Receptors for parathyroid hormone and parathyroid hormone–related protein

Chapter 28

Receptors for parathyroid hormone and parathyroid hormoneerelated protein Thomas J. Gardella, Harald Ju¨ppner and John T. Potts, Jr. Endocrine Unit, Department of Medicine and Pediatric Nephrology, MassGeneral Hospital for Children, Massachusetts General Hospital and Harvard Medical School, Boston, MA, United States

Chapter outline Introduction The PTHR1 is a class B G-protein-coupled receptor Parathyroid hormone receptor gene structure and evolution Structure of the PTHR1 gene Evolution of the parathyroid hormone receptoreligand system Mechanisms of ligand recognition and activation by parathyroid hormone receptors Basic structural properties of the PTHR1 Two-site model of ligand binding to the PTHR1 Mechanism of ligand-induced activation at the PTHR1 Conformational selectivity and temporal bias at the PTHR1 Two high-affinity PTH receptor conformational states, R0 and RG Conformation-based differences in signaling responses to PTH and PTHrP ligands

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Endosomal signaling and signal termination at the PTHR1 Ligand-directed temporal bias and therapeutic implications LA-PTH, a long-acting PTH/PTHrP analog for hypoparathyroidism Abaloparatide: a PTHrP analog for osteoporosis PTHR1 mutations in disease Jansen’s metaphyseal chondrodysplasia Other diseases linked to PTHR1 mutations Nonpeptide mimetic ligands for the PTHR1 Other receptors for parathyroid hormone and related ligands PTHR2 and PTHR3 subtypes Possible receptors for C-terminal PTH and PTHrP Conclusions References

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Introduction Parathyroid hormone (PTH) and PTH-related protein (PTHrP) mediate their principal biological actions by acting on a single cell-surface receptor, the type-1 PTH/PTHrP receptor, or PTHR1. The PTHR1 thus stands as a key regulatory molecule that is essential for normal physiology at all stages of life. The biological situation is somewhat unique in that the one receptor responds to two endogenous ligands to thereby control two disparate physiological processes: the maintenance of blood calcium and phosphate homeostasis via PTH and the timing of cell differentiation events in the developing skeleton and other tissues via PTHrP. The PTHR1 also holds interest as a prospective drug target, as pharmacologic agents that specifically modulate its activity can potentially be used to treat disturbances of bone and calcium metabolism that occur in a variety of pathologic conditions, such as osteoporosis. The endogenous PTH and PTHrP ligands are polypeptide chains of 84 and 141 amino acids, respectively, with the first 34 amino acids containing sufficient information for high-affinity binding to the PTHR1 and potent activation of downstream signaling responses. Thus synthetic PTH(1e34) and PTHrP(1e34) peptides can generally mimic most of the biological actions of the full-length molecules. Nevertheless, certain functional activities have been reported for peptides derived from the C-terminal portions of PTH and PTHrP, which has led to the notion that other receptors, distinct from the PTHR1, might exist that bind the C-terminal portion of the ligands, although no such receptor has so far been identified. For the PTHR1, much effort has been directed at elucidating the mechanisms by which the ligands PTH and PTHrP engage the receptor and stimulate signal transduction. These studies generally reveal that PTH(1e34) and PTHrP(1e34) interact

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with the PTHR1 in highly similar, yet not identical, fashions. Consistent with this, the primary structures of the ligands exhibit considerable amino acid sequence homology in the amino-terminal region, particularly at the N-terminal 1e14 portion, which is critical for receptor activation, with eight amino acid identities, while the 15e34 portion, which contributes to binding affinity, shows only a moderate level of homology, with three identities. As discussed later in this chapter, studies suggest that differences can indeed be discerned in the binding modes utilized by PTH(1e34) and PTHrP(1e34) and that such differences can lead to differences in functional effects, particularly in the duration of the signaling responses induced. Such findings further support the notion that the capacity of the PTHR1 to regulate the aforementioned two disparate types of physiological processesdthe endocrine control of mineral ion homeostasis and the paracrine control of tissue developmentdis at least partly based on differences in the mechanisms by which the two respective ligands engage the receptor. Early work conducted prior to the cloning of the PTHR1 in 1991 and which used canine or bovine renal membrane preparations or cell lines derived from bone or kidney gave initial clues as to the basic pharmacology of PTH ligand binding to endogenous receptor binding sites and the downstream signaling responses that could be activated. This work was made possible in large part by the development of peptide synthesis technology and its early application to PTH (Potts et al., 1971), which yielded most notably the PTH(1e34) peptide, as well as radioiodinated derivatives that enabled direct characterization of the binding sites used (Segre et al., 1979). Such studies further revealed that residues in both the N- and the C-terminal region were important for overall binding affinity (Nussbaum et al., 1980). Parallel studies revealed that PTH peptides induced rapid and robust increases in intracellular cAMP, and that the N-terminal residues of the ligand were critical for activating this response. That this response was mediated by a cell-surface G-protein-coupled receptor (GPCR) was indeed borne out in 1991 by the cloning of the cDNA encoding the PTHR1 from kidney- and bone-derived cell lines (Juppner et al., 1991), which revealed the seven membrane-spanning-domain protein architecture that defines all 800 or so members of the GPCR superfamily. A key goal now is to elucidate the specific mechanisms by which the PTHR1 uniquely recognizes its two specific ligands, PTH and PTHrP, and activates selected intracellular effectors in different target cells. Progress toward this goal has been made via the application of a number of molecular and pharmacological approaches, such as the use of receptor mutagenesis coupled with peptide analog design strategies to reveal sites of specific intermolecular contact and clues about conformational changes involved in activation. The use of genetically modified mice engineered so as to have a specific alteration in a selected component of the ligandereceptor response system have also helped make possible a level of analysis at the whole-animal physiological level. These experimental systems ultimately help reveal the involvement of the PTHR1 and its ligands in skeletal and mineral ion physiology as well in diseases, such that they could lead to the development of new forms of therapy targeted to the PTHR1. Broader views of the roles that the PTHR1 and its ligands play in the systemic control of metabolic processes in bone and kidney, and developing tissue, as well as the downstream processes that the receptor controls within target cells, are discussed in detail in other chapters (see Chapters 24e27, 32, and 52). This chapter then focuses on the molecular properties of the PTHR1 per se, and principally on the mechanisms by which PTH and PTHrP ligands, via their N-terminal portions, interact with this receptor and induce signal transduction processes. It will also discuss findings on the so-called type-2 PTH receptor, or PTHR2, and its endogenous ligand, tuberoinfundibular peptide of 39 residues (TIP39), as well as the possibility that other, as yet unidentified, receptors exist that can bind and potentially respond to the carboxy-terminal portion of the endogenous PTH and PTHrP ligands, which appear not to be contributing to interactions with the PTHR1.

The PTHR1 is a class B G-protein-coupled receptor The PTHR1 exhibits the general protein architecture seen in each of the class B GPCRs and thus is a single-chain integral membrane protein containing a relatively long amino-terminal extracellular domain (ECD) of w170 amino acids (after removal of residues 1e22, which act as the signal sequence), a transmembrane domain (TMD) or core region comprising the seven membrane-spanning a helices (TMs 1e7) and their interconnecting extracellular and intracellular loops (ECLs 1e3 and ICLs 1e3), and a C-terminal tail extending from the base of TM7 (wCys452) to the C terminus (Met593) (Fig. 28.1A). As a class B GPCR, the PTHR1 contains none of the hallmark amino acid residues and sequence motifs that define the three other main classes of GPCRs: the class A receptors represented by the b2Ar, the class C GPCRs represented by the calcium-sensing receptor, and class F receptors, as represented by the Frizzled receptors (Fredriksson et al., 2003). Instead, the PTHR1 exhibits a distinct pattern of amino acids that is conserved in each of the 14 or so other class B GPCRs, which include the receptors for calcitonin, secretin, glucagon, glucagon-like peptide-1 (GLP1), corticotropinreleasing factor (CRF), and several other peptide hormone ligands (Segre and Goldring, 1993). Nevertheless, comparison of the three-dimensional structures obtained for GPCR representatives from each of the main GPCR families reveals

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FIGURE 28.1 Primary structure of the human parathyroid hormone receptor type 1 protein and corresponding gene organization. (A) The human parathyroid hormone receptor type 1 (hPTHR1) protein is displayed to illustrate the domain organization and locations of selected key residues, including the eight extracellular cysteines involved in a disulfide bond network (connecting dotted lines); the four glycosylated extracellular asparagines; Pro132, at which loss-of-function mutations occur in Blomstrand’s chondrodysplasia (compound homozygous) and in failed tooth eruption (heterozygous); His223, Thr410, and Ile458, at which activating mutations occur in Jansen’s metaphyseal chondrodysplasia; cytoplasmic sites of serine and threonine phosphorylation; the four C-terminal residues involved in PDZ-domain interactions with Naþ/Hþ exchange regulatory factor 1 (NHERF) proteins; and a number of residues shown by mutagenesis and/or cross-linking studies to be involved in ligand interaction (filled shaded circles with position numbers). Also shown is the residue in each transmembrane domain helix that is the most conserved among the class B G-protein-coupled receptors (filled hexagons). (B) The intron/exon structure of the hPTHR1 gene with coding and noncoding exons indicated as filled and open boxes, respectively.

similarities not only in the basic seven-TMD protein fold that is shared across all GPCR classes, but also in some of the key interhelical contact points that occur within the hepta-helical bundle and are thought to mediate key mechanistic steps of activation (Cvicek et al., 2016). At least some aspects of function are thus likely preserved, at least topologically, between the PTHR1 and perhaps most of the 800 or so other GPCRs encoded in the human genome. While each class B GPCR responds to a peptide ligand of moderate sizedabout 30e40 amino acids in lengthdthe PTHR1 is the only class B GPCR for which the endogenous ligands extend more C-terminally, as PTH is 84 amino acids in length and PTHrP is 141 amino acids. Such C-terminal extensions are absent in the ligands identified in the genomes of fish and other nonmammalian species (Rubin and Jüppner, 1999; Mirabeau and Joly, 2013), and thus appear to be modifications that occurred later in vertebrate evolution. In any event, most if not all of the available data indicate that only the first 34 amino acids or so of PTH and PTHrP participate in binding to the PTHR1, such that PTH(1e34) and PTHrP(1e34) peptides exhibit nearly the same affinities and signaling potencies on the PTHR1 as the corresponding fulllength polypeptides (Li et al., 2012; Dong et al., 2012).

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Parathyroid hormone receptor gene structure and evolution Structure of the PTHR1 gene The gene encoding the human PTHR1 resides on chromosome 3 (locus 3p22ep21.1) and spans w26 kb of DNA (Fig. 28.1B) (McCuaig et al., 1994; Kong et al., 1994). The predicted transcript consists of 14 coding exons and 2 noncoding exons, ranging in size from 42 bp for exon M7 encoding a portion of transmembrane helix 7 to more than 400 bp for exon T encoding the C-terminal tail. The introns vary in size from 81 bp between exons M6 and M6/7 to more than 10 kb between exon S encoding the signal sequence and exon E1 encoding the N-terminal portion of the mature receptor. Three promoter regions have been identified for the gene and shown to be active at different levels in different tissues (Minagawa et al., 2001; Bettoun et al., 1997; Amizuka et al., 1997). Expression of PTHR1 mRNA can be detected in a variety of fetal and adult tissues, including the adrenals, placenta, fat, brain, spleen, liver, lung, and cardiac muscle, with strongest signals observed in adult bone and kidney (Urena et al., 1993; Tian et al., 1993; Uhlen et al., 2015). The genes for the other class B GPCRs generally exhibit a similar intron/exon organization, which is consistent with their evolution from a common ancestor gene and utilization of a similar protein design for engaging their cognate peptide ligands (Hwang et al., 2013).

Evolution of the parathyroid hormone receptoreligand system Bioinformatics-based investigations of PTH receptor as well as PTH and PTHrP ligand coding sequences present in the genomes of species representing various stages of evolution have yielded clues about how the PTH ligand and receptor system first emerged and evolved over time to provide the complex developmental and adaptive capacities established in the mammalian species. These studies suggest that PTH receptors and ligands emerged before the evolution of the first vertebrates, as apparent orthologs of the receptor as well as PTH-like ligands have been detected in the genomes of early vertebrates, such as the elephant shark and sea lamprey, as well as some invertebrates, such as the tunicate Ciona intestinalis and the amphioxus Branchiostoma floridae, which are thought to be representative of early chordates (Fig. 28.2) (Kamesh et al., 2008; Pinheiro et al., 2012; Mirabeau and Joly, 2013; On et al., 2015; Cardoso et al., 2006; Hwang et al., 2013). Sequences with at least some homology (w69% amino acid similarity) have also been identified in the genomes of several insects, such as the red flour beetle (Tribolium castaneum) and honeybee (Apis mellifera), but not in the fruit fly (Drosophila melanogaster) (Li et al., 2013; Cardoso et al., 2014). The biological function of any such invertebrate PTH receptorelike sequence remains unknown. Genome studies also suggest that chromosomal rearrangements contributed to the diversification of the PTH ligandereceptor system. At least two copies of a PTH receptor gene family member are thus found in the haploid genomes of most vertebrate species, and these can be attributed, in part, to two rounds of whole-genome duplication that are thought to have occurred during the early phases of vertebrate evolution (Fig. 28.2) (Hwang et al., 2013; On et al., 2015; Pinheiro et al., 2012; Cardoso et al., 2006, 2014). These early duplications were followed by other gene duplications as well as deletion events that occurred at different points along the evolutionary paths leading to the divergent animal groups. The genomes of teleost fish, as represented by the zebrafish, Danio rerio, thus encode three receptors, the PTHR1, PTHR2, and PTHR3, but lack a PTHR4 gene as a putative partner to the PTHR2, apparently due to a deletion event that happened in early fish evolution (Rubin and Juppner, 1999; Hwang et al., 2013). Bird genomes encode PTHR1 and PTHR3, and lack PTHR2 and PTHR4, while mammals encode PTHR1 and PTHR2, and lack PTHR3 and PTHR4. The absence of a PTHR2 in birds and a PTHR3 in primates apparently reflects separate gene deletion events that occurred at distinct times during the evolution of these two vertebrate groups. The peptide ligands, as represented in humans by PTH, PTHrP, and TIP39, the last a ligand for the PTHR2 as discussed further in a later section, are presumed to have evolved in parallel with their receptors, and thus to have stemmed from some precursor peptide ligand that emerged at or about the time that the first chordates appeared, as indeed, PTH- and/or TIP39-like coding sequences are found in the genomes of early fish, as well as amphioxus and C. intestinalis (Rubin and Juppner, 1999, Yan et al., 2012; Pinheiro et al., 2012; Trivett et al., 2005; Liu et al., 2010). These findings are consistent with the notion that the PTH ligandereceptor system is an evolutionarily ancient adaption that emerged in some precursor form before the appearance of the first vertebrates and evolved over time so as to contribute importantly not only to the successful adaption of the animal to changing environments but also to the development of higher-order organ systems, such as the skeleton (Hogan et al., 2005; Suzuki et al., 2011; Trivett et al., 2005).

Cio na int es tin ali Cephalaspidomorphi s Pe tr o my zu sm Chondrichthyes ar i Ca nu llo s rhi nc hu sm Da Actinopterygii ilii Ta nio r kif ug erio ur ub rip Amphibia es Xe no pu st rop ica Reptilia lis An oli sc aro lin Avian en Ga sis llu sg all us Monotremata Or nit ho rhy nc hu Marsupial M sa on na od tin elp us his do me Eutheria H sti ca om os ap ien s

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Deletion of PTH-L

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Deletion of PTH3R

Deletion of PTH2R and TIP39

2R 1R

Origin of PTHR

Emergence of PTH3R Emergence of PTH-L Duplication of the PTHR Origin of PTH family members

BILATERAL ANCESTOR FIGURE 28.2 Evolutionary model for parathyroid hormone receptors. Shown is a tree diagram to depict that the ancestral parathyroid hormone receptor (PTHR) sequence probably originated before the appearance of the first chordates, as represented by the tunicate Ciona intestinalis, and that two rounds of whole-genome duplication (1R and 2R) occurred before the emergence of the terrestrial vertebrates to result in three receptor subtypesdPTHR1, PTHR2, and PTHR3dwith the expected fourth subtype lost by an early gene deletion event. The PTHR2 and PTHR3 subtypes were probably lost by later gene deletion events occurring with the radiations leading to birds and mammals, respectively. Reproduced pending permission from Pinheiro, P. L., Cardoso, J. C., Power, D. M., Canario, A. V. 2012. Functional characterization and evolution of PTH/PTHrP receptors: insights from the chicken. BMC Evol. Biol. 12, 110.

Mechanisms of ligand recognition and activation by parathyroid hormone receptors Basic structural properties of the PTHR1 As in all class B GPCRs, the ECD contains six highly conserved cysteine residues that form a disulfide network (Fig. 28.1A) that maintains the tertiary fold of the ECD, which follows a tripartite helixeb sheetehelix pattern (Pioszak et al., 2008). The ECD contains four asparagines that are glycosylated during intracellular processing and transported to the plasma membrane. A feature of the ECD that is seen only in the mammalian PTHR1s, and not in the mammalian PTHR2s or any other class B GPCR, is a 44-amino-acid segmentdSer61eGly105 in the human PTHR1dlocated between the first and the second cysteine. This segment is encoded by a separate exon, called E2, and appears not to contribute to function, as assessed by site-directed mutagenesis strategies and pharmacological analyses in transfected cells (Lee et al., 1994). Other than the general seven membrane-spanning helical architecture, the class B receptors share no obvious homology with receptors from the other main GPCR subgroups, including the class A GPCRs, which comprise the largest GPCR subgroup and include the b-adrenergic receptor and rhodopsin as well-studied representatives. Nevertheless, the highresolution structures now available for a number of GPCRs from each main class, including several class B GPCRs, suggest that certain relationships of structure and function might be preserved in all GPCRs, particularly in the intramolecular interaction networks that are located within the TMD bundle and mediate processes of receptor activation and G protein coupling (Bortolato et al., 2014; Cvicek et al., 2016). Within the class B GPCRs there are about 45 amino acid residues that are strongly conserved. These conserved residues are dispersed in the seven TM helices and in the ECD and probably help define the basic three-dimensional scaffold structure of the receptor and as well may participate more directly in basic mechanisms of activation, while other more divergent residues are likely to provide the specific recognition determinants used for selective binding of the appropriate peptide ligand. Binding of a PTH agonist peptide ligand, such as PTH(1e34), to the extracellular surface of the PTHR1 induces a conformational change in the receptor that leads to G protein coupling and activation of downstream signaling responses (see Chapters 32 and 52 for an in-depth discussion of downstream signaling responses). In most target cells the PTHR1

696 PART | I Basic principles

couples strongly to G proteins containing the GaS subunit, and thus activates the adenylyl cyclase/cAMP/protein kinase A (PKA) signaling system. The agonist-stimulated PTHR1 can also activate other signaling systems, including the Gaq/ phospholipase C (PLC)/IP3/iCa2þ pathway, the Ga12/DAG/PLD pathway, and the arrestin/ERK1/2 pathway, depending potentially on the type of target cell and PTH or PTHrP ligand analog used (Gesty-Palmer et al., 2006, 2013). PTHR1 signaling is generally thought to be a highly regulated process and thus to terminate relatively soon after initial binding via mechanisms involving receptor phosphorylation and internalization (Bisello et al., 2002; Malecz et al., 1998; Qian et al., 1998), but variations on this theme now seem possible, as discussed in a later section. It is now becoming clear that ligands with different structures can bind to a receptor in different modalities so as to induce different biological outcomes in target cells, in terms of type of pathway activated as well as the duration of the response, a concept generally referred to as biased agonism (Luttrell et al., 2015). This concept has particular relevance for the PTHR1, as it may help explain, at least in part, certain differences in biological actions that have been observed for PTH and PTHrP peptides (Horwitz et al., 2003) and, moreover, could provide a strategic basis for developing new ligand analogs for the PTHR1 that have therapeutic utility.

Two-site model of ligand binding to the PTHR1 Clues regarding the overall mechanism of ligand binding and agonist-induced activation used by the PTHR1 were initially obtained from receptor mutagenesis and photoaffinity cross-linking studies, but more recently have come from the successful application of X-ray crystallography and cryo-electron microscopy (cryo-EM) approaches to the analysis of several other class B GPCRs, either as intact receptors or as the isolated ECD or TMD. Such structural information for the PTHR1, however, is so far reported only for the isolated ECD in complex with PTH(15e34) or PTHrP(12e34) peptides (Pioszak et al., 2008, 2009). The emerging structural data combined are providing valuable insights into the basic mechanisms by which the class B GPCRs engage their cognate peptide ligand and activate signal transduction. The earlier studies on binding mechanisms for the PTHR1 performed in parallel by several groups led to the so-called “two-site” model of ligandereceptor interaction. According to the model, the ligand, such as PTH(1e34), first docks to the receptor via the binding of the C-terminal domain of the ligand, approximately residues 15e34, to the ECD of the receptor. Then the amino-terminal portion of the ligand, approximately residues 1e14, engages the extracellular surface of the TMD portion of the receptor to yield the intact ligandereceptor complex (Fig. 28.3). The ECD component of the interaction contributes predominantly to the overall binding affinity of the complex, while the TMD component mediates the process of receptor activation and G protein coupling. The experimental findings that led to this model came from studies employing, often in parallel, the two complementary approaches of receptor mutagenesis and photoaffinity cross-linking. By the former approach, receptors altered at specific residues or defined regions were generated and analyzed in pharmacologic binding and signaling assays for interaction with various peptide ligand analogs with defined structural modifications such that they could be used as functional probes of specific contact sites or regions. For example, a receptor chimera strategy based on the weaker binding of PTH(7e34) to the rat versus the human PTH receptor was used to establish that the ECD of the receptor was the major site of binding for the C-terminal portion of the ligand (Juppner et al., 1994). A similar chimera strategy based on the capacity of the analog Arg2-PTH(1e34), in which the highly conserved valine-2 is replaced by a bulky arginine, to function as an antagonist on the rat PTHR1 and an agonist on the opossum PTHR1 led to the finding that residue 370 at the extracellular end of TM5, alanine and serine in the opossum and rat receptors, respectively, is a key determinant of ligand-induced activation (Goltzman et al., 1975). Other such mutagenesis studies that followed, extending also to the PTHR2, further supported the two-domain model (Bergwitz et al., 1996, 1997; Turner et al., 1998). The second approach involving the use of photoaffinity cross-linking strategies provided more direct biophysical analyses of ligand and receptor sites in close intermolecular proximity. The method involved ligands modified at a selected position with an amino acid analog containing a photolabile side chain, typically benzophenylalanine (BPA) (Zhou et al., 1997). After binding to the receptor, the complex is UV irradiated, causing the BPA group to covalently link to a site in the target protein chain within a radius of a few angstroms. Initially BPA was thought to react rather nonselectively, but was later discovered to have a propensity to react with methionines (Wittelsberger et al., 2006b). In any event, the strategy resulted in the identification of a number of cross-links that could be mapped to specific receptor residues, including between positions 1 and 2 in the ligand and Met425 at the extracellular end of TM6 (Bisello et al., 1998; Gensure et al., 2001a), between ligand residue 13 and Arg186 at the extracellular end of TM1 (Adams et al., 1998), and between ligand position 23 and Thr33 and/or Gln27 at the N-terminal end of the receptor (Mannstadt et al., 1998). A number of other contacts involving residues in the middle or C-terminal region of PTH(1e34) or PTHrP(1e36) and various sites in the receptor were also mapped, although not always to specific receptor residues (Gensure et al., 2001b, 2003; Greenberg et al., 2000; Wittelsberger et al., 2006a). The information gained from these cross-linking studies provided distance constraints that enabled development of initial molecular models of the PTHR1$PTH ligand complex (Greenberg et al., 2000; Gensure

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FIGURE 28.3 Ligand binding mechanisms at the parathyroid hormone receptor 1. (A) Sequence of PTH(1e34) highlighting the N-terminal residues Val2, Ile5, and Met8, which play critical roles in receptor activation, and the C-terminal residues Arg20, Trp23, Leu24, and Leu28, which play critical roles in receptor binding. (B) The two-domain model of ligand binding and activation at the PTHR1, as developed by cross-linking and mutagenesis data obtained for the parathyroid hormone receptor 1 (PTHR1): the C-terminal portion of PTH(1e34) first docks to the amino-terminal extracellular domain (ECD) of the receptor to provide affinity interactions, and then the amino-terminal portion of the ligand engages the transmembrane domain (TMD) of the receptor to induce conformational changes that enable G protein coupling. (C) Refinement of the two-domain model based on high-resolution X-ray crystal and cryo-electron microscopy structures of the glucagon receptor and GLP1 receptor, which are class B GPCRs structurally related to the PTHR1. The ligand, shown in red, binds as a nearly linear a helix and makes extensive contacts with exposed extracellular surfaces in both the ECD and the TMD receptor regions. Ligand binding results in an outward movement of the cytoplasmic termini of several of the TM helices, particularly TM6, to thus open a cavity that will accommodate the G protein. (C) Reproduced pending permission from Zhang, H., Qiao, A., Yang, L., Van Eps, N., Frederiksen, K. S., Yang, D., Dai, A., Cai, X., Zhang, H., Yi, C., Cao, C., He, L., Yang, H., Lau, J., Ernst, O. P., Hanson, M. A., Stevens, R. C., Wang, M. W., Reedtz-Runge, S., Jiang, H., Zhao, Q., Wu, B. 2018a. Structure of the glucagon receptor in complex with a glucagon analogue. Nature 553, 106e110.

et al., 2003; Wittelsberger et al., 2006a; Piserchio et al., 2002), although the incomplete nature of the input data left a fair amount of uncertainty. The two-site model was also found to be relevant for most if not all of the other class B GPCRs and their cognate peptide ligands, as elucidated using similar functional and cross-linking-based approaches by other groups (Parthier et al., 2009). This basic model has now been confirmed and refined by high-resolution structural data that have been obtained using X-ray crystallography or cryo-EM approaches for several of the other class B GPCRs, including the CRFR1, the glucagon receptor, and the GLP1 receptor, although not yet for the PTHR1 (de Graaf et al., 2017). A key modification used in the crystallography work, and a potential limitation in terms of data interpretation, is the introduction of thermostabilizing point mutations and insertions of heterologous protein segments known to promote crystallization at dispersed positions in the receptor TMD, which generally tends to be conformationally dynamic. One set of findings from the functional studies that helped define the TMD component of the two-domain model for the PTHR1 was that short aminoterminal PTH fragment peptides, such as PTH(1e14) and PTH(1e11), which as unmodified peptides are inert for binding

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and signaling due to a lack of stabilizing interactions with the ECD, could act as potent agonists when modified with certain helix-stabilizing and affinity enhancing substitutions. Furthermore, they could stimulate signaling via a PTHR1 construct that lacks most of the ECD (up to Glu182), about as potently as PTH(1e34) does on the intact PTHR1 (Shimizu et al., 2001). These findings highlight the functional autonomy of the N-terminal region of the ligand and the TMD of the receptor, and also suggested that ligands much smaller than PTH(1e34) that target the functionally critical TMD and thus act as potent PTH mimetics could be developed. Native PTH and PTHrP peptides are thought to bind to the PTHR1 via a sequential process that involves initial ligand interactions with the ECD followed by interactions with the TMD and the conformational rearrangements involved in activation (Fig. 28.3) (Hoare and Usdin, 2001). The nature of these conformational changes and the extent to which they may involve a higher-order folding of the complex, for example, a movement of the ECD relative to the TMD, remains an area of uncertainty, but the new class B GPCR structures now available provide clues as to the types of changes that might be possible for the PTHR1ePTH complex. The crystal structures of the isolated ECD protein in complex with the PTH(15e34) or PTHrP(12e34) fragment suggest that the two ligands bind to the ECD via similar, though not identical, mechanisms (Pioszak et al., 2009). Each peptide thus is bound to the ECD as an a helix and fits into a groove that runs along the center of the ECD. Key hydrophobic interactions occur between Trp23, Leu24, and Leu 28 in the ligand, which align along an apolar face of the helix, and complementary apolar surfaces in the receptor-binding groove. The side chain of Arg20 in the ligand makes extensive interactions with a ring of polar residues located at one end of the ECD. Residues Arg20, Trp23, Leu24, and Leu28 are well conserved in PTH and PTHrP ligands, while residues on the opposite helix face are less well conserved, are mostly polar in character, and can be mutated with relatively little effect on binding. Compared with the PTH(15e34)$ ECD structure, the PTHrP helix bends modestly at about Leu24 such that the C-terminal portions of the two helices make distinct contacts, and the C-terminal residue is offset by a few angstroms in the two structures (Pioszak et al., 2009). Interactions that occur between the N-terminal portion of the ligand, as contained in the PTH(1e14) segment, and the receptor’s TMD are of key interest as they are critically involved in receptor activation. There is no direct structural data available for the PTHR1 TMD. The aforementioned mutagenesis and cross-linking data are consistent with the ligand making multiple contacts with an extensive, solvent-exposed surface area that involves the extracellular ends of the TMD helices and the extracellular loops. In the crystal and cryo-EM structures of the TMDs of the other class B GPCRs, the extracellularly exposed surface of the TMD bundle forms a relatively large pocket that accommodates the N-terminal portion of the ligand (Liang et al., 2017; de Graaf et al., 2017). In the intact GLP1ReGLP1 (Zhang et al., 2017, 2018a) and CTRecalcitonin structures, the full-length peptides are bound as linear a helices that extend along the ECD, which is positioned above the TMD, and enter into the TMD bundle such that the N-terminal residues of the ligand helix project into the bundle such that they can trigger the conformational rearrangements involved in receptor activation (Zhang et al., 2018a) (Fig. 28.3C). It is reasonable to suggest that PTH(1e34) binds to the PTHR1 in a fashion similar to that seen for glucagon and calcitonin, but direct confirmation of any such interaction mode for PTH and the PTHR1 remains to be established by direct high-resolution methods.

Mechanism of ligand-induced activation at the PTHR1 Previous mutagenesis and biophysical cross-linking data suggest that substantial conformational changes occur within the TMD bundle of the PTHR1 during agonist-induced activation (Gardella et al., 1996; Gensure et al., 2001a; Thomas et al., 2008). Insights into the types of specific changes that might occur can be inferred by comparing the active- versus inactivestate structures obtained for the several other class B GPCRs. Such comparisons suggest that during the agonist binding and receptor activation process, the ECD of the complex rotates in a counterclockwise direction, relative to the vertical axis of the TMD bundle, by nearly 90 degrees (Zhang et al., 2018a). In addition, two segments at the extracellular ends of the TM1 and TM2 helices isomerize from b-strand conformations to a-helical conformations. Interestingly, whereas the two b strands in the inactive state receptor pair together to form a cover over the ligand-binding pocket of the TMD bundle, the induced a-helical segments extend upward and contact the midregion of the bound ligand helix to thus help stabilize the ligand and guide the N-terminal segment into the TMD pocket (Zhang et al., 2018a). The structural studies on the class B GPCRs also reveal a cluster of highly conserved polar residues within the core of the TMD bundle that form a network of H-bond and electrostatic interactions and thus act to constrain the bundle in a closed, inactive conformation. Agonist interactions within the TMD core trigger a rearrangement of this network to release the constraints and promote the outward movement of the cytoplasmic ends of several of the TM helices, particularly TM6. These movements result in a cavity on the cytoplasmic surface of the receptor that accommodates the G protein (Fig. 28.3C) (Zhang et al., 2017; Yin et al., 2017).

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While any such mechanism remains to be assessed for the PTHR1, the studies provide clues as to the types of conformational changes that are likely to occur during the PTHR1 activation process. It is worth noting that several PTHR1 residues that are located at least in the vicinity of the predicted cytoplasmic cavity used by G proteins have been implicated by mutagenesis methods to be involved in G protein interaction. These include Lys319, Val384, and Leu385 for Gaq coupling; Thr387 for Gas coupling; and Lys388 for Gas and Gaq coupling (Iida-Klein et al., 1997; Huang et al., 1996). Of further note, these studies led to the generation of the “DSEL” knock-in mouse in which the normal PTH receptor allele is replaced by a PTHR1 mutant having the GlueLyseLyseTyr sequence at positions 317e320 replaced by AspeSere GlueLeu and which is thus selectively impaired for Gaq/PLC signaling; the relatively modest effect of the mutant allele on the skeletons of these mice suggests that the Gaq/PLC signaling pathway does not play a critical role in PTH receptoremediated control of endochondral bone formation (Guo et al., 2002).

Conformational selectivity and temporal bias at the PTHR1 Two high-affinity PTH receptor conformational states, R0 and RG The aforementioned structural models are consistent with the notion that structurally distinct ligands for the PTHR1 can stabilize or induce different receptor conformations so as to result in different types of signal transduction responses. The notion that variation in receptor conformation could play an important role in determining the affinity with which certain ligands bind to the PTHR1 emerged from a series of PTH radioligand binding studies that utilized membranes from cells transfected to express the PTHR1 and were performed under conditions to favor either the G-protein-uncoupled receptor conformation or the G-protein-coupled conformation (Hoare et al., 1999b). To promote the G-protein-uncoupled state, the binding reactions were conducted in the presence of GTPgS, which binds to the G protein a subunit and causes it to dissociate from the receptor. To promote the G-protein-coupled state, the binding reactions were conducted using membranes from cells transfected to express excess Gas, typically a mutant form that binds the receptor with high affinity. The conceptual basis came from early pharmacologic studies performed on the b2-adrenergic receptor and other such prototypic GPCRs that bind small catecholamine ligands. These studies led to the classical ternary complex model of GPCR action that posits that G protein coupling promotes a high-affinity receptor conformation, while G protein uncoupling, which occurs upon GDPeGTP exchange or GTPgS binding, causes the receptor to relax to a low-affinity state and thus release the bound agonist (De Lean et al., 1980). It was thus surprisingly found that certain ligands for the PTHR1, including PTH(1e34), bound with high affinity even in the presence of GTPgS, while others, such as PTHrP(1e36) and the modified N-terminal PTH fragment M-PTH(1e14), behaved in a fashion more consistent with the classical model and dissociated rapidly upon addition of GTPgS (Hoare et al., 2001; Dean et al., 2006, 2008). The findings thus suggested that the PTHR1 could exist in two distinct high-affinity conformations, depending on the type of ligand bound: one highaffinity conformation, as bound by PTH(1e34), could exist even in the absence of a bound G protein, while the other conformation, as bound by PTHrP(1e36) and M-PTH(1e14), required G protein coupling. Consequently, the two highaffinity states, G protein uncoupled and G protein coupled, were termed R0 and RG, respectively.

Conformation-based differences in signaling responses to PTH and PTHrP ligands While the initial findings on distinct affinity PTHR1 conformations came from biochemical studies performed in cell membranes, subsequent studies in intact cells as well as animals supported a biological significance. Thus, cAMP timecourse studies performed in PTHR1-expressing cells, in which various ligands were allowed to bind to the receptor typically for 15e30 min and then all unbound ligand was removed by washout, revealed that Gas/cAMP signaling as induced by R0-selective ligands such as PTH(1e34) persisted for extended durations after washout. In contrast, cAMP signaling induced by RG-selective ligands such as PTHrP(1e36) or M-PTH(1e14) terminated more rapidly after washout (Okazaki et al., 2007; Dean et al., 2008). The results thus revealed a difference in the mode of binding and hence biological action for the structurally distinct ligands, including for PTH(1e34) and PTHrP(1e36), which until then were thought to bind to and activate the PTHR1 in similar if not identical fashions (Nissenson et al., 1988; Jüppner et al., 1988). However, an intriguing deficiency for stimulation of vitamin D synthesis was suggested for PTHrP(1e36) relative to PTH(1e34) in a human infusion study (Horwitz et al., 2003). The kinetic washout approaches thus enabled differences in modes of binding and signaling to be revealed for different PTH and PTHrP ligands. The kinetic assays initially used were performed in multiwell plate formats and involved measurement of cAMP either by radioimmunoassay of cell lysates or by a luciferasebased Glo sensor reporter that enables the nearly continuous detection of intracellular cAMP in live cells over time (Okazaki et al., 2007; Dean et al., 2008; Maeda et al., 2013). Such studies were subsequently complemented by highly

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sensitive optical methods of fluorescence resonance energy transfer (FRET) microscopy, which enabled assessment of near-instantaneous changes in intracellular cAMP production at the single-cell level (Ferrandon et al., 2009; Feinstein et al., 2011). The findings also suggest a possible mechanistic basis for the distinct biological roles that PTH and PTHrP play in biology. Thus, a sustained mode of action, as occurs with PTH, would provide a suitable means to maintain blood calcium at the levels needed for normal physiological processes, while a more transient mode of signaling, as occurs with PTHrP, would provide a more suitable means to achieve the precise temporal control of cell differentiation that is required during tissue development, as for example in the skeletal growth plates, in which a precisely timed program of chondrocyte differentiation is required for proper elongation and shape formation of the long bones (Chagin and Kronenberg, 2014; Kronenberg, 2003). The structural basis for the different modes of binding and hence signaling observed for PTH and PTHrP ligands in these studies is not completely known, but several amino acids that differ in the two ligands have been implicated. Most notably is the divergence at position 5, which is Ile in PTH and His in PTHrP, as it was thus found that the Ile5PTHrP(1e36) analog binds with higher affinity to the R0 conformation and exhibits more prolonged cAMP responses after washout than does PTHrP(1e36) (Dean et al., 2008). The divergences in the C-terminal (15e34) regions of the two ligands, which probably result in moderately altered modes of binding to the ECD (Pioszak et al., 2009), could also contribute to the differences in binding modes detected for the two ligands in the functional assays. The PTHrP(12e34) fragment was in fact shown to bind to the isolated ECD of the PTHR1 with an affinity about twofold higher than that of PTH(15e34), which supports an altered mode of binding, although the difference in affinity is opposite to that seen for the N-terminally intact ligands and the intact receptor (Dean et al., 2008). In any case, the findings on conformational selectivity for the PTHR1 provide insights into the mechanisms controlling the duration of ligand-induced signaling at the PTHR1, and furthermore have potential implications for the development of new PTHR1-based therapeutics. These two aspects are discussed in the following sections.

Endosomal signaling and signal termination at the PTHR1 Termination of G protein signaling at most GPCRs has generally been thought to occur relatively soon after initial binding of the agonist ligand and coincident with internalization of the receptor into endosomes (Drake et al., 2006). Findings on the PTHR1, as well as several other GPCRs, however, indicate that G-protein-mediated signaling can continue after most if not all of the receptor has moved into the endosomal domain. According to classical GPCR models, the process of signal termination involves a sequence of biochemical steps that include the phosphorylation of the receptor at serine and threonine residues located on exposed portions of the receptor’s C-terminal tail and intracellular loops, the recruitment of b-arrestin proteins to the phosphorylated receptor with a coincident displacement of the G protein, the assembly of the b-arrestin-associated receptor into clathrin-coated pits that then pinch off from the membrane to form vesicles, and the trafficking of the vesicles containing the receptor and ligand as cargo through the endosomal sorting system. The last step directs the cargo to pathways of degradation as mediated by lysosomes and/or the proteasome system or recycles the receptor back to the cell surface (Drake et al., 2006). Many studies provide data generally consistent with this general scenario for the PTHR1. Thus, the agonist-activated PTHR1 is indeed rapidly phosphorylated on at least seven serine and two threonine residues, as well as being ubiquitinated on at least two lysines in the C-terminal tail and intracellular loops; b-arrestin proteins are recruited, the receptor moves into endosomal vesicles and internalizes, and it can be at least partially recycled back to the cell surface (Rey et al., 2006; Tawfeek et al., 2002; Vilardaga et al., 2002; Chauvin et al., 2002; Zhang et al., 2018b). The PTHR1 also engages with certain intracellular scaffolding proteins, particularly members of the Naþ/Hþ exchange regulatory factor family of proteins via PDZ-domain-based anchoring to the receptor’s C-terminal tail (Mahon et al., 2003; Mamonova et al., 2012), which further are thought to contribute to the trafficking and signal termination events that occur following agonist binding and activation (see Chapter 27 for further information on PTHR1 trafficking events related to actions in the kidney). The initial evidence suggesting that PTHR1 could deviate from the classical model of GPCR signal termination came from studies on M-PTH(1e34) and M-PTH(1e28) analogs that contained the same set of affinity- and potency-enhancing modifications as contained in the M-PTH(1e14) analogs mentioned earlier (Okazaki et al., 2008). These M-PTH(1e34) analogs were thus found to mediate cAMP responses in PTHR1-expressing cells that lasted for as long as several hours after washout, and thus for much longer times than those induced by PTH(1e34). Microscopy studies using fluorescent PTH analogs, typically modified with tetramethylrhodamine attached to the ε-amino function of lysine-13, revealed that within 15 min of initial binding, most, if not all, of the ligand was located in cytoplasmic vessels and not on the cell surface. There was thus a spatiotemporal correlation between persistent signaling and location in vesicles, which raised the novel possibility that the agonist-activated PTHR1, at least when bound by certain modified ligands, could mediate

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prolonged Gas-mediated cAMP responses from the endosomal compartment. It was further observed that upon injection into mice, such M-PTH(1e34) and related analogs induced elevations in blood calcium that persisted for much longer durations than those induced by PTH(1e34). Importantly, the prolonged calcium responses could not be explained by a simple plasma pharmacokinetic effect, as the modified ligands disappeared from the blood, if anything, more rapidly than did PTH(1e34). The results thus suggested that the modified ligands were rapidly sequestered by binding to receptors in target cells of bone and kidney and thus continued to signal from the endosomal compartment (Okazaki et al., 2008). Parallel studies by Vilardaga and colleagues using fluorescence microscopy and FRET-based kinetic approaches to track ligandereceptor complexes in real-time in live HEK293 cells supported a novel mechanism, and also identified some of the key cytoplasmic effector proteins involved. These data thus confirmed that the ligandereceptor complexes were translocated rapidly from the plasma membrane to internalized endosomes where they remained associated with the G protein as well as adenylyl cyclase (Ferrandon et al., 2009). Prolonged signaling at the PTHR1 thus clearly correlated not only with binding to a distinct high-affinity state of the receptor, R0, but also with the formation of signaling complexes that remained stable and presumably active in endosomes. Evidence of such endosomal signaling has now been reported for a number of other GPCRs, including the calcitonin receptor (Andreassen et al., 2014), the calcium-sensing receptor, a class C GPCR (Gorvin et al., 2018), and a number of class A receptors (Godbole et al., 2017; Thomsen et al., 2016). The concept of endosomal signaling could have implications for understanding the basic processes for how these receptors function. PTHrP(1e36) was found to bind more selectively to the RG conformation of the PTHR1 and thus induces more transient cAMP responses compared with PTH(1e34) or the more strongly R0-selective analogs, such as MPTH(1e34) (Dean et al., 2008). Live-cell fluorescence tracking studies in HEK293 cells revealed that PTHrP(1e36) did not colocalize with the PTHR1 in intracellular vesicles (Ferrandon et al., 2009). The findings thus suggested that PTHrP and presumably other RG-selective ligands follow a subcellular trafficking path distinct from that used by PTH(1e34) and the longer-acting analogs and which thus involves differences in processes of signal termination. A key step in the signal termination process for the PTHR1, as induced by PTH(1e34), was thus found to be the association of the internalized receptor with a macromolecular assembly called the retromer complex (Feinstein et al., 2011), which acts at the later stages of endosomal maturation to sort vesicle cargo along pathways of either recycling or degradation. Retromer-mediated signal termination for the PTHR1 was found to be dependent on the constituent proteins, VPS26, VPS35, and SNX27, the last of which binds to the PTHR1 C tail via a PDZ-domain-directed interaction (McGarvey et al., 2016; Chan et al., 2016; Feinstein et al., 2011). The biological relevance for this PTHR1eretromer interaction, initially revealed in HEK293 cells, is supported by studies showing that targeted reduction of VP35 expression in osteoblastic MC3T3 cells prolongs the cAMP signaling response to PTH(1e34), and that mice genetically ablated for VPS35 in osteoblasts exhibit a mild osteoporotic phenotype and an enhanced anabolic response to intermittent PTH(1e34) (Xiong et al., 2016). These studies also revealed that PPP1R14C, which acts to inhibit the PP1 phosphatase, can associate with VPS35 or the internalized PTHR1 to thus result in changes in the phosphorylation status of downstream effectors, including phosphorylated cAMPresponsive element binding protein (CREB). It is thus intriguing to consider that endosomal signaling provides a means for the PTHR1 to activate specific target effectors in defined subcellular compartments so as to achieve efficient control of downstream responses; for example, the phosphorylation of a transcriptional activator such as CREB in the immediate vicinity of the nucleus to thus facilitate its access to the genome. A second mechanistic aspect of subcellular trafficking and signal termination for the PTHR1 revealed in these studies concerns the role of endosomal acidification. In general, the endosomal interior progressively acidifies from an initial pH of w7.4 to a low pH of w4.5, at which most biological ligandereceptor complexes dissociate. This acidification is mediated by the vacuolar ATPase proton pumps, which are activated by cAMP/PKA-dependent phosphorylation. PTH(1e34)induced cAMP/PKA signaling was thus found to promote vesicle acidification and hence the dissociation of the PTH$PTHR1 complexes, leading to signal termination. Thus PTHR1 signaling appears to be regulated at the level of the endosome by a negative feedback system of acidification (Gidon et al., 2014). These findings further suggest the possibility that structurally distinct ligands might bind to the PTHR1 in different fashions so as to form complexes that have differential sensitivities to endosomal acidification and hence can signal for different durations.

Ligand-directed temporal bias and therapeutic implications The profound differences in the duration of signaling now seen to be clearly possible for structurally distinct ligand analogs via binding to distinct PTHR1 conformations are consistent with the emerging GPCR concept of ligand-directed temporal bias (Grundmann and Kostenis, 2017). Thus, for the PTHR1, ligands that bind mainly to the G-protein-dependent conformation, RG, mediate transient signaling responses because the ligandereceptor complexes dissociate soon after the first round of G protein activation, hastened by endosomal acidification, whereas ligands that bind with high affinity to the

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G-protein-independent conformation, R0, mediate prolonged signaling responses as the ligandereceptor complexes remain intact upon G protein uncoupling and endosomal acidification and hence can activate multiple G protein coupling cycles (Okazaki et al., 2008). Such temporal bias for the PTHR1, as it relates to distinct PTH and PTHrP analog ligands, has relevance to strategies aimed at the development of new PTHR1-based therapeutics, as the duration of signaling at the PTHR1 can have a profound impact on physiological outcomes, particularly those relating to the balanced processed of bone anabolism and catabolism (Martin et al., 2006). Ligand-dependent temporal bias at the PTHR1 holds relevance for two key diseases that are treatable with PTHR1targeted ligands: osteoporosis and hypoparathyroidism. Because of the distinct pathophysiological mechanisms underlying these two diseasesdan imbalance in the coupled processes of bone formation bone and resorption leading to an inadequate bone structure in osteoporosis, and a deficiency of PTH production by the parathyroid glands leading to a condition of chronic hypocalcemia in hypoparathyroidismddistinct PTH ligand modes of action and hence pharmacodynamic profiles are needed to achieve effective treatment. Thus, a pulsatile mode of action is needed for osteoporosis, while a more sustained mode of action is needed for hypoparathyroidism (see Chapters 10, 23, and 70 for more detailed information on underlying mechanisms and modes of treatment for these diseases). The concept of temporal bias thus suggests that a short-acting RG-selective ligand, such as PTHrP(1e36), could have utility for osteoporosis, whereas a longer-acting R0-selective ligand, such as those in the M-PTH(1e34) series of peptides and the long-acting analog LAPTH, discussed in the next section, could have utility for hypoparathyroidism (Okazaki et al., 2008).

LA-PTH, a long-acting PTH/PTHrP analog for hypoparathyroidism Based on the initial findings with M-PTH(1e34), efforts were made to develop even longer-acting analogs that could potentially be useful for hypoparathyroidism. One new long-acting analog derived, called LA-PTH, is a hybrid peptide in which the M-PTH(1e14) portion also present in the M-PTH(1e34) is joined to a modified C-terminal 15e36 portion of PTHrP(1e36). This unique structure results in high-affinity binding to the PTHR1 R0 conformation, and hence markedly prolonged calcemic responses (Fig. 28.4AeD) (Maeda et al., 2013). The functional properties of LA-PTH suggested that it could have therapeutic utility for hypoparathyroidism, a disease for which PTH(1e34) as delivered especially by pump (Winer et al., 2014) and PTH(1e84) by subcutaneous injection by which it exhibits a prolonged pharmacokinetic profile (Mannstadt et al., 2013) have proven to be efficacious, with the latter now available as the drug Natpara. As a potentially improved form of therapy, LA-PTH was tested in several rodent models of the disease in which the parathyroid glands were either surgically removed or, in mice, ablated by diphtheria toxin treatment (Fig. 28.4D), and the results revealed that indeed the analog could normalize blood calcium levels more effectively and for longer periods of time after a single injection than could at least severalfold higher doses of PTH(1e34) or PTH(1e84) (Shimizu et al., 2016; Bi et al., 2016). The observed prolonged pharmacodynamic effects were again not explained by a prolonged pharmacokinetic profile, as the analog was cleared from the circulation if anything more rapidly than PTH(1e34). LA-PTH thus stands as a promising preclinical candidate as a new therapeutic option for hypoparathyroidism. It should be noted that several other PTH(1e34)-based analogs have been reported to induce sustained pharmacodynamic actions in animals via distinct mechanisms. These mechanisms include the prolongation of the pharmacokinetic profile, as is achieved via fusion of the C terminus of the PTH(1e34) peptide to either the fragment crystallizable (Fc) portion of IgG (Kostenuik et al., 2007) or a 20-kDa polyethylene glycol (PEG) chain (Guo et al., 2017), each of which acts to impede the rate of glomerular filtration, or, in a third analog, via a combination of modifications (Nle8, Lys27, and a C-terminal 2-kDa PEG group) that results apparently in a prolonged retention of the peptide on the cell surface due to impairment of b-arrestin-mediated receptor internalization (Krishnan et al., 2018). Such analogs could also lead to new modes of treatment for hypoparathyroidism. Whether such analogs designed to induce prolonged actions in vivo via distinct mechanisms elicit different types of downstream responses, as, for example, in the specific gene sets regulated, remains to be determined.

Abaloparatide: a PTHrP analog for osteoporosis Based on the dogma that a pulsatile administration and hence action of PTH is required to achieve an optimal increase in bone structure and strength while avoiding an excess of bone resorption, which typically occurs with a more continuous administration and action of the peptide, it was postulated that a PTHR1 ligand analog that binds selectively to the RG PTHR1 conformation, and thus induces signaling responses of short duration, would be more effective at building new

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FIGURE 28.4 Conformational selectivity and temporal bias at the parathyroid hormone receptor 1. (A) Ligands that exhibit altered modes of conformational selectivity and temporal bias at the parathyroid hormone receptor 1 (PTHR1). (B) Proposed mechanism of temporal bias. A ligand that binds with high affinity to the G-protein-independent conformation, R0, mediates prolonged signaling since the complex is stable and can interconvert over time to the biologically active G-protein-coupled conformation, RG; a ligand that binds selectively to RG induces only transient signaling since the ligand dissociates after G protein activation. (C) In HEK293 membranes and cells expressing the human PTHR1, LA-PTH binds with high affinity to R0 and thus mediates prolonged cAMP signaling responses after washout, whereas abaloparatide binds preferentially to RG and induces more transient cAMP responses. PTH(1e34) and PTHrP(1e36) exhibit intermediate selectivity profiles. (D) In a parathyroidectomized mouse model of hypoparathyroidism, a single injection of LA-PTH, assessed at several doses, results in an extended elevation in serum calcium, whereas PTH(1e34) at an even higher dose results in a more transient elevation. (C) Reproduced pending permission from Hattersley, G., Dean, T., Corbin, B. A., Bahar, H. & Gardella, T. J. 2016. Binding selectivity of abaloparatide for PTH-type-1-receptor conformations and effects on downstream signaling. Endocrinology 157, 141e149; (D) Reproduced pending permission from Bi, R., Fan, Y., Lauter, K., Hu, J., Watanabe, T., Cradock, J., Yuan, Q., Gardella, T. & Mannstadt, M. 2016. Diphtheria toxin- and GFP-based mouse models of acquired hypoparathyroidism and treatment with a long-acting parathyroid hormone analog. J. Bone Miner. Res. 31, 975e984.

bone than would an analog that binds selectively to the R0 conformation and thus induces prolonged responses. Moreover, because excess bone resorption, as occurs with continuous PTHR1 signaling, gives rise to hypercalcemia, an RG-selective analog might provide a reduced risk of this adverse event, which probably was a factor that limited the final dose approved by the US FDA to 20 mg per day rather than 40 mg, even though the latter was more effective at reducing fracture risk (Neer et al., 2001). The newer PTHrP(1e34) analog, called abaloparatide, formerly called BA058, which was approved by the FDA in 2016 as an alternative anabolic treatment for osteoporosis, is thus of interest as data from preclinical and human clinical studies suggest that it increases bone mass at least as effectively as PTH(1e34) but causes less bone resorption and hence hypercalcemia (Miller et al., 2016). Abaloparatide is administered by subcutaneous injection at a dose of 80 mg per day. As both peptide drugs act by binding to the same target PTHR1, the differences in clinical outcomes suggested the possibility that their modes of action on the receptor might not be equivalent. Abaloparatide was found to bind with relatively high selectively to the RG PTHR1 conformation and thus induce more transient cAMP signaling responses in PTHR1-expressing HEK293 cells than PTH(1e34) (Hattersley et al., 2016) (Fig. 28.4AeC). These studies thus revealed a conformation-based mode of temporal bias for abaloparatide that was distinct from that of PTH(1e34), and hence provided at least a plausible mechanism to help explain the distinct effects that the two ligands have on bone metabolism, as seen in the clinical and preclinical testing (Makino et al., 2018).

PTHR1 mutations in disease The full spectrum of diseases associated with PTHR1 mutations and their biological and clinical aspects are discussed in Chapter 58. The following sections focus on how the disease-associated mutations relate to the structure and functional properties of the receptor, particularly as they are assessed in cell and mouse model systems.

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Jansen’s metaphyseal chondrodysplasia Jansen’s metaphyseal chondrodysplasia (JMC) is a rare disease associated with skeletal abnormalities, dwarfism, and hypercalcemia, and is caused by heterozygous dominant activating mutations in the PTHR1 (Ohishi et al., 2012; Schipani et al., 1995). Five different PTHR1-activating mutations have been identified in patients with JMC. These mutations occur at the cytoplasmic termini of TM2 (Arg233 / His), TM6 (Thr410 / Pro/Arg), and TM7 (Ile458 / Arg/Lys) (Fig. 28.1A), and each results in agonist-independent cAMP signaling. Strikingly, the mutations each occur at or, in the case of the Ile458 mutations, adjacent to a residue that is involved in the conserved polar network that is present in all class B GPCRs and operates to control receptor activation and deactivation processes and particularly the outward movements of the TMD helices that allow G protein coupling (Yin et al., 2017). Several PTH and PTHrP antagonist ligand analogs, such as [Leu11, D-Trp12]PTHrP(7e34), behave as inverse agonists on the mutant receptors and thus depress their basal signaling activities (Carter et al., 2001, 2015). The mechanism by which these ligands achieve their inverse effect on the receptor’s conformational status is unknown, although the Gly12 / D-Trp substitution is required for the effect. In any case, the functional properties of the analogs suggest possible paths toward therapy for JMC, which potentially can be tested in the transgenic models of JMC that have been developed to express the PTHR1-H223R allele in osteoblasts or osteocytes, which leads to marked increases in bone mass (O’Brien et al., 2008; Calvi et al., 2001).

Other diseases linked to PTHR1 mutations Enchondromatosis (Ollier disease/Maffucci syndrome) is a rare disease characterized by cartilage tumors of the bone, and has been associated with four PTHR1 mutations, Gly121 / Glu, Ala122 / Thr, Arg150 / Cys, and Arg255 / His, each located in the ECD or ECL1 portion of the receptor. One study on the Arg150 / His mutant expressed in COS-7 cells intriguingly suggested a moderately elevated rate of basal cAMP signaling when corrected for a reduced cell-surface expression level (Hopyan et al., 2002), while another study suggested that each of the four mutations causes a loss-offunction phenotype due to effects on expression and/or binding affinity (Couvineau et al., 2008). The mechanism by which these mutations result in cartilage tumors in bone thus remains unclear, but certainly effects on the extracellular scaffold structure seem possible. Blomstrand’s chondrodysplasia is a neonatal lethal condition of markedly accelerated bone mineralization that is caused by homozygous loss-of-function mutations in the PTHR1. One coding mutation has been identified, Pro132 / Leu, which affects a conserved site in the core ECD scaffold (Zhang et al., 1998; Karaplis et al., 1998). Eiken syndrome is a very rare skeletal dysplasia that has been associated with a homozygous recessive nonsense mutation in the PTHR1, Arg485 / Stop, that leads to a shortened C-terminal tail (Duchatelet et al., 2005). The phenotype is markedly delayed ossification of the skeleton, which is opposite that of Blomstrand’s disease and seems consistent with a gain-of-function effect, albeit a mild one, since the heterozygous mutation is not associated with disease and the homozygous phenotype is distinct from that of JMC. In any case, it seems possible that the mutation leads to altered interactions with cytoplasmic effectors or scaffolding proteins, and hence diversions in subcellular trafficking and signaling, but this remains to be determined. A number of heterozygous loss-of-function mutations have been identified in individuals with defects in tooth eruption (Roth et al., 2014), which is consistent with studies in mice that show that PTHR1 signaling, as induced by PTHrP, is required for proper tooth formation (Ono et al., 2016). Among the mutations found in patients with tooth eruption defects is the Pro132 / Leu also found in Blomstrand’s disease, but in that disease the mutation is in a compound-heterozygous arrangement.

Nonpeptide mimetic ligands for the PTHR1 The capacity for PTH agonists to effectively treat osteoporosis and hypoparathyroidism raises interest in the goal of developing orally available nonpeptide mimetics for this receptor. So far two such small-molecule PTHR1 agonists have been reported. The first, AH3960, was tested only in cells and shown to stimulate cAMP formation at doses of about 10 mM or higher (Rickard et al., 2006). More recently, PCO371 was reported and shown to stimulate cAMP formation in cells at concentrations in the low-micromolar range and, moreover, to effectively raise blood calcium levels in TPTX rats (Tamura et al., 2016). Due to an unexpectedly prolonged calcium response, possibly attributable to a prolonged pharmacokinetic profile, PCO371 is under development for hypoparathyroidism, rather than osteoporosis, as treatment of the latter disease requires a transient pharmacodynamic response to avoid excess bone catabolism, whereas treatment of the former disease requires a more sustained pharmacodynamic profile to mimic the effects of the missing hormone. Several nonpeptide antagonist compounds have also been identified for the PTHR1. SW106, discovered by screening a compound library for agents that could inhibit the binding of a radiolabeled PTH(1e14) peptide analog, binds to the PTHR1 with

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FIGURE 28.5 Small-molecule agonist for the PTHR1. The compound PCO371 was developed from a lead compound identified in a high-throughput screen for parathyroid hormone receptor 1 (PTHR1) agonist ligands. In COS-7 cells expressing the human PTHR1, PCO371 is a full agonist for cAMP signaling, albeit its potency is about 3 log-orders weaker than that of PTH(1e34). In TPTX rats, PCO371 induces elevations in serum calcium that are sustained, relative to the effects of PTH(1e34), presumably because of a prolonged pharmacokinetic profile. Consequently, PCO371 is reported to be in development for hypoparathyroidism. Reproduced pending permission from Tamura, T., Noda, H., Joyashiki, E., Hoshino, M., Watanabe, T., Kinosaki, M., Nishimura, Y., Esaki, T., Ogawa, K., Miyake, T., Arai, S., Shimizu, M., Kitamura, H., Sato, H. & Kawabe, Y. 2016. Identification of an orally active small-molecule PTHR1 agonist for the treatment of hypoparathyroidism. Nat. Commun. 7, 13384.

micromolar affinity and behaves as a competitive antagonist (Carter et al., 2007, 2015). A broad set of antagonist compounds was reported by a different group, and the most effective of these antagonized the cAMP-stimulating actions of PTH(1e34) in HEK293 cells transfected with the human PTHR1, with inhibitory constants in the 10 nM range, although studies in vivo were not reported (McDonald et al., 2007). Mechanistic studies performed on SW106 and AH3960 (Carter et al., 2015), as well as on PCO371 (Tamura et al., 2016), show that each of these compounds interacts with the TMD region of the receptor, as they exhibit the same effectiveness on the PTHR1 construct that lacks the ECD as they do on the intact PTHR1. Moreover, PCO371 was found to be inactive on the human PTHR2, specifically due to a single divergent residue corresponding to proline-415 in the PTHR1, which is replaced by leucine in the PTHR2. Pro415 is otherwise highly conserved in the class B GPCRs and is located in the middle of TM6, where it is predicted to play a pivotal role in receptor activation (Zhang et al., 2017). Whether PCO371 directly binds to Pro415 or to a site within the extracellularly exposed orthosteric pocket used by the peptide ligand remains to be determined. In any event, it is now clear that smallmolecule ligands, both agonists and antagonists, can be developed for the PTHR1, and ultimately might lead to more effective therapies for PTHR1-related diseases (Fig. 28.5).

Other receptors for parathyroid hormone and related ligands PTHR2 and PTHR3 subtypes There continues to be appropriate interest in other receptors for the PTH family of ligands. Two apparent receptor subtypes distinct from the PTHR1 have been identified, as discussed in the earlier section on the evolution of this receptor/ligand family. The PTHR3 is present in vertebrate evolution as late as the avian radiation, but its function is not yet clarified or studied extensively, partly because it has been lost from the genome of higher mammals, including humans. Some cellbased tests suggest that it can function in a fashion similar to that of the PTHR1, at least in terms of ligand recognition and response properties (Rubin and Juppner, 1999; Pinheiro et al., 2012). The apparent absence of the PTHR3 in the higher vertebrates leaves open the question of biological importance. On the other hand, the PTHR2 is present in humans and other mammals, and has been characterized sufficiently to suggest a distinct ligand and biological role profile (Dimitrov et al., 2013), although it has been apparently lost in the avian lineage (Pinheiro et al., 2012). The PTHR2 was initially identified through hybridization-based screening of a human brain cDNA library for PTHR1related sequences. The identified receptor was thus found to share 51% amino acid identity with the human PTHR1 (Usdin et al., 1995), but to respond weakly to PTH and not at all to PTHrP, while the rat PTHR2 responded to neither (Hoare et al., 1999a). The search for a cognate ligand in bovine hypothalamic extracts yielded the new bioactive peptide called TIP39 (Usdin et al., 1999). TIP39 binds only weakly to the PTHR1 and lacks agonist activity. Nevertheless, TIP39 exhibits some structural homology with PTH and PTHrP (Piserchio et al., 2000), and N-terminally truncated analogs such as TIP9(7e39) bind with improved affinity such that they act as PTHR1 inhibitors (Hoare and Usdin, 2000; Jonsson et al., 2001). Continuing studies with the PTHR2 and TIP39 system since the initial discovery have emphasized its principal role in the central nervous system, with evidence suggesting that it functions to modify behavior, such as combating excessive fear

706 PART | I Basic principles

reactions, modifying pain sensations, and playing a role in positive maternal behavior, as studied in suckling rodents, perhaps by stimulating oxytocin release (Cservenak et al., 2017; Usdin et al., 2003). Distinctive other functions include a critical role in spermatid production and fertility through receptors in testicular tissue (Usdin et al., 2008). Other studies indicate the presence of TIP39 and the PTHR2 in skin with a role for keratinocyte differentiation and regulation of extracellular matrix formation and wound repair (Sato et al., 2016). There is no/little evidence to suggest a role in calcium or bone metabolism.

Possible receptors for C-terminal PTH and PTHrP Other receptors with actions on bone and calcium have been postulated to interact with the carboxyl regions of PTH and PTHrP that lie beyond the amino-terminal (1e34) segment. Indirect evidence for such a possibility is suggested by the moderate degree of sequence homology maintained in the middle and carboxy-terminal regions of ligands from human and related mammalian species and even in chicken for the PTHrP molecule and also, but to a slightly lesser, extent for PTH. Added to this is the awareness that the overall peptide length of these two molecules is considerably greater than that noted for the ligands that activate other members of the class B receptor class, raising the possibility that the C-terminal extensions of PTH and PTHrP were acquired or preserved biologically during evolution. Experimental support for a C-terminal receptor for PTH comes from the capacity of the PTH(39e84) fragment to bind and cross-link to a 90-kDa protein on the surface of ROS17-2.8 cells (Inomata et al., 1995; Takasu et al., 1996), and that the fragment also induced apoptosis in a mouse osteocytic cell line ablated for the PTHR1 (Divieti et al., 2001). For PTHrP, there is evidence from in vivo studies that the mid- or C-terminal region contributes to effects on placental calcium transport as well as tissue development, the latter involving a nuclear localization mechanism (Kovacs et al., 1996; Wu et al., 1996; Gu et al., 2012; Toribio et al., 2010; Lam et al., 1999). On the other hand, genetic data support the view that the actions of PTH and PTHrP on bone are largely attributable to actions through the PTHR1. Thus, homozygous ablation of the PTHR1 in mice gives rise to a neonatal lethal skeletal dysplasia similar to that seen with homozygous ablation of PTHrP (Lanske et al., 1996). In addition, in the very rare human disorder Blomstrand’s chondrodysplasia with homozygous loss-of-function PTHR1 alleles there is a similar lethal skeletal chondrodysplasia. Heterozygous loss-of-function mutations of PTHrP in humans give rise to brachydactyly type E (Bae et al., 2018), and such mutations of the PTHR1 give rise to failures in tooth eruption (Ono et al., 2016; Roth et al., 2014), both of which are consistent with a defect in bone development as controlled by the PTHR1. For PTH, conditions of hypoparathyroidism can be sufficiently corrected with PTH(1e34) administration (Winer et al., 2012), while the related condition of pseudohypoparathyroidism results from deficiencies in Gas (Juppner, 2015), the primary mediator of PTHR1 signaling. In any event, there remains considerable interest in the possibility of such C-terminal receptors, as potential roles in calcium and bone biology have been supported by biological data in studies extending back several decades. However, despite much effort, such putative receptors have not been cloned and therefore the molecular identities not characterized.

Conclusions Mechanisms of ligand binding and signal transduction at the PTHR1 are becoming increasingly better understood, with the high-resolution X-ray crystal and cryo-EM structures obtained for several related class B GPCRs providing particularly valuable new information. Ligand binding is thus seen to occur via the basic two-site model initially revealed by mutagenesis and cross-linking studies, but to further involve significant conformational rearrangements and molecular movements that ultimately lead to G protein coupling and activation. Structurally distinct PTH and PTHrP ligand analogs can induce or stabilize different PTHR1 conformations to thereby mediate different modes of signaling, with some ligands inducing particularly prolonged cAMP signaling responses, probably from within endosomes, while others induce more transient responses, presumably from the cell surface. Such conformation-based temporal bias for the PTHR1 suggests new approaches for the development of therapies for diseases such as hypoparathyroidism and osteoporosis. Insights into key sites in the receptor involved in function are also provided by PTHR1 mutations identified in certain skeletal disorders, including loss-of-function mutations in cases of failed tooth eruption and gain-of-function mutations in JMC. Mutations of the latter disease reveal the importance of a conserved polar network located at the base of the TMD helical bundle in controlling receptor activation and deactivation. As the views provided by such insights continue to deepen and refine, so should the capacity to design new ligand analogs for the PTHR1 that offer improved efficacy as treatments for diseases of bone and mineral metabolism.

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