Distinct agonist regulation of muscarinic acetylcholine M2-M3 heteromers and their corresponding homomers

Each subtype of the muscarinic receptor family of G protein-coupled receptors is activated by similar concentrations of the neurotransmitter acetylcholine or closely related synthetic analogs such as carbachol. However, pharmacological selectivity can be generated by the introduction of a pair of mutations to produce Receptor Activated Solely by Synthetic Ligand (RASSL) forms of muscarinic receptors. These display loss of potency for acetylcholine/carbachol alongside a concurrent gain in potency for the ligand clozapine N-oxide. Co-expression of a form of wild type human M2 and a RASSL variant of the human M3 receptor resulted in concurrent detection of each of M2-M2 and M3-M3 homomers alongside M2-M3 heteromers at the surface of stably transfected Flp-InTM T-RExTM 293 cells. In this setting occupancy of the receptors with a muscarinic antagonist was without detectable effect on any of the muscarinic oligomers. However, selective agonist occupancy of the M2 receptor resulted in enhanced M2-M2 homomer interactions but decreased M2-M3 heteromer interactions. By contrast, selective activation of the M3 RASSL receptor did not significantly alter either M3-M3 homomer or M2-M3 heteromer interactions. Selectively targeting closely related receptor oligomers may provide novel therapeutic opportunities

Spatial intensity distribution analysis: studies of G Protein-coupled receptor oligomerization

Spatial intensity distribution analysis (SpIDA) is a recently developed approach for determining quaternary structure information on fluorophore-labelled proteins of interest in situ. It can be applied to live or fixed cells and native tissue. Using confocal images, SpIDA generates fluorescence intensity histograms that are analysed by super-Poissonian distribution functions to obtain density and quantal brightness values of the fluorophore-labelled protein of interest. This allows both expression level and oligomerisation state of the protein to be determined. We describe the application of SpIDA to investigate the oligomeric state of G protein-coupled receptors (GPCRs) at steady state and following cellular challenge, and consider how SpIDA may be used to explore GPCR quaternary organisation in pathophysiology and to stratify medicines

Muscarinic receptor oligomerization

G protein-coupled receptors (GPCRs) have been classically described as monomeric entities that function by binding in a 1:1 stoichiometric ratio to both ligand and downstream signalling proteins. However, in recent years, a growing number of studies has supported the hypothesis that these receptors can interact to form dimers and higher order oligomers although the molecular basis for these interactions, the overall quaternary arrangements and the functional importance of GPCR oligomerization remain topics of intense speculation. Muscarinic acetylcholine receptors belong to class A of the GPCR family. Each muscarinic receptor subtype has its own particular distribution throughout the central and peripheral nervous systems. In the central nervous system, muscarinic receptors regulate several sensory, cognitive, and motor functions while, in the peripheral nervous system, they are involved in the regulation of heart rate, stimulation of glandular secretion and smooth muscle contraction. Muscarinic acetylcholine receptors have long been used as a model for the study of GPCR structure and function and to address aspects of GPCR dimerization using a broad range of approaches. In this review, the prevailing knowledge regarding the quaternary arrangement for the various muscarinic acetylcholine receptors has been summarized by discussing work ranging from initial results obtained using more traditional biochemical approaches to those generated with more modern biophysical techniques

GPCR homo-oligomerization

G protein-coupled receptors (GPCRs) are an extensive class of trans-plasma membrane proteins that function to regulate a wide range of physiological functions. Despite a general perception that GPCRs exist as monomers an extensive literature has examined whether GPCRs can also form dimers and even higher-order oligomers, and if such organization influences various aspects of GPCR function, including cellular trafficking, ligand binding, G protein coupling and signalling. Here we focus on recent studies that employ approaches ranging from computational methods to single molecule tracking and both quantal brightness and fluorescence fluctuation measurements to assess the organization, stability and potential functional significance of dimers and oligomers within the class A, rhodopsin-like GPCR family

Dynamic regulation of quaternary organization of the M1 muscarinic receptor by subtype-selective antagonist drugs

Although rhodopsin-like G protein-coupled receptors can exist as both monomers and non-covalently associated dimers/oligomers, the steady-state proportion of each form and whether this is regulated by receptor ligands is unknown. Herein we address these topics for the M1 muscarinic acetylcholine receptor, a key molecular target for novel cognition enhancers, by employing Spatial Intensity Distribution Analysis. This method can measure fluorescent particle concentration and assess oligomerization states of proteins within defined regions of living cells. Imaging and analysis of the basolateral surface of cells expressing some 50 molecules.microm-2 of the human muscarinic M1 receptor identified an ~75/25 mixture of receptor monomers and dimers/oligomers. Both sustained and shorter-term treatment with the selective M1 antagonist pirenzepine resulted in a large shift in the distribution of receptor species to favor the dimeric/oligomeric state. Although sustained treatment with pirenzepine also resulted in marked upregulation of the receptor, simple mass-action effects were not the basis for ligand-induced stabilization of receptor dimers/oligomers. The related antagonist telenzepine also produced stabilization and enrichment of the M1 receptor dimer population but the receptor subtype non-selective antagonists atropine and N-methylscopolamine did not. In contrast, neither pirenzepine nor telenzepine altered the quaternary organization of the related M3 muscarinic receptor. These data provide unique insights into the selective capacity of receptor ligands to promote and/or stabilize receptor dimers/oligomers and demonstrate that the dynamics of ligand regulation of the quaternary organization of G protein-coupled receptors is markedly more complex than previously appreciated. This may have major implications for receptor function and behavior

Defining the organizational structure of dopamine and muscarninic acetylcholine receptors

No abstract available

Regulation of the pro-inflammatory G protein-coupled receptor GPR84

GPR84 is an understudied rhodopsin-like class A G protein-coupled receptor which is arousing particular interest from a therapeutic perspective. Not least this reflects that gpr84 expression is significantly up-regulated following acute inflammatory stimuli and in inflammatory diseases and that receptor activation plays a role in regulating pro-inflammatory responses and migration of cells of the innate immune system such as neutrophils, monocytes, macrophages and microglia. Although most physiological responses of GPR84 reflect receptor coupling to Gαi/o -proteins, several studies indicate that agonist-activated GPR84 can also recruit arrestin adaptor proteins and this regulates receptor internalisation and desensitisation. To date, little is known on the patterns of either basal or ligand regulated GPR84 phosphorylation and how these might control these processes. Here, we consider what is known about the regulation of GPR84 signalling with a focus on how G protein receptor kinase -mediated phosphorylation regulates arrestin protein recruitment and receptor function

A molecular basis for selective antagonist destabilization of dopamine D3 receptor quaternary organization

The dopamine D3 receptor (D3R) is a molecular target for both first-generation and several recently-developed antipsychotic agents. Following stable expression of this mEGFP-tagged receptor, Spatial Intensity Distribution Analysis indicated that a substantial proportion of the receptor was present within dimeric/oligomeric complexes and that increased expression levels of the receptor favored a greater dimer to monomer ratio. Addition of the antipsychotics, spiperone or haloperidol, resulted in re-organization of D3R quaternary structure to promote monomerization. This action was dependent on ligand concentration and reversed upon drug washout. By contrast, a number of other antagonists with high affinity at the D3R, did not alter the dimer/monomer ratio. Molecular dynamics simulations following docking of each of the ligands into a model of the D3R derived from the available atomic level structure, and comparisons to the receptor in the absence of ligand, were undertaken. They showed that, in contrast to the other antagonists, spiperone and haloperidol respectively increased the atomic distance between reference α carbon atoms of transmembrane domains IV and V and I and II, both of which provide key interfaces for D3R dimerization. These results offer a molecular explanation for the distinctive ability of spiperone and haloperidol to disrupt D3R dimerization

Chemokine receptor CXCR4 oligomerization is disrupted selectively by the antagonist ligand IT1t

CXCR4, a member of the family of chemokine-activated G protein-coupled receptors, is widely expressed in immune response cells. It is involved in both cancer development and progression as well as viral infection, notably by HIV-1. A variety of methods, including structural information, have suggested the receptor may exist as a dimer or oligomer. However, the mechanistic details surrounding receptor oligomerization and its potential dynamic regulation remain unclear. Using both biochemical and biophysical means we confirm that CXCR4 can exist as a mixture of monomers, dimers and higher-order oligomers in cell membranes and show that oligomeric structure becomes more complex as receptor expression levels increase. Mutations of CXCR4 residues located at a putative dimerization interface result in monomerization of the receptor. Additionally, binding of the CXCR4 antagonist IT1t— a small, drug-like isothiourea derivative — rapidly destabilizes the oligomeric structure, while AMD3100, another well-characterized CXCR4 antagonist, does not. Although a mutation that regulates constitutive activity of CXCR4 also results in monomerization of the receptor, binding of IT1t to this variant promotes receptor dimerization. These results provide novel insights into the basal organization of CXCR4 and how antagonist ligands of different chemotypes differentially regulate its oligomerization state