Biased M1-muscarinic-receptor-mutant mice inform the design of next-generation drugs.

Cholinesterase inhibitors, the current frontline symptomatic treatment for Alzheimer's disease (AD), are associated with low efficacy and adverse effects. M1 muscarinic acetylcholine receptors (M1 mAChRs) represent a potential alternate therapeutic target; however, drug discovery programs focused on this G protein-coupled receptor (GPCR) have failed, largely due to cholinergic adverse responses. Employing novel chemogenetic and phosphorylation-deficient, G protein-biased, mouse models, paired with a toolbox of probe molecules, we establish previously unappreciated pharmacologically targetable M1 mAChR neurological processes, including anxiety-like behaviors and hyper-locomotion.

Differential regulation of β-adrenoceptor and adenosine A receptor signalling by GRK and arrestin proteins in arterial smooth muscle.

Generation of cAMP through G-coupled G protein-coupled receptor (GPCR) [e.g. β-adrenoceptor (βAR), adenosine A receptor (AR)] activation, induces arterial smooth muscle relaxation, counteracting the actions of vasoconstrictors.

No evidence for altered intracellular calcium-handling in airway smooth muscle cells from human subjects with asthma.

Background: Asthma is characterized by airway hyper-responsiveness and variable airflow obstruction, in part as a consequence of hyper-contractile airway smooth muscle, which persists in primary cell culture. One potential mechanism for this hyper-contractility is abnormal intracellular Ca(2+) handling.

Methods: We sought to compare intracellular Ca(2+) handling in airway smooth muscle cells from subjects with asthma compared to non-asthmatic controls by measuring: i) bradykinin-stimulated changes in inositol 1,4,5-trisphosphate (IP3) accumulation and intracellular Ca(2+) concentration, ii) sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) expression, iii) mechanisms of cytoplasmic Ca(2+) clearance assessed following instantaneous flash photolytic release of Ca(2+) into the cytoplasm.

Novel structural and functional insights into M3 muscarinic receptor dimer/oligomer formation.

Class A G protein-coupled receptors (GPCRs) are able to form homodimers and/or oligomeric arrays. We recently proposed, based on bioluminescence resonance energy transfer studies with the M3 muscarinic receptor (M3R), a prototypic class A GPCR, that the M3R is able to form multiple, structurally distinct dimers that are probably transient in nature (McMillin, S. M.

Nitric oxide synthesis and cGMP production is important for neurite growth and synapse remodeling after axotomy.

Nitric oxide (NO) is an important signaling molecule with a variety of functions in the CNS, including a potential role in modulating neuronal growth and synapse formation. In the present study, we used tractable, identified neurons in the CNS of the pond snail Lymnaea stagnalis to study the role of endogenous NO signaling in neuronal growth and synaptic remodeling after nerve injury. Axonal damage of L.

FRET-based detection of M1 muscarinic acetylcholine receptor activation by orthosteric and allosteric agonists.

Background And Objective: Muscarinic acetylcholine receptors (mAChRs) are 7-transmembrane, G protein-coupled receptors that regulate a variety of physiological processes and represent potentially important targets for therapeutic intervention. mAChRs can be stimulated by full and partial orthosteric and allosteric agonists, however the relative abilities of such ligands to induce conformational changes in the receptor remain unclear. To gain further insight into the actions of mAChR agonists, we have developed a fluorescently tagged M(1) mAChR that reports ligand-induced conformational changes in real-time by changes in Förster resonance energy transfer (FRET).

Structural aspects of M₃ muscarinic acetylcholine receptor dimer formation and activation.

To explore the structural mechanisms underlying the assembly and activation of family A GPCR dimers, we used the rat M(3) muscarinic acetylcholine receptor (M3R) as a model system. Studies with Cys-substituted mutant M3Rs expressed in COS-7 cells led to the identification of several mutant M3Rs that exclusively existed as cross-linked dimers under oxidizing conditions. The cross-linked residues were located at the bottom of transmembrane domain 5 (TM5) and within the N-terminal portion of the third intracellular loop (i3 loop).

Molecular mechanisms of muscarinic acetylcholine receptor-stimulated increase in cytosolic free Ca(2+) concentration and ERK1/2 activation in the MIN6 pancreatic β-cell line.

Muscarinic acetylcholine receptor (mAChR) activation of pancreatic β-cells elevates intracellular Ca(2+) and potentiates glucose-stimulated insulin secretion. In addition, it activates a number of signaling molecules, including ERK1/2, whose activation has been shown to play an important role in regulating pancreatic β-cell function and mass. The aim of this work was to determine how mAChR activation elevates intracellular Ca(2+) concentration ([Ca(2+)]( i )) and activates ERK1/2 in the pancreatic β-cell line MIN6.

[³⁵S]GTPγS binding as an index of total G-protein and Gα-subtype-specific activation by GPCRs.

On activation, G-protein-coupled receptors (GPCRs) exert many of their cellular actions through -promoting guanine nucleotide exchange on Gα subunits of heterotrimeric G proteins to release Gα-GTP and free βγ-subunits. In membrane preparations, GTP can be substituted by ³⁵S-labeled guanosine- 5'-O-(3-thio)triphosphate ([³⁵S]GTPγS) and on agonist stimulation a quasi-stable [³⁵S]GTPγS-Gα -complex forms and accumulates. Separation of [³⁵S]GTPγS-Gα complexes from free [³⁵S]GTPγS allows differences between basal and agonist-stimulated rates of [³⁵S]GTPγS-Gα complex formation- to be used to obtain pharmacological information on receptor-G-protein information transfer.

Early failure of N-methyl-D-aspartate receptors and deficient spine formation induced by reduction of regulatory heme in neurons.

An initial stage of many neurodegenerative processes is associated with compromised synaptic function and precedes synapse loss, neurite fragmentation, and neuronal death. We showed previously that deficiency of heme, regulating many proteins of pharmacological importance, causes neurodegeneration of primary cortical neurons via N-methyl-d-aspartate receptor (NMDAR)-dependent suppression of the extracellular signal-regulated kinase 1/2 pathway. Here, we asked whether the reduction of heme causes synaptic perturbation before neurite fragmentation in neuronal cultures and investigated molecular mechanisms of synaptic dysfunction in these cells.

Regulation of oxytocin receptor responsiveness by G protein-coupled receptor kinase 6 in human myometrial smooth muscle.

Oxytocin plays an important role in the progression, timing, and modulation of uterine contraction during labor and is widely used as an uterotonic agent. We investigated the mechanisms regulating oxytocin receptor (OTR) signaling in human primary myometrial smooth muscle cells and the ULTR cell-line. Oxytocin produced concentration-dependent increases in both total [(3)H]inositol phosphate accumulation and intracellular Ca(2+) concentration ([Ca(2+)](i)); however, responses were greater and more reproducible in the ULTR cell line.

G protein coupling and signaling pathway activation by m1 muscarinic acetylcholine receptor orthosteric and allosteric agonists.

The M(1) muscarinic acetylcholine (mACh) receptor is among a growing number of G protein-coupled receptors that are able to activate multiple signaling cascades. AC-42 (4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine) is an allosteric agonist that can selectively activate the M(1) mACh receptor in the absence of an orthosteric ligand. Allosteric agonists have the potential to stabilize unique receptor conformations, which may in turn cause differential activation of signal transduction pathways.

An investigation of whether agonist-selective receptor conformations occur with respect to M2 and M4 muscarinic acetylcholine receptor signalling via Gi/o and Gs proteins.

1. A range of muscarinic acetylcholine (mACh) receptor agonists (methacholine (MCh), oxotremorine-M (OXO-M), oxotremorine (OXO), arecoline (AREC), bethanechol (BETH), pilocarpine (PILO)) have been investigated with respect to their binding to, and activation of, M(2) and M(4) mACh receptors, recombinantly expressed in Chinese hamster ovary cells, to explore the possibility that these agonists may differentially affect mACh receptor-G(i/o) and -G(s) coupling. 2.

Specificity of g protein-coupled receptor kinase 6-mediated phosphorylation and regulation of single-cell m3 muscarinic acetylcholine receptor signaling.

Previously we have shown that G protein-coupled receptor kinase (GRK) 6 plays a major role in the regulation of the human M3 muscarinic acetylcholine receptor (M3 mAChR) in the human neuroblastoma SH-SY5Y. However, 30-fold overexpression of the catalytically inactive, dominant-negative K215RGRK6 produced only a 50% suppression of M3 mAChR phosphorylation and desensitization. Here, we have attempted to determine whether other endogenous kinases play a role in the regulation of M3 mAChR signaling.

Evidence for cross-talk between M2 and M3 muscarinic acetylcholine receptors in the regulation of second messenger and extracellular signal-regulated kinase signalling pathways in Chinese hamster ovary cells.

1. We have examined possible mechanisms of cross-talk between the G(q/11)-linked M(3) muscarinic acetylcholine (mACh) receptor and the G(i/o)-linked M(2) mACh receptor by stable receptor coexpression in Chinese hamster ovary (CHO) cells. A number of second messenger (cyclic AMP, Ins(1,4,5)P(3)) and mitogen-activated protein kinase (ERK and JNK) responses stimulated by the mACh receptor agonist methacholine were examined in CHO-m2m3 cells and compared to those stimulated in CHO-m2 and CHO-m3 cell-lines, expressing comparable levels of M(2) or M(3) mACh receptors.