Endosomal parathyroid hormone receptor signaling

Abstract

The canonical model for G protein-coupled receptors (GPCRs) activation assumes that stimulation of heterotrimeric G protein signaling upon ligand binding occurs solely at the cell surface and that duration of the stimulation is transient to prevent overstimulation. In this model, GPCR signaling is turned-off by receptor phosphorylation via GPCR kinases (GRKs) and subsequent recruitment of β-arrestins, resulting in receptor internalization into endosomes. Internalized receptors can then recycle back to the cell surface or be trafficked to lysosomes for degradation. However, over the last decade, this model has been extended by discovering that some internalized GPCRs continue to signal via G proteins from endosomes. This is the case for the parathyroid hormone (PTH) type 1 receptor (PTHR), which engages on sustained cAMP signaling from endosomes upon PTH stimulation. Accumulative evidence shows that the location of signaling has an impact on the physiological effects of GPCR signaling. This mini-review discusses recent insights into the mechanisms of PTHR endosomal signaling and its physiological impact.

INTRODUCTION

The PTH type 1 receptor (PTHR) is a class B G protein-coupled receptor (GPCR) and a key regulator of mineral-ion, vitamin D, and bone homeostasis in the body through the action of its two natural ligands, parathyroid hormone (PTH) and PTH-related protein (PTHrP). PTH is synthesized and secreted from the parathyroid glands as an 84 amino acid peptide; however, most studies use the biologically active N-terminal of PTH, referred to as PTH_1-34. In the kidney, PTH-mediated PTHR signaling regulates Mg²⁺, Ca²⁺, phosphate, and bicarbonate homeostasis (1). In addition, PTH plays a key role in the production of active vitamin D in the kidney by regulating the expression of 1α-hydroxylase via cAMP-responsive element (CRE) present in the 1α-hydroxylase gene promoter (2). 1α-Hydroxylase is the rate-limiting enzyme that catalyzes the hydroxylation of 25-dihydroxyvitamin D into its active form and 1,25-dihydroxyvitamin D3 in the kidney. In the bone, PTH mediates both anabolic and catabolic processes by acting on osteoblasts and osteocytes (3). PTHrP is synthesized and expressed by several different tissues, including mammary glands, skin, blood vessels, smooth muscles, bone, and kidneys, where it acts in an autocrine or paracrine fashion. PTHrP plays a fundamental role in endochondral bone development (4). Produced as a 141 amino acid protein, PTHrP can be functional at different peptide lengths, with most studies using the N-terminal portion, PTHrP_1-36. Although both ligands stimulate the same G protein subtypes and downstream signaling cascades, they exhibit different duration and location of cAMP production. Although PTH_1-34 stimulates sustained cAMP signaling from endosomes, PTHrP_1-36 can only induce transient signaling from the cell surface (5). These differences in both signaling duration and location may explain, at least in part, the different physiological roles these two ligands have.

PTHR is a clinical target for the treatment of osteoporosis with two Food and Drug Administration-approved anabolic drugs: teriparatide (PTH_1-34) and abaloparatide (ABL, a PTHrP_1-34 variant), which stimulate bone formation when injected daily subcutaneously. ABL administration, like PTHrP, results in reduced hypercalcemic effect when compared with PTH_1-34. Because both PTH_1-34 and ABL have comparable bone anabolic responses, it is possible that transient signaling from the plasma membrane may promote bone formation without causing hypercalcemia and that sustained cAMP generation from endosomes may promote bone breakdown. In agreement with this view, a PTH/PTHrP chimera, that acts as a long-acting PTH (LA-PTH) and stimulates longer cAMP responses than PTH_1-34, induces prolonged hypercalcemia in mice and monkeys (6, 7). Furthermore, a single-point mutation in PTH (R25C) found in patients with idiopathic hypoparathyroidism who present with chronic hypocalcemia, results in impaired endosomal cAMP responses and Ca²⁺ homeostasis (8). This evidence suggests that PTHR endosomal cAMP signaling has physiological relevance. This mini-review explores the most recent advances in the field of regulation and physiological impact of PTHR endosomal signaling.

HISTORICAL OVERVIEW OF PTHR ENDOSOMAL SIGNALING

Endosomal signaling of GPCRs is a relatively new concept that was first coined for the PTHR in 2009 (5) and has prompted revisiting the canonical GPCR signaling model, which assumes that ligand-receptor complex signals via G proteins exclusively from the cell surface (Fig. 1). At around the same time as the PTHR findings were published, sustained endosomal cAMP production was described for thyroid-stimulating hormone (TSH) receptor (TSHR), a stimulatory G protein (Gs)-coupled receptor regulating thyroid hormone production (10). Since then, several other GPCRs have been shown to signal from endosomes upon internalization, including other class B GPCRs, such as glucagon-like peptide 1 receptor (GLP1R), calcitonin-like receptor (CLR; 11), and pituitary adenylate cyclase-activating polypeptide type 1 receptor (PAC1R; 12, 13); and class A GPCRs, including, dopamine receptor D₁ (D1R; 14), vasopressin receptor 2 (V₂R; 15), β₂-adrenergic receptor (β2AR; 16), luteinizing hormone receptor (LHR; 17), and µ-opioid receptor (MOR; 18), among others.

Figure 1.

Modes of GPCR signaling via cAMP. In the canonical model, transient cAMP production occurs at the plasma membrane after Gs activation by ligand-bound receptor (1st pool; step 1). Receptor desensitization is initiated by the recruitment of GRKs, which phosphorylate the receptor (step 2), followed by β-arrestin recruitment (step 3), and internalization of the receptor into endosomes (step 4). In the updated model, peptide ligands such as PTH, remain bound to the receptor, inducing sustained cAMP production from endosomes (2nd pool; step 4). Acidification of endosomal compartments induces the dissociation of the ligand from the receptor (step 5), followed by receptor dephosphorylation (step 6). The receptor can then be degraded in the lysosome (step 7), trafficked to the Golgi apparatus (step 8), and/or recycled back to the plasma membrane (step 9) to initiate another round of signaling. Adapted from reference Sutkeviciute and Vilardaga (9). Created with BioRender.com. GPCR, G protein-coupled receptors; GRKs, GPCR kinases; Gs, stimulatory G protein; PTH, parathyroid hormone.

The development of optical and microscopy techniques to track GPCR signaling (19, 20) was of paramount importance to uncover the sustained signaling characteristics of PTHR. A Förster resonance energy transfer (FRET)-based sensor, containing Epac-cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP), that can be transfected into cells to measure cAMP production over time (5, 21), allowed for the discovery that PTH_1-34 causes a prolonged production of cAMP, even after a brief exposure to the ligand in HEK293 cells expressing PTHR, whereas cAMP production in response to PTHrP_1-36 is brief and limited to the cell surface (5). Further studies using confocal microscopy revealed the presence of other components of the signaling complex in Rab5-positive endosomes after PTH_1-34 stimulation (5, 22–25), including Gs subunits (α, β, γ), β-arrestins, and adenylyl cyclases (AC).

Despite both PTH and PTHrP exhibiting similar initial binding rates, PTH_1-34 dissociates very slowly from PTHR after ligand washout, whereas PTHrP_1-36 fully dissociates after a few seconds, further corroborated by experiments using a FRET sensor to investigate changes in receptor conformation and activation that showed that PTHrP_1-36 induces a transient PTHR activation, whereas receptor activation is sustained with PTH_1-34 (5). Thus, each ligand induces distinct receptor conformation upon binding: a G protein-independent high-affinity receptor conformation (R₀) that is preferentially stabilized by PTH and LA-PTH, and a G-protein dependent high-affinity receptor conformation (R_G) stabilized by PTH, LA-PTH, PTHrP, and ABL. The R₀ conformation favors extended Gs coupling and activation, which has been demonstrated by FRET and competition binding assays (5, 26) and is associated with cAMP production both from the plasma membrane and from internalized complexes in endosomes. On the other hand, ligands with a lower affinity for the R₀ conformation give raise to transient G protein activation and cAMP productions exclusively from the plasma membrane (27).

According to the canonical model for GPCR signaling, β-arrestins recruitment is key for receptor desensitization, by preventing further G-protein coupling and initiating receptor internalization. Internalized receptors are subsequently trafficked to the lysosome for degradation (Fig. 1). β-Arrestins recruitment to PTHR in response to PTH_1-34, however, does not terminate cAMP production, instead, PTHR-β-arrestin complexes are internalized into early endosome in a dynamin-dependent manner (5). Furthermore, PTHR does not colocalize with late endosome markers (5), suggesting that lysosomal degradation is an unlikely mechanism to terminate PTHR signaling. An alternative mechanism for PTHR signaling termination involving the retromer complex was described by Feinstein et al. (24). The retromer complex consists of two endosomal-bound sorting nexins, SNX1 and SNX2, and a ternary complex formed by soluble vacuolar protein sorting (Vps) Vps26, Vps29, and Vps35. The retromer is also part of the actin-SNX27-retromer-tubule (ASRT) complex, which is involved in the sorting of proteins from endosomes to the plasma membrane (28). Several minutes after stimulation, PTHR is trafficked from endosomes to the Golgi by the retromer, which terminates PTH-induced sustained cAMP signaling (24). Additional studies have also implicated SNX27, another ASRT component, to participate in PTHR desensitization by serving as a scaffold between PTHR and retromer (29–31). PTHR interacts with SNX27 via its C-terminus PDZ ligand and both proteins colocalize in endosomes after PTH_1-34 stimulation (29, 30). In conjunction with the retromer, SNX27 terminates PTHR signaling by mediating the recycling of PTHR from endosomes to the plasma membrane and not necessary to the Golgi (29). However, it is possible that recycling of PTHR to the plasma membrane occurs via the trans-Golgi network (TGN) and not directly from endosomes.

Endosomal acidification is another part of the desensitization mechanism by which sustained PTHR signaling is turned off (25). PTH-induced protein kinase A (PKA) activation results in v-ATPase phosphorylation, which in turn induces endosomal acidification, promoting the dissociation of PTH_1-34 from PTHR in the endosome and the dissociation of the PTHR-β-arrestin complexes, while inducing the assembly of inactive PTHR with the retromer (25), thus trafficking PTHR from the endosome to the Golgi and back to the plasma membrane. Although these studies shed some light on the mechanisms that terminate PTHR signaling, the exact components involved are not completely known. Understanding how PTHR is terminated can be of great importance for finding treatment options for osteoporosis. Since sustained signaling by PTH_1-34 can cause hypercalcemia, modulating signaling termination could eliminate this side effect.

REGULATION OF ENDOSOMAL SIGNALING BY Gq/11

Upon agonist stimulation, PTHR signals through both Gs and G protein q/11 (Gq/11), activating cAMP/PKA and PLC/Ca²⁺ signaling pathways, respectively. Although Gs, in conjunction with a stable PTHR-G protein, beta and gamma subunits (Gβγ)-arrestin complex in the endosome, is fundamental for sustained cAMP signaling (22), the role of Gq/11 in PTHR endosomal signaling has remained elusive. The recent generation of Gs-biased ligands, however, has allowed for a better understanding of Gq/11 role in endosomal signaling. These biased ligands have been generated by inducing PTH backbone modifications consisting of a single α→β amino acid substitution (32) that result in Gs-biased PTH analogs that stimulate cAMP production from the plasma membrane, but are unable to generate sustained endosomal cAMP due to impaired β-arrestins recruitment (33) and are deficient for Gq/11 signaling, suggesting that Gq/11 may be required for PTH-induced sustained endosomal signaling.

Accordingly, selective inhibition of Gq/11 activation decreases sustained cAMP production in response to PTH_1-34 as evidenced by FRET-based assays in HEK293 cells as well as in kidney and bone-derived cells; likewise, cells lacking endogenous Gαq/11 also exhibit decreased endosomal cAMP signaling in response to PTH_1-34 (23), suggesting that active Gq/11 is required for sustained cAMP generation. Gq/11 is also required for β-arrestin recruitment to the receptor, as FRET and bioluminescence resonance energy transfer (BRET) assays showed that the PTHR-β-arrestin interaction was impaired in Gq/11 knockout cells, in line with the initial hypothesis derived from the Gs-biased studies (33) that Gq/11 activation is required for β-arrestin recruitment. Interestingly, the Gq/11-derived Gβγ subunits, and not G protein, alpha subunit (Gα), are responsible for the modulation of PTHR-arresting interactions (23). It is well established that Gβγ subunits activate GRK2/3, which in turn induces β-arrestin recruitments to GPCR (34), so a defect in GPCR kinase (GRK)-mediated phosphorylation could potentially cause the effects observed in Gq/11 knockout cells. However, phosphoproteomic studies on PTHR revealed that PTHR phosphorylation status in Gq/11 knockout cells is not different from wild-type cells, indicating that β-arrestins recruitment impairment is not due to changes in PTHR phosphorylation status (23). The Gβγ subunits are known to activate phosphoinositide-3 kinase (PI3K), particularly PI3Kβ and PI3Kγ (35). Selective inhibition of PI3Kβ by TGX-221 results in reduced β-arrestin recruitment to the PTHR in wild type but not in Gq/11 knockout cells, suggesting that PI3Kβ activation by Gq/11-derived Gβγ is required for β-arrestins recruitment to PTHR. These findings were further confirmed by acute phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P₃, hereafter PIP3] depletion from the plasma membrane, which impairs β-arrestin recruitment to PTHR in response to PTH_1-34 (23). Thus, the Gq/11 effects on β-arrestins recruitment and endosomal cAMP production are mediated by PI3Kβ activation and subsequent PIP3 generation (Fig. 2).

Figure 2.

Regulation of PTHR endosomal signaling by Gq/11. PTH binding to PTHR activates heterotrimeric Gs (not shown) and Gq/11 proteins. Gs activation results in short cAMP production by surface adenylyl cyclase (AC; Fig. 1), whereas Gq/11 activation activates PLCβ, resulting in IP₃ production and subsequent Ca²⁺ release from the endoplasmic reticulum (step 1). Gq/11-derived βγ subunits trigger the generation of PIP3 via PI3Kβ activation (step 2), which in turn enables β-arrestin recruitment to PTHR (step 3), and the formation of PTHR-β-arrestin-Gβγ signaling complex, which remains active after internalization into endosomes (step 4), inducing sustained endosomal cAMP generation (step 5). Adapted from research and model published by White et al. (23). Created with BioRender.com. Gq/11, G protein q/11; Gs, stimulatory G protein; PIP3, Phosphatidylinositol (3,4,5)-trisphosphate; PI3K, phosphoinositide-3 kinase; PTH, parathyroid hormone; PTHR, PTH type 1 receptor.

Several questions about the role of PIP3 in β-arrestins recruitment remain unanswered, and new research is required to elucidate exactly how PIP3 mediates this process. However, these studies not only describe a novel role for Gq/11 in endosomal signaling but also provide evidence for the involvement of plasma membrane phospholipids in the regulation of endosomal PTHR signaling, which provides further insights into a rather not well-understood aspect of GPCR biology.

ALLOSTERIC MODULATION OF ENDOSOMAL PTHR SIGNALING

Numerous GPCRs can be allosterically modulated by ions, such as Na⁺ Mg²⁺, Ca²⁺, and Mn²⁺ (36–39). Studies in HEK293 cells and primary osteoblasts show that extracellular Ca²⁺ acts as a positive allosteric modulator of PTHR by increasing the time that PTH_1-34 or ABL are bound to the active conformation of the receptor, resulting in extended receptor activation and increased endosomal cAMP signaling (40). The previously mentioned disease-causing variant of PTH, PTH^R25C (8), is insensitive to extracellular Ca²⁺ and exhibits impaired endosomal cAMP production by PTHR. Thus, further supporting the role of Ca²⁺ as an allosteric modulator required for sustained signaling elicited by PTH_1-34. The extracellular loop 1 (ECL1) of PTHR contains clusters of negatively charged residues that form ionic interactions with R25 residue of PTH, which are required for Ca²⁺ positive allosteric modulation (41). Ca²⁺ likely acts as a stabilizer of PTH interaction with the receptor, thus prompting endosomal signaling (40).

Allosteric modulation by Ca²⁺ might be particularly relevant for PTHR biology. Due to Ca²⁺ release from bone, Ca²⁺ concentration can be as high as 40 mM in the bone microenvironment (42), therefore being readily available to modulate PTH action. The Ca²⁺ allostery studies in cultured cells show that increasing Ca²⁺ concentrations (0.1–10 mM) reduce the amount of ligand needed to bind and activate PTHR, thus it is likely that in the bone microenvironment the catabolic effects of PTH on Ca²⁺ rebalancing could be carried out with picomolar levels of circulating PTH. However, it is important to consider that under certain pathologies, such as hyperparathyroidism, or with excess PTH_1-34 administration, the catabolic actions of PTH could be enhanced, resulting in undesirable hypercalcemia.

PTHR can also be allosterically modulated by small organic molecules, such as ATP and other nucleotides. Extracellular ATP and cytidine monophosphate (CMP) increase the effectiveness of PTH to stimulate the production of cAMP and β-arrestins recruitment, as well as the PTH-driven activation of cAMP-responsive element-binding (CREB) protein (43). In addition, the nucleotide-induced potentiation of PTH is independent of purine receptor P2X7, and it appears that hydrolysis of a high-energy phosphoanhydride bond in ATP is not required for this effect (43). Although the exact mechanism by which extracellular nucleotides can allosterically regulate PTHR is yet to be elucidated, evidence shows that the presence of sugar phosphate is sufficient to potentiate PTH. In particular, ribose-5-phosphate (R5P), which is found in both ATP and CMP, acts as a positive allosteric modulator of PTHR by enhancing cAMP production (44). These findings are also of physiological relevance to PTHR function, as differentiated osteoblasts can release ATP in response to PTH (45), possibly triggering an autocrine mechanism in which PTH-induced ATP can act to modulate PTH-bound PTHR, resulting in a positive feedback loop of PTHR signaling in bone cells.

Allosteric modulation of PTHR is not limited to ions or small molecules, but also extends to proteins. PTHR interacts with receptor-activity modifying proteins (RAMPs), single transmembrane (TM) helix proteins that associate and regulate GPCR function (46–49). Using a series of optical approaches, a new study proposes that RAMP2 interacts specifically with PTHR and acts as an allosteric modulator by inducing a preactivation state of PTHR that allows for faster receptor activation upon ligand binding (50). RAMP2-PTHR interaction can modulate G protein signaling and β-arrestin recruitment. Since some of these effects are ligand specific, with higher effects observed for PTH_1-34 versus PTHrP_1-34 (50), perhaps RAMP2 modulates PTH-induced endosomal signaling of PTHR. Moreover, the physiological relevance of the PTHR-RAMP2 complex has been previously evidenced in RAMP2 knockout mice, resulting in impaired PTHR regulation as well as placental dysfunction (51). Further investigations are needed to have a better understanding of PTHR modulation by RAMPs in native conditions.

Using a series of signaling and structural approaches, including nuclear magnetic resonance (NMR) and molecular dynamics (MD) simulations based on the 3.0 Å cryogenic electron microscopy (cryo-EM) structure of PTHR (52), a recent study unveils allosteric interactions between PTH and PTHR that favor β-arrestins recruitment to the receptor (53). PTH, like all class B GPCRs peptide ligands, has a structured α-helical C-terminal part that is critical for binding to the large N-terminal extracellular domain (ECD) of the receptor, and a more flexible N-terminal part that inserts into the receptor’s transmembrane (TM) domain (TMD), which is critical for engaging signaling via G proteins and β-arrestins. The binding of the C-terminal of PTH to receptor’s ECD induces a conformational change in the N-terminal of PTH before binding to PTHR, followed by PTH His9 insertion into PTHR TMD, which allosterically engages receptor coupling to β-arrestins via residues in intracellular loop 3 (ICL3) of PTHR (53). Further, PTH His9 is required for β-arrestin recruitment to PTHR and endosomal phase of cAMP signaling (53). In addition, anisotropic network model (ANM) analysis revealed that His9 is part of “hot-spot” of residues in PTH_1-34 involved signal selectivity (Fig. 3A).

Figure 3.

Location bias in PTHR signaling via cAMP. A: anisotropic network model (ANM) analysis for PTH-bound receptor allowed the identification of a signal selectivity epitope in PTH. Plot of cross-correlation between PTH residues and PTHR Thr392 in one ANM mode, colored by cross-correlation value, from red (cross-correlation = +1) to blue (cross-correlation = −1). PTH residues 1–7, 9, and 10 have a high (>0.6, dashed line) cross-correlations value and correspond to the signal selectivity epitope. Originally published by Clark et al. (53) and adapted from White et al. (54). B: proposed model for PTHR location biased signaling, based on the use of biased ligands LA-PTH (endosomal signaling) and PTH^7d (surface signaling). Basolateral stimulation of PTHR in proximal tubule kidney cells induces sustained cAMP production from endosomes and promotes the increase of serum vitamin D. Endosome-generated cAMP diffuses into the nucleus (green arrow), resulting in the activation of cAMP response element-binding (CREB) protein and inducing the expression of 1-α(OH)ase, the rate-limiting enzyme of active vitamin D (1,25(OH)₂D₃) synthesis. Plasma membrane cAMP pool generated by PTHR activation might contribute to the inhibition of phosphate import. Created with BioRender.com. LA-PTH, long-acting PTH; PTH, parathyroid hormone; PTHR, PTH type 1 receptor.

PHYSIOLOGICAL RELEVANCE OF ENDOSOMAL PTHR SIGNALING

As mentioned earlier, PTH_1-34 contains a signal selectivity epitope, composed of residues 4–13 that were identified by ANM analysis (53). Out of these, residues 5, 6, 7, 9, and 10 present a high cross-correlation value with T392 of PTHR ICL3, indicating that they might be relevant for β-arrestin recruitment to the receptor (Fig. 3A). In a new study, l to d amino acid epimerization of these residues in PTH_1-34 was used to generate biased PTH analogs to further explore the role of location bias in PTHR signaling (54). Epimerization of Leu7 in PTH_1-34 (PTH^7d) results in a biased ligand that triggers sustained cAMP production exclusively from the plasma membrane and fails to recruit β-arrestins, by stabilizing an active PTHR conformation that differs from the one induced by wild-type PTH_1-34. PTH^7d shows similar cAMP production over time than LA-PTH, which engages PTHR in endosomal signaling, thus allowing the authors to explore the hypothesis that similar levels and durations of cAMP generated by the same receptor in distinct cell locations (plasma membrane and endosomes) mediate different physiological responses (54). This was verified in mice by showing that the endosomal cAMP selective ligand, LA-PTH, induced significantly more bone formation and production of the active vitamin D than PTH_7d. Further experiments in polarized MDCK cells demonstrated the failure to induce the 1-α hydroxylase in response to PTH^7d. These studies suggest that synthesis of active vitamin D in the kidney is dependent on the endosomal of PTHR signaling via cAMP. On the other hand, plasma membrane PTHR signaling may contribute to the regulation of phosphate transport in proximal tubule cells (54; Fig. 3B).

Spatial bias in GPCR signaling is an emerging topic that is altering the classic paradigms of GPCR signaling. New studies suggest that the location of cAMP generation has a differential impact in global phosphoproteomics (55), therefore, resulting in distinct physiological responses, like what has been described for PTHR location biased.

DISCUSSION

Research performed within the last decade has placed the PTHR as a prototypic GPCR for the study of endosomal signaling. Since the initial discovery that PTHR signals from endosomes after ligand-induced receptor internalization, several other GPCRs have been shown to engage in endosomal signaling, leading to a paradigm shift in the GPCR field. It is now widely accepted that endosomal cAMP production is an essential component of GPCR signaling. In the case of the PTHR, endosomal signaling is highly regulated by several mechanisms discussed in this mini-review. PTHR endosomal cAMP generation is regulated by Gq/11-dependent PI3Kβ activation (Fig. 2), in addition to several allosteric modulators, such as Ca²⁺, ATP, RAMP2. Further, PTH can also allosterically modulate PTHR endosomal signaling. Emerging evidence shows that the location of cAMP signaling, rather than duration, impacts the biological functions of PTHR. In fact, endosomal, but not plasma membrane, cAMP generation appears to be determinant for PTHR-mediated vitamin D synthesis (Fig. 3). Moreover, bacteria-derived photoactivable adenylyl cyclases (b-PAC) targeted to different organelles have been used to show differences in protein phosphorylation in response to cAMP generated from the plasma membrane and endosomes (55), further highlighting the physiological impact of GPCR location bias. Additional studies are necessary to advance the understanding of the overall consequences of endosomal signaling in PTHR biology. Location bias in GPCR signaling is also relevant to drug design. For instance, small molecules targeted to allosterically modulate the endosomal signaling of PTHR have been proposed as possible drug candidates to treat affections related to hyperactive PTHR (56). Therefore, it is crucial that future studies aimed at rational drug design consider the diverse aspects of PTHR signaling.

GRANTS

This work has been supported by NIDDK, National Institutes of Health, Grant R01 DK-116780.

DISCLAIMERS

The content of this mini-review is solely the responsibility of the author and does not represent the views of the National Institutes of Health.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author.

AUTHOR CONTRIBUTIONS

K.A.P. prepared figures; drafted manuscript; edited and revised manuscript; approved final version of manuscript.