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1521-0103/353/2/246–260$25.00 http://dx.doi.org/10.1124/jpet.114.221606 THE JOURNAL OF AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 353:246–260, May 2015 Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics

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Endogenous Allosteric Modulators of G Protein–Coupled Receptors

Emma T. van der Westhuizen, Celine Valant, Patrick M. Sexton, and Arthur Christopoulos Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria, Australia

Received December 3, 2014; accepted January 30, 2015 Downloaded from

ABSTRACT G protein–coupled receptors (GPCRs) are the largest superfam- but also a variety of ions, lipids, amino acids, , and ily of receptors encoded by the , and represent accessory proteins that display different degrees of - the largest class of current drug targets. Over the last decade specific modulatory effects. This also suggests that some GPCRs and a half, it has become widely accepted that most, if not all, may possess true “orphan” allosteric sites for hitherto un- jpet.aspetjournals.org GPCRs possess spatially distinct allosteric sites that can be appreciated endogenous modulators. Of note, the increasing targeted by exogenous substances to modulate the receptors’ identification of lipids, inflammatory peptides, biologic state. Although many of these allosteric sites are likely and GPCR-targeted autoantibodies indicates that disease context to serve other (e.g., structural) roles, they nonetheless possess plays an important role in the generation of putative endogenous appropriate properties to be serendipitously targeted by synthetic GPCR modulators. If an endogenous allosteric substance can be molecules. However, there are also examples of endogenous shown to play a role in disease, this could also serve as an impetus substances that can act as allosteric modulators of GPCRs. to pursue synthetic neutral allosteric ligands as novel therapeutic These include not only the obvious example, i.e., the G protein, agents. at ASPET Journals on June 1, 2016

Introduction The phenomenon of allostery was first described in seminal studies in the field of enzymology (Monod and Jacob, 1961; – G protein coupled receptors (GPCRs) are the largest family Monod et al., 1963), but subsequently extended to other of receptor proteins, responding to a vast range of extracel- classes of proteins (see Changeux and Edelstein, 1998, and lular mediators and widely pursued as drug targets (Bockaert Changeux, 2013). This highlights the fact that allosteric and Pin, 1999; Garland, 2013). Traditional drug discovery modulation is a ubiquitous and vital biologic process (Fenton, efforts have largely focused on or antagonists that 2008). It is also suggested that many of the mechanisms — target the orthosteric site on GPCRs that is, the (s) underlying allostery, as first formalized in the classic Monod- used by endogenous (s). However, it is now well Wyman-Changeux (Monod et al., 1965) and Koshland-Nemethy- recognized that most, if not all, GPCRs possess spatially distinct Filmer models (Koshland et al., 1966), are likely applicable to allosteric sites that can also be targeted for therapeutic benefit other protein classes, including GPCRs (Canals et al., 2011). (Christopoulos and Kenakin, 2002; May et al., 2007; Conn et al., Advantages of targeting allosteric GPCR sites include the 2009; Christopoulos, 2014; Christopoulos et al., 2014). potential for receptor subtype selectivity, either due to greater sequence divergence in allosteric pockets between receptor subtypes relative to the (necessarily) conserved orthosteric site, Portions of the work cited from the authors’ laboratories were funded by the National Health and Medical Research Council of Australia (NHMRC) [Program or due to subtype-selective cooperativity; the ability to fine tune Grant APP1055134 and Project Grants APP1026962, APP1084246, and physiologic responses in either a positive or negative direction; APP1061044]. A.C. and P.M.S. are Principal Research Fellows of the NHMRC. “ ” E.T.v.d.W. and C.V. contributed equally to this work. and a saturability, or ceiling, to the effect that may lead to dx.doi.org/10.1124/jpet.114.221606. greater on-target safety in overdose situations (May et al., 2007;

21 ABBREVIATIONS: 2-AG, 2-arachidonoylglycerol; AR, adrenoceptor; AT1, 1; [Ca ]o, extracellular ; CaSR, calcium-sensing receptor; CB, ; CCK, cholecystokinin; CCM, cholesterol consensus motif; CLR, –like; CP55,940, 2-[(1R,2R,5R)-5- 2 5 hydroxy-2-(3-hydroxypropyl) cyclohexyl]-5-(2-methyloctan-2-yl)phenol; DPDPE, [D-Pen ,D-Pen ]-enkephalin; ERK, extracellular signal-regulated 125 kinase; GPCR, G protein–coupled receptor; 5-HT, 5-hydroxytryptamine; [ I]AB-MECA, N6-(4-aminobenzyl)-9-[5-(methylcarbonyl)-b-D-ribofuranosyl] ; IP, phosphate; mAChR, muscarinic ; MCR, receptor; mGluR, metabotropic ; MRAP, accessory protein; OR, receptor; RAMP, receptor activity–modifying protein; THC, ; TSHR, thyroid-stimulating hormone receptor; WIN55212-2, (R)-(1)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo [1,2,3-de]-1,4-benzoxazin-6-yl]-1- napthalenylmethanone.

246 Endogenous GPCR Allosteric Modulators 247

Christopoulos, 2014; Conn et al., 2014). Allosteric modulators can also display distinct pharmacological properties, including the phenomena of “probe dependence” and “biased agonism/ modulation.” Both of these reflect similar conformational mechanisms: probe dependence describes the situation whereby the magnitude and direction of the allosteric effect of a given modulator can change depending on the nature of the orthosteric being used to probe receptor function, whereas biased agonism/modulation describes the allosteric change in the GPCR’s intracellular signaling preferences (i.e., receptor–transducer inter- actions) depending on the nature of the ligand being used to activate or modulate the receptor (Keov et al., 2011; Kenakin and Christopoulos, 2013). The theoretical advantages associated with GPCR allosteric modulators have spurred numerous ongoing research pro- grams in academia and industry over the last decade and a half (Conn et al., 2014). These studies are not only revealing the extent to which the theory of allostery can be put to practical use in GPCR drug discovery, but also identifying ongoing Downloaded from challenges and questions associated with the phenomenon. For example, the prevalence of probe dependence highlights the need for more broad screening of putative modulator compounds than previously anticipated, particularly for GPCRs that have more than one endogenous agonist (Wootten et al., 2013a). Similar considerations apply for biased agonists/modulators, as in many jpet.aspetjournals.org Fig. 1. (A) General modes of ligand binding to GPCRs. A domain associated cases the links between observed cellular drug effects and in vivo with binding of an orthosteric ligand at one type of GPCR may be exploited pathophysiology remain unclear (Kenakin and Christopoulos, as an allosteric site at another type of GPCR. (B) GPCR allosteric sites may 2013). Moreover, the long-term effects of allosteric GPCR arise “serendipitously” as a consequence of favorable structural features upon folding and membrane insertion, or may represent “orphan” sites for ligands on receptor regulation remain largely unknown (Lane hitherto unappreciated endogenous modulators. et al., 2013), even though many drug therapies are chronic in nature.

Interestingly, the finding that allosteric GPCR sites are a consequence of structural roles (e.g., receptor folding, trafficking) at ASPET Journals on June 1, 2016 highly prevalent is also leading to a re-evaluation of the role but possess sufficient functional groups so as to be serendipitously of many of these sites, and the possibility that they may exploited by exogenous synthetic compounds (Fig. 1B). None- represent interaction domains for unappreciated endogenous theless, it is also possible that some GPCRs may be regulated ligands. This is certainly the case for other receptor super- naturally by endogenous modulators (Fig. 1B). This review families. For instance, the function of nuclear hormone receptors considers some of the emerging data in support of the latter is characterized by the requirement for interaction with a suite possibility. of corepressor or coactivator molecules, as well as DNA, in a complex network of allosteric interactions (Burris et al., 2013). G Proteins as Endogenous Allosteric Modulators The GABAA family of ligand-gated ion channels is also modulated by endogenous neuroactive steroids (Majewska, 1992; Mitchell GPCRs are natural allosteric proteins in the sense that they et al., 2008) and proteins (Christian et al., 2013). The best respond to extracellular stimuli via a distinct interaction characterized endogenous allosteric modulator of a GPCR is, of domain (the orthosteric site) and transmit the stimulus to a course, the G protein itself (see below) but increasingly, other topographically distinct intracellular domain, the G protein–binding endogenous substances are being identified that may act as site, to transduce the signal (Christopoulos and Kenakin, 2002). allosteric GPCR modulators with greater degrees of specificity. Furthermore, the nature of the interaction is clearly allosteric This begs the question about the overall extent of endogenous because of the reciprocal effects that agonists (and inverse GPCR subtype-specific allosteric modulators. Is it possible that agonists) and G proteins exert on one another. For example, many of the allosteric sites used by synthetic small-molecule Fig. 2A illustrates the profound allosteric effect that the GPCR modulators represent “orphan” allosteric sites for as , GTP, exerts on the binding of the agonist, carbachol, yet unidentified natural modulators, or are they largely at the M2 muscarinic acetylcholine receptor (mAChR). This “serendipitous” allosteric sites with no natural ligand (Hardy interaction is characterized by negative cooperativity, because and Wells, 2004)? In the GPCR field, the latter view has agonist binding favors coupling of the nucleotide-free G protein traditionally predominated and remains logical; binding domains to form a high-affinity “ternary complex,” whereas nucleo- associated with orthosteric ligands for one type of GPCR family tide binding uncouples this complex to yield a low-affinity, (e.g., the transmembrane regions for numerous -like Gprotein–free state (De Lean et al., 1980; Ehlert, 1985). class A GPCRs) can represent serendipitous allosteric sites Further evidence of the allosteric nature of G protein–GPCR for other GPCR types (e.g., class B receptors, or class C interactions has been provided by studies in which the G protein nutrient receptors, where the orthosteric site is largely restricted has been overexpressed. For instance, overexpressing Gs or Gq to extracellular loop or N-terminal domains) (Fig. 1A). It is likely proteins increases the basal constitutive activation of the that some allosteric sites are cavities that normally arise as b2-adrenoceptor (b2-AR) or the M1/3/5 mAChRs, respectively 248 van der Westhuizen et al.

an alternative ternary complex to the classic agonist–receptor– G protein paradigm (Gurevich et al., 1997). The receptor activity–modifying proteins (RAMPs) are an- other family of accessory proteins that play a profound role in modulating the pharmacology of numerous GPCRs. These three single transmembrane-spanning proteins were initially identified as essential coupling partners for the class B calcitonin receptor–like receptor (CLR); depending on the heteromer formed between the CLR and a given RAMP, the resultant complex was revealed to be the minimal unit required to yield the pharmaco- logically defined receptors for calcitonin -related peptide or adrenomedullin (McLatchie et al., 1998; Bühlmann et al., 1999; Christopoulos et al., 1999). These studies were extended to reveal that the calcitonin receptor can also interact with RAMPs to yield distinct amylin receptors (Muff et al., 1999; Zumpe et al., 2000; Poyner et al., 2002; Hay et al., 2004, 2006; Udawela et al., 2006). Since these initial breakthroughs, further roles for RAMPs in physiology and pharmacology have been discovered. In addition to the determination of receptor phenotype, outlined Downloaded from earlier, RAMPs can also modulate the signaling preferences of GPCRs, either in terms of strength of coupling to G protein pathways (e.g., corticotropin-releasing hormone 1 receptor) Fig. 2. Allosteric effects on agonist binding due to GPCR–protein ternary (Wootten et al., 2013b) or even biasing signaling between complex formation. (A) Receptor–G protein interaction. Effects of increasing different intracellular pathways for the vasoactive intestinal concentrations of GTP on the affinity of the agonist, carbachol, at atrial polypeptide 1 receptor (Christopoulos et al., 2003) or calcitonin jpet.aspetjournals.org M2 mAChRs. Data replotted from Ehlert (1985). (B) Effects of different arrestin proteins on the binding of the agonist, isoproterenol, to the b2-AR. receptor (Morfis et al., 2008). RAMPs can also play important Data replotted from Gurevich et al. (1997). ISO, isoproterenol; NMS, chaperoning roles for receptor trafficking processes, as noted N-methylscopolamine. not only for the canonical interaction with CLR, but also for the class C extracellular calcium-sensing receptor (CaSR) (Burstein et al., 1997; Azzi et al., 2001). Inverse agonists can (Bouschet et al., 2005). Generation of genetically engineered decrease this heightened constitutive activity, whereas mouse models in recent years has unmasked key roles for the

agonist affinity and potency are generally increased under different RAMP proteins in the cardiovasacular, renal, and at ASPET Journals on June 1, 2016 the same conditions (Burstein et al., 1997; Yan et al., 2008). respiratory systems, in addition to novel roles in inflammation The tight conformational coupling between the orthosteric (Kadmiel et al., 2012; Lenhart et al., 2013; Li et al., 2014). and G protein–coupling sites is also highlighted by the fact Importantly, the RAMP-receptor interface has emerged as a that the sites show strong patterns of coevolution (Suel et al., novel druggable pocket in its own right (Sexton et al., 2009, 2003). Direct demonstration of the profound conformational 2012; Wootten et al., 2010). Figure 3 shows the recent crystal changes that occur in the process of GPCR signal trans- structure of the N-terminal region of CLR in complex with ductionuponassociationwithheterotrimericGproteinsor RAMP1 and the antimigraine drug, olcegepant (ter Haar et al., Gprotein–mimicking nanobodies has also been revealed in 2010), providing a structural basis for selective anti–calcitonin recent breakthrough structural studies (Rasmussen et al., gene-related peptide receptor ligands (Sixt et al., 2009). 2011; Ring et al., 2013). Collectively, these studies show that The characteristics of the RAMP family are likely to be G proteins can alter the conformational state of the GPCR in exemplars of a broader accessory protein paradigm for an allosteric manner to influence the binding and/or signaling GPCRs that continues to yield novel findings. For instance, the properties of agonist and antagonist ligands. melanocortin receptor accessory proteins (MRAPs) have recently

GPCR–Accessory Protein Interactions It is now well accepted that GPCRs can participate in non- canonical signaling networks via interactions with an expanding list of accessory proteins, often in a G protein–independent manner (Brady and Limbird, 2002; Sato et al., 2006; Cooray et al., 2009; Couvineau and Laburthe, 2012). Probably the best characterized example is that of the GPCR–b-arrestin interaction. Although originally identified as playing a vital role in the termination of GPCR signaling via G proteins, b-arrestins are also known to act as scaffolding proteins and novel signal transducers in their own right for certain receptors (Reiter et al., 2012). Interest- ingly, the interaction of the arrestin with the GPCR can itself modulate the properties of agonists. As shown in Fig. 2B, the Fig. 3. Crystal structure of the N-terminal region of CLR in complex with affinity of isoproterenol for the b2-AR is markedly enhanced in the accessory protein, RAMP1, and the antimigraine drug, olcegepant. the presence of different arrestin proteins, essentially forming (PDB ID 3N7S.) Endogenous GPCR Allosteric Modulators 249 been identified as single transmembrane-spanning proteins that involves the effects of sodium on GPCR sensitivity to agonists modulate the expression, trafficking, and signaling of members and inverse agonists. A seminal study by Pert and Snyder of the melanocortin receptor (MCR) family with many similar- (1973) was the first to suggest that Na1 could act as a negative ities to RAMPs and their interacting GPCRs (Novoselova et al., allosteric modulator of agonist binding while 2013). The first MRAP was identified through a genetic screen in having relatively little effect on antagonists. Subsequent studies patients with familial deficiency, and was found suggested that the effect of Na1 likely involves modulation of to be most highly expressed in the adrenal gland where it the conformational state of the receptor (Simon and Groth, facilitates the cell-surface expression of MCR2 and makes the 1975), and that the binding of certain antagonists can actually be receptor responsive to adrenocorticotropic hormone (Metherell potentiated by the ion (Appelmans et al., 1986), one of the key et al., 2005). A second homolog, MRAP2, was identified shortly characteristics exploited in the biochemical identification and thereafter and shown to be highly conserved in vertebrates validation of inverse agonism (Costa and Herz, 1989). These (Chan et al., 2009). Similar to the RAMPs, the MRAPs show observations were soon extended to numerous other class A quite a wide expression pattern, which suggests that they likely GPCRs, including those for biogenic amines, nucleosides, serve other functions that go beyond trafficking and expression. peptides, and lipids (Ericksen et al., 2009; Liu et al., 2012; Possibilities include the determination of MCR phenotype and Katritch et al., 2013); Fig. 4A shows an example of opposing signal coupling preferences (Novoselova et al., 2013). Given that effects of Na1 on the binding of an agonist (quinpirole) and approximately 20% of cases of familial glucocorticoid deficiency (epidepride) at the dopamine D2 receptor are caused by in MRAP (Metherell et al., 2005), this (Neve, 1991), which is consistent with the expectations of be- highlights the potential for targeting the MCR-MRAP interac- havior within a two-state receptor model (Christopoulos, 2014). Downloaded from tion for therapeutic purposes. However, the affinities of other known inverse agonists of the Finally, it is now widely acknowledged that GPCRs may D2 receptor are not necessarily sensitive to sodium (e.g., Vivo participate in homo- and heterodimeric/oligomeric interactions et al., 2006), suggesting that the phenomenon is also dependent with one another to yield potentially distinct and targetable phar- on ligand structure and remains an active area of research. macological entities (Milligan et al., 2003; Panetta and Greenwood, Importantly, many of the allosteric effects of sodium occur at or

2008; Milligan, 2009). Although beyond the scope of the current near physiologic concentrations (e.g., Costa and Herz, 1989) jpet.aspetjournals.org review, cooperative interactions within oligomeric receptor and thus suggest that this plays a key general role in GPCR arrays clearly highlight that GPCRs have the potential to act biology. as endogenous allosteric modulators of one another. Mutagenesis studies identified a highly conserved aspartic acid residue, D2.50, in the second transmembrane domain of Ions as Allosteric Modulators class A GPCRs as being key to the allosteric actions of sodium (Fraser et al., 1989; Horstman et al., 1990; Neve, 1991; Strader

Sodium. The functionality of many GPCRs is sensitive to et al., 1994). However, the structural basis of this interaction at ASPET Journals on June 1, 2016 modulation by various ions. One of the best-studied examples has only recently begun to be delineated as a consequence of

Fig. 4. Endogenous allosteric GPCR modulators. (A) Effects of sodium on the binding of an agonist (epitepride) and antagonist (quinpirole) at the dopa- mine D2 receptor. Replotted from Neve (1991). (B) Inhibition of the binding of the orthosteric agonist [125I]AB-MECA by the endocannabinoid 2-AG at the A3 receptor. Replotted from Lane et al. (2010). (C) Potentiation of the effects of calcium by the L-Phe at the CaSR. Replotted from Lee et al. (2007). (D) Effects on the binding of the cannabinoid agonist [3H]WIN55,212-2 by an exoge- nous orthosteric agonist, CP55,940, or an endogenous allosteric peptide, pepcan-12, at the cannabinoid CB1 receptor. Replotted from Bauer et al. (2012). 250 van der Westhuizen et al. high-resolution GPCR crystal structures revealing a coordi- Hanissian, 1990; Rodriguez et al., 1992; Schetz and Sibley, 1997, nated Na1/water cluster in the transmembrane bundle of 2001; Holst et al., 2002; Lagerstrom et al., 2003; Swaminath inactive-state GPCRs, including the adenosine A2A (Liu et al., et al., 2003). inhibits orthosteric ligand binding at D1,D2L, 2012), protease-activated receptor 1 (Zhang et al., 2012), b1-AR and D4 dopamine receptors; MC1 and MC4 melanocortin re- (Miller-Gallacher et al., 2014), and d-opioid (Fenalti et al., 2014) ceptors; b2-anda1A-ARs; and m-, k-, and d-opioid receptors (ORs) receptors. The latter structure is shown in Fig. 5A, highlighting (Stengaard-Pedersen et al., 1981; Tejwani and Hanissian, 1990; the network of amino acid side chains (green) and coordinated Rodriguez et al., 1992; Schetz and Sibley, 1997, 2001; Holst waters (red) connecting the ligand binding pocket to the sodium et al., 2002; Swaminath et al., 2002; Lagerstrom et al., 2003). ion (blue). This sodium site appears to collapse in active-state It increases the dissociation rate of antagonists from the D1 and structures, suggesting that it plays a key role in constraining D2L receptors (Schetz and Sibley, 1997, 2001), whereas it de- GPCRs in an inactive state (Wootten et al., 2013b; Katritch creases the dissociation rates of antagonists from the b2-and et al., 2014). Interestingly, the sodium site has also been a1A-ARs (Swaminath et al., 2002; Ciolek et al., 2011). Function- recently implicated in the regulation of biased agonism by the ally, zinc potentiates the effects of agonists at the MC1, MC4, d-opioid receptor (Fenalti et al., 2014) and in the actions of and b2-AR toward cAMP accumulation; however, it negatively synthetic small-molecule allosteric ligands for the m-opioid modulatestheeffectsofagonistsatthea1A-AR toward calcium receptor (Livingston and Traynor, 2014). It should be noted, signaling (Holst et al., 2002; Swaminath et al., 2002; Ciolek however, that a subset of class A GPCRs does not possess the et al., 2011). Together these results show that zinc can act as an requisite acidic residue at the 2.50 position. These GPCRs allosteric modulator of GPCRs, with positive or negative effects include the visual , which do not possess a diffusible toward orthosteric ligand binding and function. Downloaded from endogenous activator, and a number of other atypical receptors . Magnesium is another ion that has been whose functionality and/or ligands are not currently deter- implicated as an allosteric modulator at some GPCRs. Magne- mined (Katritch et al., 2014). sium increases the specific binding of opioid receptor agonists Zinc. Zinc is an important ion in the body that regulates and antagonists in a concentration-dependent manner (Pasternak the activity of many different proteins, and has been suggested et al., 1975; Rodriguez et al., 1992). Some differences are seen to act as an allosteric modulator of a number of GPCRs, between the different opioid receptor subtypes. For the m-OR jpet.aspetjournals.org including the dopamine, melanocortin, , and opioid and k-OR, magnesium increases the affinity of agonist binding receptors (Stengaard-Pedersen et al., 1981; Tejwani and without changing the number of binding sites, respectively, whereas for the d-OR, the affinity for the agonist, DPDPE 2 5 ([D-Pen ,D-Pen ]-enkephalin), was reduced with an increase in Bmax (Rodriguez et al., 1992). For antagonists, the number of binding sites doubled in the presence of magnesium, without

changing the affinity of the ligands for the ORs (Rodriguez at ASPET Journals on June 1, 2016 et al., 1992). Agonist binding affinity for b-ARs (Williams et al., 1978) or dopamine D2 receptors (Sibley and Creese, 1983) increases in the presence of magnesium, whereas no effect is observed for antagonist binding. It should be noted, however, that magnesium also plays a role in the receptor–G protein in- teraction, because it can also interact directly with the G protein itself (Birnbaumer and Zurita, 2010). Thus, some of the allosteric effects ascribed to magnesium in classic radioligand binding assays may reflect an indirect effect manifested at the level of the receptor–G protein interaction, rather than the receptor–ligand interaction. Nonetheless, a clear demonstration of magnesium acting as an allosteric modulator via a well defined allosteric site on the M2 mAChR has been documented (Burgmer et al., 1998). Other Ions. Several additional ions have been suggested to act as allosteric modulators of GPCRs; however, the evidence of a true allosteric modulation is limited. Binding of the 2 4 5 m-opioid receptor agonist, [D-Ala ,NMe-Phe ,Gly-ol ]-enkephalin, to guinea pig brain homogenates and the b- agonist, hydroxybenzylisoproterenol, to frog erythrocyte mem- branes increased in the presence of calcium or manganese (Williams et al., 1978; Rodriguez et al., 1992). Increasing the calcium concentration also affected the affinity and efficacy of Fig. 5. Structural insights into endogenous GPCR modulators. (A) High- orthosteric and allosteric ligands at the metabotropic gluta- resolution crystal structure of the d-OR in complex with the antagonist naltrindole. The sodium ion is shown in blue, the network of coordinated mate 1a receptor (mGluR1a) (Jiang et al., 2014). Copper inhibits water molecules connecting the orthosteric site and the ion are indicated the binding of the antagonist, prazosin, in COS7 cells expressing in red, and the key amino acid side chains are indicated in green ball and human a1A-ARs without altering the dissociation of prazosin stick. (PDB ID 4N6H.) (B) Crystal structure of the b2-AR in complex with from the receptor, and has complex effects on the binding and the inverse agonist timolol and two cholesterol molecules. (PDB ID 3D4S.) A putative cholesterol consensus motif that is conserved across a range of signaling of the endogenous agonist, epinephrine (Ciolek et al., GPCRs is highlighted in orange. 2011). Cobalt has also been reported to decrease the affinity of Endogenous GPCR Allosteric Modulators 251 agonist binding to m-ORs in guinea pig brain homogenates content is not always observed; although the CCK1 receptor is (Rodriguez et al., 1992). sensitive to cholesterol content, the related CCK2 receptor is Collectively, the aforementioned studies suggest that nu- not (Potter et al., 2012). This suggests that cholesterol has some merous ions may influence GPCRs allosterically. However, selectivity for different GPCRs. further work is required to conclusively establish whether ion- Substantial interest in the potential for cholesterol to mediated changes in GPCR ligand affinity, apparent number directly interact with GPCRs gained further impetus upon of receptor binding sites, and/or function are truly allosteric the solution of a 2.8-Å structure of the b2-AR simultaneously interactions and are not due to perturbations in the osmolarity bound to timolol and two molecules of cholesterol, and the of the buffers used. Moreover, there remains a paucity of identification that this binding site may contain a cholesterol studies establishing that the concentrations of many of these consensus motif (CCM) (Hanson et al., 2008). As shown in ions required to mediate allosteric effects on GPCRs are Fig. 5B, this motif comprises three residues from transmembrane routinely attained under physiologic or pathophysiological domain 4 and one aromatic residue from transmembrane conditions. domain 2. The most significant interaction is proposed to come from the highly conserved aromatic residue at position 4.50, Lipids as Allosteric Modulators which is a tryptophan in the majority of cases, and the other residues contribute additional interactions. At least 96 different Cholesterol. Given that GPCRs are integral membrane GPCRs have been proposed to contain a CCM (Hanson et al., proteins, it is not surprising that the lipid bilayer environ- 2008), although it remains to be determined to what extent ment can markedly influence their behavior. In general, there this is likely to truly constitute such a site for modulation by Downloaded from are three ways by which this can occur. The first is through cholesterol. For example, both the CCK1 and CCK2 receptors changes in the physical properties of the bilayer, such as in contain the CCM, yet only the CCK1 receptor is sensitive to terms of membrane fluidity, curvature, or stress, such that cholesterol depletion (Potter et al., 2012). Furthermore, some the conformational landscape of a given GPCR is indirectly GPCRsthatcontaintheCCMareabletobeexpressedand modulated. This has been best demonstrated in studies of display binding and function in membranes that lack cholesterol, rhodopsin, where the transition between the metarhodopsin I such as those of Escherichia coli (Oates and Watts, 2011). jpet.aspetjournals.org and II states can be substantially influenced by the physical Given that cholesterol levels can vary dramatically in and properties of the membrane (Mitchell et al., 1990; Botelho contribute directly to disease (Vance, 2012; Ridker, 2014), et al., 2002; Soubias et al., 2010). The second mechanism by their ability to influence GPCR biology may play role in which lipids influence GPCR activity is via their ability to con- pathophysiology. However, a definitive link between patho- tribute to the subcellular compartmentalization of the receptor physiological variations in membrane cholesterol and GPCR and associated effector molecules in highly ordered domains, signaling remains to be determined.

such as caveolae and lipid rafts (Chini and Parenti, 2004; Endocannabinoids. Anandamide and 2-arachidonoylgly- at ASPET Journals on June 1, 2016 Ostrom and Insel, 2004; Patel et al., 2008). The third mechanism cerol (2-AG) are the principal endogenous cannabinoid (CB) is via a direct interaction of different lipid types with specific ligands found in the brain and periphery (Castillo et al., 2012). binding domains on the GPCR itself. In all cases, probably the Although their cognate receptors are the CB1 and CB2 GPCRs, best studied example of a GPCR-modulating lipid is cholesterol potential receptor-specific interactions have also been reported (Paila and Chattopadhyay, 2010). between these substances and other classes of GPCR. For Cholesterol is an important constituent of lipid rafts, dense example, anandamide has been shown to inhibit binding of platforms that are found in lipid bilayers. The affinities of ligands to central 5-HT receptors (Kimura et al., 1998) and ligands binding to the , cholecystokinin re- mAChRs (Lagalwar et al., 1999). Effects on mAChRs were ceptor 1 (CCK1), mGluR1a, and the 5-hydroxytryptamine 1A also evident in recombinant cell lines expressing the human (5-HT1A) receptor are increased when these receptors are M1 (Christopoulos and Wilson, 2001; Lanzafame et al., 2004) present in cholesterol-rich rafts compared with when they are or M4 mAChR (Christopoulos and Wilson, 2001). More detailed expressed in cholesterol-depleted rafts (Gimpl et al., 1997; studies indicated that the effects on the M1 mAChR, at least, Eroglu et al., 2003; Prasad et al., 2009; Potter et al., 2012). reflected the ability of anandamide to inhibit antagonist binding Restoring the cholesterol content of cholesterol-depleted mem- in a noncompetitive manner by decreasing the apparent number branes increased the number of receptors in the lipid rafts and of mAChR binding sites without affecting antagonist affinity restored the binding affinity of the ligands to the receptors, in the (Lanzafame et al., 2004). However, anandamide did not change case of oxytocin and mGluR1a receptors (Gimpl et al., 1997; the competition binding profiles of classic mAChR orthosteric Eroglu et al., 2003). These results suggest that the ligand agonists or allosteric modulators, but did affect agonist function binding properties of GPCRs can depend on the sterols and the in a manner correlated with agonist efficacy. This suggests that lipid environment of the membrane. Cholesterol depletion has its actions are either via a novel allosteric site on the M1 mAChR also been shown to affect receptor signaling, where decreases in or through a membrane-perturbing effect that is sensitive to calcium mobilization for the CCK1 receptor and extracellular receptor conformation (Lanzafame et al., 2004). signal-regulated kinases 1/2 (ERK1/2) phosphorylation and In a similar fashion, 2-AG has also been shown to modulate c-Fos activation for the gonadotrophin-releasing hormone the binding of ligands to a noncannabinoid GPCR. Specifi- receptor are lost, but can be restored upon replenishment cally, the binding of both agonists and antagonists to the of the cholesterol (Navratil et al., 2003; Potter et al., 2012). was inhibited by 2-AG in a noncompet- Similarly, depletion of membrane cholesterol content reduced itive manner (Lane et al., 2010). Figure 4B illustrates the effects m-OR signaling, whereas d-OR signaling was insensitive to the of increasing concentrations of 2-AG on the binding of the 125 same manipulation (Levitt et al., 2009). Of note, the increases radiolabeled A3 agonist, [ I]AB-MECA [N6-(4-aminobenzyl)-9- in agonist affinity for GPCRs with increased membrane cholesterol [5-(methylcarbonyl)-b-D-ribofuranosyl]adenine], where it can be 252 van der Westhuizen et al. seen that most of the inhibition occurs over a very narrow et al., 1998). Functionally, inhibited oxytocin- concentration range that is not consistent with competition for promoted inositol phosphate accumulation and calcium responses a common binding site. Importantly, these effects of 2-AG were at the rat oxytocin receptor in a concentration-dependent manner not observed at the related A1 or A2A adenosine receptors, (Grazzini et al., 1998). The effects of progesterone appear suggesting a certain degree of specificity to the effect. Given that to be species-specific, since progesterone did not compete 3 adenosine A3 and mAChRs are colocalized in similar brain for [ H]oxytocin binding to the human oxytocin receptor regions as cannabinoid receptors, it is possible that elevated (Grazzini et al., 1998). However, the progesterone metab- local levels of endocannabinoids may mediate hitherto unap- olite 5b-dihydroxyprogesterone did inhibit agonist binding to preciated roles as endogenous negative allosteric modulators. the human oxytocin receptor (Grazzini et al., 1998). Likewise, in The mechanism underlying this effect, however, remains un- functional assays, inositol phosphate accumulation was inhib- clear. For instance, it may reflect an interaction with a specific ited only by 5b-dihydroxyprogesterone and not progesterone at allosteric site on each receptor, or it may reflect cooperativity the human oxytocin receptor (Grazzini et al., 1998) between orthosteric sites within an adenosine-mAChR hetero- Oleamide. Oleamide is an endogenous fatty acid amide dimeric complex. found in cerebrospinal fluid that plays a role in sleep regulation. Lipoxin A4. The arachidonic acid derivative, lipoxin A4, Its mechanism of action remains unclear, but is likely to has recently been shown to induce cannabinoid-like responses involve multiple systems, in particular ago- in mouse brain that are antagonized by CB1 receptor antago- nistic activity at CB receptors and inhibition of fatty acid amide nists, suggesting that lipoxin A4 acts at the CB1 receptor hydrolase (Leggett et al., 2004). However, allosteric effects of the (Pamplona et al., 2012). The traditional biologic target of lipoxin substance have also been reported at various 5-HT receptor Downloaded from A4 is the FPR1 (Chiang et al., 2006), subtypes. For instance, at the 5-HT2A receptor, oleamide leading to the possibility that the nonclassic eicosanoid may be potentiated the effects of agonist-stimulated inositol phosphate acting allosterically at the CB1 receptor. In agreement with the (IP) turnover in rat P11 cells without having an effect when latter possibility, the same study found that lipoxin A4 can applied alone (Thomas et al., 1997, 1998). In HeLa cells transfected 3 enhance the affinity of the agonists—anandamide, [ H]CP55,940 with 5-HT7 receptors, oleamide and 5-HT each increased cAMP

(2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl) cyclohexyl]-5- accumulation; however, when they were coapplied, a reduction jpet.aspetjournals.org (2-methyloctan-2-yl)phenol), and [3H]WIN55212-2 [(R)-(1)-[2,3- was observed in 5-HT–promoted cAMP responses, suggesting dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo [1,2,3-de]-1, an allosteric interaction (Thomas et al., 1997). Direct negative 4-benzoxazin-6-yl]-1-napthalenylmethanone]—while partially dis- cooperativity between oleamide and 5-HT was subsequently placing antagonist binding (Pamplona et al., 2012). Allosteric demonstrated in radioligand binding assays at the 5-HT7 effects of lipoxin A4 were also seen functionally, where coinjection receptor (Hedlund et al., 1999). In Xenopus oocytes expressing of subeffective doses of anandamide or 2-AG into mice brains, 5-HT2A or 5-HT2C receptors, oleamide potentiated 5-HT–promoted with lipoxin A4, potentiated the cataleptic effects of the chloride channel currents (Huidobro-Toro and Harris, 1996). at ASPET Journals on June 1, 2016 endocannabinoids (Pamplona et al., 2012). . A very recent study found that adminis- 9 Amino Acids and Peptides as Allosteric tration of D -tetrahydrocannabinol (THC), the main active Modulators component of marijuana, in rats resulted in an increase in the levels of pregnenolone, which has traditionally been viewed as Amino Acids. In addition to their obligate roles in the the inactive precursor to the generation of (Vallée synthesis of proteins or other major biomolecules, some of the et al., 2014). Surprisingly, however, pregnenolone appeared to 20 eukaryotic amino acids have also been identified as putative exert negative modulatory effects on the cannabinoid CB1 endogenous allosteric modulators of specific GPCRs. Arguably receptor through a potential allosteric mechanism. Although the best validated example is that of aromatic amino acids, pregnenolone had no effect on agonist affinity, it inhibited such as L-Phe, L-Trp, and L-Tyr, that allosterically modulate 21 CB1-mediated ERK1/2 phosphorylation and THC-induced the actions of extracellular calcium ([Ca ]o)attheCaSR changes in cellular and mitochondrial respiration. It also inhibited (Conigrave et al., 2000). These amino acids bind in the venus key in vivo effects on locomotion, temperature, catalepsy, and flytrap domain, near the orthosteric site, to potentiate the 21 analgesia commonly attributed to THC (Vallée et al., 2014). actions of [Ca ]o at a variety of intracellular pathways Taken together, many of the aforementioned studies indicate (Conigrave et al., 2000; Zhang et al., 2002; Mun et al., 2005), 21 an unappreciated complexity in the cannabinoid system as in addition to enhancing [Ca ]o-dependent suppression of a source of GPCR lipidic modulators and/or a target for novel parathyroid hormone secretion in parathyroid cells (Lee endogenous modulators. It is possible the overactivation of et al., 2007). Figure 4C illustrates an example of the effects of cannabinoid receptors, in particular, needs to be tightly L-Phe on intracellular calcium mobilization in response to regulated, thus providing a rationale for the identification of the endogenous agonist. Although the potentiating effects of different putative endogenous negative allosteric modulators. amino acids on CaSR signaling are relatively subtle, they are However, it should also be noted that further experimental nonetheless likely to be physiologically relevant, as small studies are required to validate and extend these intriguing degrees of positive allosteric modulation of the CaSR by synthetic results, but may yield rich opportunities for novel therapeutic modulators acting in the receptor’s transmembrane domain interventions. (e.g., Davey et al., 2012) are known to be clinically relevant Progesterone. Similar to observations with anandamide (Block et al., 2004; Saidak et al., 2009). at the mAChRs, the steroid hormone progesterone inhibited The aromatic amino acid L-Phe as well as the aliphatic L-Leu agonist and antagonist binding at the recombinantly expressed and L-Ile have also been suggested to allosterically modulate rat oxytocin receptor cells by reducing the maximal binding the properties of baclofen at the GABAB receptor (Kerr and capacity without changing apparent ligand affinity (Grazzini Ong, 2003), although the significance of this effect remains Endogenous GPCR Allosteric Modulators 253 unclear since no modulation by these amino acids of the inhibition of radioligand binding mediated by the orthosteric endogenous agonist GABA could be detected in either native or agonist CP55,940, pepcan-12 only partially inhibited specific recombinant systems (Urwyler et al., 2004). radioligand binding at saturating concentrations, consistent Amino Acid Metabolites. Some of the best characterized with limited negative cooperativity (Bauer et al., 2012). This orthosteric and hormones, such as serotonin, negative allosteric effect on CB1 agonist affinity is also manifested dopamine, norepinephrine/epinephrine, GABA, and , in functional studies of receptor signaling (Bauer et al., 2012). are biosynthesized from amino acids. However, other amino acid Negative allosteric modulation of the CB1 receptor may have metabolites have been suggested to act as allosteric modulators. beneficial effects in pathologic states, such as obesity and type 2 For example, the methionine metabolite has been diabetes, while avoiding on-target depression-related side effects proposed to interact with the third extracellular loop of the often attributed to classic CB1 antagonists (de Kloet and Woods, dopamine D2 receptor to negatively modulate the binding of 2009). dopamine, while exhibiting neutral allosteric properties against The M2 mAChR is the first GPCR for which synthetic the antagonist raclopride (Agnati et al., 2006). It is acknowl- allosteric modulators were identified (Lüllman et al., 1969; edged that increased plasma levels of homocysteine are strong Clark and Mitchelson, 1976). Structural studies have estab- indicators of neurologic disorders and could therefore play a lished a key role for positively charged modulators to interact detrimental role in various diseases, including dementia, with a well defined allosteric site in the extracellular vestibule Alzheimer’s, Huntington’s, and Parkinson’s diseases (Seshadri of these receptors (Dror et al., 2013). It is interesting, therefore, et al., 2002; Morris, 2003; Andrich et al., 2004; Müller, 2008). that endogenous, arginine-rich peptides were previously specu- Another notable amino acid metabolite, agmatine (decarboxyl- lated to act as natural negative allosteric modulators of this Downloaded from ated arginine), positively modulates norepinephrine-mediated receptor. For example, protamine negatively modulates the inhibition of norepinephrine release at the a2-AR, although binding of radiolabeled orthosteric antagonists at cardiac multiple modes of interaction involving both the orthosteric M2 mAChRs with many of the hallmarks associated with ex- and an allosteric site on this receptor have also been proposed tracellular small-molecule allosteric modulators (Hu et al., (Molderings et al., 2000). Biosynthesis of agmatine, and sub- 1992). However, given that this protein is not secreted into sequent activation of a2-AR, may have cardiovascular protec- the extracellular space, the specific modulatory effect at jpet.aspetjournals.org tive effects, as well as , antineurotoxic, and the M2 mAChR may not be physiologically relevant (Balhorn, antidepressant actions (Piletz et al., 2013). 2007). In contrast, dynorphin-A (1–13) and myelin basic protein Small Peptides. The antioxidant glutathione (g-L-glutamyl- display allosteric actions similar to protamine at this receptor l-cysteinyl glycine), synthesized predominantly by the liver, is (Hu and el-Fakahany, 1993), and they can be found extracel- the most abundant thiol present in mammalian cells (up to lularly. Dynorphin-A, an endogenous opioid peptide, is widely 10 mM; Meister, 1988). Recently, glutathione has also been expressed in the central nervous system, and myelin basic pro-

identified as an endogenous allosteric modulator of the CaSR, tein is a major component of nerve myelin (Gupta, 1987). High at ASPET Journals on June 1, 2016 acting in a similar location and manner as the aromatic amino expression of these two proteins may induce a dampening effect 21 acids to positively modulate the functional effects of [Ca ]o of central M2 mAChRs, thus modifying the negative feedback (Wang et al., 2006; Broadhead et al., 2011). Glutathione could effect that these autoreceptors normally play in the process of potentially be of value in the physiologic suppression of high controlling neuronal acetylcholine release. parathyroid hormone levels in various forms of hyperparathy- The M2 mAChR is also highly expressed in the periphery, roidism. Another brain-derived tripeptide of interest is Leu-Pro- particularly in the postganglionic nerves in the airways, Gly, which has also been referred to as melanotrophin release where its autoreceptor activity plays a central role in various inhibiting factor or melanocyte-stimulating hormone release- airway diseases (Barnes et al., 1988). A hallmark of asthma is inhibiting factor-1 (Horvath and Kastin, 1990). Leu-Pro-Gly the infiltration of eosinophils to the site of inflammation appears to be a positive allosteric modulator of the binding and (Gleich et al., 1995) and their degranulation to release major function of agonists at the dopamine D2 receptor (Bhargava, basic protein, a highly basic, arginine-rich protein, with a pri- 1983; Johnson et al., 1986; Ott et al., 1996; Mishra et al., 1999). mary role in activating mast cells and (Rosenberg Because enhanced sensitivity of dopamine receptors to endog- et al., 2013). It has been proposed that in asthmatics, neuronal enously released dopamine may effectively compensate for par- M2 mAChRs are dysfunctional predominantly due to the pres- tially degenerating terminals in Parkinson’s ence of major basic protein in the neuromuscular junction disease, the pursuit of positive allosteric modulators of these (Jacoby et al., 1998). This proinflammatory protein is able to receptors remains of significant interest. A third endogenous bind with micromolar affinity to the M2 mAChR and allo- peptide with potential neurologic benefits is 5-HT–moduline sterically modifies the binding properties of the receptor (Jacoby (Leu-Ser-Ala-Leu). 5-HT–moduline is released in various parts et al., 1993). This is of particular relevance, as high micro- of the brain, particularly under conditions of stress (Fillion molar concentrations of major basic protein are found in the et al., 1996; Fillion, 2000). Interestingly, it is a negative allosteric sputum of asthmatics, with possibly even higher concentra- modulator of serotonin binding (Rousselle et al., 1996) and of tions in surrounding tissues (Frigas et al., 1981; Fryer and agonist-induced synaptosomal activity (Massot et al., 1996) at Jacoby, 1992). the 5-HT1B/1D receptors, acting therefore as a potential protective agent against depression (Fillion, 2000). Allosteric Autoantibodies Large Peptides and Proteins. Figure 4D shows the effects of pepcan-12, a 12-amino-acid neuropeptide that is Over the last two decades, a number of chronic diseases have highly expressed in mouse brain (Gomes et al., 2009), on the been associated with the presence of circulating autoantibodies, binding of the orthosteric radioligand agonist [3H]WIN55212-2 either as initiators or amplifiers of the disease. A common at the cannabinoid CB1 receptor. In contrast to the complete feature is that most GPCR-directed autoantibodies bind to parts 254 van der Westhuizen et al. of extracellular domains of the receptors, often in an allosteric cancer patients with paraneoplastic cerebellar ataxia contains mode. Although most autoantibodies have been characterized antibodies that react with mGluR1 (Sillevis Smitt et al., 2000; for their ability to induce agonist-like activity at their target Coesmans et al., 2003). The cerebrospinal fluid of patients with GPCR, a small number have also been classified as antagonists, limbic encephalitis contains autoantibodies against GABAB inhibiting the GPCR signaling processes (Table 1). Interestingly, receptors (Lancaster et al., 2010; Boronat et al., 2011), whereas some autoantibodies show very specific, noncanonical GPCR sera from patients with basal ganglia encephalitis with dominant activity, ranging from permanent agonist-like activity, such as movements and psychiatric disorders contain autoantibodies for the thyroid-stimulating hormone receptor (TSHR) in hyper- against the dopamine D2 receptor (Dale et al., 2012). The thyroidism from Grave’s disease (Kendall-Taylor et al., 1975), to pathogenesis of debilitating disorders such as a complete inability to desensitize their target receptors, as with may in some instances involve a repertoire of autoantibodies the b1-AR, M2 mAChR, or even angiotensin 1 (AT1) receptors in specifically targeting both M1 and M2 mAChRs (Borda et al., myocarditis and dilated cardiomyopathy (Wallukat et al., 1991, 2004; Jones et al., 2014). Most of these GPCR-directed 1999; Wallukat and Wollenberger, 1991; Xia and Kellems, 2013). autoantibodies have recently been isolated from patients but It should be noted, however, that the majority of these studies to remain to be fully characterized in terms of binding epitope date have not delved into the precise allosteric mechanisms and pharmacological activity. mediating the GPCR-autoantibody interaction. Autoantibodies Targeting the Cardiovascular Sys- Autoantibodies Targeting the Central and Periph- tem. Arguably, GPCR-directed autoantibodies have been eral Nervous System. It is now becoming evident that im- better studied with regard to their contributions to cardio- mune responses affecting neurons of the central or peripheral vascular diseases (Unal et al., 2012; Wallukat and Schimke, Downloaded from nervous system can result in a broad spectrum of neurologic 2014). One of the major GPCR targets for autoantibodies in syndromes ranging from encephalitis to Alzheimer’sdiseaseand vascular diseases is the AT1 receptor. Antibodies against the schizophrenia (van Coevorden-Hameete et al., 2014). The last AT1 receptor have been found in patients with renal trans- few years have seen the emergence of several lines of evidence plant rejections associated with vascular pathology (Dragun suggesting that GPCR-directed autoantibodies are indeed in- et al., 2005; Dragun, 2007), in pre-eclamptic women (Wallukat volved in an ever-increasing number of neurologic disorders et al., 1999; Herse and LaMarca, 2013; Xia and Kellems, jpet.aspetjournals.org (Fig. 6). For instance, the sera or the cerebrospinal fluid of 2013), in hypertensives (Fu et al., 2000; Xia and Kellems, Hodgkin’s lymphoma, lung cancer, ovarian cancer, and breast 2013), as well as in systemic sclerotic patients (Riemekasten

TABLE 1 Selected examples of putative endogenous GPCR allosteric modulators at ASPET Journals on June 1, 2016 Modulator Targets Ions Na+ Various Zn2+ Various 2+ Mg m-OR, k-OR, b1-AR Mn2+ m-OR, d-OR, k-OR, b-AR Amino acids and derivatives L-Phe/L-Trp/L-Tyr CaSR L-Leu/L-Ile/L-Phe GABABR Homocysteine D2R Agmatine a2D-AR Peptides Melanotrophin release inhibiting factor 1 D2R 5-HT–moduline 5-HT1B/1D Glutathione CaSR Larger peptides/proteins Pepcan-12 CB1R Dynorphin-A M2 mAChR Protamine M2 mAChR Myelin basic protein M2 mAChR Major basic protein M2 mAChR Lipids Oleamide 5-HT2C/2AR(s), 5-HT1AR, 5-HT7R Anandamide M1/4 mAChR(s), M1 mAChR, hA3R 2-AG hA3R Arachidonic acid mAChR(s) Lipoxin A4 CB1R Progesterone (P4) rOTR 5b-Dihydroprogesterone hOTR Cholesterol Rhodopsin, b2-AR, OTR, A2AR, 5-HT1A Pregnenolone CB1R Autoantibodies IgG 5-HT4R, a1-AR, AT1R, b1-AR, b2-AR, CaSR, D2R, ETAR, GABABR, M1 mAChR, M2 mAChR, M3 mAChR, mGluR1, mGluR5, TSH-R

A2AR, ; AT1R, angiotensin II receptor type 1; CB1R, type 1; D2R, D2;ETAR, type A; hA3R, human adenosine A3 receptor; hOTR, human oxytocin receptor; 5-HT1AR, serotonin 5HT1a receptor; 5-HT2C/2AR(s), serotonin 5-HT2c/2a receptors; 5-HT7R, serotonin 5-HT7 receptor; OTR, oxytocin receptor; rOTR, rat oxytocin receptor; TSH-R, . Endogenous GPCR Allosteric Modulators 255 Downloaded from

Fig. 6. Examples of central nervous system and cardiovascular disorders mediated by endogenous autoantibodies targeting various GPCRs. D2R, ;ETAR, endothelin type A receptor. jpet.aspetjournals.org et al., 2011). In all cases, these autoantibodies were able to to cause hyperthyroidism (the most common form of Grave’sdis- induce agonist-like activity at the AT1 receptor, promoting ease; Orgiazzi et al., 1976) or inactivating it, causing hypothy- chronic activation. Some cardiovascular diseases generate roidism (Endo et al., 1978). Recently, neutral TSHR-directed a highly heterogeneous antibody repertoire, such as hyper- antibodies have also been identified (Morshed et al., 2010). Some

tension and systemic sclerosis. For example, some patients patients can swing between hyper- and hypothyroidism depend- at ASPET Journals on June 1, 2016 with hypertension have also shown high levels of antibodies ing on the levels of stimulating or blocking TSHR autoantibodies targeting the a1-AR (Fu et al., 1994; Luther et al., 1997; (McLachlan and Rapoport, 2013). In the sera of patients suffering Wenzel et al., 2008), whereas patients suffering from systemic from Sjögren’s syndrome, a systemic autoimmune disease sclerosis may be positive for antibodies against the endothelin characterized by the severe impairment of salivary and lacrimal endothelin type A receptor (Becker et al., 2014). Additionally, secretions, M3 mAChR autoantibodies have been identified 5-HT4 receptor autoantibodies were found in the sera of some (Bacman et al., 1998). Interestingly, these antibodies, which patients with systemic erythematosus (Eftekhari et al., are directed toward the extracellular loop 3 domain of the 2000). This subtype of serotonin receptor plays a critical role M3 mAChR (Koo et al., 2008), exhibit antagonist-like activity in regulating atrial arrhythmias in the developing heart, explain- (Jin et al., 2012). In the sera of complex regional pain syndrome ing why children of women with 5-HT4 receptor autoantibodies patients, the presence of autoantibodies with agonist-like activity develop neonatal lupus-induced congenital heart block. Finally, against the b2-AR and/or the M2 mAChR has also been identified among the cardiovascular diseases associated with GPCR-directed (Kohr et al., 2011). Upon targeting the extracellular loop 2 of autoantibodies, cardiomyopathy is a common pathology (Wallukat these receptors, the autoantibodies can stimulate the release and Schimke, 2014). Antibodies for two specific cardiac GPCRs, of intracellular calcium. Recently, a blocking autoantibody the b1-AR and the M2 mAChR, are a common feature. IgGs against the CaSR has been isolated and reported to cause targeting and activating either or even both receptors simulta- acquired hypocalciuric hypercalcemia (Kifor et al., 2003). The neously (Elies et al., 1996) have been isolated in patients with autoantibody itself is able to inhibit the calcium-mediated idiopathic cardiomyopathy (Wallukat and Wollenberger, 1987; Fu signaling pathways of the CaSR, reducing IP accumulation as et al., 1993), peripartum cardiomyopathy (Warraich et al., 2005; well as ERK1/2 phosphorylation. In contrast, a different autoan- Stavrakis et al., 2009; Stavrakis et al., 2011; Liu et al., 2014), and tibody was identified in a patient suffering from acquired in Chagas-induced cardiomyopathy (Sterin-Borda et al., 1976; hypocalciuric hypercalcemia that displayed biased allosteric Borda et al., 1984; Elies et al., 1996; Wallukat et al., 2010; Muñnoz- modulation—namely, a potentiation of Gq-mediated IP accumu- Saravia et al., 2012). lation but inhibition of Gi-mediated ERK1/2 phosphorylation Autoantibodies in Other Systemic Disorders. In addi- (Makita et al., 2007). Patients suffering autoimmune polyendo- tion to nervous and cardiovascular system disorders, autoim- crine syndrome type 1 have also shown high levels of CaSR- mune thyroid disease is another established example involving directed autoantibodies that activate, rather than inhibit, the GPCR-directed autoantibodies (Rapoport et al., 1998). In Grave’s receptor (Gavalas et al., 2007; Kemp et al., 2009). In patients disease, the autoimmune pathogenesis is well characterized with allergic asthma, autoantibodies showing antagonist-like and attributed to the ability of autoantibodies to bind the activity at the b2-AR have been isolated (Turki and Liggett, ectodomain of the TSHR, and either chronically activating it 1995). Finally, in Chagas disease, digestive manifestations 256 van der Westhuizen et al. sometimes occur, leading to a syndrome called megaviscera Although most current drug discovery programs are pursuing (Haberland et al., 2013). Autoantibodies isolated from Chagasic positive or negative allosteric modulators as therapeutic agents, patients with megacolon are able to bind and activate the it is possible that neutral allosteric ligands can find a niche if it M2 mAChR, increasing the basal tone of the colon as well as can be demonstrated that a given pathophysiological state is inhibiting intracellular cAMP signaling (Sterin-Borda et al., mediated by aberrant endogenous allosteric modulation; in this 2001). case, the neutral allosteric ligand could be used to block the binding of the endogenous allosteric modulator while sparing Concluding Remarks the binding and signaling of the endogenous orthosteric agonist. It is acknowledged that many of these considerations currently The study of endogenous allosteric modulators of GPCRs remain theoretical, but, as has been proven repeatedly over the represents a burgeoning field with some provocative findings last decade and a half, the study of allosteric modulation of that, in many instances, require further validation. Nonethe- GPCRs continues to yield new insights and exciting opportunities less, the concept is important and raises a number of issues. for improving human health. The first, as alluded to in the Introduction, is whether there should be an expectation that most GPCR allosteric sites Authorship Contributions represent orphan sites for hitherto unidentified modulators. Wrote or contributed to the writing of the manuscript: van der This is probably not the case except in the broadest sense, Westhuizen, Valant, Sexton, Christopoulos. i.e., GPCRs are structurally similar yet highly dynamic in the References types of substances that they interact with; as highlighted in Agnati LF, Ferré S, Genedani S, Leo G, Guidolin D, Filaferro M, Carriba P, Casadó V, Downloaded from Fig. 1A, an orthosteric domain on one type of GPCR can be Lluis C, Franco R, et al. (2006) Allosteric modulation of dopamine D2 receptors by homocysteine. J Proteome Res 5:3077–3083. serendipitously exploited as an allosteric site on another type Andrich J, Saft C, Arz A, Schneider B, Agelink MW, Kraus PH, Kuhn W, and Müller of GPCR. However, as noted for other receptor superfamilies, T (2004) Hyperhomocysteinaemia in treated patients with Huntington’s disease homocysteine in HD. Mov Disord 19:226–228. there are examples of endogenous substances that indeed ap- Appelmans N, Carroll JA, Rance MJ, Simon EJ, and Traynor JR (1986) Sodium ions pear to play key roles in allosteric modulation of some GPCRs. increase the binding of the antagonist peptide ICI 174864 to the delta-opiate re- ceptor. Neuropeptides 7:139–143. These types of observations raise a second important issue Azzi M, Piñeyro G, Pontier S, Parent S, Ansanay H, and Bouvier M (2001) Allosteric jpet.aspetjournals.org for future studies—namely, the validation and classification of effects of G protein overexpression on the binding of beta-adrenergic ligands with distinct inverse efficacies. Mol Pharmacol 60:999–1007. endogenous allosteric substances. 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