Abstract
The G(i)-coupled somatostatin 2A receptor (sst2A) mediates many of the neuromodulatory and neuroendocrine actions of somatostatin (SS) and is targeted by the SS analogs used to treat neuroendocrine tumors. As for other G protein-coupled receptors, agonists stimulate sst2A receptor phosphorylation on multiple residues, and phosphorylation at different sites has distinct effects on receptor internalization and uncoupling. To elucidate the spatial and temporal regulation of sst2A receptor phosphorylation, we examined agonist-stimulated phosphorylation of multiple receptor GPCR kinase sites using phospho-site-specific antibodies. SS increased receptor phosphorylation sequentially, first on Ser-341/343 and then on Thr-353/354, followed by receptor internalization. Reversal of receptor phosphorylation was determined by the duration of prior agonist exposure. In acutely stimulated cells, in which most receptors remained on the cell surface, dephosphorylation occurred only on Thr-353/354. In contrast, both Ser-341/343 and Thr-353/354 were rapidly dephosphorylated when cells were stimulated long enough to allow receptor internalization before agonist removal. Consistent with these observations, dephosphorylation of Thr-353/354 was not affected by either hypertonic sucrose or dynasore, which prevent receptor internalization, whereas dephosphorylation of Ser-341/343 was completely blocked. An okadaic acid- and fostriecin-sensitive phosphatase catalyzed the dephosphorylation of Thr-353/354 both intracellularly and at the cell surface. In contrast, dephosphorylation of Ser-341/343 was insensitive to these inhibitors. Our results show that the phosphorylation and dephosphorylation of neighboring GPCR kinase sites in the sst2A receptor are subject to differential spatial and temporal regulation. Thus, the pattern of receptor phosphorylation is determined by the duration of agonist stimulation and compartment-specific enzymatic activity.
Highlights
Calcium [1, 2]
GPCR signaling and trafficking are stringently regulated by receptor phosphorylation, which usually occurs on multiple Ser and Thr residues in the receptor C terminus or third intracellular loop
Because measurement of receptor phosphorylation at individual phospho-sites is experimentally difficult in intact cells, the detailed regulation of GPCR phosphorylation has been investigated for only a few receptors and is not fully understood with any GPCR
Summary
Materials—Cell culture reagents and geneticin (G-418) were purchased from Invitrogen. The specificity of the phospho-site-directed antibodies for phosphorylated sst2A receptor was validated in Western blots and in an in-cell ELISA (see below). In-cell Enzyme-linked Immunoassay for Receptor Phosphorylation—SS14-induced phosphorylation of the sst2A receptor was measured in intact, fixed cells using phospho-site-specific monoclonal antibodies in a protocol similar to that described previously [29]. CHO-K1 cells stably expressing the wild type or a mutant sst2A receptor were washed twice with warm F12LH medium (Ham’s F-12 medium containing 5 mg/ml lactalbumin hydrolysate and 20 mM HEPES, pH 7.4) and were stimulated at 37 °C with SS14 for the times specified. In experiments in which cells were treated with inhibitors, we controlled for effects on receptor levels and cell loss by incubating fixed cells with monoclonal anti-HA antibodies instead of the phospho-specific antibodies. Differences between treatment groups were analyzed using either an unpaired t test or twoway analysis of variance, as appropriate. p values Ͻ 0.05 were considered statistically significant
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