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

Abstract

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. 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 a of and to that PTHrP1-141 induces cAMP signaling in an endocytosis-dependent to that for cAMP were in the of a measure of the of cAMP during a time as to the of cAMP that from the of its production and Furthermore, the to results with of sustained cAMP induced by PTHrP1-141 only for PTHrP1-141 not for of sustained cAMP response by receptor is this to via PTHR from early (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, T.N. S. T.J. et the generation of cAMP by PTH Chem. Biol. PubMed Scopus Google Scholar, C. et signaling by of and Chem. Biol. PubMed Scopus Google Scholar, M. et receptor of endosomal PTH receptor signaling via Chem. Biol. PubMed Scopus Google Scholar, S. et in cAMP generation biological to PTH type 1 receptor PubMed Scopus Google Scholar, S. et of endosomal cAMP generation by parathyroid hormone S. PubMed Scopus Google Scholar). the to that of cAMP response kinetics in real time in single The results the by PTHrP1-141 in sustained signaling and how this from the transient with the N-terminal and to courses of cAMP production in single cell PTHR that stimulation with PTHrP1-141 induced a sustained cAMP response that was in both and duration to that induced by PTH1-34 and from the cAMP response mediated by PTHrP1-36 and cAMP accumulation assays to that time courses of sustained cAMP production mediated by the two native and were and a in the hormone and a of PTHrP1-141 to the release of intracellular calcium (iCa2+) from the and activation by have that activation is for endosomal cAMP generation by PTH1-34 S. et of endosomal cAMP generation by parathyroid hormone S. PubMed Scopus Google a of cAMP generation by this the molecular basis for the of PTHrP1-141 to cAMP and iCa2+ signaling mediated by PTHrP1-36 were to be caused by to G protein coupled of PTHR G and were to the stabilization of a receptor this by using cells a PTHR in of from single cells the of the kinetics of receptor activation in response to (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). in mediated by an agonist receptor from an to an active and of receptor activation for a of the stabilization of signaling receptor (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, 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, J.P. efficacy at G protein-coupled receptors using Biol. PubMed Scopus Google Scholar). of a of PTHrP1-36, PTHrP1-141 to individual cells a in the time for receptor activation the stabilization of PTHR and the of we interactions via in cells and The was that earlier studies that PTH1-34 and PTHrP1-36 for recruitment of both and S. et in cAMP generation biological to PTH type 1 receptor PubMed Scopus Google Scholar, S. T.J. et of to the spatiotemporal of parathyroid hormone signaling via biased Chem. 2019; PubMed Scopus Google Scholar, J.P. Krasel C. S. Lohse M.J. of the parathyroid hormone receptor Biol. Chem. PubMed Scopus Google Scholar). with of PTH1-34 in of with the receptor that was In contrast, using PTHrP1-141 to promote this that the sustained signaling for PTHrP1-141 in a led to the of receptor a in PTHR endosomal of receptor internalization and recycling in single cells the PTHR with a that fluorescence in the in in internalization and recycling in response to PTHrP1-141 PTHrP1-36 to PTH1-34 and via have that of a translocation of complexes from the cell surface and the sustained phase of cAMP generation the 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, S. et in cAMP generation biological to PTH type 1 receptor PubMed Scopus Google Scholar). we the cAMP response stimulation with PTHrP1-141 in cells and to a protein of receptor internalization the and duration of cAMP production by PTH1-34 and on cAMP mediated by PTHrP1-141 and that native PTHrP not promote sustained signaling in an endocytosis-dependent on the development of PTH that stimulate sustained cAMP production from the cell surface to of active ligand–receptor complexes at the cell was via cAMP time courses using a PTHR the sustained phase of cAMP generation for not for with its to from intracellular S. et in cAMP generation biological to PTH type 1 receptor PubMed Scopus Google Scholar). this to PTHrP1-141 induces cAMP signaling via ligand–receptor complexes that are to the cell Indeed, of the at agonist rapidly cAMP to in cells with PTHrP1-141 in with PTH1-34 and findings demonstrate that PTHrP1-141 sustained cAMP via active ligand–receptor complexes to the cell with receptor cAMP signaling by time of β-arrestin with PTHR in cells and with PTH1-34 PTHrP1-141 for are the for and The the of the response by the the by time courses of internalization and recycling of PTHR with in response to by in single The the are from and time courses of cAMP in single PTHR cells with to in response to PTH1-34 and PTHrP1-141 are the for cells cells and the the the to individual and the by with time courses of cAMP in single PTHR cells for with PTH1-34 are the of cells and as in with of PTHR of PTH1-34 are the of cells and parathyroid PTH this we that the of PTHrP1-141 not in PTHrP1-36 the hormone to to the cell surface via interactions with on membrane as with this was the in the and duration of cAMP production in response to PTHrP1-141 in the of used as a to PTHrP1-141 and interactions and The of was by its of on cAMP induced by PTH1-34 PTHrP1-36 and of on cAMP cAMP time courses in single PTHR cells in response to 1 with are the of and cells for and cells for and cells for The is in the the of individual from by proposed for signaling of native The cAMP signaling mediated by PTHrP1-141 be by plasma that retain PTHrP1-141 at the cell surface of with parathyroid PTH of on cAMP 1 the from in of with by in a these a of our understanding on how act on the PTHR by that native PTHrP1-141 is biased sustained PTHR signaling via cAMP at the plasma The results a PTHrP1-141 an active receptor conformation that coupling and signaling through the with are for an of PTHrP and as a to receptor by the cell hormone and are in are in and be in the of the of (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, T.N. S. T.J. et the generation of cAMP by PTH Chem. Biol. PubMed Scopus Google Scholar, C. et signaling by of and Chem. Biol. PubMed Scopus Google Scholar, J.P. efficacy at G protein-coupled receptors using Biol. PubMed Scopus Google Scholar, et and of parathyroid hormone-related Chem. PubMed Google Scholar, S. T.J. et as an in protein and active parathyroid hormone-related protein PubMed Scopus Google Scholar, Feinstein T.N. J.P. signaling from a PTH PubMed Scopus Google Scholar, M. J.P. Krasel C. M. Lohse M.J. of the parathyroid hormone receptor signaling by protein and PubMed Scopus Google Scholar, J.P. Potts J.T. T.J. of parathyroid hormone (PTH) and protein (PTHrP) for of the Endocrinol. PubMed Scopus Google Scholar, et complex parathyroid hormone receptor Chem. PubMed Scopus 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 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 a of and to that PTHrP1-141 induces cAMP signaling in an endocytosis-dependent to that for cAMP were in the of a measure of the of cAMP during a time as to the of cAMP that from the of its production and Furthermore, the to results with of sustained cAMP induced by PTHrP1-141 only for PTHrP1-141 not for of sustained cAMP response by receptor is this to via PTHR from early (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, T.N. S. T.J. et the generation of cAMP by PTH Chem. Biol. PubMed Scopus Google Scholar, C. et signaling by of and Chem. Biol. PubMed Scopus Google Scholar, M. et receptor of endosomal PTH receptor signaling via Chem. Biol. PubMed Scopus Google Scholar, S. et in cAMP generation biological to PTH type 1 receptor PubMed Scopus Google Scholar, S. et of endosomal cAMP generation by parathyroid hormone S. PubMed Scopus Google Scholar). the to that of cAMP response kinetics in real time in single The results the by PTHrP1-141 in sustained signaling and how this from the transient with the N-terminal PTHrP1-36. and to courses of cAMP production in single cell PTHR that stimulation with PTHrP1-141 induced a sustained cAMP response that was in both and duration to that induced by PTH1-34 and from the cAMP response mediated by PTHrP1-36 and cAMP accumulation assays to that time courses of sustained cAMP production mediated by the two native and were and a in the hormone and a of PTHrP1-141 to the release of intracellular calcium (iCa2+) from the and activation by have that activation is for endosomal cAMP generation by PTH1-34 S. et of endosomal cAMP generation by parathyroid hormone S. PubMed Scopus Google a of cAMP generation by this the molecular basis for the of PTHrP1-141 to cAMP and iCa2+ signaling mediated by PTHrP1-36 were to be caused by to G protein coupled of PTHR G and were to the stabilization of a receptor this by using cells a PTHR in of from single cells the of the kinetics of receptor activation in response to (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). in mediated by an agonist receptor from an to an active and of receptor activation for a of the stabilization of signaling receptor (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, 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, J.P. efficacy at G protein-coupled receptors using Biol. PubMed Scopus Google Scholar). of a of PTHrP1-36, PTHrP1-141 to individual cells a in the time for receptor activation the stabilization of PTHR and the of we interactions via in cells and The was that earlier studies that PTH1-34 and PTHrP1-36 for recruitment of both and S. et in cAMP generation biological to PTH type 1 receptor PubMed Scopus Google Scholar, S. T.J. et of to the spatiotemporal of parathyroid hormone signaling via biased Chem. 2019; PubMed Scopus Google Scholar, J.P. Krasel C. S. Lohse M.J. of the parathyroid hormone receptor Biol. Chem. PubMed Scopus Google Scholar). with of PTH1-34 in of with the receptor that was In contrast, using PTHrP1-141 to promote this that the sustained signaling for PTHrP1-141 in a led to the of receptor a in PTHR endosomal of receptor internalization and recycling in single cells the PTHR with a that fluorescence in the in in internalization and recycling in response to PTHrP1-141 PTHrP1-36 to PTH1-34 and via have that of a translocation of complexes from the cell surface and the sustained phase of cAMP generation the 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, S. et in cAMP generation biological to PTH type 1 receptor PubMed Scopus Google Scholar). we the cAMP response stimulation with PTHrP1-141 in cells and to a protein of receptor internalization the and duration of cAMP production by PTH1-34 and on cAMP mediated by PTHrP1-141 and that native PTHrP not promote sustained signaling in an endocytosis-dependent on the development of PTH that stimulate sustained cAMP production from the cell surface to of active ligand–receptor complexes at the cell was via cAMP time courses using a PTHR the sustained phase of cAMP generation for not for with its to from intracellular S. et in cAMP generation biological to PTH type 1 receptor PubMed Scopus Google Scholar). this to PTHrP1-141 induces cAMP signaling via ligand–receptor complexes that are to the cell Indeed, of the at agonist rapidly cAMP to in cells with PTHrP1-141 in with PTH1-34 and findings demonstrate that PTHrP1-141 sustained cAMP via active ligand–receptor complexes to the cell with receptor this we that the of PTHrP1-141 not in PTHrP1-36 the hormone to to the cell surface via interactions with on membrane as with this was the in the and duration of cAMP production in response to PTHrP1-141 in the of used as a to PTHrP1-141 and interactions and The of was by its of on cAMP induced by PTH1-34 PTHrP1-36 and of on cAMP cAMP time courses in single PTHR cells in response to 1 with are the of and cells for and cells for and cells for The is in the the of individual from by proposed for signaling of native The cAMP signaling mediated by PTHrP1-141 be by plasma that retain PTHrP1-141 at the cell surface of with parathyroid PTH of on cAMP 1 the from in of with by in a these a of our understanding on how act on the PTHR by that native PTHrP1-141 is biased sustained PTHR signaling via cAMP at the plasma The results a PTHrP1-141 an active receptor conformation that coupling and signaling through the with are for an of PTHrP and as a to receptor by the cell hormone to courses of cAMP production in single cell PTHR that stimulation with PTHrP1-141 induced a sustained cAMP response that was in both and duration to that induced by PTH1-34 and from the cAMP response mediated by PTHrP1-36 and cAMP accumulation assays to that time courses of sustained cAMP production mediated by the two native and were and a in the hormone and a of PTHrP1-141 to the release of intracellular calcium (iCa2+) from the and activation by have that activation is for endosomal cAMP generation by PTH1-34 S. et of endosomal cAMP generation by parathyroid hormone S. PubMed Scopus Google a of cAMP generation by this the molecular basis for the of PTHrP1-141 to cAMP and iCa2+ signaling mediated by PTHrP1-36 were to be caused by to G protein coupled of PTHR G and were to the stabilization of a receptor this by using cells a PTHR in of from single cells the of the kinetics of receptor activation in response to (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). in mediated by an agonist receptor from an to an active and of receptor activation for a of the stabilization of signaling receptor (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, 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, J.P. efficacy at G protein-coupled receptors using Biol. PubMed Scopus Google Scholar). of a of PTHrP1-36, PTHrP1-141 to individual cells a in the time for receptor activation the stabilization of PTHR and the of we interactions via in cells and The was that earlier studies that PTH1-34 and PTHrP1-36 for recruitment of both and S. et in cAMP generation biological to PTH type 1 receptor PubMed Scopus Google Scholar, S. T.J. et of to the spatiotemporal of parathyroid hormone signaling via biased Chem. 2019; PubMed Scopus Google Scholar, J.P. Krasel C. S. Lohse M.J. of the parathyroid hormone receptor Biol. Chem. PubMed Scopus Google Scholar). with of PTH1-34 in of with the receptor that was In contrast, using PTHrP1-141 to promote this that the sustained signaling for PTHrP1-141 in a led to the of receptor a in PTHR endosomal of receptor internalization and recycling in single cells the PTHR with a that fluorescence in the in in internalization and recycling in response to PTHrP1-141 PTHrP1-36 to PTH1-34 and via have that of a translocation of complexes from the cell surface and the sustained phase of cAMP generation the 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, S. et in cAMP generation biological to PTH type 1 receptor PubMed Scopus Google Scholar). we the cAMP response stimulation with PTHrP1-141 in cells and to a protein of receptor internalization the and duration of cAMP production by PTH1-34 and on cAMP mediated by PTHrP1-141 and that native PTHrP not promote sustained signaling in an endocytosis-dependent on the development of PTH that stimulate sustained cAMP production from the cell surface to of active ligand–receptor complexes at the cell was via cAMP time courses using a PTHR the sustained phase of cAMP generation for not for with its to from intracellular S. et in cAMP generation biological to PTH type 1 receptor PubMed Scopus Google Scholar). this to PTHrP1-141 induces cAMP signaling via ligand–receptor complexes that are to the cell Indeed, of the at agonist rapidly cAMP to in cells with PTHrP1-141 in with PTH1-34 and findings demonstrate that PTHrP1-141 sustained cAMP via active ligand–receptor complexes to the cell with receptor this we that the of PTHrP1-141 not in PTHrP1-36 the hormone to to the cell surface via interactions with on membrane as with this was the in the and duration of cAMP production in response to PTHrP1-141 in the of used as a to PTHrP1-141 and interactions and The of was by its of on cAMP induced by PTH1-34 PTHrP1-36 and The the from in of with by these a of our understanding on how act on the PTHR by that native PTHrP1-141 is biased sustained PTHR signaling via cAMP at the plasma The results a PTHrP1-141 an active receptor conformation that coupling and signaling through the with are for an of PTHrP and as a to receptor by the cell hormone and are in and are in are in and be in the of the of are in and be in the of the of (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, T.N. S. T.J. et the generation of cAMP by PTH Chem. Biol. PubMed Scopus Google Scholar, C. et signaling by of and Chem. Biol. PubMed Scopus Google Scholar, J.P. efficacy at G protein-coupled receptors using Biol. PubMed Scopus Google Scholar, et and of parathyroid hormone-related Chem. PubMed Google Scholar, S. T.J. et as an in protein and active parathyroid hormone-related protein PubMed Scopus Google Scholar, Feinstein T.N. J.P. signaling from a PTH PubMed Scopus Google Scholar, M. J.P. Krasel C. M. Lohse M.J. of the parathyroid hormone receptor signaling by protein and PubMed Scopus Google Scholar, J.P. Potts J.T. T.J. of parathyroid hormone (PTH) and protein (PTHrP) for of the Endocrinol. PubMed Scopus Google Scholar, et complex parathyroid hormone receptor Chem. PubMed Scopus Google Scholar). (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, T.N. S. T.J. et the generation of cAMP by PTH Chem. Biol. PubMed Scopus Google Scholar, C. et signaling by of and Chem. Biol. PubMed Scopus Google Scholar, J.P. efficacy at G protein-coupled receptors using Biol. PubMed Scopus Google Scholar, et and of parathyroid hormone-related Chem. PubMed Google Scholar, S. T.J. et as an in protein and active parathyroid hormone-related protein PubMed Scopus Google Scholar, Feinstein T.N. J.P. signaling from a PTH PubMed Scopus Google Scholar, M. J.P. Krasel C. M. Lohse M.J. of the parathyroid hormone receptor signaling by protein and PubMed Scopus Google Scholar, J.P. Potts J.T. T.J. of parathyroid hormone (PTH) and protein (PTHrP) for of the Endocrinol. PubMed Scopus Google Scholar, et complex parathyroid hormone receptor Chem. PubMed Scopus Google Scholar). The that have of with the of this Martin for the protein to this S. and and S. and and and S. S. and was by S. from the of and from the of and and of the of from the and the of of from the The is the of the and not the of the of

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