G‐protein‐coupled receptor dephosphorylation at the cell surface

Abstract

Abbreviations: GPCR, G-protein-coupled receptor; GRK, G-protein-coupled receptor kinase; PKA, protein kinase A; PKC, protein kinase C Agonist-induced desensitization of G-protein-coupled receptors (GPCRs) usually involves phosphorylation of the receptor by G-protein-coupled receptor kinases (GRKs) or second messenger-dependent protein kinases such as PKC and PKA. Phosphorylation by GRKs promotes arrestin binding to the receptor, which not only uncouples receptor and G protein but also targets the receptor to clathrin-coated pits for internalization. The internalized GPCR is either trafficked to lysosomes for degradation or alternatively is dephosphorylated by phosphatase enzymes in an acidic endosomal compartment before being recycled to the membrane for further rounds of agonist stimulation. 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). They show that when internalization of the phosphorylated b 2 -adrenoceptor is blocked, either by expression of a dominant-negative mutant form of dynamin or with hypertonic sucrose, the receptor still undergoes dephosphorylation of both PKA and GRK sites. Indeed, the rate of dephosphorylation of the receptor is the same whether or not the receptor is able to internalize. Furthermore, they show that the b 2 -adrenoceptor still undergoes dephosphorylation following selective phosphorylation of PKA site Ser262 subsequent to the addition of a very low concentration of isoprenaline (300 pM), which does not induce receptor internalization. These results show conclusively that the b 2 -adrenoceptor does not have to internalize in order to become dephosphorylated (Figure 1). Does this apply to other GPCRs? 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...

Quantitative Modeling of GRK-Mediated β2AR Regulation

“…There has been some controversy about the possibility of plasma membrane dephosphorylation of the GRK phosphorylated receptor and the recycling of the phosphorylated receptor [30] , [34] , [49] – [51] . Recently we have shown that (i) β2AR can be dephosphorylated when internalization is blocked with either hypertonic sucrose treatment or through the use of a dominant negative form of dynamin [30] , and (ii) that dephosphorylation occurs with no detectable internalization [16] .…”

Characterization of β2-Adrenergic Receptor Dephosphorylation: Comparison with the Rate of Resensitization

“…Although our prior study showed that PKA and possibly GRK site dephosphorylations occurred at the plasma membrane, several important questions remained (Kelly, 2006). First, to what extent does functional resensitization of agonist stimulation of the ␤ 2 AR and adenylyl cyclase correlate with the rate constants for dephosphorylation of the PKA and GRK sites?…”

Encoding the β-Arrestin Trafficking Fate of Ghrelin Receptor GHSR1a: C-Tail-Independent Molecular Determinants in GPCRs

“…During the desensitization process, G-protein-coupled receptor kinase (GRK) phosphorylated GPCRs undergo clathrin-mediated endocytosis as a result of β-arrestin-binding. − It was initially hypothesized for endosomal recycling receptors, with the β2-adrenergic receptor (β2AR) serving as a role model for other GPCRs, that a downstream dephosphorylation/resensitization step was either initiated or accompanied by an acidic-induced loss of ligand binding. This view, however, was broadened to include the discovery that resensitization relevant phosphatases are active in nonendosomal cell compartments including those at the plasma membrane. − Thus, β-arrestin-based receptor trafficking information appears susceptible to modification even before the receptor has an opportunity to internalize from the plasma membrane, and to assess it requires measurement techniques applicable over both short and long times. In addition, if we use the beta2-adrenergic receptor (β2AR) and rhodopsin as templates for characterizing the GHSR1a, then the principal locations to assess trafficking information are in its C-tail and ICL2 (Figure b–e). ,, Generation of a GHSR1/arrestin active space-filling model (Figure c, lower panels) by corresponding residue substitution of its ICL2 into the rhodopsin ICL2 (Figure c, upper panels) suggests conservation of the ability to bind the active arrestin cleft as well as demonstrating conservation of a short N-capped proline α helix (Figure a, rhodopsin; Figure d, GHSR1a) within 2.4 Å of a hydrophobic patch containing arrestin leucine 250 (Figure e.…”