A Novel Function of the M₂ Muscarinic Receptor

Abstract

The M₂ muscarinic receptor is a prototypic class A G protein-coupled receptor. Interestingly, Fasciani et al. recently identified an internal translation start site within the M₂ receptor mRNA, directing the expression of a C-terminal receptor fragment. Elevated during cellular stress, this polypeptide localizes to mitochondria where it inhibits oxidative phosphorylation.

Introduction

Following their activation by extracellular ligands, G protein-coupled receptors (GPCRs) can interact with and activate distinct functional classes of heterotrimeric G proteins, resulting in the activation or inhibition of numerous intracellular signaling pathways. GPCRs represent by far the largest family of plasma membrane receptors [1]. For example, the human genome codes for ~800 individual GPCRs [1]. Most importantly, GPCRs represent excellent drug targets, as mirrored by the fact that about 1/3 of all drugs in current clinical use target one or more of these receptors [1].

The five muscarinic acetylcholine receptor subtypes (M₁-M₅) are prototypic class A GPCRs (receptors with high structural homology to rhodopsin) [2]. The M₁-M₅ muscarinic receptors regulate a wide array of peripheral and central functions including heart rate and numerous sensory and cognitive processes [2]. During the past decades, the M₂ muscarinic receptor (M2R) has served as an excellent model system to explore the molecular mechanisms underlying muscarinic receptor function including ligand binding, coupling selectivity, and receptor oligomerization. The M2R is highly expressed in the heart and various regions of the CNS [2]. In the heart, acetylcholine-mediated activation of M2Rs plays a key role in the regulation of heart rate by mediating the inhibitory cardiac effects of parasympathetic stimulation [2].

Early studies with genetically engineered “split” M₂ and M₃ muscarinic receptors indicated that GPCRs are composed of multiple structural subdomains that can fold independently of each other and combine to form functional receptors (reviewed in [3]). This finding is consistent with the concept, first proposed by Popot and Engelman more than three decades ago, that the folding of eucaryotic membrane proteins occurs in two consecutive steps [4]. Step 1 involves the formation of individually stable folding domains across the lipid bilayer, which, in step 2, combine to form a functional transmembrane protein [4].

A novel mechanism that directs the expression of a C-terminal M2R fragment

Interestingly, Fasciani et al. [5] recently showed, for the first time, that an IRES (internal ribosome entry site) element can drive the expression of a GPCR fragment that can modulate distinct cellular functions. Specifically, the authors identified an IRES sequence located within the third intracellular loop (i3 loop) of the human M2R that directs the expression of a C-terminal M2R fragment (amino acids 368–466) (Figure 1). This M2R fragment, referred to as ‘M2R-tail’, contains the C-terminal portion of the i3 loop, transmembrane domains 6 and 7, and the cytoplasmic C-terminal receptor sequence (Figure 1).

Figure 1. Scheme illustrating the generation, localization, and function of the M2R-tail fragment.

The M₂ muscarinic receptor (M2R) gene (CHRM2; human gene name) codes for two distinct transcripts. Translation from the canonical AUG translation start site yields the full-length M2R that is trafficked to the cell surface. Under starvation stress, initiation of translation from an internal ribosome entry site (IRES) results in the generation of the M2R-tail receptor fragment (amino acids 368–466; human M2R sequence). The M2R-tail protein is transported to the inner mitochondrial membrane, where it interacts with ATP synthase, resulting in the inhibition of mitochondrial respiration and impaired ATP production. Created with BioRender.

IRES elements are RNA sequences that function to recruit ribosomes for the initiation of translation in a cap-independent manner from internal mRNA sites [6]. Interestingly, the novel IRES element discovered by Fasciani et al. [5] is located within the cDNA (the vast majority of IRES sequences are found in UTR regions) and consists of only 30 bases. In contrast, the average IRES element is 174 bases long [7].

It should also be noted that Fasciani et al. [5] did not address the possibility that the M2R i3 loop sequence itself has promoter activity. Since promoter sequences are usually less well conserved, this issue deserves further investigation.

Mitochondrial localization and inhibitory function of the M2R-tail fragment

Studies with cultured cells showed that the full-length, wild-type (wt) M2R was predominantly localized to the plasma membrane, as expected [5]. In contrast, the M2R-tail fragment was trafficked mostly to mitochondria [5]. Biophysical and electron microscopy studies indicated that the M2R-tail protein was predominantly localized to the inner mitochondrial membrane.

Interestingly, starvation-induced stress led to a significant increase in IRES-mediated production of the M2R-tail fragment, associated with a marked increase in mitochondrial localization [5]. Importantly, the presence of the M2R-tail protein in the inner mitochondrial membrane resulted in reduced mitochondrial oxygen consumption and decreased reactive oxygen species (ROS) production. Biophysical studies indicated that the M2R-tail fragment can specifically interact with the F_oF₁ ATP-synthase complex, suggesting that binding of the M2R-tail protein to this enzyme inhibits its activity, resulting in the observed functional changes.

Studies with M2R knock-in mice in which the wt M2R carried a C-terminal tdTomato sequence demonstrated that the M2R-tail fragment was also produced in vivo with preferential localization to mitochondria [5]. In addition, studies with genetically engineered pluripotent stem cells showed that suppression of the expression of the M2R-tail fragment promoted cell proliferation and cellular respiration. This finding supports the concept that the M2R-tail protein most likely functions to reduce mitochondrial oxygen consumption in vivo under certain physiological conditions.

Interestingly, full-length GPCRs are present not only on the cell surface but also in various intracellular compartments, including endosomes and mitochondria, indicative of non-canonical roles for this receptor family [8]. Moreover, truncated forms of GPCRs can be generated via alternative splicing. Long deemed physiologically irrelevant, these receptor isoforms have been shown to regulate cellular functions that differ from those linked to the activation of the full-length receptors [9].

Areas of future research

The study by Fasciani et al. [5] raises several important questions. Through which mechanism is the M2R-tail fragment selectively targeted to the inner mitochondrial membrane? What other factors regulate the expression of the M2R-tail protein besides starvation stress? Is the expression of this receptor fragment altered under certain pathophysiological conditions? More generally, are IRES sequences also operative in other GPCRs? If yes, what are the functional and physiological roles of the encoded receptor fragments?

Since the M2R is enriched in cardiomyocytes, it would be of particular interest to explore whether the expression of the M2R-tail fragment affects the function of this cell type. The generation and phenotypic analysis of genetically engineered M2R mutant mice that carry a mutation that suppresses the expression of the M2R-tail protein would greatly facilitate studies of the physiological roles of this C-terminal receptor fragment.

Concluding remarks

In sum, the study by Fasciani et al. [5] opens an entire new field in membrane receptor/GPCR research. Most likely, future studies in this area will lead to novel insights into how GPCR fragments generated from IRES elements modulate cellular activity. This information could guide the development of novel strategies to modulate distinct cellular functions for therapeutic purposes [10].