Abstract
Parathyroid hormone (PTH) and parathyroid hormone–related peptide (PTHrP), acting through the osteoblast PTH1 receptor (PTH1R), play important roles in bone remodeling. Intermittent administration of PTH(1-34) (teriparatide) leads to bone formation, whereas continuous administration paradoxically leads to bone resorption. Activation of PTH1R promotes regulation of multiple signaling pathways, including Gs/cAMP/protein kinase A, Gq/calcium/protein kinase C, β-arrestin recruitment, and extracellular signal-related kinase (ERK)1/2 phosphorylation, as well as receptor internalization, but their role in promoting anabolic and catabolic actions of PTH(1-34) are unclear. In the present investigation, a collection of PTH(1-34) and PTHrP(1-34) peptide analogs were evaluated in orthogonal human PTH1R (hPTH1R) functional assays capturing Gs- and Gq-signaling, β-arrestin recruitment, ERK1/2 phosphorylation, and receptor internalization to further define the patterns of PTH1R signaling that they stimulate and further establish peptide domains contributing to agonist activity. Results indicate that both N- and C-terminal domains of PTH and PTHrP are critical for activation of signaling pathways. However, modifications of both regions lead to more substantial decreases in agonist potency and efficacy to stimulate Gq-signaling, β-arrestin recruitment, ERK1/2 phosphorylation, and receptor internalization than to stimulate Gs-signaling. The substantial contribution of the peptide C-terminal domain in activation of hPTH1R signaling suggests a role in positioning of the peptide N-terminal region into the receptor J-domain. Several PTH and PTHrP peptides evaluated in this study promote different patterns of biased agonist signaling and may serve as useful tools to further elucidate therapeutically relevant PTH1R signaling in osteoblasts. With a better understanding of therapeutically relevant signaling, novel biased peptides with desired signaling could be designed for safer and more effective treatment of osteoporosis.
Introduction
Parathyroid hormone [PTH(1-84)] and parathyroid hormone-related peptide [PTHrP(1-141)], acting through the parathyroid hormone receptor (PTH1R1), play important roles in facilitating calcium homeostasis and bone remodeling (Fitzpatrick and Bilezikian, 1996). PTH1R is a class B G protein-coupled receptor (GPCR) family receptor possessing a long extracellular N terminus important for hormone binding and an intracellular 130 amino acid (AA) C terminus containing protein interaction domains for cytosolic adaptor and effector proteins regulating receptor expression and signaling (Vilardaga et al., 2011). The N-terminal 34 AAs of both peptides contain the minimal sequence required for PTH1R activation and signal transduction (Gensure et al., 2005). Mechanisms accounting for PTH and PTHrP binding and activation of PTH1R have been widely investigated and support a two-site model involving initial rapid binding of the peptide C-terminal region (AAs 23-34) to the PTH1R N terminus, followed by a slower interaction of the peptide N terminus (AAs 1-14) with the J-domain containing transmembrane α-helices and interconnecting extracellular loops (Gardella and Jüppner, 2001; Castro et al., 2005; Gensure et al., 2005). Activation of PTH1R leads to regulation of multiple signaling pathways, including Gs/adenylyl cyclase/cAMP/protein kinase A (PKA)- (Abou-Samra et al., 1992), Gq/11/phospholipase Cβ/calcium/protein kinase C (PKC)- (Abou-Samra et al., 1992), and G12/13 /Ras homolog gene family, member A/phospholipase D-signaling (Wang and Stern, 2010), as well as G protein–dependent and G protein–independent and β-arrestin–dependent activation of extracellular signal-related kinase (ERK)1/2 (Syme et al., 2005; Gesty-Palmer et al., 2006). PTH1R-mediated activation of PKC also occurs through a phospholipase Cβ (PLCβ)-independent mechanism (Takasu et al., 1999).
PTH(1-34) (teriparatide) is the only anabolic drug on the US market for treatment of osteoporosis (Kawai et al., 2011). Although intermittent administration of PTH(1–34) promotes bone formation and reduces the incidence of fracture, continuous administration promotes bone resorption and hypercalcemia (Tam et al., 1982; Hock and Gera, 1992; Dempster et al., 1993). Anabolic effects of intermittent PTH(1-34) are attributable to PTH1R-mediated increases in osteoblast number, resulting from combined enhancement of preosteoblast differentiation and reduction of apoptosis of mature osteoblasts (Jilka, 2007). The catabolic effect of continuous PTH(1-34) is attributable to increased expression of osteoblast receptor activator of nuclear factor κ-B ligand and decreased expression of the receptor activator of nuclear factor κ-B ligand decoy protein osteoprotegerin, leading to enhanced osteoclastogenesis (Ma et al., 2001). The contribution of osteoblast PTH1R signaling pathways to these divergent actions of PTH(1-34) is unclear and requires further elucidation.
Biased agonists are widely described for GPCRs and are characterized by the capacity to activate only part of the total repertoire of signaling activated by endogenous ligand because of stabilization of distinct active receptor conformations coupling to activation of discrete signaling pathways (Kenakin, 2007, 2011; Urban et al., 2007). Several PTH and PTHrP analogs have been previously described that promote PTH1R differential signaling, including [Trp1]PTHrP(1-36) and [Bpa1]PTHrP(1-36), reported to activate human PTH1R (hPTH1R)-mediated Gs- and not Gq-signaling, β-arrestin recruitment, ERK1/2 phosphorylation, or receptor internalization (Bisello et al., 2002; Gesty-Palmer et al., 2006, 2009). Similarly, [Gly1,Arg19]PTH(1-28) is reported to stimulate Gs- and not Gq-signaling (Takasu et al., 1999; Yang et al., 2007). In addition, [D-Trp12,Tyr34]PTH(7-34)NH2 is a reported β-arrestin biased agonist promoting β-arrestin–dependent ERK1/2 activation and an inverse agonist for Gs-signaling (Gesty-Palmer et al., 2006, 2009). In vivo studies of biased PTH peptides on bone formation suggest that Gs-signaling plays a predominant role and Gq-signaling plays little or no role (Rixon et al., 1994; Hilliker et al., 1996; Whitfield et al., 1996; Yang et al., 2007). Furthermore, a recent report suggests that PTH1R-mediated Gq/11 signaling plays a role in restraining the anabolic action of intermittent PTH(1-34) (Ogata et al., 2011). However, others report that PTH1R-mediated Gq-signaling also contributes to anabolic effects of intermittently administered PTH(1-34) (Murrills et al., 2004; Rhee et al., 2006; Guo et al., 2010). In addition, β-arrestin has been reported to be required for mediating intermittent PTH1R-mediated bone formation without enhancing osteoclastogenesis in wild-type mice, but not in mice with β-arrestin ablated (Ferrari et al., 2005; Getsy-Palmer et al., 2009; Pierroz et al., 2009). Furthermore, [D-Trp12,Tyr34]PTH(7-34) has been reported to mediate bone formation found absent in β-arrestin knockout mice, suggesting that β-arrestin signaling is sufficient (Gesty-Palmer et al., 2009). In light of these different conclusions, the exact PTH1signaling pathways participating in bone reformation require further elucidation.
In this study, several PTH and PTHrP peptide analogs were tested in orthogonal hPTH1R assays capturing Gs- and Gq-signaling, β-arrestin recruitment, ΕRK1/2 phosphorylation, and receptor internalization to comprehensively characterize domains of PTH and PTHrP peptides contributing to activation of different signaling pathways and further establish patterns of differential signaling promoted. Information from this investigation should help guide design of additional biased PTH1R agonists to serve as valuable tools to further elucidate therapeutically relevant PTH1R-mediated signaling. Furthermore, this information could help to guide design of peptide analogs with better therapeutic profiles for treatment of osteoporosis.
Materials and Methods
Materials.
Human PTH(1-34) (H-4835), PTHrP(1-34) (H-6630), PTH(1-37) (H-5974), PTHrP(1-37) (H-5494), PTH(1-31) (H-2274), PTH(1-31)NH2 (H-3408), [Cyclo(Glu22-Lys26),Leu27]PTH(1-31)NH2 (H-4058), [Tyr1]PTH(1-34) (H-3092), [Nle8,18, Tyr34]PTH(1-34) (H-9110), PTH(2-38) (H-1316), PTHrP(1-16) (H-6575), PTHrP(7-34)NH2 (H-9100), and [Asn10,Leu11,D-Trp12]PTHrP(7-36)NH2 (H3274) were purchased from Bachem (San Diego, CA). Bovine PTH(3-34) (H-3088), [Nle8,18,Tyr34]PTH(3-34)NH2 (N-1130), [Tyr34]PTH(7-34)NH2 (N-1110), [Nle8,18,Tyr34]PTH(7-34)NH2 (H-9120], and [D-Trp12,Tyr34]PTH(7-34)NH2 (H-9115) were purchased from Bachem (San Diego, CA). PTH(7-34) (HOR-266) was purchased from Prospec (East Brunswick, NJ). Human MPTH(1-28), ([Aib1,3, Gln10, Har11, Ala12, Trp14, Arg19]PTH(1-28)) and (Gly1,Arg19)PTH(1-28) were purchased from Anaspec (Fremont, CA). Human PTH(1-28), [Trp1]PTHrP(1-36) [Bpa1]PTHrP(1-36), MPTH(1-14) ([Aib1,3,Gln10,Har11,Ala12,Trp14]PTH(1-14)NH2), ZP2307 ([AC5C1-Aib3,Leu8,Gln10,Har11,Ala12,Trp14,Asp17]PTH(1-17)NH2 and [Aib1,3, Nle8,Gln10,Har11]PTH(1-11)NH2 were all synthesized using standard peptide synthesis and purification methods. Specifically, molecular identities of the synthesized peptides were determined using high-resolution mass spectrometry. Matrix-assisted laser desorption ionization time-of-flight hybrid mass spectrometer was used to confirm that synthetic peptides were assembled correctly. All the mass spectra showed accurate molecular weight matching the expected or the theoretical molecular weight calculated on the basis of the chemical formula of the peptides. The purity and the homogeneity of the synthetic peptides were determined using analytical reversed-phase high-pressure liquid chromatography performed using a C18 column. The semi-preparative and analytical chromatograms from the reversed-phase high-pressure liquid chromatography runs confirmed purity of >90% for the synthetic peptides. Analytical data are shown in Supplemental Table 1.
cAMP Accumulation Assay.
PTH1R agonist–mediated stimulation of intracellular cAMP accumulation was measured using the competitive immunoassay Hunter eXpress cAMP assay kit based on a β−galactosidase enzyme complementation platform (DiscoveRX, Fremont, CA) (Olsen and Eglen, 2007). In brief, 100-μl aliquots of thawed Chinese hamster ovary (CHO)-K1 cells expressing recombinant hPTH1R were added to 96-well microtiter plates (30,000 cells/well), and plates were incubated at 37°C [5% CO2, 95% relative humidity (RH)] for 24–48 hours before performance of the assay. Test peptides dissolved in 100% dimethyl sulfoxide (DMSO) were diluted to desired concentrations with cell assay buffer and added to appropriate wells, followed by incubation of assay plates for 30 minutes at 37°C (5% CO2, 95% RH). The rest of the assay was performed according to the manufacturer’s specifications, and the chemiluminescent signal was measured using an Analyst HT plate reader (Molecular Devices, Sunnyvale, CA). Data are expressed in arbitrary luminescence units (ALU). The mean basal signal was 5000 ALU, and the mean signal obtained with a maximal concentration of PTH(1-34) was 69,000 ALU.
Inositol Phosphate Accumulation Assay.
hPTH1R-mediated stimulation of inositol monophosphate (IP1) accumulation was measured using the CisBio IPOne TRF (time-resolved fluorescence) assay kit (Bedford, MA) (Trinquet et al., 2011) and using the same CHO-K1 clone stably expressing recombinant hPTH1R used for cAMP accumulation assays (DiscoveRX). In brief, 100-μl aliquots of cells (30,000 cells/well) were added to 96-well assay microtiter plates and incubated for 48 hours at 37°C (5% CO2, 95% RH). Cell media (65 μl) were then removed, 35 μl of test peptide originally solubilized in 100% DMSO and diluted to the appropriate concentration with stimulation buffer was added to appropriate wells, assay plates were incubated for 90 minutes at 37°C (5% CO2, 95% RH), and cells were lysed for 2 hours with lysis buffer containing antibodies. A total of 70 μl of lysed cells was transferred to a half volume white 96-well microtiter plate, and fluorescent signal was measured using an Analyst HT plate reader (Molecular Devices) at wavelengths of 665 and 620 nm. Data are expressed as arbitrary fluorescence units (AFU). The mean blank was 16,000 AFU, and the mean signal in the presence of a maximal concentration of PTH(1-34) was 32,000 AFU.
β-Arrestin-2 Recruitment Assay.
PTH1R-mediated stimulation of β-arrestin recruitment was measured directly using the Pathfinder assay kit (DiscoveRX). This assay is based on β-galactosidase enzyme complementation technology in which β-arrestin-2 is fused to an N-terminal deletion mutant of β-galactosidase [enzyme acceptor (EA) protein] and hPTH1R is fused to a smaller weakly binding 42 AA complementing Prolink fragment. Agonist activation of the receptor promotes direct interaction with β-arrestin-2 and the hPTH1R and complementation of the two β-galactosidase fragments to form functional enzyme (McGuinness et al., 2009; Yin et al., 2009; Bassoni et al., 2012). In brief, vials of frozen CHO-K1 cells stably expressing recombinant hPTH1R were thawed in a 37°C water bath for 10 seconds and diluted in prewarmed media, 100-μl aliquots of cell suspension were added to 96-well assay microtiter plates (8,000 cells/well), and plates were incubated for 48 hours at 37°C (5% CO2, 95% RH) before performing assays. Test peptide solubilized in 100% DMSO and diluted to the desired concentrations with cell media was added to appropriate wells, and plates were incubated for 90 minutes at 37°C (5% CO2, 95% RH). The rest of the assay was performed according to the manufacturer’s specifications, and the chemiluminescent signal was measured using an Analyst HT plate reader (Molecular Devices). Data are expressed as ALU; the mean blank was 32,000 ALU, and the mean signal in the presence of a maximal concentration of PTH(1-34) was 650,000 ALU.
ERK1/2 Phosphorylation Assay.
The CisBio Cellul’erk TRF assay kit was used to measure PTH1R-mediated ERK1/2 phosphorylation in the same CHO-K1 clone stably expressing hPTH1R (DiscoveRX) used for cAMP and IP1 accumulation assays. This assay is based on a competitive sandwich enzyme-linked immunosorbent assay platform using an anti–phospho-ERK1/2 antibody labeled with a TRF acceptor d2 fluorophore and an anti-ERK1/2 antibody labeled with TRF donor Eu3+-cryptate fluorophore. In brief, 50-μl aliquots of cells (15,000 cells/well) were added to 96-well assay microtiter plates and incubated overnight at 37°C (5% CO2, 95% RH), cell media was removed, and cells were incubated with phosphate-buffered saline for 20 minutes at room temperature. Test peptide solubilized in 100% DMSO and diluted to the desired concentration with phosphate-buffered saline was added to appropriate wells, assay plates were incubated for 15 minutes at room temperature, and then cells were lysed for 30 minutes. The remainder of the assay was performed according to the manufacturer’s specifications, fluorescent signal was measured at wavelengths 665 and 620 nm using an Analyst HT plate reader (Molecular Devices), and data are expressed as AFU. The mean blank was 6,000 AFU, and the mean signal in the presence of a maximal concentration of PTH(1-34) was 10,000 AFU.
hPTH1R Internalization Assay.
Agonist stimulation of PTH1R endocytosis into early endosomes was measured using the DiscoveRX PathHunter Total Internalization eXpress assay kit (DiscoveRX) based on a β-galactosidase enzyme complementation platform (Olson and Eglen, 2007). Specifically, when U2OS cells stably expressing hPTH1R tagged with the Prolink interact with EA-tagged endosomes during receptor internalization, enzyme complementation occurs leading to generation of a chemiluminescent product. This assay measures both β-arrestin–dependent and –independent receptor internalization. In brief, vials of frozen U2OS cells stably expressing hPTH1R were thawed in a 37°C water bath for 30 seconds and diluted in prewarmed media; 100-μl aliquots of cell suspension were added to 96-well assay microtiter plates (8333 cells/well), and plates were incubated for 24 hours at 37°C (5% CO2, 95% RH). Test peptide solubilized in 100% DMSO and diluted to desired concentrations with cell assay buffer were added to appropriate wells containing media, and plates were incubated for 3 hours at 37°C (5% CO2, 95% RH). The rest of the assay was performed according to the manufacturer’s specifications, and chemiluminescent signal was measured using an Analyst HT plate reader (Molecular Devices). Data are expressed as ALU; the mean blank was 300,000 ALU, and the mean signal in the presence of a maximal concentration of PTH(1-34) was 130,000 ALU.
Data Analysis.
Agonist EC50 and antagonist IC50 values were determined by fitting dose-response data to a three-parameter sigmoidal function with use of a nonlinear least squares curve fitting program (GraphPad Prism; GraphPad Software, San Diego, CA), and the data for the time course of PTH(1-34) and PTHrP(1-34) stimulation of β-arrestin recruitment were fit to a hyperbolic function using the same curve-fitting program. The statistical significance of differences between EC50 values obtained for PTH(1-34) and other PTH and PTHrP peptides (P < 0.001) in the same hPTH1R functional assay was determined using an unpaired two-tailed Student’s t test with GraphPad Prism, and the statistical significance of differences in efficacy promoted by different peptide analogs [percentage of the maximal response promoted by the highest concentration of PTH(1-34); P < 0.05] was determined using a paired one-tail Student’s t test with GraphPad Prism. The statistical significance of differences in EC50 values and efficacy values (P < 0.05) for a given test peptide obtained in PTH1R functional assays was determined using one-way analysis of variance, followed by a Tukey’s posttest, with use of GraphPad Prism. The correlation between agonist activity (Emax/EC50) of various PTH and PTHrP peptide analogs obtained in different hPTH1R functional assays and data were subjected to Deming (Mode II) linear regression analysis using GraphPad Prism. Ligand bias (β factor) was quantified using a method described by Rajagopal et al. (2011), in which the equiactive comparison method was used. With use of this approach, the concentrations of agonist required for an equiactive response in one signaling pathway (pathway 1) and another signaling pathway (pathway 2) are extrapolated from the three-parameter sigmoidal curve fit of the concentration-response data (Rajagopal et al., 2011). A bias factor representing the relative stabilization of one signaling pathway over another is given by the equation:
The statistical significance of differences in calculated β factors for different peptide analogs (P < 0.05) was determined using one-way analysis of variance, followed by a Tukey posttest, with use of GraphPad Prism.
Results
PTH/PTHrP Peptide Analog Stimulation of PTH1R-Mediated Gs-Signaling.
Agonist activation of hPTH1R receptor expressed in different cells commonly results in Gs coupling, stimulation of adenylyl cyclase, and increases of intracellular cAMP, followed by stimulation of PKA and phosphorylation of select intracellular substrates. Measurement of PTH and PTHrP peptide analog stimulation of PTH1R-mediated cAMP accumulation in a CHO-K1 cell clone stably expressing recombinant hPTH1R was accomplished using the DiscoveRX Hunter eXpress enzyme-linked immunosorbent assay-based enzyme complementation cAMP assay. Representative concentration-response curves for PTH(1-34), [Tyr1]PTH(1-34), PTHrP(1-34), [Trp1]PTHrP(1-36), PTH(2-38), PTH(3-34), and MPTH(1-14) stimulation of hPTH1R-mediated cAMP accumulation are shown in Fig. 1A, and EC50 values of 0.23, 0.39, 0.86, 3.6, 1.0, 241, and 0.33 nM were determined, respectively.
A collection of several N- and C-terminal truncated PTH and PTHrP peptide analogs with select AA substitutions was evaluated for agonist activity in the cAMP accumulation assay, and Table 1 summarizes the results. All nontruncated peptide analogs, including PTH(1-37), PTHrP(1-37), PTHrP(1-34), [Tyr1]PTH(1-34), [Nle8,18,Tyr34]PTH(1-34), [Trp1]PTHrP(1-36), and [Bpa1]PTHrP(1-36), as well as PTH(2-38), were full agonists in promoting cAMP accumulation with potencies similar to that of PTH(1-34). The N-terminal truncated peptide analogs PTH(3-34) and [Nle8,18,Tyr34]PTH(3-34)NH2 displayed partial agonist activity and were 288- and 93-fold less potent than was PTH(1-34), respectively, indicating that removal or substitution of the first AA of PTH(1-34) or substitution of the first AA of PTHrP(1-36) does not alter agonist activity but removal of the first two AAs of PTH(1-34) markedly decreases agonist activity, in agreement with previous studies (Gensure et al., 2005). Several additional PTH and PTHrP peptide analogs with deletion of the first seven N-terminal AAs, including [Nle8,18,Tyr34]PTH(7-34)NH2, [Tyr34]PTH(7-34)NH2, PTHrP(7-34)NH2, PTH(7-34), [Asn10,Leu11,D-Trp12]PTHrP(7-36)NH2, and [D-Trp12,Tyr34]PTH(7-34)NH2, were also found to be inactive in the cAMP accumulation assay. Furthermore, [D-Trp12,Tyr34]PTH(7-34)NH2 completely antagonized stimulation of cAMP accumulation elicited by 0.8 nM PTH(1-34), with an IC50 value of 1 μM (Fig. 1B). None of the inactive peptides reduced basal cAMP accumulation consistent with inverse agonist activity (unpublished data).
The C-terminal truncated peptide analogs, including PTH(1-31), PTH(1-31)NH2, MPTH(1-28), and [Gly1Arg19]PTH(1-28), stimulated cAMP accumulation with similar potency and efficacy as PTH(1-34), whereas PTHrP(1-16) was found to be inactive. Additional C-terminal truncated PTH peptide analogs containing AA substitutions designed to introduce steric hindrance and increase peptide α-helicity and side group interactions were also tested. ZP2307 (Neerup et al., 2011) and [Aib1,3,Nle8, Gln10,Har11]PTH(1-11)NH2 (Caporale et al., 2010) were full agonists displaying 10- and 28-fold lower potencies than PTH(1-34), whereas the potency and efficacy of MPTH(1-14) (Okazaki et al., 2008) were similar to those of PTH(1-34).
PTH/PTHrP Peptide Analog Stimulation of PTH1R-Mediated Gq-Signaling.
Agonist activation of hPTH1R expressed in some cells leads to Gq-coupling, activation of PLCβ and hydrolysis of membrane-associated phosphatidylinositol species to form inositol phosphates, increases in intracellular calcium, and activation of calcium-dependent enzymes, including PKC (Abou-Samra et al., 1992; Taylor and Tovey, 2012). PTH and PTHrP peptide analog stimulation of PTH1R-mediated IP1 accumulation was measured as a parameter of activation of hPTH1R-mediated Gq-signaling and was accomplished using the CisBio IPone TRF assay and the same CHO-K1 clone stably expressing hPTH1R used for cAMP assays. Representative concentration-response curves for stimulation of hPTH1R-mediated IP1 accumulation by PTH(1-34), PTHrP(1-34), [Tyr1]PTH(1-34), PTH(2-38), MPTH(1-28), ZP2307, and MPTH(1-14) are shown in Fig. 2, and EC50 values of 19; 27; >10,000; >10,000; 13; 9014; and 1212 nM were determined, respectively. The agonist activity of these and additional peptides in the hPTH1R IP1 accumulation assay are summarized in Table 1.
The full-length peptides, including PTH(1-34), PTHrP(1-34), and [Nle8,18,Tyr34]PTH(1-34), possess similar agonist activity in the IP1 accumulation assay (Table 1). However, peptides with N-terminal modification or truncation, including [Bpa1]PTHrP(1-36), [Trp1]PTHrP(1-36), PTH(2-38), PTH(3-34), [Nle8,18,Tyr34]PTH(3-34), and peptide analogs with the first seven AA residues deleted, were found to be inactive. With regard to C-terminal modified peptides, both PTH(1-31) and [Cyclo(Glu22-Lys26),Leu27]PTH(1-31)NH2 displayed similar agonist potency as PTH(1-34), but peptide analogs with more extensive C-terminal truncation and AA substitutions, including PTH(1-28), [Gly1,Arg19]PTH(1-28), ZP2307, and MPTH(1-14), were weaker agonists in comparison with PTH(1-34), whereas [Aib1,3,Nle8,Gln10,Har11]PTH(1-11)NH2 was inactive. Of interest, MPTH(1-28) displayed similar agonist activity as PTH(1-34) in contrast to PTH(1-28), suggesting that introduction of N-terminal AA substitutions maximizing J-domain interaction enhance agonist activity. However, peptide analogs with more extensive C-terminal truncation and similar AA substitutions did not display enhanced Gq agonist activity. Collectively, these results suggest that the first native N-terminal AA of both PTH and PTHrP are absolutely required for stimulating hPTH1R-mediated Gq agonist activity and that C-terminal truncation of PTH(1-34) past PTH(1-31) also results in a marked loss in agonist activity. In addition, the introduction of unnatural AAs into the N terminus of PTH designed to presumably optimize interaction with the PTH1R J-domain increases agonist activity for only peptide analogs with moderate C-terminal truncation, such as MPTH(1-28), but not for more extensively C-terminally truncated peptides, including ZP2307, MPTH(1-14), and [Aib1,3,Nle8,Gln10,Har11]PTH(1-11).
PTH/PTHrP Peptide Analog Stimulation of PTH1R-Mediated β-Arrestin-2 Recruitment.
Agonist activation of hPTH1R not only stimulates Gs- and Gq-signaling, but also stimulates β-arrestin recruitment; β-arrestin-dependent signaling, including activation of ERK1/2, β-arrestin-mediated hPTH1R internalization; and desensitization (Bisello et al., 2002; Gesty-Palmer et al., 2006; Vilardaga et al., 2011). Agonist stimulation of hPTH1R-mediated β-arrestin recruitment in CHO-K1 cells stably expressing recombinant hPTH1R was measured using the DiscoveRX PathFinder assay kit based on the β-galactosidase enzyme complementation platform (Olson and Eglen, 2007; Bassoni et al., 2012). Representative concentration-response curves for stimulation of hPTH1R-mediated β-arrestin recruitment by PTH(1-34), [Tyr1]PTH(1-34), PTHrP(1-34), [Bpa1]PTHrP(1-36), [Trp1]PTHrP(1-36), PTH(1-28), ZP2307, and MPTH(1-14) are shown in Fig. 3A, and EC50 values of 0.65, 33, 0.87, 11, 36, 44, 98, and 17 nM were obtained, respectively. [D-Trp12,Tyr34]PTH(7-34)NH2 did not stimulate hPTH1R-mediated β-arrestin recruitment but completely antagonizes stimulation promoted by 15 nM PTH(1-31)NH2 with an IC50 value of 275 nM (Fig. 3B).
The time courses of hPTH1R-mediated β-arrestin recruitment elicited by maximally effective concentrations of PTH(1-34) (100 nM) and PTHrP(1-34) (100 nM) were evaluated to determine whether the kinetics of β-arrestin recruitment vary between different peptides (Fig. 4). In addition, although initial testing of [D-Trp12,Tyr34]PTH(7-34)NH2 up to a final assay concentration of 10 μM indicated that this PTH peptide analog was inactive in the β-arrestin recruitment assay (Fig. 3B), it was further tested to determine whether longer assay incubation periods may result in an agonist response. Both time courses of PTH(1-34) and PTHrP(1-34) stimulation of hPTH1R-mediated β-arrestin recruitment were similar and hyperbolic. In contrast, [D-Trp12,Tyr34]PTH(7-34)NH2 was inactive during the duration of the 3-hour assay incubation period, confirming that this PTH peptide analog does not promote PTH1R-mediated β-arrestin recruitment in CHO-K1 cells expressing recombinant hPTH1R.
The same collection of PTH and PTHrP analogs tested in the cAMP accumulation assay was tested in the β-arrestin recruitment assay, and the results are summarized in Table 1. Full-length peptides, including PTH(1-37), PTHrP(1-37), PTHrP(1-34), and [Nle8,18,Tyr34]PTH(1-34), displayed similar potency and efficacy as PTH(1-34). The N-terminally modified peptides [Tyr1]PTH(1-34), PTH(2-38), [Trp1]PTHrP(1-36), and [Bpa1]PTHrP(1-36) also promoted hPTH1R-mediated β-arrestin recruitment but were less potent (21−, 61-, 12-, and 6-fold, respectively) and possess lower intrinsic activity than does PTH(1-34) (56, 15, 42, and 50%, respectively). Peptide analogs containing additional N-terminal truncation and AA substitutions, including PTH(3-34), [Nle8,18,Try34]PTH(3-34)NH2, [Nle8,18,Tyr34]PTH(7-34)NH2, [D-Trp12,Tyr34]PTH(7-34)NH2, PTHrP(7-34), and PTH(7-34), did not promote an agonist response. With regard to C-terminally truncated and modified PTH peptide analogs, PTH(1-31), PTH(1-31)NH2, MPTH(1-28), and [Cyclo(Glu22, Lys26),Leu27]PTH(1-31)NH2 were all full agonists with potencies similar to that of PTH(1-34). In contrast, PTH(1-28), [Gly1,Arg19]PTH(1-28), ZP2307, MPTH(1-14), and [Aib1,3,Nle8,Gln10,Har11]PTH(1-11)NH2 were 33-, 58-, 82-, 16-, and 451-fold less potent full agonists than PTH(1-34), respectively. However, MPTH(1-28) was found to be equipotent with PTH(1-34), indicating that N-terminal AA substitutions increasing the α-helical structure of the N-terminal region counteract the negative effects of C-terminal truncation for PTH(1-28) but not for shorter peptide analogs. These results suggest that Ser1 and Ala1 of PTH and PTHrP, respectively, are important for potent full agonist activity in the hPTH1R β-arrestin recruitment assay and that C-terminal truncation past PTH(1-31) moderately decreases agonist potency to stimulate β-arrestin recruitment with the exception of MPTH(1-28). Furthermore, peptide modifications negatively impacting agonist activity in stimulating Gq-signaling have a similar effect on activity to promote β-arrestin recruitment without altering activity to stimulate hPTH1R-mediated Gs-signaling.
PTH/PTHrP Peptide Analog Stimulation of ERK1/2 Phosphorylation.
Activation of hPTH1R expressed in osteoblasts and osteosarcoma cells leads to phosphorylation and activation of ERK1/2 by mechanisms that can involve both G protein–dependent signaling and G protein–independent and β-arrestin–dependent signaling (Syme et al., 2005; Gesty-Palmer et al., 2006). PTH1R-mediated stimulation of ERK1/2 phosphorylation was measured using the CisBio TRF Cellul’Erk assay kit and the same CHO-K1 cell stable clone expressing recombinant hPTH1R used for cAMP and IP1 accumulation assays. Representative concentration-response curves for PTH(1-34), [Tyr1]PTH(1-34), PTHrP(1-34), [Trp1]PTHrP(1-36), MPTH(1-28), ZP2307, and MPTH(1-14) are shown in Fig. 5A, and EC50 values of 21; >10,000; 5; >10,000; 3; 151; and 114 nM were determined, respectively. [D-Trp12, Tyr34]PTH(7-34)NH2 was inactive but antagonized stimulation of ERK1/2 phosphorylation promoted by 30 nM PTH(1-34) with an IC50 value of 38 nM (Fig. 5B).
The time courses of PTH(1-34) and PTHrP(1-34) stimulation of hPTH1R-mediated ERK1/2 phosphorylation in CHO-K1 cells stably expressing hPTH1R consist of two temporal components: a rapid transient phase peaking at 10 minutes after peptide addition and a slower, sustained component remaining elevated above baseline 2 hours after peptide addition (Fig. 6). The amplitude of the initial phase of stimulation promoted by PTHrP(1-34) is approximately 60% of that of PTH(1-34), whereas the sustained component was similar. PTH(2-38) in the same experiment did not stimulate ERK1/2 phosphorylation during the 2-hour incubation period. In two additional time course studies, [Trp1]PTHrP(1-36) promoted a small, rapid increase in ERK1/2 activation without promoting sustained ERK1/2 activation. Although [D-Trp12,Tyr34]PTH(7-34)NH2 is reported to stimulate ERK1/2 activation in HEK293 cells expressing recombinant hPTH1R (Gesty-Palmer et al., 2006), this peptide analog was inactive during a during a two-hour incubation period (unpublished data).
The possible contribution of PTH1R-mediated Gs/PKA and/or Gq/PKC signaling pathways to stimulation of ERK1/2 phosphorylation in CHO-K1 cells expressing recombinant hPTH1R was investigated using the PKA-selective inhibitor H89 and the PKC-selective inhibitor chelerythrine chloride. Preincubation of CHO-K1 cells expressing hPTH1R with 10 μM H89 partially reduced the amplitude of the rapid temporal component of PTH1R-mediated ERK1/2 phosphorylation but did not appreciably alter the slower, sustained component of phosphorylation (Fig. 7A). Preincubation of CHO-K1 cells with 1 μM chelerythrine chloride did not affect either temporal component of PTH(1-34) stimulation of PTH1R-mediated ERK1/2 phosphorylation (Fig. 7B). These results suggest that activation of PTH1R-mediated Gs-signaling leading to PKA activation contributes in part to the more transient, rapid component of ERK1/2 activation, whereas Gq-signaling leading to PKC activation does not contribute to either temporal component of activation.
Several of the PTH and PTHrP peptide analogs evaluated in hPTH1R cAMP, IP1, and β-arrestin recruitment assays were evaluated for ability to stimulate hPTH1R-mediated ERK1/2 phosphorylation, and the results are summarized in Table 1. PTH(1-37), PTHrP(1-34), PTHrP(1-37), [Nle8,18,Tyr34]PTH(1-34), PTH(1-31), and [Cyclo(Glu22-Lys26)Leu27]PTH(1-31)NH2 displayed similar agonist potencies as PTH(1-34). Of interest, PTHrP(1-34) and PTHrP(1-37) displayed partial agonist activity in stimulating hPTH1R-mediated ERK1/2 phosphorylation, compared with PTH(1-34). [Tyr1]PTH(1-34), [Bpa1]PTHrP(1-36), [Trp1]PTHrP(1-36), and PTH(2-38), all of which are found to display weak partial agonist activity in the β-arrestin recruitment assay, were found to be inactive in the ERK1/2 phosphorylation assay. Furthermore, all PTH and PTHrP peptide analogs with the deletion of two or more N-terminal AAs were also found to be inactive. Regarding C-terminally truncated and modified peptide analogs, MPTH(1-28) displayed similar agonist potency and efficacy as PTH(1-34), whereas PTH(1-28), [Gly1,Arg19]PTH(1-28), ZP2307, and MPTH(1-14) displayed lower potency and efficacy than PTH(1-34), with the exception that the statistical significance of the potentially lower efficacy of [Gly1,Arg19]PTH(1-28) could not be assessed, because only two EC50 determinations were made. Finally, [Aib1,3,Nle8,Gln10,Har11]PTH(1-11)NH2 was found to be inactive in stimulating hPTH1R-mediated ERK1/2 phosphorylation. These results indicate that both N- and C-terminal domains of PTH(1-34) and PTHrP(1-34) contribute to hPTH1R-mediated stimulation of ERK1/2 phosphorylation similar to stimulation of Gq-signaling and β-arrestin recruitment. Introduction of unnatural AAs into more extensively C-terminally truncated PTH peptides, with the exception of MPTH(1-28), did not enhance agonist activity in the ERK1/2 phosphorylation assay.
PTH/PTHrP Peptide Analog Stimulation of hPTH1R Internalization.
The capacity of PTH and PTHrP peptide analogs to promote hPTH1R internalization in U2OS cells stably expressing recombinant receptor was measured using the DiscoveRX PathHunter total (β-arrestin dependent and independent) internalization assay based on β-galactosidase enzyme complementation taking place when the larger EA portion of β-galactosidase constitutively expressed on the surface of endosomes interacts with PTH1R tagged with Prolink. Several cell backgrounds were evaluated during development of this assay, including CHO-K1 cells, but U2OS cells expressing recombinant hPTH1R consistently provided the largest agonist window (communication with DiscoveRX). Representative concentration-response curves for PTH(1-34), [Tyr1]PTH(1-34), PTHrP(1-34), [Trp1]PTHrP(1-36), PTH(1-28), ZP2307, and MPTH(1-14) are shown in Fig. 8, and EC50 values of 1, 50, 4, 120, 275, 1423, and 239 nM were determined, respectively. The potency and efficacy of these and several other PTH and PTHrP peptide analogs are summarized in Table 1.
PTHrP(1-34) and [Nle8,18,Tyr34]PTH(1-34) were found to display similar potency and efficacy to promote receptor internalization as PTH(1-34). The N-terminal modified PTHrP peptide analogs [Trp1]PTHrP(1-36) and [Tyr1]PTH(1-34) stimulated hPTH1R internalization with lower potency (21- and 53-fold) and efficacy (48 and 75%), whereas the related peptide analog [Bpa1]PTHrP(1-36), in addition to PTH(2-38) and PTH(3-34), was inactive. Regarding C-terminally modified peptides, PTH(1-31) and [Cyclo(Glu22-Lys26)Leu27]PTH(1-31)NH2 were found to have similar agonist activity as PTH(1-34), whereas PTH(1-28), [Gly1,Arg19]PTH(1-28), ZP2307, and MPTH(1-14) were less potent (53-, 136-, 342-, and 47-fold lower, respectively) than PTH(1-34) and [Aib1,3,Nle8,Gln10,Har11]PTH(1-11) was inactive. Of interest, PTH(1-28), [Gly1,Arg19]PTH(1-28), ZP2307, and MPTH(1-14) stimulated significantly higher levels of hPTH1R internalization than did PTH(1-34). Conversely, MPTH(1-28) displayed similar potent agonist activity as PTH(1-34) but was significantly less efficacious. These results suggest that substitution of the first AA of PTH or PTHrP or removal of the first AA of PTH results in a complete loss of ability to promote internalization. In addition, additional C-terminal truncation of PTH from PTH(1-31) and introduction of AA substitutions optimizing J-domain interaction with the exception of MPTH(1-28) result in a decrease in agonist potency while increasing the capacity to promote hPTH1R internalization.
Comparison of Potencies of PTH/PTHrP Peptide Analogs to Stimulate hPTH1R-Mediated Signaling Pathways.
To facilitate comparison of the agonist potency of PTH/ PTHrP peptides in the different hPTH1R functional assays, EC50 values were converted to pEC50 values and plotted for each peptide analog (Fig. 9). With regard to full-length peptides, the observed agonist potency rank order for PTH(1-34), PTHrP(1-34), and [Nle8,18,Tyr34]PTH(1-34) was cAMP accumulation = β-arrestin recruitment = internalization > ERK1/2 phosphorylation = IP1 accumulation (Fig. 9A). The potency rank order of N-terminally modified peptides [Tyr1]PTH(1-34) and [Trp1]PTHrP(1-36) was cAMP accumulaltion = β-arrestin > internalization and no agonist activity in the IP1 accumulation and ERK1/2 phosphorylation assays. [Bpa1]PTHrP(1-36) displayed a similar agonist profile as [Trp1]PTHrP(1-36) and [Tyr1]PTH(1-34), with the exception that it does not promote hPTHR1 internalization.
Agonist potency profiles determined for N-terminally truncated PTH peptide analogs are shown in Fig. 9B. As previously discussed, removal of the Ser1 of PTH does not impact agonist activity in the cAMP assay, reduces agonist potency and efficacy to promote β-arrestin recruitment, and results in a complete loss of agonist activity in IP1 accumulation, ERK1/2 phosphorylation, and receptor internalization assays. Furthermore, removal Ser1 and Val2 of PTH(1-34) [(PTH(3-34) and [Nle8,18,Tyr34]PTH(3-34)] also results in a significant reduction in agonist potency and efficacy to stimulate Gs-signaling. Figure 9B clearly shows that N-terminal truncation of PTH and PTHrP has a greater negative impact on stimulation of hPTH1R-mediated Gq-signaling, β-arrestin recruitment, ERK1/2 phosphorylation, and receptor internalization than on stimulation of Gs-signaling. Furthermore, all PTH/PTHrP analogs with deletion of seven N-terminal amino acids were found to be inactive in all five hPTH1R functional assays, which is consistent with the proposed requirement of these AAs for interaction with the hPTH1R J-domain (Table 1).
The agonist potency profiles for C-terminally truncated and modified PTH and PTHrP peptide analogs tested in all five hPTH1R functional assays are shown in Fig. 9C. Removal of the last three C-terminal AAs of PTH(1-34) does not greatly alter agonist activity in any of the five hPTH1R functional assays, compared with PTH(1-34). PTH peptides with further truncation and AA substitutions, including PTH(1-28), [Gly1, Arg19]PTH(1-28), ZP2307, and MPTH(1-14), generally have the potency rank order of cAMP > β−arrestin = ERK1/2 > receptor internalization > IP1 accumulation, whereas MPTH(1-28) possesses a similar assay potency rank order as PTH(1-34). Finally, although [Aib1,3,Nle8,Gln10,Har11]PTH(1-11)NH2 has only a modest decrease in agonist potency in the cAMP assay, a more significant decrease in potency to stimulate β-arrestin recruitment, and no agonist activity in IP1 accumulation, ERK1/2 phosphorylation and receptor internalization assays were observed. These results suggest that PTH(1-34) peptide analogs with C-terminal truncation of six AAs and substitution of hydrophobic AAs with unnatural AAs increasing N-terminal α-helicity and side chain interactions [e.g., MPTH(1-28)] helps to maintain agonist potency to promote Gq-signaling, β-arrestin recruitment, and ERK1/2 phosphorylation [e.g., MPTH(1-28)]. However, agonist activity of more extensively truncated peptide analogs with these modifications is not maintained. Of importance, these results indicate that C-terminal AAs of PTH(1-34) generally considered to be critical in primarily mediating binding to the PTH1R N terminus also contribute to peptide docking into the J-domain and subsequent stimulation of both G protein–dependent and G protein–independent PTH1R-mediated signaling.
Biased PTH/PTHrP Peptide Analogs.
The correlation of the different PTH and PTHrP peptide analogs to stimulate hPTH1R-mediated cAMP accumulation, IP1 accumulation, β-arrestin recruitment, ERK1/2 phosphorylation, and receptor internalization was also determined for peptides found to be active in all five hPTH1R functional assays (Fig. 10). This was accomplished by comparing calculated EMax/EC50 values for PTH and PTHrP peptide analogs within each assay pairing and performing a Deming linear regression analysis of the data. Several of the PTH and PTHrP peptide analogs evaluated in this study were found to stimulate hPTH1R-mediated Gs-signaling but were devoid of agonist activity in other functional assays, consistent with biased agonist activity. Additional peptide analogs were also found to display more subtle differences in efficacy and/or potency in cAMP accumulation, IP1 accumulation, β-arrestin recruitment, ERK1/2 phosphorylation, and receptor internalization assays, and the equiactive comparison method recently described by Rajagopal et al. (2011) was used to quantify biased agonism for these peptides. With use of this approach, concentrations of peptide analog and PTH(1-34) required for an equiactive response for one signaling pathway versus another are extrapolated from fits of each concentration-effect curve, and a bias factor, referred to as β (equation described in Materials and Methods), was calculated. The bias factor represents the relative ability of a given PTH/PTHrP peptide analog to stabilize one hPTH1R-mediated signaling pathway over another on a logarithmic scale, compared with PTH(1-34) (Rajagopal et al., 2011).
PTH/PTHrP peptide analogs displaying statistically significant (P < 0.05) bias, compared with PTH(1-34), for stimulating hPTH1R-mediated Gs-signaling versus Gq-signaling, compared with PTH(1-34), include the C-terminally modified peptides [Gly1,Arg19]PTH(1-28) and MPTH(1-14) (Fig. 11A). PTH and PTHrP peptide analogs promoting significant biased hPTH1R-mediated Gs-signaling over β-arrestin recruitment include PTH(2-38), [Tyr1]PTH(1-34), [Gly1,Arg19]PTH(1-28), MPTH(1-14), and [Aib1,3,Nle8,Gln10,Har11]PTH(1-11)NH2 (Fig. 11B). None of the peptide analogs evaluated displayed agonist bias for stimulation of hPTH1R-mediated Gs-signaling versus ERK phosphorylation (unpublished data), but several peptide analogs, including [Gly1,Arg19]PTH(1-28), [Tyr1]PTH(1-34), MPTH(1-14), ZP2307, [Trp1]PTHrP(1-36), and PTH(1-28) were found to promote significant biased Gs-signaling over receptor internalization, compared with PTH(1-34) (Fig. 11C), whereas [Cyclo(Glu22-Lys26)Leu27]PTH(1-31)NH2 and PTHrP(1-34) showed a slight bias for activating receptor internalization over Gs-signaling. No significant signaling bias was observed for any PTH/PTHrP peptide analogs evaluated for simulation of PTH1R-mediated Gq-signaling versus β-arrestin recruitment, Gq-signaling versus ERK1/2 phosphorylation, Gq-signaling versus internalization, β-arrestin recruitment versus ERK1/2 phosphorylation, β-arrestin recruitment versus receptor internalization, or ERK1/2 phosphorylation versus receptor internalization (unpublished data), compared with PTH(1-34).
Discussion
Studies of N- and C-terminally modified PTH/PTHrP peptide analogs in PTH1R radioligand binding and functional assays capturing Gs- and Gq-signaling suggest the N-terminal domain (AA 1-14) is important for stimulating PTH1R-mediated G protein signaling, and the C-terminal domain (AA23-34) contributes to receptor binding and PLCβ-independent PKC activation (Gensure et al., 2005; Vilardaga et al., 2011). Peptide domains contributing to stimulation of hPTH1R-mediated β-arrestin recruitment, ERK1/2 phosphorylation, and receptor internalization are not well defined. Therefore, several PTH/PTHrP peptide analogs were evaluated in orthogonal functional assays capturing not only PTH1R-mediated Gs- and Gq-signaling, but also β-arrestin recruitment, ERK1/2 activation, and receptor internalization to provide a more comprehensive understanding of regions contributing to hPTH1R signaling and further define patterns of biased agonist activity. All hPTH1R assays except receptor internalization were performed using a CHO-K1 clone stably expressing hPTH1R and another engineered CHO-K1 clone expressing prolink-tagged hPTH1R and β-arrestin-EA was used for the β-arrestin recruitment assay. It is possible that levels of hPTH1R expression in these two CHO-K1 clones and U2OS cells used for the internalization assay may differ, which could impact agonist efficacy. Similar to a study using HEK293 cells expressing hPTH1R (Gesty-Palmer et al., 2006), stimulation of hPTH1R-mediated ERK1/2 phosphorylation in CHO-K1 cells consists of two temporal components consisting of rapid transient G-dependent and slower, sustained β-arrestin–dependent phases. Strong correlations between PTH/PTHrP peptide EC50 values for stimulation of Gs/Gq-signaling and β-arrestin recruitment and β-arrestin recruitment and receptor internalization were noted, implying they are likely to be interdependent signaling pathways.
Structural Domains Involved in Stimulation of Different PTH1R-Mediated Signaling Pathways.
PTH(1-34) and PTHrP(1-34) display similar agonist activities in all hPTH1R CHO-K1functional assays, with a potency rank order of cAMP = β-arrestin recruitment = internalization > ERK1/2 phosphorylation = IP1. Higher concentrations of PTH(1-34) are reportedly required for stimulation of hPTH1R-mediated Gq- than for Gs-signaling (Taylor and Tovey, 2012), which was also observed in this study. The significance of the observation that PTHrP(1-34) and PTHrP(1-37) are partial agonists in the ERK1/2 phosphorylation assay is unclear. PTHrP(1-34) was found to promote similar levels of receptor internalization as PTH(1-34).
Evaluation of N-terminally modified peptides, including [Trp1]PTHrP(1-36), [Tyr1]PTH(1-34), and PTH(2-34), indicates that the first native AA of either PTH and PTHrP is not required for stimulation of Gs agonist activity but plays a critical role in facilitating stimulation of Gq, β-arrestin recruitment, ERK1/2 phosphorylation, and internalization. In contrast, Val2 of PTH is required for agonist activity in all hPTH1R functional assays, as previously reported for Gs- and Gq-signaling (Takasu et al., 1999). The inability of [Bpa1]PTHrP(1-36) to promote hPTH1R internalization has been reported by others (Bisello et al., 2002). Results of this study do not support previous claims that [Trp1]PTHrP(1-36) and [Bpa1]PTHrP(1-36) are solely Gs-biased agonists (Bisello et al., 2002, Gesty-Palmer et al., 2006) and that [D-Trp12,Tyr34]PTH(7-34)NH2 is a β-arrestin–biased agonist capable of stimulating ERK1/2 phosphorylation (Gesty-Palmer et al., 2006, 2009). Furthermore, PTH(7-34)NH2 does not promote receptor internalization in CHO-K1 cells, as reported for DTC cells expressing hPTH1R (Sneddon et al., 2004). The differences between the results of these studies may be attributable to a combination of expression of hPTH1R in different cell backgrounds (e.g., CHO-K1 versus HEK293), different levels of receptor expression, or use of different assay methods. For example, β-arrestin recruitment has been previously measured by translocation of GFP-tagged arrestin (Bisello et al., 2002; Gesty-Palmer et al., 2006), whereas the β-arrestin recruitment assay directly measures binding of β-arrestin to agonist activated receptor and is not impacted by receptor reserve and agonist EC50 values correlate well with receptor affinity, enabling a clear separation of affinity and efficacy of GPCR agonists (Nickolls et al., 2011).
Evaluation of the agonist activity of C-terminally modified PTH peptides indicates that removal of the last six AA residues of PTH(1-34) does not affect ability to stimulate Gs-signaling but does decrease agonist activity in all the other hPTH1R assays. Furthermore, the efficacy of peptides with truncation past PTH(1-31) to stimulate hPTH1R-mediated ERK phosphorylation and receptor internalization is decreased and increased, respectively. In contrast, the agonist profile of MPTH(1-28) is similar to that of PTH(1-34), suggesting that N-terminal AA substitutions increasing α-helicity of the N-terminal domain (Okazaki et al., 2008) help maintain agonist activity of PTH(1-28) analogs. However, introduction of the same AA substitutions into more extensive C-terminal truncated peptides helps predominately to maintain agonist activity to stimulate Gs-signaling, as reported by others (Shimizu et al., 2001, 2004; Caporale et al., 2009, 2010; Neerup et al., 2011). The structural basis for the greater capacity of PTH(1-28), [Gly1,Arg19]PTH(1-28), ZP2307, and MPTH(1-14) to stimulate receptor internalization is unclear but may be a result of C-terminal AAs present in PTH(1-31) providing a restraint on receptor internalization.
The structural basis for the differences in the negative impact of PTH/PTHrP peptide N- and C-terminal modifications on stimulation of PTH1R-mediated Gs-signaling, compared with other signaling pathways, is unclear. However, our experimental results are consistent with previous reports that the first two AAs play an important role in mediating peptide activation of the J-domain and Gs-signaling (Gensure et al., 2005), in addition to activation of other hPTH1R signaling pathways evaluated. Although the high resolution X-ray crystal structure of PTH(15-34) and PTHrP(12-34) bound to the extracellular domain of the hPTH1R receptor have been reported (Pioszak and Xu, 2008, 2009), structural details regarding the interaction between the peptide N-terminal and receptor J-domains are not available. Although the two-site model of PTH1R activation posits that the C-terminal domain of PTH (AA 23-34) predominately mediates binding of peptide to the extracellular N terminus (Vilardaga et al., 2011), our results also suggest that the C-terminal domain also plays an important role in activation of all hPTH1R signaling pathways and receptor internalization. Vilaradaga et al. (2011) have suggested that the two-site model may be an oversimplification and hypothesize that bound peptide may adopt a folded, rather than an extended, conformation after binding to receptor and that the ligand-binding surface of receptor N- and J-domains are in close proximity to each other in the agonist bound state. Additional structural studies of PTH(1-34) and PTHrP(1-34) bound to full-length hPTH1R will help to further elucidate the roles that agonist peptide N- and C-terminal domains play in activating the J-domain.
Patterns of hPTH1R-Mediated Signaling by PTH and PTHrP Peptide Analogs.
Previous in vivo studies using reported biased PTH/PTHrP peptide analogs suggest that PTH1R-mediated Gs-signaling plays a predominant role in anabolic actions of intermittent PTH(1-34) (Rixon et al., 1994; Rhee et al., 2006; Yang et al., 2007), whereas Gq-signaling does not. Furthermore, [D-Trp12,Tyr34]PTH(7-34)NH2, a proposed β-arrestin biased agonist, is reported to promote bone formation that is lost with ablation of β-arrestin (Gesty-Palmer et al., 2009). However, we observe that patterns of hPTH1R-mediated signaling promoted by some of these peptide analogs are more complex or different from those in previous reports. During revision of this article, van der Lee et al. (2012) reported that [D-Trp12,Tyr34]PTH(7-34)NH2 does not stimulate β-arrestin recruitment or ERK1/2 phoshorylation in CHO-K1 cells expressing recombinant hPTH1R but does antagonize agonist response, similar to results of this study (van der Lee et al., 2013). Thus, inconsistencies regarding therapeutically relevant hPTH1R signaling required for bone formation exist and further studies using PTH/PTHrP peptide analogs with well-defined signaling patterns are required to further address this important question.
Several PTH/PTHrP peptide analogs evaluated in this study promote differential PTH1R-mediated signaling consistent with biased agonism; however, the contribution of differences in strength of signaling cannot be ruled out. The use of the same CHO-K1 cells for all functional assays, with the exception of internalization, lessens the possibility that differences are solely attributable to the latter phenomenon. All of the peptides promoting differential signaling activated Gs-signaling, and none displayed a reversal in potency or efficacy rank order in the other PTH1R functional assays, which is considered to be a hallmark of biased GPCR signaling but is not a requirement (Kenakin 2007, 2011; Urban et al., 2007). One question arising from this study is whether biased PTH/PTHrP peptide analogs can be designed that exclusively activate other signaling pathways but not Gs-signaling.
A prerequisite to additional efficacy studies using well-defined signaling biased PTH/PTHrP peptide analogs to ascertain therapeutically relevant hPTH1R signaling pathways will be to confirm biased signaling patterns of peptide analogs with use of therapeutically important osteoblasts. In addition, in vitro efficacy studies of the effect of different biased peptides on osteoblast proliferation, differentiation, apoptosis, and function could then be used to guide which biased PTH/PTHrP peptides should be prioritized for in vivo studies of rodent bone formation. Potentially useful peptide analogs for this purpose could include [Trp1]PTHrP, [Tyr1]PTH(1-34), [Bpa1]PTHrP(1-36), MPTH(1-14), and [Aib1,3,Nle8,Gln10,Har11]PTH(1-11). In addition, we have recently discovered a PTH analog with potent Gs-biased agonist activity that would also be useful for these studies (Gineste et al., personal communication). After therapeutically desirable PTH1R-mediated signaling is further established in in vivo models of osteoporosis, peptides promoting restricted desirable increases in bone formation without resorption could potentially be designed to provide a safer and more effective treatment of osteoporosis. Indeed, discovery of biased GPCR agonists with superior therapeutic profiles has recently created a valuable opportunity for the pharmaceutical industry (Reiter et al., 2012).
Acknowledgments
The authors thank Cyrille Gineste, Amit Galande, and Patrick Kibler for the synthesis and purification of [Trp1]PTHrP(1-36), [Bpa1]PTHrP (1-36), PTH(1-28), ZP2307, MPTH(1-14), and [Aib1,3,Nle8,Gln10, Har11]PTH(1-11)NH2, and Gregory Stauber for help with data analyses.
Authorship Contributions
Participated in research design: Cupp, Nayak, Adem, Thomsen.
Conducted experiments: Cupp.
Performed data analysis: Cupp, Nayak, Thomsen.
Wrote or contributed to the manuscript: Thomsen.
Footnotes
- Received September 6, 2012.
- Accepted March 14, 2013.
↵This article has supplemental material available at jpet.aspetjournals.org.
Abbreviations
- AA
- amino acid
- AFU
- arbitrary fluorescence units
- ALU
- arbitrary luminescent units
- CHO
- Chinese hamster ovary (cells)
- DMSO
- dimethyl sulfoxide
- EA
- enzyme acceptor
- ERK
- extracellular signal-related kinase
- GPCR
- G protein–coupled receptor
- hPTH1R
- human PTH1R
- IP1
- inositol monophosphate
- PKA
- protein kinase A
- PKC
- protein kinase C
- PLCβ
- phospholipase Cβ
- PTH
- parathyroid hormone
- PTH1R
- PTH1 receptor
- PTHrP
- PTH-related peptide
- RH
- relative humidity
- TRF
- time-resolved fluorescence
- Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics