Identification of key phosphorylation sites in PTH1R that determine arrestin3 binding and fine-tune receptor signaling.
The parathyroid hormone receptor 1 (PTH1R) is a member of family B of G-protein-coupled receptors (GPCRs), predominantly expressed in bone and kidney where it modulates extracellular Ca2+homeostasis and bone turnover. It is well established that phosphorylation of GPCRs constitutes a key event in regulating receptor function by promoting arrestin recruitment and coupling to G-protein-independent signaling pathways. Mapping phosphorylation sites on PTH1R would provide insights into how phosphorylation at specific sites regulates cell signaling responses and also open the possibility of developing therapeutic agents that could target specific receptor functions. Here, we have used mass spectrometry to identify nine sites of phosphorylation in the C-terminal tail of PTH1R. Mutational analysis revealed identified two clusters of serine and threonine residues (Ser489-Ser495 and Ser501-Thr506) specifically responsible for the majority of PTH(1-34)-induced receptor phosphorylation. Mutation of these residues to alanine did not affect negatively on the ability of the receptor to couple to G-proteins or activate extracellular-signal-regulated kinase 1/2. Using fluorescence resonance energy transfer and bioluminescence resonance energy transfer to monitor PTH(1-34)-induced interaction of PTH1R with arrestin3, we show that the first cluster Ser489-Ser495 and the second cluster Ser501-Thr506 operated in concert to mediate both the efficacy and potency of ligand-induced arrestin3 recruitment. We further demonstrate that Ser503 and Thr504 in the second cluster are responsible for 70% of arrestin3 recruitment and are key determinants for interaction of arrestin with the receptor. Our data are consistent with the hypothesis that the pattern of C-terminal tail phosphorylation on PTH1R may determine the signaling outcome following receptor activation.
Institut für Pharmakologie und Klinische Pharmazie, Philipps-Universität Marburg, 35043 Marburg, Germany.|Institut de Génomique Fonctionnelle (IGF), University of Montpellier, CNRS UMR5203, 34094 Montpellier, France.|INSERM U1091, 34094 Montpellier, France.
Diana
D
Zindel
Institut für Pharmakologie und Klinische Pharmazie, Philipps-Universität Marburg, 35043 Marburg, Germany.
Sandra
S
Engel
The Protein and Nucleic Acid Chemistry Laboratory, University of Leicester, Hodgkin Building, Lancaster Road, Leicester LE1 9HN, U.K.
Andrew R
AR
Bottrill
Institut de Génomique Fonctionnelle (IGF), University of Montpellier, CNRS UMR5203, 34094 Montpellier, France.|INSERM U1091, 34094 Montpellier, France.
Jean-Philippe
JP
Pin
Institut de Génomique Fonctionnelle (IGF), University of Montpellier, CNRS UMR5203, 34094 Montpellier, France.|INSERM U1091, 34094 Montpellier, France.
Laurent
L
Prézeau
Institute of Molecular, Cell and Systems Biology, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
Andrew B
AB
Tobin
Institut für Pharmakologie und Klinische Pharmazie, Philipps-Universität Marburg, 35043 Marburg, Germany.
Moritz
M
Bünemann
Institut für Pharmakologie und Klinische Pharmazie, Philipps-Universität Marburg, 35043 Marburg, Germany.
Cornelius
C
Krasel
MRC Toxicology Unit, University of Leicester, Leicester LE2 2RG, U.K.
Adrian J
AJ
Butcher
D000595:Amino Acid Sequence; D019390:Arrestins / Q000378:metabolism; D060807:Bioluminescence Resonance Energy Transfer Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010766:Phosphorylation; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000737:chemistry / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
D019390:Arrestins; D044168:Receptor, Parathyroid Hormone, Type 1; D043562:Receptors, G-Protein-Coupled; C082333:arrestin3
G-protein-coupled receptor (GPCR); arrestin; parathyroid hormone receptor 1 (PTH1R); phosphorylation