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

Abstract

Termination of signaling of activated G protein-coupled receptors (GPCRs) is essential for maintenance of cellular homeostasis. It is well established that ␤-arrestin redistributes to phosphorylated GPCRs and thereby facilitates desensitization of classical G protein-dependent signaling. ␤-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 ␤-arrestin-dependent GPCR signaling. We identify protein phosphatase 1␤ (PP1␤) as a phosphatase for the cluster of phosphorylated threonines ( 353 TTETQRT 359 ) within the sst 2A somatostatin receptor carboxyl terminus that mediates ␤-arrestin binding using siRNA knock-down screening. We show that PP1␤-mediated sst 2A dephosphorylation is initiated directly after receptor activation at or near the plasma membrane. As a functional consequence of diminished PP1␤ 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 ␤-arrestin-dependent GPCR signaling in that recruitment of PP1␤ to activated GPCRs facilitates GPCR dephosphorylation and, hence, leads to disruption of the ␤-arrestin-GPCR complex. Desensitization of GPCR2 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 (1). Agonist-induced phosphorylation allows binding of ␤-arrestin to the receptor (2). It is well established that ␤-arrestin promotes desensitization of G protein signaling and induces receptor internalization. More recent work has established that ␤-arrestin binding in turn stimulates a second wave of signaling (3,4).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 (5-7). Loss of retinal degeneration C causes disturbance of light-signal transduction and leads to light-dependent retinal degeneration (7). At the same time, a PP2-related phosphatase that dephosphorylates the ␤ 2 -adrenergic receptor was identified and named GPCR phosphatase (8, 9). 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 (8, 9). However, later it was shown that inhibition of ␤ 2 -adrenergic receptor internalization with dominant negative dynamin or hypertonic sucrose did not affect the rate of receptor dephosphorylation. Similar, ...

Mechanisms of Regulation of Olfactory Transduction and Adaptation in the Olfactory Cilium

“…On one hand, GPCR phosphorylation by PKA and GRK3 increases internalization, on the other hand, GPCR dephosphorylation results in exocytosis and resensitization [45], [62]. Thus, inhibition of dephosphorylation increases GPCR internalization (Figure 7A) and explains the similar results obtained using okadaic acid, the recycling inhibitor monensin, and IBMX in the levels of STA (Figures 2 and 4).…”

Illuminating somatostatin analog action at neuroendocrine tumor receptors

“…Interestingly, a subsequent study found that PP1 rather than PP2A dephosphorylated Thr353/354 in HEK293 cells 109 . The finding that different phosphatases act on sst 2A in different cell lines suggests that the phosphatases that catalyze sst 2A dephosphorylation may be cell specific.…”

Regulation of heterologously expressed 5‐HT1B receptors coupling to potassium channels in AtT‐20 cells

“…Agonist‐induced internalization of 5‐HT 1B receptors in fibroblasts can be reduced by antibodies to GRK2/3, providing indirect evidence for an agonist‐induced receptor phosphorylation (Janoshazi et al ., ). While it is often assumed that such receptor phosphorylation commits receptors to being internalized, reversal of agonist‐induced phosphorylation of both μ‐opioid and SST 2 receptors at the plasma membrane has been demonstrated, indicating that dephosphorylation of 5‐HT 1B receptors could potentially explain the reversal of agonist‐induced desensitization observed (Pöll et al ., ; Doll et al ., ). Unfortunately, there is no direct evidence in any system that 5‐HT 1B receptor phosphorylation is changed by agonist binding, and no evidence that phosphorylation mediates desensitization.…”