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Rapid Dephosphorylation of G Protein-coupled Receptors by Protein Phosphatase 1β Is Required for Termination of β-Arrestin-dependent Signaling

  • Florian Pöll
    Affiliations
    Institute of Pharmacology and Toxicology, University Hospital, Friedrich Schiller University, D-07747 Jena, Germany
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  • Christian Doll
    Affiliations
    Institute of Pharmacology and Toxicology, University Hospital, Friedrich Schiller University, D-07747 Jena, Germany
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  • Stefan Schulz
    Correspondence
    To whom correspondence should be addressed: Institute of Pharmacology and Toxicology, University Hospital, Friedrich Schiller University, Drackendorfer Str. 1, D-07747 Jena, Germany. Tel.: 49-3641-9325650; Fax: 49-3641-9325652;
    Affiliations
    Institute of Pharmacology and Toxicology, University Hospital, Friedrich Schiller University, D-07747 Jena, Germany
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Open AccessPublished:July 27, 2011DOI:https://doi.org/10.1074/jbc.M111.224899
      Termination of signaling of activated G protein-coupled receptors (GPCRs) is essential for maintenance of cellular homeostasis. It is well established that β-arrestin redistributes to phosphorylated GPCRs and thereby facilitates desensitization of classical G protein-dependent signaling. β-Arrestin in turn serves as a scaffold to initiate a second wave of signaling. Here, we report a molecular mechanism that regulates the termination of unconventional β-arrestin-dependent GPCR signaling. We identify protein phosphatase 1β (PP1β) as a phosphatase for the cluster of phosphorylated threonines (353TTETQRT359) within the sst2A somatostatin receptor carboxyl terminus that mediates β-arrestin binding using siRNA knock-down screening. We show that PP1β-mediated sst2A dephosphorylation is initiated directly after receptor activation at or near the plasma membrane. As a functional consequence of diminished PP1β activity, we find that somatostatin- and substance P-induced but not epidermal growth factor-induced ERK activation was aberrantly enhanced and prolonged. Thus, we demonstrate a novel mechanism for fine tuning unconventional β-arrestin-dependent GPCR signaling in that recruitment of PP1β to activated GPCRs facilitates GPCR dephosphorylation and, hence, leads to disruption of the β-arrestin-GPCR complex.

      Introduction

      Desensitization of GPCR
      The abbreviations used are: GPCR
      G protein-coupled receptor
      PP
      protein phosphatase
      SS-14
      somatostatin
      sst
      somatostatin receptor.
      signaling causes a reduction of receptor response to repeated or long-lasting stimuli. It usually involves agonist-induced phosphorylation of cytoplasmic parts of the receptor by GRKs or second messenger-dependent protein kinases such as protein kinase A or protein kinase C (
      • Marchese A.
      • Paing M.M.
      • Temple B.R.
      • Trejo J.
      ). Agonist-induced phosphorylation allows binding of β-arrestin to the receptor (
      • Krupnick J.G.
      • Benovic J.L.
      ). It is well established that β-arrestin promotes desensitization of G protein signaling and induces receptor internalization. More recent work has established that β-arrestin binding in turn stimulates a second wave of signaling (
      • Lefkowitz R.J.
      • Shenoy S.K.
      ,
      • Defea K.A.
      ).
      Although the regulation of agonist-induced phosphorylation has been studied in detail for many GPCRs, the molecular mechanisms and functional consequences of receptor dephosphorylation are far from understood. Earlier studies have identified retinal degeneration C as the phosphatase required for rhodopsin dephosphorylation in Drosophila melanogaster. The catalytic domain of retinal degeneration C exhibits high homology to PP1, PP2, and PP3 (
      • Steele F.R.
      • Washburn T.
      • Rieger R.
      • O'Tousa J.E.
      ,
      • Byk T.
      • Bar-Yaacov M.
      • Doza Y.N.
      • Minke B.
      • Selinger Z.
      ,
      • Vinós J.
      • Jalink K.
      • Hardy R.W.
      • Britt S.G.
      • Zuker C.S.
      ). Loss of retinal degeneration C causes disturbance of light-signal transduction and leads to light-dependent retinal degeneration (
      • Vinós J.
      • Jalink K.
      • Hardy R.W.
      • Britt S.G.
      • Zuker C.S.
      ). At the same time, a PP2-related phosphatase that dephosphorylates the β2-adrenergic receptor was identified and named GPCR phosphatase (
      • Pitcher J.A.
      • Payne E.S.
      • Csortos C.
      • DePaoli-Roach A.A.
      • Lefkowitz R.J.
      ,
      • Krueger K.M.
      • Daaka Y.
      • Pitcher J.A.
      • Lefkowitz R.J.
      ). It was proposed that GPCR phosphatase is tethered to vesicular membranes and that receptors have to internalize into an acidic endosomal compartment to become dephosphorylated (
      • Pitcher J.A.
      • Payne E.S.
      • Csortos C.
      • DePaoli-Roach A.A.
      • Lefkowitz R.J.
      ,
      • Krueger K.M.
      • Daaka Y.
      • Pitcher J.A.
      • Lefkowitz R.J.
      ). However, later it was shown that inhibition of β2-adrenergic receptor internalization with dominant negative dynamin or hypertonic sucrose did not affect the rate of receptor dephosphorylation. Similar, D1 dopamine receptor dephosphorylation was not blocked in the presence of concanavalin A, which also inhibits receptor internalization (
      • Gardner B.
      • Liu Z.F.
      • Jiang D.
      • Sibley D.R.
      ). More recent studies have shown that phosphatase inhibitors such as okadaic acid and calyculin A can block the dephosphorylation of a number of GPCRs including the β2-adrenergic receptor, D1 dopamine receptor, parathyroid hormone receptor 1, thromboxane A receptor, and the vasopressin receptor 1. However, a specific phosphatase has not been identified so far (
      • Gardner B.
      • Liu Z.F.
      • Jiang D.
      • Sibley D.R.
      ,
      • Chauvin S.
      • Bencsik M.
      • Bambino T.
      • Nissenson R.A.
      ,
      • Innamorati G.
      • Sadeghi H.
      • Birnbaumer M.
      ,
      • Spurney R.F.
      ,
      • Tran T.M.
      • Friedman J.
      • Baameur F.
      • Knoll B.J.
      • Moore R.H.
      • Clark R.B.
      ).
      In the present study, we have examined the mechanism and function of receptor dephosphorylation using the sst2A somatostatin receptor as model. Earlier, we have defined the carboxyl-terminal 353TTETQRT359 motif as primary phosphorylation and β-arrestin-binding site of the sst2A receptor (
      • Tulipano G.
      • Stumm R.
      • Pfeiffer M.
      • Kreienkamp H.J.
      • Höllt V.
      • Schulz S.
      ). Additionally, agonist-induced phosphorylation occurs on two nearby serine residues, Ser341 and Ser343 (
      • Ghosh M.
      • Schonbrunn A.
      ). We have then generated phosphosite-specific antibodies, which enabled us to study the spatial and temporal dynamics of agonist-induced sst2A phosphorylation in detail (
      • Pöll F.
      • Lehmann D.
      • Illing S.
      • Ginj M.
      • Jacobs S.
      • Lupp A.
      • Stumm R.
      • Schulz S.
      ). Here, we have used chemical protein phosphatase inhibitors and siRNA knock-down screening to identify PP1β as the GPCR phosphatase that catalyzes rapid dephosphorylation of the residues Thr353, Thr354, Thr356, and Thr359. The dephosphorylation of these threonine residues occurs at or near the plasma membrane. Our findings suggest that PP1β-dependent GPCR dephosphorylation plays an essential role in the termination of β-arrestin-dependent signaling.

      DISCUSSION

      Desensitization of GPCR signaling is essential for maintenance of cellular homeostasis. For many GPCRs, agonist-dependent regulation involves rapid phosphorylation of a series of phosphate acceptor sites within the carboxyl-terminal tail of the receptor. This phosphorylation facilitates binding of β-arrestin, which in turn mediates desensitization of G protein-dependent signaling. In addition, β-arrestin serves as scaffold to facilitate receptor internalization and to initiate a second wave of signaling (
      • DeWire S.M.
      • Ahn S.
      • Lefkowitz R.J.
      • Shenoy S.K.
      ,
      • Luttrell L.M.
      • Gesty-Palmer D.
      ). However, until now the mechanisms involved in the negative feedback regulation of unconventional β-arrestin-dependent signaling remained elusive. Given the fact that β-arrestin-dependent signaling is initiated by its binding to phosphorylated GPCRs, we hypothesized that receptor dephosphorylation by specific GPCR phosphatases would lead to disruption of the GPCR-β-arrestin complex and, hence, facilitate termination of β-arrestin-dependent signaling.
      In the present study, we used a combination of phosphosite-specific antibodies and siRNA screening to identify PP1β as bona fide GPCR phosphatase for the sst2A somatostatin receptor. Inhibition of PP1β expression resulted in increased agonist-driven receptor phosphorylation at the 353TTETQRT359 motif and facilitated detection of phosphorylated receptors at the plasma membrane shortly after agonist exposure, indicating that PP1β catalyzes dephosphorylation directly after receptor activation at or near the plasma membrane. Remarkably, dephosphorylation of Ser341/Ser343 occurred at a much slower rate than dephosphorylation of the four threonine residues. It has been reported that sst2A internalization is a prerequisite for Ser341/Ser343 dephosphorylation, but not for Thr353/Thr354 or Thr356/Thr359 dephosphorylation (
      • Ghosh M.
      • Schonbrunn A.
      ). These findings strongly suggest that Ser341/Ser343 dephosphorylation occurs via a mechanism distinct from rapid PP1β-dependent 353TTETQRT359 dephosphorylation. Hence, GPCR dephosphorylation occurs in distinct temporal and spatial dynamics for different phosphorylation sites.
      PP1 is composed of a catalytic subunit bound to one or more regulatory subunits. At present, >40 different regulatory PP1 subunits are known, which determine substrate specificity and subcellular localization of PP1s (
      • Virshup D.M.
      • Shenolikar S.
      ). It remains open to question whether a single or multiple regulatory PP1 subunits are required for efficient GPCR dephosphorylation and whether PP1β is recruited directly to phosphorylated GPCRs or via additional adapter proteins.
      In previous work, we have clearly demonstrated that GPCR kinase 2/3-driven phosphorylation of the 353TTETQRT359 motif is essential for β-arrestin binding. We have also shown that sst2A receptor stimulation leads to both Gi protein-dependent and β-arrestin-dependent ERK activation (
      • Pöll F.
      • Lehmann D.
      • Illing S.
      • Ginj M.
      • Jacobs S.
      • Lupp A.
      • Stumm R.
      • Schulz S.
      ,
      • Lesche S.
      • Lehmann D.
      • Nagel F.
      • Schmid H.A.
      • Schulz S.
      ). In the present work, we found that inhibition of PP1β expression resulted in a robust increase in β-arrestin-dependent ERK activation in SS-14-treated cells. This effect was selective. It was not seen after exposure to EGF or after inhibition of PP1α or PP1γ expression under otherwise identical conditions, suggesting that diminished PP1 activity does not directly lead to a general enhancement of ERK excitability. Nevertheless, this effect was not unique. Similar to that seen with the sst2A receptor, ERK activation was also robustly enhanced and prolonged after exposure of NK1-expressing cells to substance P suggesting that not only the sst2A receptor but also other GPCRs require functional PP1β for termination of their β-arrestin-dependent ERK signaling.
      In conclusion, we identify PP1β as the first GPCR phosphatase for the sst2A receptor. Our findings indicate that PP1β-mediated 353TTETQRT359 dephosphorylation is initiated directly after receptor activation at or near the plasma membrane. In contrast, Ser341/Ser343 dephosphorylation occurs at a much slower rate by a distinct yet unknown mechanism. We also discovered a novel mechanism for fine tuning unconventional β-arrestin-dependent GPCR signaling in that engagement of PP1β facilitates GPCR dephosphorylation. Dephosphorylation in turn leads to disruption of the β-arrestin-GPCR complex and thereby limits β-arrestin-dependent ERK signaling. We propose that this could be a common mechanism for many GPCRs.

      Acknowledgments

      We thank Heidrun Guder and Heike Stadler for excellent technical assistance.

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