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Biased GPCR signaling by the native parathyroid hormone–related protein 1 to 141 relative to its N-terminal fragment 1 to 36

Open AccessPublished:August 03, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102332
      The parathyroid hormone (PTH)–related protein (PTHrP) is indispensable for the development of mammary glands, placental calcium ion transport, tooth eruption, bone formation and bone remodeling, and causes hypercalcemia in patients with malignancy. Although mature forms of PTHrP in the body consist of splice variants of 139, 141, and 173 amino acids, our current understanding on how endogenous PTHrP transduces signals through its cognate G-protein coupled receptor (GPCR), the PTH type 1 receptor (PTHR), is largely derived from studies done with its N-terminal fragment, PTHrP1-36. Here, we demonstrate using various fluorescence imaging approaches at the single cell level to measure kinetics of (i) receptor activation, (ii) receptor signaling via Gs and Gq, and (iii) receptor internalization and recycling that the native PTHrP1-141 displays biased agonist signaling properties that are not mimicked by PTHrP1-36. Although PTHrP1–36 induces transient cAMP production, acute intracellular Ca2+ (iCa2+) release and β-arrestin recruitment mediated by ligand–PTHR interactions at the plasma membrane, PTHrP1-141 triggers sustained cAMP signaling from the plasma membrane and fails to stimulate iCa2+ release and recruit β-arrestin. Furthermore, we show that the molecular basis for biased signaling differences between PTHrP1-36 and properties of native PTHrP1-141 are caused by the stabilization of a singular PTHR conformation and PTHrP1-141 sensitivity to heparin, a sulfated glycosaminoglycan. Taken together, our results contribute to a better understanding of the biased signaling process of a native protein hormone acting in conjunction with a GPCR.

      Keywords

      Abbreviations:

      βarr (β-arrestin), GAG (glycosaminoglycan), PTH (parathyroid hormone), PTHR (PTH receptor)
      Upon its activation, the parathyroid hormone (PTH) receptor (PTHR) triggers both Gs/cAMP/PKA and Gq/Ca2+/PKC signaling cascades. Developments in recording GPCR-signaling cascade in individual cells in real time using optical approaches during the decade of the ‘00s (
      • Vilardaga J.P.
      • Bunemann M.
      • Krasel C.
      • Castro M.
      • Lohse M.J.
      Measurement of the millisecond activation switch of G protein-coupled receptors in living cells.
      ,
      • Vilardaga J.P.
      • Bünemann M.
      • Feinstein T.N.
      • Lambert N.
      • Nikolaev V.O.
      • Engelhardt S.
      • et al.
      GPCR and G proteins: drug efficacy and activation in live cells.
      ) have revealed that PTH1-34 and PTHrP1-36 differ markedly by the duration and cellular localization of the cAMP response (
      • Ferrandon S.
      • Feinstein T.N.
      • Castro M.
      • Wang B.
      • Bouley R.
      • Potts J.T.
      • et al.
      Sustained cyclic AMP production by parathyroid hormone receptor endocytosis.
      ). Brief stimulation with PTHrP1-36 induces only transient cAMP production from the cell surface that is rapidly desensitized upon recruitment of β-arrestins (βarrs), cytosolic adapter proteins that canonically act to occlude further G protein coupling and promote translocation of the ligand–receptor complex from the cell surface to early endosomes. In contrast, PTH1-34 causes an additional sustained phase of cAMP generation via PTH–PTHR–βarr complexes that remain active in early endosomes. Thus, this distinction in the spatiotemporal cAMP profiles of PTH and PTHrP was proposed to be the underlying determinant responsible for their biological specificity.
      Mature forms of PTH and PTHrP are originally synthesized and secreted as 84 aa and 141 aa proteins, respectively. Early reports demonstrating that their respective N-terminal part, PTH1-34 and PTHrP1-36, retain their full capacity to stimulate adenylyl cyclase in cAMP accumulation assays led to the utilization of these N-terminal fragments in most studies. Indeed, it was PTH1-34 and PTHrP1-36 that were used in the aforementioned work that revealed differences in the time courses and subcellular locations of cAMP production by these two peptides. In contrast to these earlier findings of transient signaling by PTHrP1-36, a recent publication proposed sustained endosomal cAMP generation induced by full-length PTHrP1-141 (
      • Ho P.W.M.
      • Chan A.S.
      • Pavlos N.J.
      • Sims N.A.
      • Martin T.J.
      Brief exposure to full length parathyroid hormone-related protein (PTHrP) causes persistent generation of cyclic AMP through an endocytosis-dependent mechanism.
      ). The authors employed a combination of radioimmunoassays and chemical inhibitors to suggest that PTHrP1-141 induces prolonged cAMP signaling in an endocytosis-dependent manner analogous to that observed for PTH1-34; however, cAMP experiments were performed in the presence of phosphodiesterase inhibition, which provided a measure of the cumulative levels of cAMP produced during a defined time interval, as opposed to the dynamic levels of cAMP that result from the net effects of its production and breakdown. Furthermore, the chemical compounds utilized to inhibit endocytosis generated inconsistent results with experiments showing no reduction of sustained cAMP responses induced by PTHrP1-141 or PTH1-34, while others showed only reduction for PTHrP1-141 but not for PTH1-34. Reduction of PTH1-34-induced sustained cAMP response by blocking receptor endocytosis is expected given this ligand’s established ability to signal via internalized PTHR from early endosomes (
      • Ferrandon S.
      • Feinstein T.N.
      • Castro M.
      • Wang B.
      • Bouley R.
      • Potts J.T.
      • et al.
      Sustained cyclic AMP production by parathyroid hormone receptor endocytosis.
      ,
      • Feinstein T.N.
      • Wehbi V.L.
      • Ardura J.A.
      • Wheeler D.S.
      • Ferrandon S.
      • Gardella T.J.
      • et al.
      Retromer terminates the generation of cAMP by internalized PTH receptors.
      ,
      • Gidon A.
      • Al-Bataineh M.M.
      • Jean-Alphonse F.G.
      • Stevenson H.P.
      • Watanabe T.
      • Louet C.
      • et al.
      Endosomal GPCR signaling turned off by negative feedback actions of PKA and v-ATPase.
      ,
      • Jean-Alphonse F.G.
      • Wehbi V.L.
      • Chen J.
      • Noda M.
      • Taboas J.M.
      • Xiao K.
      • et al.
      beta2-adrenergic receptor control of endosomal PTH receptor signaling via Gbetagamma.
      ,
      • White A.D.
      • Peña K.A.
      • Clark L.J.
      • Maria C.S.
      • Liu S.
      • Jean-Alphonse F.G.
      • et al.
      Spatial bias in cAMP generation determines biological responses to PTH type 1 receptor activation.
      ,
      • White A.D.
      • ean-Alphonse F.G.
      • Fang F.
      • Peña K.A.
      • Liu S.
      • König G.M.
      • et al.
      Gq/11-dependent regulation of endosomal cAMP generation by parathyroid hormone class B GPCR.
      ). These considerations motivated the necessity to implement alternative methods that permit analysis of real-time cAMP response kinetics in real time in single cells. The results unveil the mechanism by which PTHrP1-141 engages in sustained signaling and how this differs from the transient effects observed with the N-terminal fragment PTHrP1-36.

      Results and discussion

      We utilized FRET to record real-time courses of cAMP production in single HEK293 cell stably expressing PTHR (HEK-PTHR). We found that brief stimulation with PTHrP1-141 induced a sustained cAMP response that was similar in both magnitude and duration to that induced by PTH1-84 or PTH1-34 and clearly distinct from the short-lived cAMP response mediated by PTHrP1-36 (Figs. 1A and S1). We next applied Glo-sensor cAMP accumulation assays to verify that time courses of sustained cAMP production mediated by the two native hormones, PTH1-84 and PTHrP1-141, were similar (Fig. 1B) and without a significant difference in the hormone concentration dependence (Fig. 1, C and D). We observed a striking inability of PTHrP1-141 to efficiently induce the release of intracellular calcium (iCa2+) from the endoplasmic reticulum (Fig. 1, E and F), indicating defective Gq activation by PTHrP1-141. We have previously shown that Gq activation is required for endosomal cAMP generation by PTH1-34 (
      • White A.D.
      • ean-Alphonse F.G.
      • Fang F.
      • Peña K.A.
      • Liu S.
      • König G.M.
      • et al.
      Gq/11-dependent regulation of endosomal cAMP generation by parathyroid hormone class B GPCR.
      ), suggesting a differential location of cAMP generation by this ligand. Moreover, the molecular basis for the failure of PTHrP1-141 to mimic cAMP and iCa2+ signaling responses mediated by PTHrP1-36 were unlikely to be caused by different binding affinities to either G protein coupled (RG) or uncoupled (R0) states of PTHR (Fig. 1, G and H) but were rather due to the stabilization of a distinct receptor conformation. We tested this theory by using cells expressing a FRET-based PTHR sensor (scheme in Fig. 1I). Time-resolved determination of intramolecular FRET changes recorded from single cells allows the analysis of the kinetics of receptor activation in response to ligand binding (
      • Vilardaga J.P.
      • Bunemann M.
      • Krasel C.
      • Castro M.
      • Lohse M.J.
      Measurement of the millisecond activation switch of G protein-coupled receptors in living cells.
      ). A decrease in FRET mediated by an agonist reflects receptor switching from an inactive to an active conformation, and distinct time-constants of receptor activation measured for a saturating concentration of agonists indicate the stabilization of distinct signaling receptor conformations (
      • Vilardaga J.P.
      • Bunemann M.
      • Krasel C.
      • Castro M.
      • Lohse M.J.
      Measurement of the millisecond activation switch of G protein-coupled receptors in living cells.
      ,
      • Ferrandon S.
      • Feinstein T.N.
      • Castro M.
      • Wang B.
      • Bouley R.
      • Potts J.T.
      • et al.
      Sustained cyclic AMP production by parathyroid hormone receptor endocytosis.
      ,
      • Vilardaga J.P.
      Studying ligand efficacy at G protein-coupled receptors using FRET.
      ). As expected, perfusion of a saturating concentration of PTH1-34, PTHrP1-36, or PTHrP1-141 to individual cells triggered a decrease in FRET; however, the significantly distinct time constants (τ) for receptor activation indicated the stabilization of distinct PTHR conformations (Fig. 1, I and J).
      Figure thumbnail gr1
      Figure 1Signaling properties of PTHrP1-141. A, time courses of cAMP in single HEK293 cells stimulated for 30 s with 1 nM ligands. Data are the mean ± SEM of n = 37 (PTHrP1-36), n = 21 (PTHrP1-141), n = 6 (PTH1-84), and n = 7 (PTH1-34) cells. B, time courses of cAMP in HEK-293 cells after washout of ligands measured by Glo-sensor assay. Data are the mean ± SEM of n = 3 experiments. C and D, relationship between cAMP responses in HEK-293 cells after washout of a range of ligand concentrations (C) and corresponding EC50 values (D). Data represent the integrated response determined by measuring the area under the curve of experiments shown in panel (B) and are the mean ± SD of n = 3 experiments. ns, not significant with p = 0.097 by t test. E, intracellular Ca2+ mobilization measurements in single HEK-293 cells stably expressing PTHR. Data are the mean ± SEM for n = 44 (PTH1-34), n = 42 (PTHrP1-36), and n = 48 (PTHrP1-141) cells. F, scatter plots with the mean ± SD of data shown in panel (C). ∗∗∗∗p < 0.0001 determined by one-way ANOVA with Tukey–Kramer post hoc test. (G and H) competition binding at equilibrium with [125I]-PTH1-15 and [125I]-PTH1-34 as radioligands to detect the RG (E) and R0 (F) states of PTHR, respectively. Data are mean ± SD from N = 2 independent experiments with duplicate wells for each concentration. I and J, kinetics of PTHR activation. Normalized activation kinetics of PTHR determined by FRET ratio changes from HEK293 cells expressing the receptor sensor (scheme) (G), and time constant (τ) of PTHR activation determined by fitting curves in panel (A) to a monoexponential decrease (H). Mean ± SEM of n = 25 (PTH1-34), n = 6 (PTHrP1-36), and n = 9 (PTHrP1-141) cells. ∗p = 0.0114, ∗∗∗p = 0.0003, and ∗∗∗∗p < 0.0001 determined by one-way ANOVA with Tukey–Kramer post hoc test. PTH, parathyroid hormone; PTHR, PTH receptor.
      To assess the role of βarr recruitment, we measured PTHR–βarr interactions via FRET in cells transiently expressing PTHRCFP and βarr-2YFP. The βarr2 isoform was randomly selected, given that earlier studies demonstrated that PTH1-34 and PTHrP1-36 displayed equal potencies (EC50 values) for recruitment of both β-arr1 and β-arr2 (
      • White A.D.
      • Peña K.A.
      • Clark L.J.
      • Maria C.S.
      • Liu S.
      • Jean-Alphonse F.G.
      • et al.
      Spatial bias in cAMP generation determines biological responses to PTH type 1 receptor activation.
      ,
      • Liu S.
      • Jean-Alphonse F.G.
      • White A.D.
      • Wootten D.
      • Sexton P.M.
      • Gardella T.J.
      • et al.
      Use of backbone modification to enlarge the spatiotemporal diversity of parathyroid hormone receptor-1 signaling via biased agonism.
      ,
      • Vilardaga J.P.
      • Krasel C.
      • Chauvin S.
      • Bambino T.
      • Lohse M.J.
      • Nissenson R.A.
      Internalization determinants of the parathyroid hormone receptor differentially regulate beta-arrestin/receptor association.
      ). Consistent with previous studies, addition of PTH1-34 resulted in significant association of βarr with the receptor that was stably maintained following ligand washout (Fig. 2A). In contrast, analogous experiments using PTHrP1-141 failed to promote this interaction (Fig. 2A), suggesting that the sustained signaling observed for PTHrP1-141 occurs in a βarr-independent manner. This finding led us to test the role of receptor internalization, a key step in PTHR endosomal signaling. Measurements of receptor internalization and recycling in single cells stably expressing PTHRSEP, the PTHR N-terminally tagged with a pH-sensitive GFP (super-ecliptic pHluorin SEP) that exhibits fluorescence intensity reduction in the acidic environment encountered in endosomes (scheme in Fig. 2B), showed reduced internalization and faster recycling in response to PTHrP1-141 or PTHrP1-36 when compared to PTH1-34 (Figs. 2B and S2). We next determined whether internalized PTHrP1-141-PTHR can signal via cAMP. We have previously shown that expression of a dominant-negative dynamin mutant, DynK44A, effectively blocks translocation of PTH–PTHR complexes from the cell surface and blunts the sustained phase of cAMP generation without affecting the forskolin response (
      • Ferrandon S.
      • Feinstein T.N.
      • Castro M.
      • Wang B.
      • Bouley R.
      • Potts J.T.
      • et al.
      Sustained cyclic AMP production by parathyroid hormone receptor endocytosis.
      ,
      • White A.D.
      • Peña K.A.
      • Clark L.J.
      • Maria C.S.
      • Liu S.
      • Jean-Alphonse F.G.
      • et al.
      Spatial bias in cAMP generation determines biological responses to PTH type 1 receptor activation.
      ). Accordingly, we compared the cAMP response following brief stimulation with PTHrP1-141 in HEK-PTHR control cells and those transiently expressing DynK44A fused to a red fluorescent protein (DynK44ARFP) (Fig. 2C). Strikingly, blockade of receptor internalization significantly reduced the magnitude and duration of cAMP production by PTH1-34 (Fig. 2, C and E) but had no effect on cAMP mediated by PTHrP1-141 (Fig. 2, D and E), indicating that native PTHrP does not promote sustained signaling in an endocytosis-dependent manner. We recently reported on the development of Gs-biased PTH analogs that stimulate sustained cAMP production exclusively from the cell surface due to retention of active ligand–receptor complexes at the cell surface. This was experimentally confirmed via cAMP time courses using a cell-impermeable PTHR antagonist, which completely abolished the sustained phase of cAMP generation for GS-biased peptides but not for PTH1-34, consistent with its ability to signal from intracellular compartments (
      • White A.D.
      • Peña K.A.
      • Clark L.J.
      • Maria C.S.
      • Liu S.
      • Jean-Alphonse F.G.
      • et al.
      Spatial bias in cAMP generation determines biological responses to PTH type 1 receptor activation.
      ). We thus utilized this same approach to test whether PTHrP1-141 likewise induces prolonged cAMP signaling via ligand–receptor complexes that are localized to the cell surface. Indeed, addition of the cell-impermeable antagonist at 15 min following agonist washout rapidly reduced cAMP levels to baseline in cells treated with PTHrP1-141 but had no effect in those stimulated with PTH1-34 (Fig. 2, F and G). These findings demonstrate that PTHrP1-141 promotes sustained cAMP responses via active ligand–receptor complexes localized to the cell surface, which appear inconsistent with experiments showing receptor internalization.
      Figure thumbnail gr2
      Figure 2Endosomal cAMP signaling by PTHrP1-141. A, time course of β-arrestin interaction with PTHR in HEK293 cells transiently expressing PTHRCFP and βarr-2YFP treated with 10 nM PTH1-34 (black) or PTHrP1-141 (red) for 30 s. Data are the mean ± SEM for n = 40 (PTH1-34) and n = 49 (PTHrP1-141) cells. The scatter plot shows the mean ± SD of the integrated response determined by measuring the area under the curve (a.u.c.) ∗∗∗∗p < 0.0001 by t test. B, time courses of internalization and recycling of PTHR tagged with super-ecliptic pHluorin (PTHRSEP) in response to 100 nM ligand measured by time-lapse confocal microscopy in single cells. The schematic illustrates the measured values. Data are mean ± SEM from n = 12 (PTH1-34) and n = 51 (PTHrP1-141) cells. C–E, time courses of cAMP in single HEK-293 PTHR cells transiently expressing with DynK44ARFP compared to control in response to PTH1-34 (C) and PTHrP1-141 (D). Data are the mean ± SEM for n = 14 cells (PTH1-34 control), n = 9 cells (PTH1-34 DynK44A), n = 8 (PTHrP1-141 control), and n = 12 (PTHrP1-141 DynK44A) cells. E, the scatter plot represents the area under the curve (a.u.c.) corresponding to individual values and the mean ± SD. ∗∗p = 0.0017 determined by one-way ANOVA with Tukey–Kramer post hoc test. F, time courses of cAMP in single HEK-293 PTHR cells stimulated for 30 s with 10 nM PTH1-34 (black) or PTHrP1-141. Data are the mean ± SEM of n = 32 (PTH1-34) cells and n = 46 (PTHrP1-141) cells. G, similar experiments as in panel (E) with addition of cell-impermeable PTHR antagonist 15 min after washout of PTH1-34 or PTHrP1-141. Data are the mean ± SEM of n = 50 (PTH1-34) cells and n = 37 (PTHrP1-141) cells. PTH, parathyroid hormone; PTHR, PTH receptor.
      To reconcile this apparent incompatibility, we hypothesized that the highly positively charged domain of PTHrP1-141 (88KKKKGKPGKRKEQEKKKRRTR108), not present in PTHrP1-36 or PTH, permits the hormone to attach to the cell surface via interactions with polyanionic glycosaminoglycans (GAGs) present on membrane glycoproteins such as heparan sulfate proteoglycan. Consistent with this theory was the significant reduction in the magnitude and duration of cAMP production in response to PTHrP1-141 in the presence of soluble heparin used as a decoy to prevent potential PTHrP1-141 and GAGs interactions (Fig. 3, A and B). The selective effect of heparin was verified by its lack of inhibitory action on cAMP induced by either PTH1-34 or PTHrP1-36 (Fig. 3A, and Table 1).
      Figure thumbnail gr3
      Figure 3Effect of heparin on cAMP production. A, cAMP time courses in single HEK-293 PTHR cells in response to 1 nM ligands preincubated with 10 nM heparin. Data are the mean ± SEM of n = 51 (control) and n = 52 (heparin) cells for PTHrP1-141; n = 25 (control) and n = 39 (heparin) cells for PTHrP1-36; n = 39 (control) and n =46 (heparin) cells for PTH1-34. The statistical analysis is in . B, corresponding scatter plots representing the area under the curve (a.u.c) of individual values from (A). ∗∗∗∗p < 0.0001 determined by t test. C, proposed mechanism for location-biased signaling of native PTHrP1-141. The continuous cAMP signaling mediated by PTHrP1-141 can be controlled by plasma membrane–anchored glycosaminoglycans that hypothetically retain PTHrP1-141 at the cell surface thus permitting reactivation of recycled receptors. Created with BioRender.com. PTH, parathyroid hormone; PTHR, PTH receptor.
      Table 1Effect of heparin on cAMP production
      LigandsControlHep, 1 nMp ValueControlHep, 10 nMp Value
      PTHrP1-141100 ± 37 (33)51 ± 30 (24)< 0.0001100 ± 37 (51)38 ± 22 (52)< 0.0001
      PTHrP1-36100 ± 26 (15)129 ± 44 (9)0.056100 ± 47 (25)92 ± 33 (39)0.39
      PTH1-34100 ± 44 (43)91 ± 31 (23)0.38100 ± 25 (39)91 ± 35 (46)0.18
      The area under the curve (a.u.c) from data in Figure 3. Mean value ± SD of (N) experiments with p values determined by t test.
      Abbreviations: Hep, heparin.
      Collectively, these data prompt a reinterpretation of our previous understanding on how hormones act on the PTHR by providing compelling evidence that native PTHrP1-141 is biased toward sustained PTHR signaling via cAMP at the plasma membrane. The results support a model where PTHrP1-141 stabilizes an active receptor conformation that impairs βarr coupling and Gq signaling possibly through the interaction with GAG. Future experiments are needed for an extended characterization of PTHrP and GAG interaction as a possible means to reactivate recycled receptor by the cell surface–anchored hormone (Fig. 3C).

      Experimental procedures

      Materials and methods are detailed in SI Appendix.

      Data availability

      Source data are stored in Excel 2013 and will be deposited in the institutional repository of the University of Pittsburgh (http://d-scholarship.pitt.edu/).

      Supporting information

      This article contains supporting information (
      • Vilardaga J.P.
      • Bunemann M.
      • Krasel C.
      • Castro M.
      • Lohse M.J.
      Measurement of the millisecond activation switch of G protein-coupled receptors in living cells.
      ,
      • Feinstein T.N.
      • Wehbi V.L.
      • Ardura J.A.
      • Wheeler D.S.
      • Ferrandon S.
      • Gardella T.J.
      • et al.
      Retromer terminates the generation of cAMP by internalized PTH receptors.
      ,
      • Gidon A.
      • Al-Bataineh M.M.
      • Jean-Alphonse F.G.
      • Stevenson H.P.
      • Watanabe T.
      • Louet C.
      • et al.
      Endosomal GPCR signaling turned off by negative feedback actions of PKA and v-ATPase.
      ,
      • Vilardaga J.P.
      Studying ligand efficacy at G protein-coupled receptors using FRET.
      ,
      • Hammonds R.G.
      • McKay Jr., P.
      • Winslow G.A.
      • Diefenbach-Jagger H.
      • Grill V.
      • Glatz J.
      • et al.
      Purification and characterization of recombinant human parathyroid hormone-related protein.
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      • Li J.
      • Dong S.
      • Townsend S.D.
      • Dean T.
      • Gardella T.J.
      • Danishefsky S.J.
      • et al.
      Chemistry as an expanding resource in protein science: fully synthetic and fully active human parathyroid hormone-related protein (1-141).
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      • Wehbi V.L.
      • Stevenson H.P.
      • Feinstein T.N.
      • Calero G.
      • Romero G.
      • Vilardaga J.P.
      Noncanonical GPCR signaling arising from a PTH receptor-arrestin-Gbetagamma complex.
      ,
      • Castro M.
      • Dicker F.
      • Vilardaga J.P.
      • Krasel C.
      • Bernhardt M.
      • Lohse M.J.
      Dual regulation of the parathyroid hormone (PTH)/PTH-related peptide receptor signaling by protein kinase C and beta-arrestins.
      ,
      • Dean T.
      • Vilardaga J.P.
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      • Gardella T.J.
      Altered selectivity of parathyroid hormone (PTH) and PTH-related protein (PTHrP) for distinct conformations of the PTH/PTHrP receptor.
      ,
      • McGarvey J.C.
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      Actin-sorting nexin 27 (SNX27)-retromer complex mediates rapid parathyroid hormone receptor recycling.
      ).

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      We thank Dr T. John Martin for providing the PTH1-141 protein to initiate this work.

      Author contributions

      I. S. and J. P. V. conceptualization; J. P. V. methodology; K. A. P., F. G. J. A., T. J. G., and J. P. V. validation; K. A. P., A. D. W., S. S., I. P. C., and F. G. J. A. investigation; A. D. W. and J. P. V. writing–original draft; K. A. P. and J. P. V. writing–review & editing; S. S. visualization; T. J. G. and J. P. V. supervision.

      Funding and additional information

      This research was supported by grant Award Numbers T32GM133332 (to S. S.) from the National Institute of General Medical Sciences (NIGMS), DK116780 and DK122259 (to J. P. V.) from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), United States of the US National Institutes of Health (NIH), United States, R01HD100468 from the NIH, and grant number CHE-9808188 (to the Center of Molecular Analysis, Department of Chemistry, Carnegie Mellon University) from the National Science Foundation ( NSF ), United States. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

      Supporting information

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