PerspectivePhysiology

Making Muscles Grow by G Protein–Coupled Receptor Signaling

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Science Signaling  29 Nov 2011:
Vol. 4, Issue 201, pp. pe45
DOI: 10.1126/scisignal.2002670

Abstract

Activation of G protein–coupled receptors is involved in regulating many cellular responses, but less is known regarding the role of these receptors in the differentiation and maintenance of skeletal muscle. New findings implicate the inhibitor subunit Gαi2 as a vital mediator of myofiber maturation and growth, operating through multiple signaling pathways to selectively stimulate protein synthesis or inhibit cytokine-dependent protein turnover.

G protein–coupled receptors (GPCRs) constitute the largest family of membrane proteins and are responsible for transmitting most cellular responses upon binding to hormones, neurotransmitters, or ions. Equally impressive is the realization that more than half of all known existing pharmaceutical compounds target these receptors, accentuating their importance in pathophysiological conditions [reviewed in (1, 2)].

Structurally, GPCRs consist of seven transmembrane regions, with an extracellular N-terminal segment and an intracellular C-terminal tail. Coupled to this receptor is a heterotrimeric G protein complex composed of α, β, and γ subunits. In an inactive state, Gα is bound to guanosine diphosphate (GDP) and associates with Gβ and Gγ subunits. Agonist binding to the transmembrane receptor leads to stimulation of the G protein complex (Gαs) and an exchange of guanosine 5′-triphosphate (GTP) for GDP, resulting in a conformational change that dissociates Gαs from the Gβγ heterodimer. Free Gα-GTP and Gβγ subunits then activate an effector, such as adenylate cyclase, to produce small-molecule second messengers, such as cAMP (cyclic adenosine monophosphate), which stimulates protein kinase A (PKA) and other downstream signaling events. In addition, Gαi inhibitor subunits, which include the Gαi1, Gαi2, and Gαi3 isoforms, block adenylate cyclase and cAMP production (1). Of the various physiological events regulated by GPCRs, only a few reports have linked the activities of these receptors to skeletal muscle homeostasis. The study conducted by Minetti and colleagues (3) identifies a role for the Gαi2 subunit as a regulator of skeletal muscle maturation and growth (Fig. 1).

Fig. 1

The GPCR subunit Gαi2 promotes skeletal muscle differentiation and cell growth. The Gαi2 subunit of GPCRs has multiple signaling activities that regulate skeletal muscle growth. These include (left) stimulating differentiation through the transcription factor NFAT; (center) promoting hypertrophy through selective stimulation of PKC and mTOR, which individually activate protein translation; and (right) inhibiting atrophy by blocking the pro-cachectic effects of TNF-α, which acts through myogenin to induce E3 ubiquitin ligases that promote muscle proteolysis.

CREDIT: Y. HAMMOND/SCIENCE SIGNALING

These investigators have played a pivotal role in defining the major molecular mechanisms that regulate skeletal muscle cell size (4). Catabolic factors, such as glucocorticoids, promote muscle atrophy by stimulating the expression of genes encoding muscle-specific E3 ubiquitin ligases, muscle ring finger (MuRF) 1 and Atrogin-1 (also known as MAFbx), which ubiquitinate and target myofibrillar proteins for proteasomal-mediated degradation (5, 6). These ubiquitin ligases are opposed by growth factors, such as insulin-like growth factor 1 (IGF-1), which promotes myofiber hypertrophy through the activation of the phosphoinositide-3 kinase (PI3K)–Akt pathway and mTOR (mammalian target of rapamycin), which stimulates protein synthesis to expand the myofibrillar muscle cell compartment (79). How GPCRs function within the landscape of these counteracting signaling pathways is a question that up to now had remained unexplored.

To investigate this connection, Minetti and co-workers tested the effects of the GPCR agonist, lysophosphatidic acid (LPA), on human primary skeletal myotubes. LPA enhanced myotube size, which depended on the activity of the Gαi2 subunit, as shown by pharmacological inhibitor treatment or small interfering RNA (siRNA)–mediated depletion. To further address the question of specificity, the authors used a constitutively active mutant of Gαi2 in which a leucine residue was substituted for glutamine at position 205 (Q205L). This mutant inhibited adenylate cyclase activity and, when expressed in an adenoviral cassette, mimicked LPA activity by eliciting growth of myotubes, which correlated with an increased protein synthesis rate and type II myosin heavy chain content. Moreover, viral delivery of a similar transgene produced similar results in adult mouse myofibers.

Although GPCRs can signal through the PI3K-Akt pathway (10), the hypertrophic activity of Gαi2 was independent of Akt but still required mTOR. The data also supported the possibility that Gαi2-induced myotube hypertrophy was mediated by protein kinase C (PKC), which activates p70S6K and protein translational machinery by indirectly phosphorylating and inactivating glycogen synthase kinase (GSK) 3β. Because GSK-3β is an inhibitor of the transcription factor NFAT (nuclear factor of activated T cells), which itself is required in the differentiation of myoblasts to myotubes (11, 12), the authors speculated that Gαi2 function might extend to regulating the maturation process of myocytes. Indeed, in both cultured myoblasts and in response to acute toxin-induced injury in mouse muscle, constitutive expression of the Gαi2 active mutant led to enhanced myogenesis, which at least in vitro was connected to a Gαi2-PKC–GSK-3β-NFAT pathway.

A hypertrophic response in muscle cells can also be mediated by reducing the expression of genes encoding atrophy-inducing factors. This is similar to how Akt-mediated phosphorylation leads to the inactivation of the FoxO family of transcription factors, which directly increase the expression of genes encoding the E3 ubiquitin ligases MuRF1 and Atrogin-1 (13, 14). Thus, Minetti and colleagues tested the ability of Gαi2 to impinge on the catabolic response factor, tumor necrosis factor–α (TNF-α) (15). In human myotube cultures, LPA and Gαi2 abrogated TNF-α−mediated reduction in myotube size and induction of E3 ubiquitin ligases, suggesting that Gαi2 signaling could maintain myotube size by preventing the actions of pro-cachectic factors such as TNF-α. Gαi2 action on TNF-α seems to intersect with an atrophy mechanism that increases the activity of class II histone deacetylases (HDACs) on the promoter of the gene encoding the myogenic responsive factor, myogenin (16), which directly binds to the promoters of genes encoding MuRF1 and Atrogin-1 and stimulates transcription (13, 14). Addition of TNF-α to myotubes increased Myogenin messenger RNA abundance, which was reversed upon expression of Gαi2 in a PKC-independent manner. Furthermore, the activity of HDAC4 and -5, which is required for increased expression of mRNAs encoding myogenin and E3 ubiquitin ligase (16), was also reduced in the presence of Gαi2, again irrespective of PKC. Because TNF-α can inhibit HDAC activity and abundance (17, 18), it is possible that Gαi2 mediates its inhibition on myogenin further upstream by inhibiting the TNF-α signaling pathway independently of PKC.

Collectively, Minetti and colleagues have revealed a potentially new important player in the hypertrophy and atrophy arena. Until now, Akt was the only protein that was known to promote myotube hypertrophy through two mechanisms: by stimulating protein synthesis through mTOR activity while inhibiting FoxO to reduce E3 ubiquitin ligase abundance, preventing myofibrillar protein turnover. New data suggest that Gαi2 also induces muscle hypertrophy through a dual action mechanism: by promoting mTOR and protein synthesis in an Akt-independent mechanism while inhibiting atrophy in response to TNF-α. Akt also has pro-myogenic activity (19), and similarly, Gαi2 appears equally capable of stimulating myocyte differentiation through a PKC–GSK-3β–NFAT pathway.

As is often the case in studies in which a new molecule is implicated in a critical aspect of tissue homeostasis, one is left asking for more, prompting several interesting unresolved questions. For instance, is it possible that the effects of Gαi2 on PKC and GSK-3β are in part mediated through mTOR, in addition to acting directly on mTOR substrates that control protein translation? As for the regulation of myogenesis, because mRNA abundance of the transcription factor Pax7 was increased in satellite cells isolated from injured muscles overexpressing Gαi2, is it possible that in addition to regulating NFAT, Gαi2 also functions at an earlier stage of the myogenic program to affect satellite cell activation or commitment? With regard to the inhibitory activity on TNF-α, one wonders whether Gαi2 is capable of blocking nuclear factor-κB (NF-κB), which is activated by TNF-α and also promotes myofiber atrophy by inducing the transcriptional activation of genes encoding E3 ubiquitin ligases (20). Although these and other questions remain to be addressed, it is nevertheless tempting to project Gαi2 as a potential therapeutic target capable of sparing muscle mass or even stimulating growth under numerous chronic conditions of muscle wasting.

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