|Signaling Via G-Protein-Coupled Receptors||Papers|
Neurotransmitters may act on two distinct classes of receptors to transmit signals: ligand-gated ion channels and G-protein coupled receptors. The latter is discussed in this article, with focus on norepinephrine, acetylcholine, glutamate, dopamine and serotonin (5-HT). For ion channels, the binding of neurotransmitters (ligands) results in the opening of channels, allowing ions to pass through. In this case, signals are transmitted via ions, particularly the calcium ions that control all kinds of enzymes. For G-protein coupled receptors, signals are transmitted via G proteins.
G-protein coupled receptors do not possess a pore for ion passage. They mediate cell signaling via the G protein, which consists of three subunits: α, β and γ. Before activation, these subunits are linked together. Gβ and Gγ are tightly bound, whereas Gα may dissociate from Gβγ, depending on whether it is bound by GDP (guanosine diphosphate) or GTP (guanosine triphosphate). In the resting state, Gα is bound by GDP, facilitating the assembly of three subunits. After activation, the GDP bound to Gα will be replaced by GTP, promoting dissociation of Gα from Gβγ. The separated Gα and Gβγ can then act on specific targets, known as effectors. The GTP on Gα cannot last long, because Gα has the enzymatic activity to hydrolyze it into GDP, thereby returning to the resting state (Figure 1).
Gα has several isoforms, including Gαs (stimulatory), Gαi (inhibitory), Gαo (other) and Gαq. They play distinct roles in cell signaling by acting on different effectors.
G Protein-coupled Receptors
A G protein-coupled receptor (GPCR) is characterized by seven transmembrane α helices (Figure 2). It does not form an ion-conducting pore, but may act on ion channels via separated Gα or Gβγ. In addition to ion channels, the G-protein can also act on enzymes. For instance, Gαs stimulates the production of cyclic AMP (cAMP) from ATP by directly activating the enzyme adenylate cyclase (also known as adenylyl cyclase). Gαi inhibits the production of cAMP by inactivating adenylate cyclase.
Another important signaling pathway is mediated by Gαq which can activate phospholipase C (PLC) to cleave a phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), into diacyl glycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) (Figure 3). DAG may activate protein kinase C while IP3 can induce the release of Ca2+ ions from the intracellular store (Figure 4). The GPCRs coupled to Gαq include group I metabotropic glutamate receptors (mGluR1 and mGluR5), dopamine's D1 receptor, muscarinic acetylcholine receptors (M1, M3 and M5) and α1-adrenergic receptor.
Norepinephrine (NE), also called noradrenaline, is a neurotransmitter produced mainly in the locus coeruleus (a small brain region within the brainstem). In adult humans, the locus coeruleus contains less than 50,000 noradrenergic neurons that release primarily NE. However, they project to widespread brain areas, including hippocampus, cerebral cortex, basolateral amygdala and striatum (Hansen, 2017; Ferrucci et al., 2013), where NE may bind to specific receptors, triggering a series of signaling cascades. The NE receptors are often referred to as "adrenergic receptors" (ARs).
ARs have two main groups: α and β. α is divided into two types: α1 and α2. β is divided into three types: β1, β2 and β3. They are coupled to different G proteins:
Gαq mediates the release of Ca2+ ions from the intracellular store, as described above. The signaling cascades mediated by Gαs and Gαi are illustrated in the following figure.
Acetylcholine (ACh) has five muscarinic receptors, M1 - M5, which belong to G protein-coupled receptors. M1, M3 and M5 are coupled to Gαq while M2 and M4 are coupled to Gαi. Activation of Gαi inactivates adenylyl cyclase (AC), reducing cAMP levels, thereby suppressing the activity of protein kinase A (PKA).
Gαq can activate PLC to cleave PIP2 into DAG and IP3, thereby activating protein kinase C (PKC) and inducing the release of Ca2+ ions from the intracellular store. PKC has been shown to inhibit hyperpolarization-activated cyclic nucleotide-gated (HCN) channels which underlie the h current (Ih) (Williams et al., 2015). The HCN channels play key roles in regulating alpha rhythms. Consistent with these findings, ACh has been demonstrated to induce alpha rhythms in sensory thalamic nuclei by acting on M3 receptors (Lörincz et al., 2008).
M1 receptors are highly expressed in the granule cells of dentate gyrus and the pyramidal cells of CA3 and CA1. Importantly, they are distributed preferentially on the extrasynaptic membrane of pyramidal cell dendrites and spines (Yamasaki et al., 2010), suggesting that their major targets are not located at synapses. Rather, ACh could regulate extrasynaptic NMDA receptors by acting on M1 receptors to facilitate memory retrieval.
Glutamate has eight types of metabotropic receptors that belong to G protein-coupled receptors. They are classified into three groups:
Group I mGluRs are coupled to Gαq whereas Group II and III are coupled to Gαi and Gαo. As described above, Gαq can activate PLC to cleave PIP2, producing DAG and IP3. Subsequently, DAG may activate protein kinase C (PKC) while IP3 induces the release of Ca2+ ions from the intracellular store. Coupling with Gαi/o leads to inhibition of adenylyl cyclase and other signaling pathways (Niswender and Conn, 2010).
mGluR5 plays important roles in gene transcription by regulating the transcription factors, cyclic AMP responsive element binding protein (CREB) and nuclear factor κB (NF-κB). CREB can be activated by protein kinase A (PKA) and CaMKIV, both are under the regulation of Ca2+ ions induced by IP3 (Wang and Zhuo, 2012). NF-κB is regulated by PKC subtypes β and δ through PKC-associated kinase (PKK) (Muto et al., 2002; Kim et al., 2014).
mGluR5 is critical for the toxicity exerted by beta amyloid oligomers (AβOs). The signaling cascade involves Ca2+-stimulated coactivation of Fyn and Pyk2, which in turn activate GSK-3β and RhoA kinase, leading to hyperphosphorylation of CRMP2 and consequently synapse loss (see this article).
Dopamine has five known receptor subtypes, designated as D1 - D5. D1 and D5 receptors are alike, while D2, D3 and D4 receptors have similar properties. Their signaling pathways are summarized in Figure 9.
D1-like receptors are coupled to Gαs and Gαq. D2-like receptors are coupled to Gαi. Their downstream signaling has been described in previous sections. These pathways are sometimes referred to as "canonical pathways", which apply to a variety of neurotransmitters. In addition to canonical pathways, the D3 receptor (D3R) can also activate the PI3K/Akt pathway through Gβγ (Collo et al., 2014).
The D2 receptor (D2R) also mediates a non-canonical pathway via coupling with β-arrestin 2 (βArr2) (Bozzi and Borrelli, 2013). In this pathway, D2R stimulates the formation of a complex containing βArr2, protein phosphatase 2A (PP2A) and Akt such that PP2A can dephosphorylate and inactivate Akt (Beaulieu et al., 2005). Akt may activate mTOR which is a risk factor for various diseases, including neurodegeneration, diabetes and cancer (see this article).
Serotonin (5-HT)-induced signaling
There are at least 7 families of 5-HT receptors. Except for 5-HT3, all others belong to G-protein-coupled receptors (Rojas and Fiedler, 2016). This section will focus on 5-HT1 and 5-HT2 receptors, which are the most studied. 5-HT1 has five subtypes: A, B, D, E and F while 5-HT2 has three subtypes: A, B and C. Note that there is no 5-HT1C receptor because the originally nemed 5-HT1C was found to have more in common with the 5-HT2 family and thus redesignated as the 5-HT2C receptor (Wikipedia).
In neurons, activation of the 5-HT1A receptor (5-HT1AR) stimulates Gβγ signaling which promotes the activity of adenylyl cyclase types 2 (AC2) that in turn augments cAMP level and protein kinase A (PKA) activity (Figure 10). In non-neuronal cells, activation of 5-HT1ARs produces the opposite effect, namely, reduction of PKA activity, by stimulating the Gαi/o signaling (Rojas and Fiedler, 2016).
Gβγ also participates in the activation of the phosphoinositide-3-kinase (PI3K)-Akt pathway. Akt plays an important role in regulating the activity of glycogen synthase kinase 3 (GSK-3). Phosphorylation of GSK-3α and GSK-3β on Ser-21 and Ser-9, respectively by Akt may become inactive. GSK-3 is also under the regulation of PKA via direct and indirect pathways. In the direct pathway, PKA physically associates with and phosphorylates both forms of GSK-3, α and β on Ser-21 and Ser-9, respectively (Fang et al., 2000). In the indirect pathway, PKA may inhibit Src (a non-receptor tyrosine kinase) via phosphorylation of C-terminal Src kinase (Csk) which can inactivate Src (Trepanier et al., 2013). Importantly, Src can phosphorylate GSK-3 at Y216, leading to its activation even when Akt is active (Goc et al., 2014). Therefore, Src plays a dominant role in GSK-3 activation. Inhibition of PKA would augment both Src and GSK-3 activities.
5-HT2A receptors (5-HT2ARs) play crucial roles in schizophrenia which is a mental disorder characterized by hallucination, avolition and cognitive dysfunction. They are the target of atypical antipsychotics (Beaulieu, 2012) and a class of hallucinogenic drugs called psychedelics (Nichols, 2016). Activation of 5-HT2AR may trigger both canonical and non-canonical pathways.
In the canonical pathway, Gq stimulates phospholipase C (PLC) to cleave PIP2 into DAG and IP3. DAG may activate protein kinase C (PKC) while IP3 can induce the release of Ca2+ ions from the intracellular store. The elevated Ca2+ concentration in the cytosol may further activate Ca2+-dependent adenylyl cyclase (AC), types 1 and 8 (Wong et al., 1999; Wang et al., 2003), to increase the production of cAMP, thereby enhancing the activity of PKA. PKC may stimulate the expression of brain-derived neurotrophic factor (BDNF) via the ERK-CREB pathway (Ferraguti et al., 1999; Wang et al., 2012).
The non-canonical pathway is mediated by β-arrestin which may recruit protein phosphatase 2A (PP2A) to dephosphorylate and inactivate Akt, thereby enhancing GSK-3 activity (Polter and Li, 2011). IP3 activation increases intracellular Ca2+ level which can stimulate synergistic coactivation of Src and Pyk2 (Heidinger et al., 2002; Brody and Strittmatter, 2018), which also augment GSK-3 activity (Hartigan et al., 2001). However, Ca2+ may attenuate GSK-3 activity via the AC/cAMP/PKA pathway (Figure 11).
Coupling with GIRK Channels
A neurotransmitter receptor, whether it is a ligand-gated ion channel or G protein-coupled receptor (GPCR), can be activated by only one type of neurotransmitters. For instance, the GABAA or GABAB receptor can be activated only by GABA, while the NMDA or mGluR5 receptor responds only to glutamate. Although they could be activated by other agonists which, however, are not endogenous neurotransmitters. In contrast, a variety of neurotransmitters (adenosine, GABA, serotonin, etc.) can act on their specific GPCRs to open the G protein-gated inwardly rectifying potassium (GIRK) channels which play important roles in conscious perception as well as other physiological functions (Mayfield et al., 2015).
Basic Properties of GIRK Channels
The G protein coupled with GIRK channels comprises Gαi (or Gαo) and Gβγ. Upon activation, the Gαi subunit dissociates from Gβγ. GIRK channels do not interact directly with Gα. Instead, they are activated by Gβγ (Luscher and Slesinger, 2010). A few neurotransmitter receptors have been demonstrated to couple with GIRK channels, including adenosine's A1 receptor, melatonin's MT1 or MT2 receptor, and GABA's GABAB receptor (Figure 12). Activation of GIRK channels causes K+ ions to flow outward, resulting in membrane hyperpolarization. Hence, the opening of GIRK channels has inhibitory effects on neuronal firing. This property can be used to suppress faster oscillations (e.g., alpha and theta rhythms) within the infra-slow oscillation (ISO).
Suppression of Alpha Oscillations Within ISO
The infra-slow oscillation (ISO) typically contains alpha oscillations and a long silence period. Experiments have demonstrated that this silence period is caused by GIRK channels which can reduce neuronal excitability (Figure 13).
How Alcohol Reduces Conscious Level
Alpha oscillations play a critical role in consciousness (more info). Since activation of GIRK channels can suppress the alpha oscillations nested within ISO, higher GIRK channel activity should result in lower conscious level. This simple mechanism can explain how alcohol reduces conscious level.
A GIRK channel is formed by four subunits (Figure 14). Four different GIRK channel subunits have been identified: GIRK1-GIRK4. A GIRK channel may contain four identical GIRK2, or mixed GIRK1/2, GIRK1/3, GIRK1/4, or GIRK2/3. Binding with PIP2 is required to stabilize the GIRK channel in its open state. Gβγ can enhance their binding, thus activating the GIRK channel. Similarly, alcohol can also interact directly with the GIRK channel to enhance its binding with PIP2 (Bodhinathan and Slesinger, 2014). thereby increasing the activity of GIRK channels.
Author: Frank Lee