Changes in Membrane Cholesterol Differentially Influence Preferential and Non-preferential Signaling of the M1 and M3 Muscarinic Acetylcholine Receptors.

Pavel Michal,Vladimír Doležal,Esam E El-Fakahany

doi:10.1007/s11064-014-1325-z

Abstract

We have found earlier that changes in membrane cholesterol content have distinct impact on signaling via the M1, M2, or M3 receptors expressed in CHO cells (CHO-M1 through CHO-M3). Now we investigated whether gradual changes in membrane cholesterol exerts differential effects on coupling of the M1 and M3 muscarinic receptors to preferential signaling pathways through Gq/11 and non-preferential Gs G-proteins signaling. Changes in membrane cholesterol resulted in only marginal alterations of antagonist and agonist affinity of the M1 and M3 receptors, and did not influence precoupling of either subtype. Changes in membrane cholesterol did not influence parameters of carbachol-stimulated GTP-γ35S binding in CHO-M1 membranes while reduction as well as augmentation of membrane cholesterol lowered the efficacy but increased the potency of carbachol in CHO-M3 membranes. Gradual increase or decrease in membrane cholesterol concentration dependently attenuated agonist-induced inositolphosphates release while only cholesterol depletion increased basal values in both cell lines. Similarly, membrane cholesterol manipulation modified basal and agonist-stimulated cAMP synthesis via Gs in the same way in both cell lines. These results demonstrate that changes in membrane cholesterol concentration differentially impact preferential and non-preferential M1 and M3 receptor signaling. They point to the activated G-protein/effector protein interaction as the main site of action in alterations of M1 receptor-mediated stimulation of second messenger pathways. On the other hand, modifications in agonist-stimulated GTP-γ35S binding in CHO-M3 membranes indicate that in this case changes in ligand-activated receptor/G-protein interaction may also play a role.

Highlights

Muscarinic receptors belong to the family of G-protein coupled receptors (GPCR) that are the most abundant and pharmacologically targeted plasma membrane receptors [9, 22]
We have found earlier that changes in membrane cholesterol content have distinct impact on signaling via the M1, M2, or M3 receptors expressed in CHO cells (CHO-M1 through CHO-M3)
In the present experiments we explored the influence of gradual changes in membrane cholesterol concentration on individual steps of signal transduction via M1 muscarinic receptors, including agonist binding, activation of G-proteins, and resulting stimulation of intracellular signaling pathways

Summary

Introduction

Muscarinic receptors belong to the family of G-protein coupled receptors (GPCR) that are the most abundant and pharmacologically targeted plasma membrane receptors [9, 22]. A common structural feature of GPCR is the extracellular N-terminus, seven membrane spanning domains, three extracellular, three intracellular loops, and intracellular C-terminus. There are five subtypes of muscarinic receptors denoted as M1-M5 and encoded by five different genes [2,3,4,5, 31]. Individual muscarinic receptor subtypes share a high degree of homology in the transmembrane domains while the extracellular and intracellular loops are less conserved [13,14,15]. The N-terminal part of the third intracellular loop represents the contact domain for interaction with G-proteins [12, 37]. Higher variability of this domain enables selectivity of interaction with different G-proteins

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Conclusion