Molecular determinants of ACTH receptor for ligand selectivity

Molecular and Cellular Endocrinology 503 (2020) 110688

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Molecular determinants of ACTH receptor for ligand selectivity Yingkui Yang , Carroll M. Harmon

T

∗

Department of Surgery, State University of New York at Buffalo, USA

ARTICLE INFO

ABSTRACT

Keywords: POMC ACTH receptor GPCR ACTH α-MSH

The adrenocorticotropic hormone (ACTH) receptor, known as the melanocortin-2 receptor (MC2R), plays a key role in regulating adrenocortical function. ACTH receptor is a subtype of the melanocortin receptor family which is a member of the G-protein coupled receptor (GPCR) superfamily. ACTH receptor has unique characteristics among MCRs. α-MSH, β-MSH, γ-MSH and ACTH are agonists for MCRs but only ACTH is the agonist for ACTH receptor. In addition, the melanocortin receptor accessory protein (MRAP) is required for ACTH receptor expression at cell surface and function. In this review, we summarized the information available on the relationship between ACTH and ACTH receptor and provide the latest understanding of the molecular basis of the ACTH receptor responsible for ligand selectivity and function.

1. Introduction Adrenocorticotropic hormone (ACTH) is a polypeptide hormone produced and secreted by the anterior pituitary gland (Li et al., 1942). ACTH is classified as a member of the melanocortins (MCs) and stimulates secretion of glucocorticoid steroid hormones from adrenal cortex cells through ACTH receptor which is primarily found in the zona fasciculata of the adrenal cortex. The adrenocorticotropic hormone (ACTH) receptor, also known as the melanocortin-2 receptor (MC2R), is critical for ACTH-mediated adrenal glucocorticoid release (Yang and Harmon, 2017; Mountjoy et al., 1992; Chhajlani and Wikberg, 1992). ACTH binds to ACTH receptor and stimulates the adrenal glands to produce glucocorticoids which include cortisol and corticosterone. In addition, ACTH receptor is required for fetal and neonatal adrenal gland development (Roebuck et al., 1980; Lohse and First, 1981; McNulty et al., 1981; Chida et al., 2007). Deficiency of the ACTH receptor resulted in familial glucocorticoid deficiency (FGD) (Clark and Weber, 1998; Clark et al., 2005; Elias et al., 1999; Huebner et al., 1999; Chan et al., 2008). Patients with ACTH receptor deficiency have high levels of serum ACTH and low levels of cortisol due to an impaired adrenal responsiveness to ACTH (Habeb et al., 2013; Meimaridou et al., 2013a, 2013b; Abuduxikuer et al., 2019). The melanocortin system consists of three components which are melanocortin peptides; melanocortin receptors; and two endogenous antagonists, agouti-signaling protein (ASIP) (agouti in rodents) and agouti-related protein (AGRP). Melanocortin peptides are composed of

ACTH as well as α−, β−, and γ−melanocyte stimulating hormone (MSH) (Tatro and Reichlin, 1987; Tatro, 1996; Bicknell, 2002; Cooper et al., 1996; Clark et al., 1978; De Wied and Jolles, 1982; Catania et al., 1996; Gantz and Fong, 2003). To date, five melanocortin receptors have been cloned and each receptor has its own unique pattern of tissue expression and function (Gantz and Fong, 2003). ACTH binds to ACTH receptor, and, as with the other melanocortin peptides and receptors, stimulates adenylate cyclase, thereby elevating the second messenger, cellular cyclic AMP (cAMP). ACTH is the only known physiologic ligand for ACTH receptor, while α−, β−, and γ-MSH and ACTH are physiologic ligands for MC1R, MC3R, MC4R and MC5R (Schioth et al., 1996). The amino acid sequences of the melanocortins are shown in Fig. 1. ACTH is composed of 39 amino acid residues and shares the first 13 amino acids with MSH. They share the core sequence “His6-Phe7-Arg8Trp9” which was identified as important residues for ligand binding and activities at human (h)MC1R, hACTH receptor, hMC3R, hMC4R and hMC5R (Holder et al., 2002a, 2002b, 2003a, 2003b; Todorovic et al., 2005; Haskell-Luevano et al., 1997; Shizume et al., 1954; Goldman and Hadley, 1970). The ACTH receptor gene sequence was reported in 1992 by two research groups (Mountjoy et al., 1992; Chhajlani and Wikberg, 1992). ACTH receptor shares a nearly 40% homology with other melanocortin receptor subtypes, but it is unique among melanocortin receptor subtypes (Schioth et al., 1996). While ligand selectivity for melanocortin receptor family is usually achievable, it is often difficult to obtain selectivity among subtypes of the receptor family. There is no selective

Abbreviations: hACTH RECEPTOR, human Melanocortin 2 receptor; hMC3R, human melanocortin-3 receptor; GPCR, G-protein coupled receptor; α−MSH, αmelanocyte stimulating hormone; TM, transmembrane domains ∗ Corresponding author. Division of Pediatric Surgery, State University of New York at Buffalo, 875 Ellicott St room 8030C, USA. E-mail address: [email protected] (Y. Yang). https://doi.org/10.1016/j.mce.2019.110688 Received 30 July 2019; Received in revised form 3 December 2019; Accepted 16 December 2019 Available online 19 December 2019 0303-7207/ © 2019 Published by Elsevier B.V.

Molecular and Cellular Endocrinology 503 (2020) 110688

Y. Yang and C.M. Harmon

Fig. 1. Amino acid sequences of melanocortin peptides. The core sequences of the peptides are denoted in black fond.

agonist or antagonist developed so far for ACTH receptor because detailed molecular basis of ACTH receptor for ligand selectivity is unclear. The design of ligands that provide receptor selectivity has emerged as a new paradigm for drug discovery of G protein-coupled receptors. For decades, the development of ligands in traditional GPCR-based drug discovery has focused on targeting the primary endogenous ligand (orthosteric) binding site of the receptor, guiding the development of the most classical orthosteric agonists, inverse agonists, and antagonists. However, many receptor subtypes in GPCR families often exhibit a highly conserved orthosteric binding site, such as a single ligand can interact with several receptor subtypes simultaneously, leading to the activation of the multiple receptor subtype, sometimes with opposing of their signaling profiles, resulting in side effects (Wootten et al., 2018; Seyedabadi et al., 2019). Although MCRs often share similar binding sites in their structures, it is often expected to design and synthesize ligands which can selectively bind to a specific receptor subtype and alter this specific receptor subtype function without affecting other receptor subtype. Designing and developing a selective agonist or antagonist for ACTH receptor remains a challenge task because the molecular basis of ACTH receptor responsible for ligand selectivity is not fully understood. ACTH is not only an agonist for ACTH receptor but it is also an agonist for other MCRs. All melanocortin ligands shared the same H6F7R8W9 motif, which is important for MCR binding and stimulation. Development of the ACTH receptor selective agonist or antagonist is challenging due to the conserved amino acid sequences of the MCRs and of their structural similarity in the seven-transmembrane GPCR fold. The limited structural variations of the endogenous melanocortin ligands further reduce options in the design of ACTH receptor ligands for achieving receptor subtype selectivity. If they are not selective, these peptides could be the source of severe undesirable effects due to the numerous specific roles of the five MCRs. Designing molecules that possess both melanocortin receptor selectivity and melanocortin receptor subtype selectivity from the melanotropin core sequence H6F7R8W9 has been difficult. Therefore, a better understanding of the molecular determinants of ACTH receptor responsible for ACTH selectivity is essential for our comprehensive understanding of ACTH receptor function and for the development of the selective ACTH receptor agonist or antagonist.

Kwon et al., 1994). Agouti is a competitive antagonist at MC1R and MC4R in which agouti inhibited MSH induced cAMP production in a dose dependent manner. However, the inhibitory pattern of agouti on ACTH mediated cAMP production at ACTH receptor is different. Agouti significantly diminished ACTH mediated cAMP production at ACTH receptor and the maximum response plateau is well below the levels attained in the absence of agouti which is described as noncompetitive antagonism (Ollmann et al., 1997; Yang et al., 1997a). Agouti-related protein (AGRP) is a potent antagonist for MC3R and MC4R (Jackson et al., 2002; McNulty et al., 2001; Millhauser et al., 2003). AGRP inhibits MSH mediated cAMP production in a dose dependent manner at MC3R and MC4R. The sequences of human agouti protein (ASIP) and AGRP are shown in Fig. 3. Sequence similarity between ASIP and AGRP is their Cys-rich C terminal domains. These Cys-rich domains are also involved in melanocortin receptor selectivity (McNulty et al., 2005). Detailed study showed that the extracellular loop 2 and 3 of the MC4R are crucial for AGRP selectivity. Replacement of the EXL2 and EXL3 of the MC4R with the corresponding regions of the MC1R significantly reduced AGRP antagonist activity (Yang et al., 1999). However, the molecular mechanism of agouti and AGRP for MCR subtype selectivity is still not fully identified. Two ACTH receptor antagonists were developed and reported. Studies showed that the peptide GPS1573 (Nle-P-DF-R-DW-F-K-A-V-GK-K-R-R NH2) and the peptide GPS1574 (Nle-[E-DF-R-DW-F-K]-A-V-GK-K-R-R) significantly inhibited ACTH mediated cAMP production at ACTH receptor in vitro. GPS1574 also significantly inhibits ACTH mediated corticosterone increase in the neonatal rat (Bouw et al., 2014; Goldenberg et al., 2018). Further experiment indicated that pretreatment with GPS1574 inhibited ACTH (Li et al., 1942; Yang and Harmon, 2017; Mountjoy et al., 1992; Chhajlani and Wikberg, 1992; Roebuck et al., 1980; Lohse and First, 1981; McNulty et al., 1981; Chida et al., 2007; Clark and Weber, 1998; Clark et al., 1978, 2005; Elias et al., 1999; Huebner et al., 1999; Chan et al., 2008; Habeb et al., 2013; Meimaridou et al., 2013a, 2013b; Abuduxikuer et al., 2019; Tatro and Reichlin, 1987; Tatro, 1996; Bicknell, 2002; Cooper et al., 1996; De Wied and Jolles, 1982; Catania et al., 1996; Gantz and Fong, 2003; Schioth et al., 1996; Holder et al., 2002a, 2002b, 2003a, 2003b; Todorovic et al., 2005; Haskell-Luevano et al., 1997; Shizume et al., 1954; Goldman and Hadley, 1970; Wootten et al., 2018; Seyedabadi et al., 2019; Harris et al., 2014; Navarro et al., 2016; Dores and Baron, 2011) induced acute corticosterone response in rat pups (Nensey et al., 2016). Recently, Dr. Galac group reported that the compound BIM22A229 is a potent ACTH receptor antagonist (Sanders et al., 2018). However, we still do not know whether these antagonists are ACTH receptor subtype specific. Further experiment is therefore warranted to test whether these peptides are ACTH receptor selective and they have no effect on other MCRs. More studies may be needed to develop the selective ACTH receptor antagonists.

2. ACTH receptor ligands The endogenous agonist ACTH comes from proopiomelanocortin (POMC) which is a complex precursor protein that is proteolytically cleaved to a variety of biologically active and important neuroendocrine peptides (Harris et al., 2014; Navarro et al., 2016). The POMC gene, encodes a polypeptide hormone precursor, is located on chromosome 2p23.3 and is expressed in the pituitary gland. This polypeptide hormone precursor undergoes extensive, tissue-specific, posttranslational processing through prohormone convertases and s cleaved to multiple peptide hormones which include the highly potent opioid peptide β-endorphin and its intermediate β-lipotropin, adrenocorticotropic hormone (ACTH), and various melanotropic peptides including α-, β-, and γ-melanocyte-stimulating hormone (MSH) (Dores and Baron, 2011; Dores et al., 2014, 2016; Fridmanis et al., 2017) (Fig. 2). There are two endogenous antagonists for the melanocortin receptor family but the ligand selectivity is differ for a specific melanocortin receptor subtype. Antagonist agouti is a potent antagonist for MC1R, ACTH receptor and MC4R (McNulty et al., 2005; Bultman et al., 1992;

3. ACTH receptor accessory protein ACTH receptor has two unique characteristics compared to that of other MCRs. One is that ACTH is a sole endogenous agonist for ACTH receptor. The second characteristic is that ACTH receptor was not expressed in heterologous cell lines while other MCRs did. Previous studies indicate that the functioned ACTH receptor can only be expressed in a limited number of cell types, such as the Y6 and OS3 cell lines that are derived from a mouse adrenocortical tumor and that had been 2

Molecular and Cellular Endocrinology 503 (2020) 110688

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Fig. 2. Schematic representation of the proopiomelanocortin precursor and the products of its proteolysis. Melanocortin peptides are colored green. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) Fig. 3. Amino acid sequences of agouti and AGRP. ASIP 80–132 has 53 amino acid residues. Five disulfide bonds are present in ASIP 80–132 which are 93–108, 100–114, 107–125,111-132, 116–123. Human AGRP (87–132) has 46 amino acid residues. Five disulfide bonds are also present in AGRP (87–132) which are 87–102, 94–108, 101–119,105129, 110–117.

a significant amount of receptor expression was seen at the plasma membrane (Roy et al., 2007; Metherell et al., 2005). MRAP was identified to be required for both surface expression and signaling of the receptor (Sebag and Hinkle, 2007, 2009, 2010; Rouault et al., 2017; Hinkle and Sebag, 2009; Novoselova et al., 2013; Jackson et al., 2015; Clark et al., 2016; Clark and Chan, 2019). MRAPs are identified to be a class of single-pass transmembrane domain accessory protein and highly expressed in the adrenal gland (Metherell et al., 2005). Fig. 4 shows the MC2R/MRAP1 heterodimer involved in the receptor expression and signaling. Deficiency of MRAP cause familial glucocorticoid (GC) deficiency (FGD2), where the adrenal gland fails to respond to ACTH and to produce cortisol. In MRAP knock-out mice, most pf MRAP−/− mice died at birth but could be rescued by administration of corticosterone to pregnant mice. Survived MRAP−/− mice developed isolated GC deficiency with normal mineralocorticoid and catecholamine production (Novoselova et al., 2018). 4. Structural features of ligand ACTH for the receptor activity ACTH is composed of 39 amino acid residues and shares the first 13 amino acids with MSH. They share the core sequence “His6-Phe7-Arg8Trp9” which was identified as important residues for ligand binding and activity at hMC1R, hMC2R, hMC3R, hMC4R and hMC5R (Holder et al., 2002a, 2002b, 2003a, 2003b; Todorovic et al., 2005; Haskell-Luevano et al., 1997; Shizume et al., 1954; Goldman and Hadley, 1970). Extensive studies have been performed to determine the molecular basis of ACTH for ACTH receptor function in the last several decades. In early years, ACTH1-14, ACTH11-19 and ACTH11-24 were reported to increase corticosterone levels in the isolated adrenal cell system and ACTH11-24 was also reported to play an important role in adipocyte function (Nakamura, 1972; Goverde and Smals, 1984; Opmeer et al., 1978; Elliott et al., 1977). But in other reports, ACTH1124 significantly decreased ACTH1-39 mediated cAMP production in the isolated adrenal cells (Seelig et al., 1971; Schwyzer et al., 1971). The explanation for these variations is that different method of adrenal cell preparation and a separate receptor may be involved in these peptide activities. Since ACTH receptor was cloned, it greatly advanced our understanding of the ligand receptor interactions between ACTH and

Fig. 4. Schematic representation of MRAP1 structure and domains. Panel A represents MRAP1 anti-parallel homodimer. Panel B represents the location and function of MRAP1 domains for MC2R trafficking and/or signaling.

selected as being ACTH resistant. Studies indicate that in most cell types, transiently transfected ACTH receptor is retained in the ER (Noon et al., 2002; Roy et al., 2007), whereas in Y6 or OS3 cells ACTH receptor was found to be localized to the cell surface and forms a functional ACTH-responsive receptor (Elias et al., 1999; Yang et al., 1997a). This finding suggests that an adrenal specific accessory factor might play a key role in facilitating ACTH receptor trafficking to cell surface. Dr. Clark's group identified that melanocortin receptor accessory protein (MRAP) is required for the trafficking of ACTH receptor to the cell surface. This discovery has made a big significant contribution to ACTH receptor research. When MRAP and ACTH receptor were co-expressed, 3

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Fig. 5. Two dimensional representation of the seven transmembrane structure of the MC2R. The amino acid residues involved in ligand binding and signaling are denoted by black highlight. The amino acid residue involved in ACTH or MSH binding and signaling is denoted in gray highlight.

ACTH receptor. It was reported that ACTH1-24 possesses higher binding affinity and potency compared to that of ACTH 1–39 which is a full length of endogenous agonist. ACTH1-17 and ACTH1-16 possess full agonist activities but the potencies of these two peptides were significantly decreased (Chen et al., 2007). Further studies indicate that the peptides without first six amino acids (ACTH 6–24) lost their biological activities (Chen et al., 2007). Extensive studies were performed and showed that two regions in ACTH1-39 are crucial for ligand binding and signaling at ACTH receptor (Schioth et al., 1996; Kapas et al., 1996; Cammas et al., 1995). The first region in ACTH is H6F7R8W9, the second one is K15K16R17. Mutation of the ACTH residue H6F7R8W9 or ACTH1-14 (delete ACTH15-17) significantly decreased ligand mediated cAMP production at ACTH receptor. Mutation of the amino acid residues K15K16R17 in ACTH significantly decreased agonist potency (Liang et al., 2013). Every amino acid (except glycine) can occur in two isomeric forms due to the possibility of forming two different enantiomers (stereoisomers) around the central carbon atom. These are called L- and D-forms. Further experiments indicate that the role of Phe7 in ACTH at ACTH receptor is different from other MCR subtype. Substitution of Phe7 with DPhe7 at ACTH1-24 significantly decreased cAMP production at ACTH receptor. However, the effect of this substitution is opposite at MC1R, MC3R, MC4R and MC5R. Substitution of Phe7 with DPhe7 at α-MSH (NDP-MSH) or at ACTH significantly increased both ligand binding affinity and potency at MC1R, MC3R, MC4R and MC5R. In addition, the activity of amino acid residue DNal7 in ACTH at ACTH receptor is different from that of MC3R and MC4R. D-2′-naphthylalanine7 (DNal (2′) is a bulky aromatic amino acid. Replacement of DPhe7 with DNal (2′)7 at MTII (SHU9119) switches ligand from agonist to antagonist at MC3R and MC4R(79). However, substitution of DNal7 (2′) in ACTH did not switch agonist ACTH to antagonist at ACTH receptor, suggesting that an aromatic side chain in position 7 of α-MSH is crucial for receptor activation at MC3R and MC4R but not at ACTH receptor (Yang et al., 2015).

5. Structural features of ACTH receptor for ligand binding and selectivity The melanocortin receptor family consists of a single polypeptide featuring seven α-helical TM domains, three extracellular loops, and three intracellular loops. This family has five MCR subtype and shares significant sequence similarity at the transmembrane regions of the receptors (Yang and Harmon, 2003, 2017). All MCRs share the similar conserved amino acids, aspartic acid–arginine–tyrosine (DRY) motif at the junction of the TM3 domain and contain a C-terminal cysteine which is a common feature for other GPCR. Conserved amino acid residues in the TM region of the receptor have been proposed to be involved in the melanocortin receptor selectivity based on other GPCR receptor research (Venkatakrishnan et al., 2013). Extensive studies have been performed to examine the molecular basis of the melanocortin receptors responsible for ligand binding and signaling (Yang et al., 1997b, 2000; Chen et al., 2006). The results indicate that conserved residues in TM of melanocortin receptors are involved in MSH binding and signaling (Haskell-Luevano et al., 1996). Two major binding sites were identified to be involved in endogenous ligand binding. The first is a predominantly ionic binding site composed of receptor residues in TM2 and TM3 of the MCRs. These receptor residues were identified to collectively interact with the single ligand amino acid Arg8 of NDP-MSH or ACTH via ionic, amino-aromatic, and hydrogen bond interactions. The second binding sites are a hydrophobic one consisting of a series of aromatic residues (phenylalanine and tyrosine residues) spanning TM4, 5, and 6. These functionally interconnected receptor residues participate in aromatic-aromatic interactions with residues DPhe7 and Trp9 of NDP-MSH or ACTH (Yang et al., 1997b, 2000, 2013; Chen et al., 2006; Haskell-Luevano et al., 1996). ACTH receptor share many structural features with other MCRs. The transmembrane domains (TM) of the ACTH receptor share more conserved amino acid residues with other MCRs compared to that of the 4

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“donor receptor” into an “acceptor receptor” results in the transfer of a specific functional property that is characteristic for the donor receptor. Dr. Fridmanis group used both MC4R and ACTH receptor as the template to generate the chimeric receptors. Their results indicate that MSH becomes agonist when the TM5, TM6 and TM7 and IX3, EX3 and C terminal of the ACTH receptor were replaced with the corresponding regions of the MC4R (Fridmanis et al., 2010, 2014). However, Dr. Hinkle group reported that TM2 and TM3 of the ACTH receptor is important for αMSH selectivity using ACTH receptor-based chimeric receptors. MSH becomes an agonist when the TM2, EX1 and TM3 of the ACTH receptor were replaced with the corresponding regions of the MC4R (Hinkle et al., 2011). From results above, we did not obtain clear picture about which region of the ACTH receptor is crucial for MSH binding and signaling. It appears that the conformation of ACTH receptor is important for ligand selectivity because there is no single domain that of the receptor is responsible for ligand selectivity. Furthermore, from these studies, we could not exclude other regions of the ACTH receptor are not involved in ligand selectivity because some chimeric receptors were not expressed at cell surface. Therefore, which region of the ACTH receptor is involved in MSH selectivity is still not clear. The advantage of using chimeric receptor approach is that we can identify which region of the receptor is involved in ligand selectivity but the disadvantage is that such big replacement of the receptor would alter the tertiary structure of the receptor and the receptor is unable to be expressed at cell surface and their functions can't be determined. Based on above studies, we tried to change the receptor structure as little as possible in order to gain insight into which region of the ACTH receptor is responsible for the ligand selectivity. We replaced only one TM domain (TM2, TM3, TM4, TM5, or TM6) at a time without disruption of the adjacent intracellular or ELs. We generated the chimeric receptors using MC4R as a template. We replaced individual MC4R TM region with the corresponding region of the ACTH receptor. In all cases, these replacements had little or no effect on the receptor surface expression. Agonist NDP-MSH is able to induce cAMP production at these MC4R based chimeric receptors. Using these chimeric receptors, we have identified that the TM3 region of the ACTH receptor is crucial for ligand selectivity (Yang et al., 2015). To further determine ACTH receptor ligand selectivity, another chimeric receptor was generated. We individually replaced the TM3 of the ACTH receptor with the corresponding region of MC3R and the receptor function was evaluated (Yang et al., 2019). Our study show that ACTH receptor TM3 plays an important role in ligand selectivity. The replacement of ACTH receptor TM3 with that of the MC3R switches DPhe-ACTH1-17 from no activity at ACTH receptor wild type to agonist. Sequence analysis between ACTH receptor and MC3R shows that there are four amino acid residues in the upper region of the ACTH receptor TM3 are different. We then substituted single amino acid in the ACTH receptor TM3 with the corresponding one of the MC3R. Our result indicates that replacement of L112 to I112 in ACTH receptor (L112I) dramatically altered ACTH receptor pharmacological characteristics. ACTH1-15, which has no agonist activity at ACTH receptor WT, becomes a full agonist at L112I. α-MSH also becomes a full agonist at L112I although its potency is less than that of ACTH1-17 or ACTH1-15 (Yang et al., 2019). This study provides a new picture how ACTH receptor selects ACTH or α-MSH. One amino acid residue L112 in TM3 of the ACTH receptor controls ACTH or MSH ligand selectivity (Fig. 6). This result is also consistent with the results from other MCRs in which the non-conserved amino acid residues in TM3 of the MCRs are key for MCR subtype ligand selectivity. In the last several years, many selective agonists for a specific receptor subtype have been developed. Extensive studies have been performed to determine the molecular basis of the MCR responsible for these ligand selectivity. The results demonstrated that non-conserved residues present in the TM3 of the MCRs play pharmacologically important roles in determining receptor ligand selectivity for a specific receptor subtype. Synthetic nonpeptide compound N- (3R)-1 4-tetrahydroisoquinolinium-3-ylcarbonyl -(1R)-1-(4-chlorobenzyl)-2- 4-cyclohexyl-4-(1H-1,2,4-triazol-1-ylmethyl) piperidin-1-yl -2-oxoethylamine (THIQ) is a synthetic small compound

Fig. 6. The role of the TM3 in MC2R for ligand selectivity. Panel A shows the comparison of amino acid sequence in TM3 of the MCRs. Panel B shows that ACTH1-15 or α-MSH is unable to induce cAMP production at ACTH receptor wild type but induced cAMP production at MC2R mutation L112I in a dose response manner.

extracellular loops and intracellular loops. There are 68 charged or aromatic amino acid residues located in TM regions of the ACTH receptor which shares 29 conserved amino acid residues with other melanocortin receptors (Figs. 5 and 7). To determine the molecular determinants of human ACTH receptor for ligand binding and signaling, the receptor mutagenesis study was used to examine which amino acid in the receptor is involved in ACTH activity. Conserved amino acid residues in TM region of ACTH receptor were substituted with alanine and the receptor function was examined. Mutations of glutamine acids 80 and aspartic acid 103 and 107 in TM3 of the ACTH receptor and mutation of phenylalanine 235 and histidine 238 in TM6 of the ACTH receptor resulted in significantly decrease in endogenous agonist binding and receptor signaling(75). Besides conserved amino acid residues of the ACTH receptor, some unique amino acid residues in TMs of ACTH receptor are also identified to be involved in ACTH binding and signaling (Chen et al., 2007). Mutation of amino acid residue D104 and F108 in TM3, and F168 in TM4 in TM6 significantly decreased ACTH binding and signaling. As shown in Fig. 5, amino acid residues in TM2 and TM3 of ACTH receptor are involved in ACTH binding. These conserved amino acid residues are predominantly receptor ionic binding site. These receptor residues collectively interacted with the single ligand amino acid Arg8 of ACTH via ionic, amino-aromatic, and hydrogen bond interactions. Amino acid residues in TM4, 5 and 6 of the receptor are composed of hydrophobic binding sites and these conserved amino acid residues participate in aromatic-aromatic interactions with residue and Trp9 of ACTH. To determine which region of ACTH receptor is crucial for ligand selectivity, the chimeric receptor approach was used. Chimeric receptor studies are mainly used as gain-of-function studies. This approach expects to find out whether the substitution of a distinct region from a 5

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which is a selective MC4R agonist. Mutations of the conserved residues, D122 and D126 in the TM3 of the melanocortin MC4 receptor, reduce the binding affinity and potency of both NDP-MSH and THIQ. However, mutation of non-conserved residues, I129T and L125 in the TM3 of the MC4R, significantly reduces the potency of THIQ but not on binding affinity or potency of the NDP-MSH (Yang et al., 2000; Haskell-Luevano et al., 2001; Pogozheva et al., 2005), demonstrating the role of nonconserved amino acid residues play an important role in ligand selectivity at a specific receptor subtype. This compound utilized conserved amino acid residues in TM domains of the MC4R for receptor specific binding and used non-conserved residues in TM domains for receptor subtype selective binding (Pogozheva et al., 2005; Yang et al., 2009). SHU9119 is an agonist for hMC1R and hMC5R but is an antagonist for hMC3R and hMC4R. The substitution of leucine 165 (L165) in MC3R or L133 in MC4R to methionine did not alter receptor NDP-MSH binding affinity but led to complete conversion of SHU9119 activity from antagonist to agonist, demonstrating that this L165 in hMC3R or L133 in MC4R is important for ligand selectivity (Yang et al., 2002). All these results suggest that specific amino acid residue in TM3 of the MC receptors plays a key role in ligand selectivity for a specific receptor subtype. The conserved amino acid residues in TMs form an orthosteric binding sites for endogenous ligand binding and non-conserved residues provide allosteric binding site for the selective agonist binding for a specific MCR subtype. Receptor modeling is a new approach for elucidating ACTH receptor structure and function. Based on current MCRs work, electrostatic and hydrophobic forces have been proposed to be involved in ACTH binding and receptor activation at ACTH receptor. We docked different ACTH analogues into the ACTH receptor model which was developed by Dr. Mosberg group (http://mosberglab.phar.umich.edu/resources/). In this model, D103 and D107 of the ACTH receptor have been proposed to form an ionic interaction with Arg8 of ACTH1-17. In the model, the positively charged Arg8 of peptides is located close to the extracellular surface of the receptor, and it participates in the H-bond network with acidic residues from TM3 (Asp103and Asp107). Amino acid residue Phe aromatic ring is situated on the bottom of the receptor pocket. This Phe7 residue is most essential for ligand docking into ACTH receptor binding pocket although this residue has no direct interaction with receptor residue. As show in Fig. 7, the structure and conformation of the amino

acid residue, Phe, DPhe or DNal (2′) are different and therefore their positions in binding pocket are also different. The residue of DPhe or DNal (2′) in ligand may prevent residue Arg8 interacting with D103 and D107. The positively charged Arg8 of peptides is located close to the extracellular surface of the receptor, and it participates in the H-bond network with acidic residues from TM3 (D103and D107). The Phe7 residue's aromatic ring is situated on the bottom of the receptor pocket and plays a key role for ACTH binding and activity. As shown in Fig. 7B, the position of Phe7 in ACTH1-17 is away from ionic binding site. However, the positions of DPhe is very close to ionic binding site, suggesting that the position of DPhe7 in ACTH may prevent residue Arg8 interacting with D103 and D107 (Fig. 7C) (Yang et al., 2015). The position of DNal7 in ACTH1-17 is similar to that of DPhe7 in ACTH which is very close to ionic binding site, suggesting that the position of DPhe7 or DNal7 in ACTH may prevent residue Arg8 interacting with D103 and D107 (Fig. 7D). This provides evidence that the position of Phe in ACTH influences ligand binding affinity. That may be the reason for DPhe in ACTH analogue has low potency at ACTH receptor. Furthermore, DPhe7-ACTH1-17 resulted in loss of ligand binding affinity and potency at ACTH receptor WT further support this theory. However, the model presented in Fig. 7 does not include interaction with MRAP1 and may oversimplify the docking of ACTH to the receptor. Further experiment is needed to identify the role of MRAP1 in ACTH and ACTH receptor interaction. This article aims to provide a comprehensive perspective of the structural knowledge gained so far in the molecular basis of the ACTH receptor for ligand binding and signaling. Although a substantial amount of information on ACTH receptor structure-activity relationship has emerged during the last several decades, there are still several issues that remain unresolved, including elucidation of the ACTH receptor responsible for antagonist agouti selective binding and entire crystal structure of ACTH receptor. A more precise identification of such structural features might allow to design the highly specific therapeutic strategies, which activates the mutated ACTH receptor or block putative deleterious ACTH effects on these particular tissues. Crystals of ACTH receptor also will aid to clarify many aspects on ACTH receptor function that may be translated in the near-term to human therapeutics.

Fig. 7. Computer modeling of MC2R and ACTH analogues interaction. Panel A. ligand receptor interaction between ACTH and MC2R from outside of the receptor; Panel B. ligand receptor interaction between Phe7-ACTH1-17 and MC2R; Panel C. ligand receptor interaction between DPhe7-ACTH1-17 and MC2R; Panel D. ligand receptor interaction between DNal7-ACTH1-17 and MC2R. The amino acid residue Phe7, D-Phe7 and DNal-7 in ligand ACTH are highlighted in green color. The amino acid residue Asp103 (D103) and Asp 107 (D107) in ACTH receptor are highlighted in red color. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) 6

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Y. Yang and C.M. Harmon

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