N-Terminal Phosphorylation of Parathyroid Hormone (PTH) Abolishes

Letters pubs.acs.org/acschemicalbiology

N‑Terminal Phosphorylation of Parathyroid Hormone (PTH) Abolishes Its Receptor Activity Amit Kumar,† Mohanraj Gopalswamy,† Clare Wishart,§ Mathias Henze,† Lennart Eschen-Lippold,‡ Dan Donnelly,§ and Jochen Balbach*,#,† †

Institut für Physik, Biophysik and #Mitteldeutsches Zentrum für Struktur und Dynamik der Proteine (MZP), Martin-Luther-Universität Halle-Wittenberg, D-06120 Halle (Saale), Germany § School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, U.K. ‡ Department of Stress and Developmental Biology, Leibniz Institute of Plant Biochemistry, D-06120 Halle (Saale), Germany S Supporting Information *

ABSTRACT: The parathyroid hormone (PTH) is an 84-residue peptide, which regulates the blood Ca2+ level via GPCR binding and subsequent activation of intracellular signaling cascades. PTH is posttranslationally phosphorylated in the parathyroid glands; however, the functional significance of this processes is not well characterized. In the present study, mass spectrometric analysis revealed three sites of phosphorylation, and NMR spectroscopy assigned Ser1, Ser3, and Ser17 as modified sites. These sites are located at the N-terminus of the hormone, which is important for receptor recognition and activation. NMR shows further that the three phosphate groups remotely disturb the α-helical propensity up to Ala36. An intracellular cAMP accumulation assay elucidated the biological significance of this phosphorylation because it ablated the PTH-mediated signaling. Our studies thus shed light on functional implications of phosphorylation at native PTH as an additional level of regulation.

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it is synthesized as pre-pro-parathyroid hormone of 115 residues and released into the blood after proteolysis as an 84-residue peptide.24 In early 1984, phosphorylation of mature PTH(1−84) during biosynthesis in human and bovine parathyroid glands was detected by HPLC methods. The phosphorylated form of PTH accounts for 10−20% of total hormone present under physiological conditions.25 In later studies, a “new” form of PTH was identified in blood circulation, corresponding to less than 15% of whole PTH. In cancer patients it may raise up to 65% of whole PTH.26,27 This “new” form failed to be detectable by a second generation of PTH immunoassay, and a posttranslational modification in the region comprising residues 12−18 was suggested26,28 and correlated to previous findings by Rabbani et al.25 These authors suggested some structural alterations in the N-terminal binding epitope (region 15−20) of PTH.27,28 Therefore, the present studies have focused on the posttranslational modification of the peptide hormone. Currently there is no information available about the exact site of modification, the effect of phosphorylation on receptor binding, modulation of the intracellular signaling, or physiological significance of this modification.

eterotrimeric GTP-binding protein (G protein) coupled receptors (GPCRs) form the largest family of transmembrane proteins.1,2 A vast diversity of signaling molecules, including hormones, neurotransmitters, chemokines, ions, tastants, and odorants3 binds to GPCRs before translating effective information across the membrane.4−6 Recent structural studies shed new light on the mechanism of activation of signaling cascades by class A and B GPCRs.4,5,7 Parathyroid hormone (PTH) receptor 1 (PTH1R) and 2 (PTH2R) belong to the class B subfamily of GPCRs and function as agonist receptors for peptide hormones including PTH, PTH related peptide (PTHrP), and tuberoinfundibular peptide (TIP39).8−13 The signaling cascade via heterotrimeric G proteins modulates the flow of secondary messengers, including cAMP, inositol trisphosphate, diacylglycerol, or cGMP.14−16 These secondary messengers activate the intracellular signaling pathways, which in turn modulate cell function, including the skeletal, endocrine, cardiovascular, or nervous systems.17−19 Thus, PTH(1−34) and PTH(1−84) are used as drugs against osteoporosis.20,21 Despite our broad understanding of mechanisms of intracellular signaling, less is known about the interaction of PTH with its receptor,5 including the inactivation mechanism. Many proteins are posttranslationally modified via phosphorylation and dephosphorylation,22 such that one state is the active form, while the other state is inactive.23 PTH also undergoes posttranslational modifications during maturation; for instance, © XXXX American Chemical Society

Received: June 6, 2014 Accepted: August 26, 2014

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dx.doi.org/10.1021/cb5004515 | ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology

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Here we show that human PTH(1−84) gets phosphorylated at three sites, Ser1, Ser3, and Ser17, and its physiological relevance in ablating the activation of both PTH1 and PTH2 receptors. Therefore, we anticipate that this posttranslational modification provides an additional level of regulation of GPCR activation. In order to follow phosphorylation and to remain close to in vivo conditions, human PTH(1−84) (hPTH(1−84)) was treated with crude cell lysate prepared from bovine parathyroid glands or human embryonic kidney (HEK) 293 cells. Phosphorylation of hPTH(1−84) was confirmed by autoradiography for the metabolic incorporation of radiolabeled ATP (32P ATP). Autoradiography gives a strong band for phosphorylated hPTH(1−84) by both lysate of parathyroid glands (Figure 1) or HEK 293 cells (Supplementary Figure S1). These studies are in line with the previously reported PTH phosphorylation analyzed by HPLC.25

Figure 2. MALDI-TOF mass spectrometric analysis of hPTH(1−84) phosphorylation. hPTH(1−84) was added to the lysate from (A) parathyroid gland and (B) HEK 293 cells and incubated for 3 h.

phosphorylation by chemical shift changes (Figure 3A and Supplementary Figure S3A; gray in Figure 3E and Supplementary Figure S3C). No chemical shift changes were observed for all C-terminal serines or their neighbor residues upon peptide phosphorylation, and therefore we assigned the third phosphoserine to Ser1. The N-terminus typically shows no NMR cross peak in 2D 1H−15N HSQC spectrum. In a control experiment, phosphorylated hPTH(1−84) was treated with non-specific alkaline phosphatase. This dephosphorylation caused the reappearance of all NMR cross peaks at their native chemical shift position (Figure 3B). This also led to the disappearance of all three resonances in the 1D 31P NMR spectrum corresponding to the phosphoserines (Figure 3D). In summary, mass spectrometry and 1D 31P and 2D 1H−15N HSQC NMR confirmed that both lysates identically phosphorylate hPTH(1−84) without degradation or any other posttranslational modification. Next, we assayed whether the status of the phosphorylation of hPTH(1−84) correlates with the cellular function by measuring hPTH(1−84)-induced cAMP accumulation in stable HEK 293 cell lines expressing recombinant PTH1R or PTH2R.32 In the control experiment we examined the hPTH(1−84)-mediated cAMP signaling cascade via PTH1R (Figure 4A) and PTH2R (Figure 4B). This activation led to an elevation of the cAMP level inside the cells in a concentrationdependent fashion (black in Figure 4) similar to previously reported PTH1R receptor activation.10,14 Interestingly, hPTH(1−84) also activated the PTH2R-mediated elevation of cAMP level, which has been reported so far only for hPTH(1−34)33 and rat PTH(1−84).34 However, there is a 25fold difference in the potency of hPTH(1−84) between these two receptors. hPTH(1−84) displayed 9.89 ± 0.09 and 8.50 ± 0.13 pEC50 values for the PTH1R and PTH2R, respectively. Subsequently, we examined the accumulation of cAMP resulting from phosphorylated hPTH(1−84). Surprisingly, the phosphorylation of hPTH(1−84) almost completely ablated receptor activation (blue in Figure 4). The same assay was performed with the dephosphorylated form of hPTH(1−84) achieved after addition of alkaline phosphatase to the phosphorylated hPTH(1−84). This treatment rescued the

Figure 1. Phosphorylation of hPTH(1−84) by parathyroid gland extract stained by Coomassie brilliant blue (A) or autoradiography (B). Lane 1: protein molecular weight marker (kDa). Lane 2: hPTH(1−84) incubated with parathyroid gland extract. Lane 3: hPTH(1−84). Lane 4: parathyroid gland extract.

To evaluate the extent of phosphorylation, the experiments were carried out in a similar way with parathyroid gland or HEK 293 cells lysate but 31P ATP was used instead of a radiolabel (32P-ATP). Phosphorylation of the hPTH(1−84) was analyzed by MALDI-TOF tandem mass spectrometry. Control untreated hPTH(1−84) resulted in a peak at 9551.2 m/z (theoretical mol wt, 9549.70 Da) corresponding to unmodified uniformly labeled 15N peptide. The 15N labeling is required for subsequent NMR experiments. After 3 h of lysate treatment, a single peak corresponding to three phosphate groups attached to hPTH(1−84) was observed (Figure 2A and B). The 1D 31P NMR spectrum revealed three resonances (at −6, −8, and −20 ppm) corresponding to phosphoserines (Figure 3C and Supplementary Figure S3B), confirming the analysis of mass spectrometry. For identification of the sites of phosphorylation and subsequent conformational rearrangements, NMR spectroscopy was used.29,30 Sequence-specific backbone resonance assignments of hPTH(1−84) were accomplished using standard triple resonance NMR experiments31 (Supplementary Figure S2). 15N-Labeled hPTH(1−84) was incubated with parathyroid gland and HEK 293 cell lysate for 3 h. Subsequently, 2D 1H−15N HSQC spectra were recorded (Figure 3A and Supplementary Figure S3A). Upon phosphorylation, the local environment changed, making the chemical shift a sensitive tool to identify the respective serine residue. A detailed analysis of these HSQC spectra revealed that Ser3 and Ser17 specifically changed their chemical shift values, whereas Ser48, Ser62, Ser66, and Ser83 remain unchanged (Figure 3A and Supplementary Figure S3A). Interestingly, identical residues of hPTH(1−84) changed the chemical shift value with lysate of parathyroid gland or HEK 293 cells. Additionally, various neighboring residues of the N-terminal serine sense the B



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Figure 3. NMR spectroscopic analysis of phosphorylated and dephosphorylated 15N hPTH(1−84) recorded at pH 5.3 and 25 °C. (A) Overlaid 2D 1 H−15N HSQC spectra of phosphorylated hPTH(1−84) carried out with lysate of parathyroid glands (red) and untreated peptide hormone (black). (B) Overlaid 2D 1H−15N HSQC spectra of dephosphorylated hPTH(1−84) achieved by alkaline phosphatase treatment (red) and untreated peptide hormone (black). All cross peaks reappeared at their native chemical shift positions following this dephosphorylation. (C) 1D 31P NMR spectrum of the sample for panel A, where asterisks mark 31P signals arising from three phosphoserines. (D) 1D 31P NMR spectrum of the sample for panel B with missing peaks of phosphoserines. Resonances in the 1D 31P spectrum around 0 to +4 or −10 to −14 ppm arise from the cell extract. (E) Phosphorylated serines are labeled in red on the primary amino acid sequence of hPTH(1−84), and residues sensing the phosphorylation by chemical shift changes are marked in gray.

residues 15−34 (PDB codes 1ZWB39 and 3C4M,40 respectively). The 10th and 11th residues of parathyroid hormone PTH(1−12) are important for stabilizing its helical conformation. Substitutions at position 7, 10, and 11 markedly decreased cAMP generation.14 The presented NMR and mass spectrometry data revealed that 3 out of 7 serines at positions 1, 3, and 17 get phosphorylated. The phosphorylation resulted in detected chemical shift changes of residues between 2 and 36, including those critical residues important for receptor activation. These structural rearrangements are not sufficient to prevent binding to the receptor because phosphorylated hPTH(1−84) could still interact with the isolated ECDPTH1R. However, receptor activation on cultured cells was observed only with nonphosphorylated hPTH(1−84).

activity of hPTH(1−84) and yielded Emax values, relative to hPTH(1−84), of 98.58 ± 2.24% (pEC50 9.97 ± 0.08) and 99.35 ± 2.28% (pEC50 8.56 ± 0.13) for PTH1R and PTH2R, respectively (red in Figure 4). These results confirmed the phosphorylation-mediated inactivation of the peptide hormone. Cell lysate did not have any effect on the receptors activation (green in Figure 4). Additionally we analyzed whether phosphorylated hPTH(1−84) interacts with the receptor. Analogous to earlier NMR detected PTH1R binding studies12 the N-terminal extracellular domain of parathyroid hormone receptor 1 (ECD-PTH1R) was added to 15N-labeled hPTH(1− 84). Both forms of hPTH(1−84), phosphorylated or not, showed binding to the ECD-PTH1R (Figure 5). In summary, we present a detailed analysis of the peptide hormone hPTH(1−84) phosphorylation and its modulated response toward the PTH1 and PTH2 receptors. The hPTH(1−84) is composed of three distinct regions: residues ∼1−15 are important for interacting with transmembrane helices and activation of the receptor,35 ∼15−37 are involved in receptor recognition,36 whereas the function of residues ∼37− 84 is not clear.37 At a residue level the first, second, fourth, fifth, seventh, and eighth residue of PTH are important components of receptor activation.38 Both the free PTH peptide and the receptor-bound state show α-helical secondary structure for

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METHODS

Protein Expression and Purification. Human PTH(1−84) was cloned and purified according to the previously reported procedure.15 The expression plasmids for hPTH(1−84) with C-terminal His-tags are based on the pET SUMO adapt vector. Plasmids were transformed into E. coli BL21 codon+ cells. For isotope labeling M9 minimal medium was supplemented with 15N NH4Cl. hPTH(1−84) was purified by soluble fraction using Ni-NTA affinity chromatography. The purified SUMO fusion peptide was cleaved with 50−150 μg/mL C



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°C for 30 min) fetal bovine serum (FBS) and 5% sodium pyruvate at 37 °C in a humidified chamber with 5% CO2. Parathyroid glands or exponentially growing cells were lysed in the presence of protease inhibitor, 2x kinase assay buffer (20 mM MOPS pH 7.5, 15 mM MgCl2, 10 mM EGTA, and 2 mM DTT), and phosphatase inhibitors (0.25 mM activated Na3VO4, 0.5 mM NaF, 15 mM β-glycerophosphate). Bovine parathyroid glands were lysed by homogenization, and HEK 293 cells were lysed by sonication at 10% amplitude for 3 s pulses, repeated three times. Lysed cells were centrifuged at 12000 rpm for 15 min at 4 °C. The pellet was discarded, and the clear supernatant was used for the kinase reaction. Kinase assays were performed by incubating 30 μg of hPTH(1−84) with lysate (1−2 mg mL−1 total protein concentration), 32P ATP (5 μCi 32P-γ-ATP and cAMP (5 μM). This reaction mixture was incubating for 3 h at 37 °C. After the reaction samples were analyzed by 15% SDS PAGE and detected by autoradiography. NMR Spectroscopy and Mass Spectrometry. The lysate was obtained as mentioned above in the presence of kinase assay buffer, protease inhibitors, and phosphatase inhibitors. For the NMR experiment, lysate (15−20 mg mL−1) was mixed with 30 μM of 15 N-labeled hPTH(1−84), 100 μM ATP, and 5 μM cAMP. This reaction mixture was incubated for 3 h at 37 °C. Subsequently, the reaction samples were dialyzed using a 1 kDa cutoff membrane in 10 mM BisTris, 300 mM Na2SO4, 0.02% NaN3, pH 5.3 and 4 °C for 17 h in order to remove the unincorporated ATP and small molecules. This dialysis was also performed to minimize buffer effects to facilitate a proper chemical shift analysis of the NMR cross-peaks. All spectra were recorded on a Bruker 800 MHz Avance III spectrometer equipped with a CP-TCI cryoprobe at 25 °C. The same samples were used for detecting the phosphorus attached to the hPTH(1−84) (1D 31 P NMR) on a Bruker 600 MHz Avance II spectrometer. Spectra were processed using the programs NMRPipe and NMR Draw. The same samples were analyzed by MALTI-TOF tandem mass spectrometry. cAMP Accumulation Assay. HEK 293 cells stably expressing hPTH1R/hPTH2R were prepared as earlier reported.32 These cells were washed twice with PBS, harvested by centrifugation (3000g), and diluted to a density of 6.67 × 105 cells/mL in stimulation buffer (HBSS, 5 mM HEPES, 0.1% BSA, pH 7.4). Alexa Fluor 647-anticAMP antibody was diluted into adequate cell suspension at 0.005% (v/v), and the suspension was seeded at a density of 4 × 103 cells/well into a 96-well plate (6 μL/well). hPTH(1−84) was diluted to the desired concentration using stimulation buffer supplemented with 1 mM IBMX. Cells were treated with 6 μL of a single concentration of peptide hormone per well, performed in triplicate. Receptor stimulation was carried out at 37 °C for 30 min. Receptor stimulation was terminated upon the addition of 12 μL of detection mix containing 0.00044% (v/v) Eu-W8044 labeled streptavidin and 0.00133% (v/v) biotin-cAMP. Plates were incubated at RT for 1 h before fluorescence measurements were taken using a VICTOR X4 2030 plate reader (PerkinElmer Life and Analytical Sciences). All experiments were performed in triplicate and repeated independently three times. GraphPad Prism 6.01 was used for data analysis.

Figure 4. hPTH(1−84)-induced cAMP accumulation in HEK 293 cells stably expressing wild type receptors (A) PTH1R and (B) PTH2R. Symbols in the figures represent the mean of at least three independent experiments. Values for Emax were calculated as a percentage of the maximal response of hPTH(1−84): (black ■) hPTH(1−84), (blue ●) phosphorylated hPTH(1−84), (red ▲) dephosphorylated hPTH(1−84), and (green ▼) HEK 293 cells extract.

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Figure 5. Binding analysis of hPTH(1−84) to the extracellular domain of parathyroid hormone receptor 1 (ECD-PTH1R). (A) Overlaid 2D 1 H−15N HSQC spectra of hPTH(1−84) in its free (black) and bound state to ECD-PTH1R (red). (B) Overlaid 2D 1H−15N HSQC spectra of phosphorylated hPTH(1−84) in its free (black) and bound state to EDC-PTH1R (red). Both experiments were performed at hPTH(1− 84) to ECD-PTH1R ratio of 1:2.

ASSOCIATED CONTENT

* Supporting Information S

Additional figures and tables as described in the text. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

SUMO protease (1:100 ratio). hPTH(1−84) was further purified by S-75 gel filtration chromatography. Preparation of Lysates and Kinase Assay. Human embryonic kidney 293 (HEK 293) cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated (56

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. D



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ACKNOWLEDGMENTS This work has been supported by grants from the DFG (GRK 1026, SFB TRR102), the BMBF (ProNet-T3), the state Sachsen-Anhalt (Exzellenznetzwerk Biowissenschaften), and ERDF by the EU

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N-Terminal Phosphorylation of Parathyroid Hormone (PTH) Abolishes

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