Biased GPCR signaling by the native parathyroid hormone–related protein 1 to 141 relative to its N-terminal fragment 1 to 36
2022; Elsevier BV; Volume: 298; Issue: 9 Linguagem: Inglês
10.1016/j.jbc.2022.102332
ISSN1083-351X
AutoresKarina A. Peña, Alex D. White, Sofya Savransky, Ignacio Portales Castillo, Frédéric Jean‐Alphonse, Thomas J. Gardella, Ieva Sutkevičiu̅tė, Jean‐Pierre Vilardaga,
Tópico(s)Metabolism, Diabetes, and Cancer
ResumoThe 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. 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. 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 (1Vilardaga 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.Nat. Biotechnol. 2003; 21: 807-812Crossref PubMed Scopus (359) Google Scholar, 2Vilardaga 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.Mol. Endocrinol. 2009; 23: 590-599Crossref PubMed Scopus (68) Google Scholar) have revealed that PTH1-34 and PTHrP1-36 differ markedly by the duration and cellular localization of the cAMP response (3Ferrandon S. Feinstein T.N. Castro M. Wang B. Bouley R. Potts J.T. et al.Sustained cyclic AMP production by parathyroid hormone receptor endocytosis.Nat. Chem. Biol. 2009; 5: 734-742Crossref PubMed Scopus (396) Google Scholar). 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 (4Ho 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.Biochem. Pharmacol. 2019; 169113627Crossref PubMed Scopus (6) Google Scholar). 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 (3Ferrandon S. Feinstein T.N. Castro M. Wang B. Bouley R. Potts J.T. et al.Sustained cyclic AMP production by parathyroid hormone receptor endocytosis.Nat. Chem. Biol. 2009; 5: 734-742Crossref PubMed Scopus (396) Google Scholar, 5Feinstein 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.Nat. Chem. Biol. 2011; 7: 278-284Crossref PubMed Scopus (169) Google Scholar, 6Gidon 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.Nat. Chem. Biol. 2014; 10: 707-709Crossref PubMed Scopus (58) Google Scholar, 7Jean-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.Nat. Chem. Biol. 2017; 13: 259-261Crossref PubMed Scopus (38) Google Scholar, 8White 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.Sci. Signal. 2021; 14eabc5944Crossref PubMed Scopus (8) Google Scholar, 9White 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.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 7455-7460Crossref PubMed Scopus (18) Google Scholar). 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 discussionWe 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 (9White 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.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 7455-7460Crossref PubMed Scopus (18) Google Scholar), 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 (1Vilardaga 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.Nat. Biotechnol. 2003; 21: 807-812Crossref PubMed Scopus (359) Google Scholar). 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 (1Vilardaga 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.Nat. Biotechnol. 2003; 21: 807-812Crossref PubMed Scopus (359) Google Scholar, 3Ferrandon S. Feinstein T.N. Castro M. Wang B. Bouley R. Potts J.T. et al.Sustained cyclic AMP production by parathyroid hormone receptor endocytosis.Nat. Chem. Biol. 2009; 5: 734-742Crossref PubMed Scopus (396) Google Scholar, 10Vilardaga J.P. Studying ligand efficacy at G protein-coupled receptors using FRET.Met. Mol. Biol. 2011; 756: 133-148Crossref PubMed Scopus (18) Google Scholar). 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).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 (8White 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.Sci. Signal. 2021; 14eabc5944Crossref PubMed Scopus (8) Google Scholar, 11Liu 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.J. Am. Chem. Soc. 2019; 141: 14486-14490Crossref PubMed Scopus (14) Google Scholar, 12Vilardaga 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.J. Biol. Chem. 2002; 277: 8121-8129Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). 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 (3Ferrandon S. Feinstein T.N. Castro M. Wang B. Bouley R. Potts J.T. et al.Sustained cyclic AMP production by parathyroid hormone receptor endocytosis.Nat. Chem. Biol. 2009; 5: 734-742Crossref PubMed Scopus (396) Google Scholar, 8White 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.Sci. Signal. 2021; 14eabc5944Crossref PubMed Scopus (8) Google Scholar). 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 (8White 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.Sci. Signal. 2021; 14eabc5944Crossref PubMed Scopus (8) Google Scholar). 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 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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 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 Table 1. 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 1Effect of heparin on cAMP productionLigandsControlHep, 1 nMp ValueControlHep, 10 nMp ValuePTHrP1-141100 ± 37 (33)51 ± 30 (24)< 0.0001100 ± 37 (51)38 ± 22 (52)< 0.0001PTHrP1-36100 ± 26 (15)129 ± 44 (9)0.056100 ± 47 (25)92 ± 33 (39)0.39PTH1-34100 ± 44 (43)91 ± 31 (23)0.38100 ± 25 (39)91 ± 35 (46)0.18The 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. Open table in a new tab 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 proceduresMaterials and methods are detailed in SI Appendix.Data availabilitySource 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 informationThis article contains supporting information (1Vilardaga 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.Nat. Biotechnol. 2003; 21: 807-812Crossref PubMed Scopus (359) Google Scholar, 5Feinstein 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.Nat. Chem. Biol. 2011; 7: 278-284Crossref PubMed Scopus (169) Google Scholar, 6Gidon 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.Nat. Chem. Biol. 2014; 10: 707-709Crossref PubMed Scopus (58) Google Scholar, 10Vilardaga J.P. Studying ligand efficacy at G protein-coupled receptors using FRET.Met. Mol. Biol. 2011; 756: 133-148Crossref PubMed Scopus (18) Google Scholar, 13Hammonds 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.J Biol Chem. 1989; 264: 14806-14811Abstract Full Text PDF PubMed Google Scholar, 14Li 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).Angew Chem Int Engl. 2012; 51: 12263-12267Crossref PubMed Scopus (20) Google Scholar, 15Wehbi 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.Proc Natl Acad Sci U S A. 2013; 110: 1530-1535Crossref PubMed Scopus (110) Google Scholar, 16Castro 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.Endocrinology. 2002; 143: 3854-3865Crossref PubMed Scopus (38) Google Scholar, 17Dean T. Vilardaga J.P. Potts Jr., J.T. Gardella T.J. Altered selectivity of parathyroid hormone (PTH) and PTH-related protein (PTHrP) for distinct conformations of the PTH/PTHrP receptor.Mol Endocrinol. 2008; 22: 156-166Crossref PubMed Scopus (160) Google Scholar, 18McGarvey J.C. Xiao K. Bowman S.L. Mamonova T. Zhang Q. Bisello A. et al.Actin-sorting nexin 27 (SNX27)-retromer complex mediates rapid parathyroid hormone receptor recycling.J Biol Chem. 2016; 291: 10986-11002Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). 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 (1Vilardaga 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.Nat. Biotechnol. 2003; 21: 807-812Crossref PubMed Scopus (359) Google Scholar, 2Vilardaga 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.Mol. Endocrinol. 2009; 23: 590-599Crossref PubMed Scopus (68) Google Scholar) have revealed that PTH1-34 and PTHrP1-36 differ markedly by the duration and cellular localization of the cAMP response (3Ferrandon S. Feinstein T.N. Castro M. Wang B. Bouley R. Potts J.T. et al.Sustained cyclic AMP production by parathyroid hormone receptor endocytosis.Nat. Chem. Biol. 2009; 5: 734-742Crossref PubMed Scopus (396) Google Scholar). 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 (4Ho 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.Biochem. Pharmacol. 2019; 169113627Crossref PubMed Scopus (6) Google Scholar). 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 (3Ferrandon S. Feinstein T.N. Castro M. Wang B. Bouley R. Potts J.T. et al.Sustained cyclic AMP production by parathyroid hormone receptor endocytosis.Nat. Chem. Biol. 2009; 5: 734-742Crossref PubMed Scopus (396) Google Scholar, 5Feinstein 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.Nat. Chem. Biol. 2011; 7: 278-284Crossref PubMed Scopus (169) Google Scholar, 6Gidon 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.Nat. Chem. Biol. 2014; 10: 707-709Crossref PubMed Scopus (58) Google Scholar, 7Jean-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.Nat. Chem. Biol. 2017; 13: 259-261Crossref PubMed Scopus (38) Google Scholar, 8White 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.Sci. Signal. 2021; 14eabc5944Crossref PubMed Scopus (8) Google Scholar, 9White 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 p
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