Analysis of Ligand-stimulated CC Chemokine Receptor 5 (CCR5) Phosphorylation in Intact Cells Using Phosphosite-specific Antibodies

Abstract

Human CC chemokine receptor 5 (CCR5), a member of the superfamily of G protein-coupled receptors, regulates the activation and directed migration of leukocytes and serves as the main coreceptor for the entry of R5 tropic strains of human immunodeficiency viruses. We have previously shown that RANTES/CCL5 binding to CCR5 induces GPCR kinase (GRK)-and protein kinase C (PKC)-mediated phosphorylation of four distinct C-terminal serine residues. To study these phosphorylation events in vivo, we have generated monoclonal antibodies, which specifically react only with either phosphorylated or nonphosphorylated CCR5. These phosphosite-specific antibodies reveal that following ligand stimulation of the receptor serine 337 is exclusively phosphorylated by a PKC-mediated mechanism, while GRKs phosphorylate serine 349. GRK-mediated receptor phosphorylation proceeds in a regular time-dependent manner (t1 ⁄2 ϳ2 min) with an apparent EC 50 of 5 nM. In contrast, PKC phosphorylates serine 337 at 50-fold lower concentrations and in a very rapid, albeit transient manner. Protein phosphatases that are active at neutral pH and are inhibited by okadaic acid rapidly dephosphorylate phosphoserine 337, but less efficiently phosphoserine 349, in intact cells and in an in vitro assay. Immunofluorescence microscopy demonstrates that phosphorylated receptors accumulate in a perinuclear compartment, which resembles recycling endosomes. This study is the first to analyze in detail the spatial and temporal dynamics of GRK-versus PKCmediated phosphorylation of a G protein-coupled receptor and its subsequent dephosphorylation on the level of individual phosphorylation sites. G protein-coupled receptors (GPCR)1 comprise the largest known family of signal-transducing proteins and respond to a large variety of external stimuli (1, 2). The receptors relay the information encoded by the ligand through the activation of heterotrimeric guanine nucleotide-binding proteins and intracellular effector molecules. Many GPCR undergo a process of rapid desensitization, which involves ligand-induced phosphorylation of serine and threonine residues located in the third intracellular loop or C-terminal domain by two different families of protein kinases. (i) GPCR kinases (GRKs) specifically phosphorylate only the agonist-occupied GPCR and thus mediate agonist-specific or homologous receptor phosphorylation (3, 4). (ii) In contrast, the second messenger-activated kinases, such as cyclic AMP-dependent protein kinase and protein kinase C (PKC), potentially phosphorylate both the ligand-bound GPCR and multiple other receptors in a heterologous manner. Receptor phosphorylation enhances the affinity of the agonistoccupied receptor for interaction with arrestin which interdicts signal transduction between the receptor and G proteins by steric mechanisms. The nonvisual arrestins, ␤-arrestin-1 and ␤-arrestin-2, also promote clathrin-mediated endocytosis of phosphorylated receptors and have been implicated in crosstalk with other signaling pathways. Once internalized, GPCR...

Non-visual GRKs: are we seeing the whole picture?

“…Despite the lack of apparent selectivity between GRKs, phosphopeptide mapping identified distinct phosphorylation profiles between different GRKs, suggesting the possibility of differential regulation. More recently, an elegant study using phospho-specific antibodies directed against four individual serine-containing epitopes within the C-terminal tail of the chemokine receptor CCR5 highlighted the distinct stimulus strength and temporal patterns of PKC-and GRK-mediated phosphorylation [50]. Overall, there is increasing evidence for specificity of GRK -GPCR interactions when endogenous kinases are investigated in physiological settings.…”

Chemokine receptor internalization and intracellular trafficking

“…The lower endocytosis rate of wild-type CCR5 may keep the gate open for receptor resensitization at the plasma membrane. Indeed, an unknown GPCR phosphatase, active at neutral pH and hence different from the endosome-associated phosphatases, was shown to trigger rapid CCR5 dephosphorylation at the plasma membrane [127]. In contrast to the palmitoylation-deficient CCR5 deletion mutant, a considerable fraction of wild-type CCR5 trafficked by clathrinindependent routes into caveolin-containing vesicular structures in HeLa transfectants in response to CCL4 or CCL5 [3].…”

Agonist-induced Endocytosis of CC Chemokine Receptor 5 Is Clathrin Dependent

“…Down-modulation of cell sur- face CCR5 is due to rapid agonist-induced endocytosis and accumulation of the receptors inside the cell. CCR5 was found to be internalized into the perinuclear recycling compartment and presumed to use a clathrin-dependent pathway (Amara et al, 1997;Mack et al, 1998;Pollok-Kopp et al, 2003). ␤-Arrestins have been shown not only to influence agonist-mediated endocytosis of CCR5 but also to be required for this process (Aramori et al, 1997;Fraile-Ramos et al, 2003).…”

“…In RBL-CCR5 cells, this activation is also apparent as agonistinduced cell spreading ( Figure 1B). We and others have shown that agonist treatment of CHO-CCR5 or RBL-CCR5 cells leads to rapid CCR5 internalization (Mack et al, 1998;Olbrich et al, 1999;Pollok-Kopp et al, 2003). To investigate the mechanisms responsible for this internalization in more detail, we focused on the events occurring in the first few minutes after agonist addition.…”