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Phosphorylation of intracellular serine and threonine residues is the most important post translational modification of G protein-coupled receptors (GPCRs) also called heptahelical or seven transmembrane receptors (7TMR). After agonist exposure, these receptors acquire an active conformation, which is recognized by a family of highly specialized GPCR kinases (GRKs). Agonist-driven phosphorylation by GRKs regulates acute receptor desensitization, arrestin recruitment, internalization, post-activation signaling, long-term tolerance and drug addiction. Phosphosite-specific 7TM antibodies are designed to specifically detect agonist-activated GPCRs. In fact, recent work shows that ligand profiling using phosphosite-specific 7TM antibodies provides valuble information on ligand bias beyond that obtained with conventional ß-arrestin recruitment assays. Phosphosite-specific 7TM antibodies are novel tools for GPCR research that can be used to:

  • profile agonist properties of novel GPCR ligands
  • decipher the phosphorylation barcode of GPCRs
  • determine the spatial and temporal dynamics of receptor phosphorylation
  • identify relevant kinases and phosphatases for GPCR phosphorylation and dephosphoryation

Lifecycle3

Schematic representation of the G protein-coupled receptor phosphorylation / dephosphorylation cycle. GRK, G protein-coupled receptor kinase; PKC, protein kinase C; cPP1, catalytic subunit of protein phosphatase 1; R*, activated GPCR; CCP, clathrin-coated pit. 

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Agonist-induced Serine401 phosphorylation of the Neurotensin Receptor
pS401-NTS1 (phospho-Neurotensin Receptor 1...
Serine401 (S401) is major phosphorylation site of the Neurotensin Receptor 1 (NTS1). The pS401-NTS1 antibody detects phosphorylation in response to agonists. S401 phosphorylation is likely to be involved in efficient ligand sequestration...
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Agonist-induced Serine403/Serine404 phosphorylation of the Neurotensin Receptor 1
pS403/pS404-NTS1 (phospho-Neurotensin Receptor...
Serine403/Serine404 (S403/S404) is major phosphorylation site of the Neurotensin Receptor 1 (NTS1). The pS403/pS404-NTS1 antibody detects phosphorylation in response to agonists. S403/S404 phosphorylation is likely to be involved in...
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Agonist-induced Serine409/Serine410 phosphorylation of the Neurotensin Receptor 1
pS409/pS410-NTS1 (phospho-Neurotensin Receptor...
Serine409/Serine410 (S409/S410) is major phosphorylation site of the Neurotensin Receptor 1 (NTS1). The pS409/pS410-NTS1 antibody detects phosphorylation in response to agonists. S409/S410 phosphorylation is likely to be involved in...
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Agonist-induced Serine351/Serine354 phosphorylation of the Neuropeptide Y
pS351/pS354-NPY2 (phospho-Neuropeptide Y...
Serine351/Serine354 (S351/S354) is a major phosphorylation site of the Neuropeptide Y Receptor 2. The pS351/pS354-NPY2 antibody detects phosphorylation in response to high- and low-efficacy agonists but not after PKC activation....
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Agonist-induced Serine354/Threonine356 phosphorylation of the Neuropeptide Y receptor 2
pS354/pT356-NPY2 (phospho-Neuropeptide Y...
Serine354Threonine356 (S354/T356) is a major phosphorylation site of the Neuropeptide Y Receptor 2. The pS354/pT356-NPY2 antibody detects phosphorylation in response to high- and low-efficacy agonists but not after PKC activation....
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Agonist-induced Serine369/Serine374 phosphorylation of the Neuropeptide Y receptor 2
pS369/pS374-NPY2 (phospho-Neuropeptide Y...
Serine369/Serine374 (S369/S374) is a major phosphorylation site of the Neuropeptide Y Receptor 2. The pS369/pS374-NPY2 antibody detects phosphorylation in response to high- and low-efficacy agonists but not after PKC activation....
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Agonist-induced Threonine362/Serine363 phosphorylation of the Neuropeptide Y receptor 1
pT362/pS363-NPY1 (phospho-Neuropeptide Y...
Threonine362/Serine363 (T362/S363) is a major phosphorylation site of the Neuropeptide Y Receptor 1. The pT362/pS363-NPY1 antibody detects phosphorylation in response to high- and low-efficacy agonists but not after PKC activation....
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gonist-induced Threonine357/Serine360 phosphorylation of the Neuropeptide Y receptor 1
pT357/pS360-NPY1 (phospho-Neuropeptide Y...
Threonine357/Serine360 (T357/S360) is a major phosphorylation site of the Neuropeptide Y Receptor 1. The pT357/pS360-NPY1 antibody detects phosphorylation in response to high- and low-efficacy agonists but not after PKC activation....
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Agonist-induced Serine346/Serine349/Threonine352 phosphorylation of the NPS Receptor
pS346/pS349/pT352-NPSR (phospho-NPS Receptor...
Serine346/Serine349/threonine352 (S346/S349/T352) is major phosphorylation site of the NPS Receptor (NPSR). The pS346/pS349/pT352-NPRS antibody detects phosphorylation in response to agonists. S346/S349/T352 phosphorylation is likely to...
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Agonist-induced Serine361 phosphorylation of the Neuropeptide FF Receptor 1
pS361-NPFF1 (phospho-Neuropeptide FF Receptor 1...
Serine361 (S361) is major phosphorylation site of the Neuropeptide FF Receptor 1 (NPFF1). The pS361-NPFF1 antibody detects phosphorylation in response to agonists. S361 phosphorylation is likely to be involved in efficient ligand...
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Agonist-induced Serine385/Serine387 phosphorylation of the Neuropeptide Receptor 1
pS385/pS387-NPFF1 (phospho-Neuropeptide FF...
Serine385/Serine387 (S385/S387) is major phosphorylation site of the Neuropeptide FF Receptor 1 (NPFF1). The pS385/pS387-NPFF1 antibody detects phosphorylation in response to agonists. S385/S387 phosphorylation is likely to be involved...
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Agonist-induced Serine313 phosphorylation of the MRGPRX2 Mas-related Receptor
pS313-MRGPRX2 (phospho-MRGPRX2 Mas-related...
Serine313 (S313) is a major phosphorylation site of the MRGPRX2 Mas-related receptor. The pS313-MRGPRX2 antibody detects phosphorylation in response to high- and low-efficacy agonists and after PKC activation. S313 phosphorylation is a...
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Agonist-induced Serine327/Serine328 phosphorylation of the MRGPRX2 Mas-related Receptor
pS327/pS328-MRGPRX2 (phospho-MRGPRX2...
Serine327/Serine328 (S327/S329) is a major phosphorylation site of the MRGPRX2 Mas-related receptor. The pS327/pS328-MRGPRX2 antibody detects phosphorylation in response to high- and low-efficacy agonists and after PKC activation....
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Agonist-induced Serine320/Threonine324 phosphorylation of the Leukotriene Receptor BLT2.
pS320/pT324-BLT2 (phospho-Leukotriene Receptor...
Serine320/Threonine324 (S320/T324) is a major phosphorylation site of the BLT2 receptor. The pS320/pT324-BLT2 antibody detects phosphorylation in response to high- and low-efficacy agonists and after PKC activation. S320/T324...
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pT329/pT330-BLT2 (phospho-Leukotriene Receptor BLT2 Antibody)
pT329/pT330-BLT2 (phospho-Leukotriene Receptor...
Threonine329/Threonine330 (T329/T330) is a major phosphorylation site of the BLT2 receptor. The pT329/pT330-BLT2 antibody detects phosphorylation in response to high- and low-efficacy agonists and after PKC activation. T329/T330...
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Agonist-induced Threonine338 phosphorylation of the Hydroxycarboxylic Acid Receptor 2.
pT338-HCA2 (phospho-Hydroxycarboxylic Acid...
Threonine338 (T338) is major phosphorylation site of the Hydroxycarboxylic Acid Receptor 2 (HCA2). The pT338-HCA2 antibody detects phosphorylation in response to agonists. T338 phosphorylation is likely to be involved in efficient ligand...
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For further reading refer to:

Kliewer A, Reinscheid RK, Schulz S. Emerging Paradigms of G Protein-Coupled Receptor Dephosphorylation. Trends Pharmacol Sci. 2017 Jul;38(7):621-636. doi:10.1016/j.tips.2017.04.002. Epub 2017 May 4. Review. PubMed PMID: 28478994.

Miess E, Gondin AB, Yousuf A, Steinborn R, Mösslein N, Yang Y, Göldner M, Ruland JG, Bünemann M, Krasel C, Christie MJ, Halls ML, Schulz S, Canals M. Multisite phosphorylation is required for sustained interaction with GRKs and arrestins during rapid μ-opioid receptor desensitization. Sci Signal. 2018 Jul 17;11(539). pii: eaas9609. doi: 10.1126/scisignal.aas9609. PubMed PMID: 30018083.

Kliewer A, Schmiedel F, Sianati S, Bailey A, Bateman JT, Levitt ES, Williams JT, Christie MJ, Schulz S. Phosphorylation-deficient G-protein-biased μ-opioid receptors improve analgesia and diminish tolerance but worsen opioid side effects. Nat Commun. 2019 Jan 21;10(1):367. doi: 10.1038/s41467-018-08162-1. PubMed PMID: 30664663; PubMed Central PMCID: PMC6341117.

Mann A, Moulédous L, Froment C, O'Neill PR, Dasgupta P, Günther T, Brunori G, Kieffer BL, Toll L, Bruchas MR, Zaveri NT, Schulz S. Agonist-selective NOP receptor phosphorylation correlates in vitro and in vivo and reveals differential post-activation signaling by chemically diverse agonists. Sci Signal. 2019 Mar 26;12(574). pii: eaau8072. doi: 10.1126/scisignal.aau8072. PubMed PMID: 30914485; PubMed Central PMCID: PMC6934085.

Saaber F, Schütz D, Miess E, Abe P, Desikan S, Ashok Kumar P, Balk S, Huang K, Beaulieu JM, Schulz S, Stumm R. ACKR3 Regulation of Neuronal Migration Requires ACKR3 Phosphorylation, but Not β-Arrestin. Cell Rep. 2019 Feb 5;26(6):1473-1488.e9. doi: 10.1016/j.celrep.2019.01.049. PubMed PMID: 30726732.

Glück L, Loktev A, Moulédous L, Mollereau C, Law PY, Schulz S. Loss of morphine reward and dependence in mice lacking G protein-coupled receptor kinase 5. Biol Psychiatry. 2014 Nov 15;76(10):767-74. doi: 10.1016/j.biopsych.2014.01.021. Epub 2014 Feb 3. PubMed PMID: 24629717; PubMed Central PMCID: PMC4119866.

For further reading refer to: Kliewer A, Reinscheid RK, Schulz S. Emerging Paradigms of G Protein-Coupled Receptor Dephosphorylation. Trends Pharmacol Sci. 2017 Jul;38(7):621-636.... read more »
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For further reading refer to:

Kliewer A, Reinscheid RK, Schulz S. Emerging Paradigms of G Protein-Coupled Receptor Dephosphorylation. Trends Pharmacol Sci. 2017 Jul;38(7):621-636. doi:10.1016/j.tips.2017.04.002. Epub 2017 May 4. Review. PubMed PMID: 28478994.

Miess E, Gondin AB, Yousuf A, Steinborn R, Mösslein N, Yang Y, Göldner M, Ruland JG, Bünemann M, Krasel C, Christie MJ, Halls ML, Schulz S, Canals M. Multisite phosphorylation is required for sustained interaction with GRKs and arrestins during rapid μ-opioid receptor desensitization. Sci Signal. 2018 Jul 17;11(539). pii: eaas9609. doi: 10.1126/scisignal.aas9609. PubMed PMID: 30018083.

Kliewer A, Schmiedel F, Sianati S, Bailey A, Bateman JT, Levitt ES, Williams JT, Christie MJ, Schulz S. Phosphorylation-deficient G-protein-biased μ-opioid receptors improve analgesia and diminish tolerance but worsen opioid side effects. Nat Commun. 2019 Jan 21;10(1):367. doi: 10.1038/s41467-018-08162-1. PubMed PMID: 30664663; PubMed Central PMCID: PMC6341117.

Mann A, Moulédous L, Froment C, O'Neill PR, Dasgupta P, Günther T, Brunori G, Kieffer BL, Toll L, Bruchas MR, Zaveri NT, Schulz S. Agonist-selective NOP receptor phosphorylation correlates in vitro and in vivo and reveals differential post-activation signaling by chemically diverse agonists. Sci Signal. 2019 Mar 26;12(574). pii: eaau8072. doi: 10.1126/scisignal.aau8072. PubMed PMID: 30914485; PubMed Central PMCID: PMC6934085.

Saaber F, Schütz D, Miess E, Abe P, Desikan S, Ashok Kumar P, Balk S, Huang K, Beaulieu JM, Schulz S, Stumm R. ACKR3 Regulation of Neuronal Migration Requires ACKR3 Phosphorylation, but Not β-Arrestin. Cell Rep. 2019 Feb 5;26(6):1473-1488.e9. doi: 10.1016/j.celrep.2019.01.049. PubMed PMID: 30726732.

Glück L, Loktev A, Moulédous L, Mollereau C, Law PY, Schulz S. Loss of morphine reward and dependence in mice lacking G protein-coupled receptor kinase 5. Biol Psychiatry. 2014 Nov 15;76(10):767-74. doi: 10.1016/j.biopsych.2014.01.021. Epub 2014 Feb 3. PubMed PMID: 24629717; PubMed Central PMCID: PMC4119866.

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