7TM receptor signalling
The ghrelin receptor belongs to the large family of 7 transmembrane segment receptors (7TM receptors), which are localized in the plasma membrane and transduce extracellular signals to intracellular responses. In addition to peptide hormones such as ghrelin, these extracellular stimuli also include large proteins, lipids, small organic molecules, ions and photons. Importantly a large percentage of drugs on the market targets the 7TM receptor superfamily, including some anti-histamines and β-blockers (Rask-Andersen et al., 2011). To differentiate the extracellular stimuli from this large array of 7TM receptors, cells have developed a sophisticated intracellular signalling network, governed by G–protein-dependent and G–protein-independent mechanisms. The G-protein is a heterotrimer composed of Gα and Gβγ subunits, which when inactive is assembled and bound to GDP. Upon receptor activation the GDP is exchanged with GTP, thereby allowing the Gα and Gβγ subunits to dissociate and initiate their respective signalling cascades. There are four highly studied Gα-mediated signalling pathways: (i) Gαs activates adenylate cyclase, leading to cAMP accumulation and cAMP-mediated responses whereas (ii) Gαi/o inhibits adenylate cyclase and thereby the cAMP accumulation; (iii) Gαq/11 activates PLC leading to inositol tris-1,4,5-phosphate [IP3; in the rest of this review article we will refer to the inositol phosphates (IP3, IP2, IP) as IP, IP is the most predominant form of the IPs measured in experiments of Gαq and subsequently PLC activation] and DAG production; and (iv) Gα12/13 activates Rho guanine exchange factors, resulting in activation of Rho associated kinases, and cytoskeletal rearrangement. The Gβγ subunits have also been shown to regulate signalling pathways such as calcium release (Stehno-Bittel et al., 1995), PLB, adenylate cyclase, but also MAPK, nucleic histone deacetylase 5 and adipocyte enhancer-binding protein 1 among others (Tang and Gilman, 1991; Crespo et al., 1994; Zhang et al., 1996; Park et al., 1999; Spiegelberg and Hamm, 2005).
The most commonly described G–protein-independent signalling pathway involves phosphorylation of the 7TM receptor by GPCR kinases (GRKs) followed by β-arrestin adaptor recruitment. β-Arrestins are able to activate various kinases such as Src, Akt and MAPK (e.g. ERK 1/2 and p38; Violin and Lefkowitz, 2007). β-Arrestin recruitment is also one of the possible pathways for internalization of most 7TM receptors, initiating processes of intracellular trafficking, which determine whether receptors are recycled or degraded during chronic agonist exposure (Zhang et al., 1998; Hanyaloglu and von Zastrow, 2008).
Ghrelin receptor mediated signalling
The ghrelin receptor was originally discovered to induce calcium release during an investigation into its growth hormone-releasing properties (Howard et al., 1996). When its endogenous ligand, ghrelin, was eventually detected (Kojima et al., 1999) more thorough studies of its signal transduction properties demonstrated a ghrelin receptor-dependent elevation of the PLC endproduct IP. Ghrelin has also been reported to activate other downstream signalling pathways, such as CREB-mediated transcription, in a dose-dependent manner – presumably through Gαq, as CREB can be activated by calcium calmodulin kinase (Dash et al., 1991; Kojima et al., 1999; Holst et al., 2003; 2004). In addition to the Gαq-coupled signalling the ghrelin receptor couples to Gα12/13 and thereby activates RhoA kinase. The combined actions of Gαq and Gα12/13 are responsible for the majority of the ghrelin-induced activation of serum response element (SRE), whereas Gαi coupling is not relevant for this pathway (Sivertsen et al., 2011), as the signal is unaffected by pertussis toxin. However Gαi/o coupling has been demonstrated in GTPγS assays in model systems (Bennett et al., 2009) as well as in isolated lipid discs (Damian et al., 2012; Mary et al., 2012). Furthermore, activation of the ghrelin receptor leads to the recruitment of the clathrin adaptor-related protein complex 2 (AP2), or β-arrestins, in a manner that is independent of G-protein coupling (Damian et al., 2012; Mokrosinski et al., 2012). Stimulation of the ghrelin receptor also induces ERK1/2 phosphorylation in a dose-dependent manner. This process has been shown to be dependent on PKC stimulation and phosphatidylcholine accumulation in a G–protein-dependent manner. In contrast β-arrestin does not play a role in this signalling, because a dominant negative mutant of β-arrestin failed to decrease ERK1/2 phosphorylation (Mousseaux et al., 2006; Chu et al., 2007). This wide range of signalling possibilities has allowed the development of ligands with functionally biased signalling properties (Figure 2).
Figure 2. Multiple signalling pathways of the ghrelin receptor. Dotted arrows represent not fully verified signalling pathways, and black full arrows indicate pathways that have been described or suggested for the ghrelin receptor. The ghrelin receptor is able to signal through three different G-proteins, for example Gαq, Gαi/o, Gα12/13 in addition to G–protein-independent arrestin coupling and internalization. Gαq, that activates PLC and leads to increased IP and DAG formation can induce an increase in Ca2+ signalling. However, the pharmacological profiling of different ghrelin receptor agonists indicates that the Ca2+ signalling and IP accumulation originate from separate signalling pathways (Holst et al., 2005), which explains the dotted line. In addition, Gαq coupling may also lead to CRE-mediated transcription activity and probably contributes to the SRE-mediated transcriptional activity. Finally, ligand-mediated Gαq coupling may also stimulate ERK1/2 phosphorylation. Gα12/13 activates RhoA and ROCK resulting in SRE transcription. Gαi/o, generally inhibits adenylate cylase to decrease cAMP accumulation; however, this has not been shown for the ghrelin receptor and it is possible that it can couple to Gαi/o and induce Ca2+ release. Ligand activation of the ghrelin receptor induces recruitment of β-arrestin, which might lead to receptor internalization. Both the constitutive and ligand activation of the receptor induce internalization of the receptor. β-Arrestins might lead to ERK phosphorylation but this is still uncertain.
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Unusually, ligand binding is not required for significant ghrelin receptor activation. In the absence of any ligand present, the receptor signals with almost 50% constitutive activity as measured in IP accumulation assays. This property has been demonstrated for several different signalling pathways including SRE- and CREB-mediated transcriptional activity (Holst et al., 2004). Recruitment of arrestin has also been shown to occur in a ligand-independent manner in heterologous expression systems, whereas in isolated lipid discs the arrestin recruitment is ligand dependent (Mary et al., 2012; Mokrosinski et al., 2012). Internalization of the ghrelin receptor can occur in a ligand-independent manner, but is dependent on receptor constitutive activity and domains within the C-terminal tail (Holliday et al., 2007). The recent demonstration of ligand-independent AP2 recruitment to the ghrelin receptor may therefore contribute to these basal receptor internalization properties (Damian et al., 2012). Interestingly, however, some signalling pathways, such as ERK phosphorylation and Gαi coupling, do require receptor activation by exogenous agonists (Holst et al., 2004; Damian et al., 2012). Recent studies have shown that the constitutive activity of the ghrelin receptor is an intrinsic property of the receptor; when the receptor is embedded in lipid discs it can induce IP accumulation and GTPγS binding and these signals can be reduced by addition of the inverse agonist or increased by the agonist (Damian et al., 2012). Thus, the scope for functionally selective ghrelin receptor ligands may also include those that might differentially modulate constitutively active receptor signalling rather than ghrelin-stimulated responses, known as ‘protean’ agonism (Ganguli et al., 1998; Gbahou et al., 2003).
The ghrelin receptor has been shown to dimerize both with other 7TM receptors as heterodimers and with itself as a homodimer. The interface responsible for the dimerization has not been studied for this receptor family. However, it has been demonstrated that the dopamine D1 receptor (Jiang et al., 2006), dopamine D2 receptor (Kern et al., 2012), melanocortin MC3 receptor (Rediger et al., 2012) and the 5-HT2C receptor (Schellekens et al., 2013) are co-expressed with the ghrelin receptor under physiological conditions. Importantly it has been shown that co-expression of the ghrelin receptor with either the MC3, the D1 or the 5HT2C receptor leads to decreased ghrelin-mediated signalling in heterologous expression systems. In addition α-melanocyte stimulating hormone (α-MSH) signalling is enhanced by co-expression of the ghrelin receptor with the MC3 receptor (Rediger et al., 2012).
Ligand development for the ghrelin receptor was initially focused on agonists to increase growth hormone secretion. Several high-potency efficacious agonists – based on either peptide or non-peptide scaffolds – were studied in clinical trials, when the appetite promoting effects of the ghrelin system was revealed and ligands to block the function of ghrelin subsequently became the major focus. The substance P analogue ([D-Arg1, D-Phe5, D-Trp7,9, Leu11]-substance P), was the first antagonist and inverse agonist to be identified for the ghrelin receptor. Since then, several other ligands for the ghrelin receptor have been discovered. Based on a conserved motif wFwLL (where wFwLL denotes one letter abbreviations of amino acids, using the small letters to denote the D-form and capital letters to denote the L-form.) of the substance P analogue, a series of inverse agonists has been developed (Holst et al., 2003). The penta-peptide wFwLL in itself binds to the ghrelin receptor with submicromolar affinity. However, it has the potential to act both as an agonist with a high potency and as an inverse agonist with a slightly lower potency. Modification by the N-terminal elongation of this motif with an alanine (AwFwLL) produced a more effective partial agonist whereas addition of lysine (KwFwLL) produced an inverse agonist (Holst et al., 2007). Thus, even subtle changes in the structure of this motif can give rise to large variations ranging from negative to positive efficacy. This family of peptides interacts with the ghrelin receptor through a different subset of receptor interaction points compared with the endogenous ligand ghrelin, as shown by mutational analysis and computational docking simulations (Holst et al., 2006). More recently, in an attempt to develop a treatment for obesity, small-molecule antagonists have been discovered for the ghrelin receptor. However, the molecular pharmacological properties of these compounds have yet to be fully characterized (Xin et al., 2006; Rudolph et al., 2007; Palus et al., 2011).
Using mutation studies in combination with computational chemistry, the interaction of ligands with the ghrelin receptor has also been studied for several agonist compounds and for the previously mentioned peptide-based inverse agonists. The most important ligand interaction site described in the ghrelin receptor is a glutamic acid in the extracellular part of TM III. This is pivotal for the binding and function of both ghrelin and almost all ghrelin receptor ligands. Furthermore, mutations of aromatic and positively charged residues in TM VI affect the potency of ghrelin significantly (Feighner et al., 1998; Holst et al., 2006; 2009). Apart from these residues, no other substitutions in the binding pocket affect ghrelin-induced activation, indicating that ghrelin only makes a few key interactions within the centre of the binding pocket of the receptor in addition to potential interactions in the extracellular loops (ECLs; Holst et al., 2006; 2009). However, the constitutive activity of the ghrelin receptor is diminished by substitutions in several other receptor domains, including substitutions of aromatic and charged residues in the extracellular part of TM III, TM VI and VII (Holst et al., 2004; Goze et al., 2010). These studies demonstrate the importance of a hydrophobic cluster connecting the extracellular parts of TM VI and VII for the activation of the receptor. Finally, inverse agonists generally require interactions deep in the transmembrane-binding pocket compared with the interactions made by agonists, which occur more superficially towards the extracellular ends of the TM domains (Holst et al., 2007; 2009).