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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 Threoine339/Serine341 phosphorylation of GPR132
pT339/pS341-GPR132 (phospho-GPR132 Antibody)
Threonine339/Serine341 (T339/S341) is major phosphorylation site of human GPR132 receptor. The pT339/pS341-GPR132 antibody detects phosphorylation in response to agonists. T339/S341 phosphorylation is likely to be involved in efficient...
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Agonist-induced Serine335/Serine337 phosphorylation of the Sphingosine 1-Phosphate Receptor 3
pS335/pS337-S1P3 (phospho-Sphingosine...
Serine335/Serine337 (S335/S337) is major phosphorylation site of the Sphingosine 1-Phosphate Receptor 3 (S1P3). The pS335/pS337-S1P3 antibody detects phosphorylation in response to agonists. S335/S337 phosphorylation is likely to be...
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pS335/pS336-CB2 (phospho-Cannabinoid Receptor 2 Antibody)
pS335/pS336-CB2 (phospho-Cannabinoid Receptor 2...
Serine335/Serine336 (S335/S336) is major phosphorylation site of the Cannabinoid Receptor 2 (CB2). The pS335/pS336-CB2 antibody detects phosphorylation in response to agonists. S335/S336 phosphorylation is likely to be involved in...
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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 Threonine370/Threonine371 phosphorylation of the CXC Chemokine Receptor 5
pT370/pT371-CXCR5 (phospho-CXC Chemokine...
Threonine370/Threonine371 (T370/T371) is a major phosphorylation site of the CXCR5 receptor. The pT370/pT371-CXCR5 antibody detects phosphorylation in response to high- and low-efficacy agonists but not after PKC activation. T370/T371...
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Agonist-induced Threonine367/Serine368 phosphorylation of the CXC Chemokine Receptor 5
pT367/pS368-CXCR5 (phospho-CXC Chemokine...
Threonine367/Serine368 (T367/S368) is a major phosphorylation site of the CXCR5 receptor. The pT367/pS368-CXCR5 antibody detects phosphorylation in response to high- and low-efficacy agonists but not after PKC activation. T367/S368...
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Agonist-induced Serine716/Threonine717 phosphorylation of the Thyrotropin Receptor
pS716/pT717-TSHR (phospho-Thyrotropin Receptor...
Serine716/Threonine717 (S716/T717) is major phosphorylation site of the Thyrotropin Receptor (TSHR). The pS716/pT717-TSHR antibody detects phosphorylation in response to agonists. S716/T717 phosphorylation is likely to be involved in...
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Agonist-induced Threonine503/Serine504 phosphorylation of the Parathyroid Hormone Receptor 1
pT503/pS504-PTH1 (phospho-Parathyroid Hormone...
Threonine503/Serine504 (T503/S504) is major phosphorylation site of the Parathyroid Hormone Receptor 1 (PTH1). The pT503/pS504-PTH1 antibody detects phosphorylation in response to agonists. T503/S504 phosphorylation is likely to be...
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pS493/pS495-PTH1 (phospho-Parathyroid Hormone Receptor 1 Antibody)
pS493/pS495-PTH1 (phospho-Parathyroid Hormone...
Serine493/Serine495 (S493/S495) is major phosphorylation site of the Parathyroid Hormone Receptor 1 (PTH1). The pS493/pS495-PTH1 antibody detects phosphorylation in response to agonists. S493/S495 phosphorylation is likely to be involved...
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Agonist-induced Serine435/Serine436 phosphorylation of the Endothelin Receptor B
pS435/pS436-ETB (phospho-Endothelin Receptor B...
Serine435/Serine436 (S435/S436) is major phosphorylation site of the Endothelin Receptor B (ETB). The pS435/pS436-ETB antibody detects phosphorylation in response to agonists. S435/S436 phosphorylation is likely to be involved in...
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Agonist-induced Threonine311/Serine314 phosphorylation of the M4 Muscarinic Acetylcholine Receptor
pT311/pS314-M4 (phospho-M4 Muscarinic...
Threonine311/Serine314 is a major phosphorylation site of the M4 Muscarinic Acetylcholine Receptor. The pT311/pS314-M4 antibody detects phosphorylation in response to high-efficacy agonists but not after PKC activation. T311/S314...
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Agonist-induced Serine296/Serine297 phosphorylation of the M4 Muscarinic Acetylcholine Receptor
pS296/pS297-M4 (phospho-M4 Muscarinic...
Serine296/Serine297 is a major phosphorylation site of the M4 Muscarinic Acetylcholine Receptor. The pS296/pS297-M4 antibody detects phosphorylation in response to high-efficacy agonists but not after PKC activation. S296/S297...
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Agonist-induced Serine385/Serine386 phosphorylation of the M3 Muscarinic Acetylcholine Receptor
pS385/pS386-M3 (phospho-M3 Muscarinic...
Serine385/Serine386 is a major phosphorylation site of the M3 Muscarinic Acetylcholine Receptor. The pS385/pS386-M3 antibody detects phosphorylation in response to high-efficacy agonists but not after PKC activation. S385/S386...
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Agonist-induced Serine359/Threonine361 phosphorylation of the M3 Muscarinic Acetylcholine Receptor
pS359/pT361-M3 (phospho-M3 Muscarinic...
Serine359/Threonine361 is a major phosphorylation site of the M3 Muscarinic Acetylcholine Receptor. The pS359/pT361-M3 antibody detects phosphorylation in response to high-efficacy agonists but not after PKC activation. S359/T361...
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Agonist-induced Serine332/Serine333/Serine334 phosphorylation of the M3 Muscarinic Acetycholine Receptor.
pS332/pS333/pS334-M3 (phospho-M3 Muscarinic...
Serine332/Serine333/Serine334 is a major phosphorylation site of the M3 Muscarinic Acetylcholine Receptor. The pS332/pS333/pS334-M3 antibody detects phosphorylation in response to high-efficacy agonists but not after PKC activation....
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Agonist-induced Serine315/Serine320 phosphorylation of the M2 Muscarinic Acetycholine Receptor
pS315/pS320-M2 (phospho-M2 Muscarinic...
Serine315/Serine320 is a major phosphorylation site of the M2 Muscarinic Acetylcholine Receptor. The pS315/pS320-M2 antibody detects phosphorylation in response to high-efficacy agonists but not after PKC activation. S315/S320...
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7 From 28

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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