Differential phosphorylation and dephosphorylation of β₂‐adrenoceptor sites Ser262 and Ser355,356

Abstract

1 Activated b 2 -adrenoceptors are rapidly desensitized by phosphorylation of Ser262 by protein kinase A (PKA) and of Ser355,356 by G-protein-coupled receptor kinase (GRK). We sought to determine whether the phosphorylation and subsequent dephosphorylation of these sites had similar kinetics and requirements for receptor endocytosis. 2 The phosphorylation of the PKA and GRK sites were measured using antibodies that recognize phosphoserine 262 and phosphoserine 355,356. Endocytosis in stably transfected HEK293 cells was blocked by inducible expression of dominant-negative dynamin-1 K44A or by treatment with hypertonic sucrose. 3 The phosphorylation of the GRK site Ser355,356 during a 10 mM isoprenaline treatment rapidly reached a steady state, and the extent of kinetics of phosphorylation were unaffected by dynamin-1 K44A expression, and minimally by hypertonic sucrose. 4 In contrast, phosphorylation of the PKA site Ser262 during a 10 mM isoprenaline treatment peaked after 2 min and then rapidly declined, while inhibition of endocytosis enhanced and prolonged phosphorylation. Treatment with 300 pM isoprenaline, a concentration too low to provoke endocytosis, also resulted in prolonged PKA site phosphorylation. 5 The dephosphorylation of these sites was measured after removal of agonist. Significant dephosphorylation of phosphoserines 262 and 355,356 was observed under conditions of very low endocytosis, however dephosphorylation of the GRK site was greater if antagonist was present after removal of agonist. 6 The results indicate that the kinetics of b 2 -adrenoceptor GRK and PKA site phosphorylation are distinct and differently affected by endocytosis, and that receptor dephosphorylation can occur either at the plasma membrane or in internal compartments.

Reprogramming of G protein-coupled receptor recycling and signaling by a kinase switch

“…4B). Given that PKA inhibition has no effect on B2AR internalization (24)(25)(26)(27)32), this increase in surface B2AR on PKA inhibition likely reflects an increase in B2AR recycling rates from steady state (Fig. 3D).…”

“…Interestingly, B2AR itself contains two cytoplasmic consensus PKA sites that are phosphorylated in response to an agonist (2,23,24). One of these two sites, Ser-261/262, located in the third cytoplasmic loop, is not involved in endocytic trafficking (24)(25)(26)(27); thus, we focused on Ser-345/346 in the cytoplasmic tail, which has been implicated in surface delivery of B2AR (20). We first detected iso-induced B2AR phosphorylation at this site, using a phospho-specific Ser346 antibody.…”

Processive phosphorylation: Mechanism and biological importance

“…The β 2 -adrenergic receptor is phosphorylated by PKA at Ser262 in the third intracellular loop and by GRK at Ser355 and Ser256 in the C-terminus. The kinetics of PKA and GRK phosphorylation are different in this case, and the events are differentially affected by interfering with endocytosis [32]. PKA phosphorylation of β 1 -or β 2 -adrenergic receptors can also redirect the G protein coupling specificity, so that the receptors have reduced affinity for G s and increased affinity for G i .…”

G‐protein‐coupled receptor dephosphorylation at the cell surface

“…These GPCR regulatory pathways were identified largely as a result of the seminal studies by the Lefkowitz laboratory in the U.S.A. (reviewed in Lefkowitz, 2004), and have reached the status of dogma in the field of GPCR biology. However, some new studies, including one in this issue of the British Journal of Pharmacology (Iyer et al, 2005), suggest that the role of internalization in GPCR dephosphorylation is less clear than previously thought. This work is particularly elegant because the authors have developed antibodies to detect b 2 -adrenoceptor phosphorylation by PKA at Ser262 in the receptor's third intracellular loop and by GRKs at Ser355,356 in the COOH-terminus (Tran et al, 2004).…”

“…Apparently it does; a recent study (Gardiner et al, 2001) shows that the agonist-phosphorylated G s -coupled D 1 dopamine receptor undergoes the same rate of dephosphorylation whether or not it is able to undergo internalization. Furthermore, a very recent study shows that the agonist-activated G q -coupled thyrotropin-releasing hormone receptor is rapidly dephosphorylated at the cell membrane in mouse embryo fibroblast cells from arrestin-knockout animals where these receptors are unable to undergo significant internalization (Jones & Hinkle, 2005).How to reconcile these recent findings with the earlier results (Yu et al, 1993;Pippig et al, 1995;Krueger et al, 1997)? Iyer et al (2005 suggest that the interpretation of earlier studies on the b 2 -adrenoceptor is made difficult because the 32 P labelling technique reflects a combination of PKA and GRK phosphorylation of the receptor.…”