2 receptor engages with the two Gα proteins in the same orientation. Thermodynamic studies of the free energy of binding of the receptors to the Gα proteins showed that the M1 and M2 receptors bind more strongly to their cognate Gα proteins compared to their non-cognate ones, which is in line with previous experimental studies on the M3 receptor. A detailed analysis of receptor–G protein interactions showed some cognate-complex-specific interactions for the M2:Gαi complex; however, G protein selectivity determinants are spread over a large overlapping subset of residues. Conserved interaction between transmembrane helices 5 and 6 far away from the G-protein-binding receptor interface was found only in the two cognate complexes and not in the non-cognate complexes. An analysis of residues implicated previously in G protein selectivity, in light of the cognate and non-cognate structures, shaded a more nuanced role of those residues in affecting G protein selectivity. The simulation of both cognate and non-cognate receptor–G protein complexes fills a structural gap due to difficulties in determining non-cognate complex structures and provides an enhanced framework to probe the mechanisms of G protein selectivity exhibited by most GPCRs.

Down-regulation of the G-proteins Gqα and G11α by transfected human M3 muscarinic acetylcholine receptors in Chinese hamster ovary cells is independent of receptor down-regulation

1995 ◽  
Vol 310 (2) ◽  
pp. 559-563 ◽  
Author(s):  
E van de Westerlo ◽  
J Yang ◽  
C Logsdon ◽  
J A Williams
Keyword(s):  
G Proteins ◽  
Chinese Hamster Ovary ◽  
Acetylcholine Receptors ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Muscarinic Acetylcholine Receptors ◽  
Phosphoinositide Hydrolysis ◽  
Down Regulation ◽  
Ovary Cells ◽  
Muscarinic Acetylcholine

Chinese hamster ovary cells stably transfected with human M3 muscarinic acetylcholine receptors show a 40-50% reduction in the immunoreactive G-proteins Gq alpha and G11 alpha when stimulated with the cholinergic agonist carbachol. This effect is seen after 9 h, is maximal after 24 h, and occurs over a range of carbachol concentrations that activate phosphoinositide hydrolysis in these cells. The effect is specific for Gq alpha family proteins as Gs alpha was slightly increased after carbachol treatment and G13 alpha was unchanged. Using a urea gel system, we were able to resolve Gq alpha and G11 alpha, both of which were down-regulated by carbachol. An M3 receptor mutant, with C-terminal threonines changed to alanines as described previously, binds ligand and activates phosphoinositide hydrolysis normally but is not down-regulated in response to carbachol. This receptor, however, induces Gq alpha/G11 alpha down-regulation similarly to wild-type M3 receptors, indicating that G-protein down-regulation is not directly coupled to receptor down-regulation. Thus down-regulation of Gq alpha and G11 alpha may contribute to heterologous desensitization particularly at longer times of agonist exposure.

Characterization of the gonadotrophin-releasing hormone receptor in αT3-1 pituitary gonadotroph cells

1993 ◽  
Vol 136 (1) ◽  
pp. 51-NP ◽  
Author(s):  
L. Anderson ◽  
G. Milligan ◽  
K. A. Eidne
Keyword(s):  
G Protein ◽  
Inositol Phosphate ◽  
Rank Order ◽  
Second Messenger ◽  
Time Dependent ◽  
Binding Studies ◽  
Gnrh Analogues ◽  
Α Subunit ◽  
Releasing Hormone ◽  
Gonadotrophin Releasing Hormone

ABSTRACT The present study has characterized the gonadotrophin-releasing hormone (GnRH) receptor in immortalized αT3-1 pituitary gonadotroph cells. GnRH and GnRH analogues produced both a dose- and time-dependent increase in total inositol phosphate (IP) accumulation. The rank order of potency of these analogues was the same as that obtained in parallel receptor-binding studies in αT3-1 cells. These responses were abolished following pretreatment with a GnRH antagonist. The use of a specific inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) assay demonstrated a rapid but short-lived rise in Ins(1,4,5)P3 production. Intracellular calcium ([Ca2+]i) was subsequently measured in αT3-1 cells using dual wavelength fluorescence microscopy combined with dynamic video imaging. GnRH produced a biphasic rise in [Ca2+]i. The initial calcium transient was complete within seconds while the smaller secondary plateau phase lasted several minutes. G-protein involvement in the IP response to GnRH in αT3-1 cells was investigated using sodium fluoride (NaF) and pertussis toxin (PTx) which activate and inactivate G-proteins respectively. Like GnRH, NaF produced a dose- and time-dependent increase in IP accumulation. Activation of phospholipase C in these cells by either GnRH or NaF was PTx-insensitive, suggesting that the G-protein involved was neither Gi nor Go but more probably Gq. Immunoblot analysis of αT3-1 cell membranes using antisera raised against the predicted C-terminal decapeptide of the α subunit of Gq demonstrated the presence of Gq in αT3-1 cells. Collectively these results show that the GnRH receptors expressed in αT3-1 cells are coupled to the phosphatidylinositol second messenger pathway via a specific G-protein. αT3-1 therefore represents a convenient model in which to study GnRH-related second messenger pathways. Journal of Endocrinology (1993) 136, 51–58

New thoughts on the role of the βγ subunit in G protein signal transduction

2000 ◽  
Vol 78 (5) ◽  
pp. 537-550 ◽  
Author(s):  
Barbara Vanderbeld ◽  
Gregory M Kelly
Keyword(s):  
Signal Transduction ◽  
G Protein ◽  
G Proteins ◽  
G Protein Coupled Receptors ◽  
Limited Information ◽  
Α Subunit ◽  
Downstream Effectors ◽  
Receptor Signal ◽  
G Protein Coupled

Heterotrimeric G proteins are involved in numerous biological processes, where they mediate signal transduction from agonist-bound G-protein-coupled receptors to a variety of intracellular effector molecules and ion channels. G proteins consist of two signaling moieties: a GTP-bound α subunit and a βγ heterodimer. The βγ dimer, recently credited as a significant modulator of G-protein-mediated cellular responses, is postulated to be a major determinant of signaling fidelity between G-protein-coupled receptors and downstream effectors. In this review we have focused on the role of βγ signaling and have included examples to demonstrate the heterogeneity in the heterodimer composition and its implications in signaling fidelity. We also present an overview of some of the effectors regulated by βγ and draw attention to the fact that, although G proteins and their associated receptors play an instrumental role in development, there is rather limited information on βγ signaling in embryogenesis.Key words: G protein, βγ subunit, G-protein-coupled receptor, signal transduction, adenylyl cyclase.

scholarly journals Preparation of a First 18F-Labeled Agonist for M1 Muscarinic Acetylcholine Receptors

Molecules ◽  
2020 ◽  
Vol 25 (12) ◽  
pp. 2880 ◽  
Author(s):  
Boris D. Zlatopolskiy ◽  
Felix Neumaier ◽  
Till Rüngeler ◽  
Birte Drewes ◽  
Niklas Kolks ◽  
...  
Keyword(s):  
Acetylcholine Receptors ◽  
Radiochemical Yield ◽  
Muscarinic Acetylcholine Receptors ◽  
Preparative Scale ◽  
Muscarinic Acetylcholine ◽  
Pet Tracer ◽  
Positron Emission ◽  
Diagnostic Applications ◽  
M1 Muscarinic ◽  
Subtype Selective

M1 muscarinic acetylcholine receptors (mAChRs) are abundant in postsynaptic nerve terminals of all forebrain regions and have been implicated in the cognitive decline associated with Alzheimer’s disease and other CNS pathologies. Consequently, major efforts have been spent in the development of subtype-selective positron emission tomography (PET) tracers for mAChRs resulting in the development of several 11C-labeled probes. However, protocols for the preparation of 18F-labeled mAChR-ligands have not been published so far. Here, we describe a straightforward procedure for the preparation of an 18F-labeled M1 mAChR agonist and its corresponding pinacol boronate radiolabeling precursor and the non-radioactive reference compound. The target compounds were prepared from commercially available aryl fluorides and Boc protected 4-aminopiperidine using a convergent reaction protocol. The radiolabeling precursor was prepared by a modification of the Miyaura reaction and labeled via the alcohol-enhanced Cu-mediated radiofluorination. The developed procedure afforded the radiotracer in a non-decay-corrected radiochemical yield of 17 ± 3% (n = 3) and in excellent radiochemical purity (>99%) on a preparative scale. Taken together, we developed a straightforward protocol for the preparation of an 18F-labeled M1 mAChR agonist that is amenable for automation and thus provides an important step towards the routine production of a 18F-labeled M1 selective PET tracer for experimental and diagnostic applications.

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