The chemokine receptor CXCR4 is a widely expressed G protein-coupled receptor that has been implicated in a number of diseases including human immunodeficiency virus, cancer, and WHIM syndrome, with the latter two involving dysregulation of CXCR4 signaling. To better understand the role of phosphorylation in regulating CXCR4 signaling, tandem mass spectrometry and phospho-specific antibodies were used to identify sites of agonist-promoted phosphorylation. These studies demonstrated that Ser-321, Ser-324, Ser-325, Ser-330, Ser-339, and two sites between Ser-346 and Ser-352 were phosphorylated in HEK293 cells. We show that Ser-324/5 was rapidly phosphorylated by protein kinase C and G protein-coupled receptor kinase 6 (GRK6) upon CXCL12 treatment, whereas Ser-339 was specifically and rapidly phosphorylated by GRK6. Ser-330 was also phosphorylated by GRK6, albeit with slower kinetics. Similar results were observed in human astroglia cells, where endogenous CXCR4 was rapidly phosphorylated on Ser-324/5 by protein kinase C after CXCL12 treatment, whereas Ser-330 was slowly phosphorylated. Analysis of CXCR4 signaling in HEK293 cells revealed that calcium mobilization was primarily negatively regulated by GRK2, GRK6, and arrestin3, whereas GRK3, GRK6, and arrestin2 played a primary role in positively regulating ERK1/2 activation. In contrast, GRK2 appeared to play a negative role in ERK1/2 activation. Finally, we show that arrestin association with CXCR4 is primarily driven by the phosphorylation of far C-terminal residues on the receptor. These studies reveal that site-specific phosphorylation of CXCR4 is dynamically regulated by multiple kinases resulting in both positive and negative modulation of CXCR4 signaling.
"The mechanism by which GRKs determine whether to promote GPCR desensitization or G protein-independent signaling remains unclear. Several studies have focused on the GRK subfamily that mediates desensitization or GRK/β-arrestin signaling
[46,47,65,66]. It has been shown that the phosphorylation of AT1AR by GRK2 and GRK3 induces GPCR desensitization and internalization, whereas phosphorylation by GRK5 leads to β-arrestin-dependent ERK activation
[Show abstract][Hide abstract] ABSTRACT: Desensitization is a physiological feedback mechanism that blocks detrimental effects of persistent stimulation. G protein-coupled receptor kinase 2 (GRK2) was originally identified as the kinase that mediates G protein-coupled receptor (GPCR) desensitization. Subsequent studies revealed that GRK is a family composed of seven isoforms (GRK1-GRK7). Each GRK shows a differential expression pattern. GRK1, GRK4, and GRK7 are expressed in limited tissues. In contrast, GRK2, GRK3, GRK5, and GRK6 are ubiquitously expressed throughout the body. The roles of GRKs in GPCR desensitization are well established. When GPCRs are activated by their agonists, GRKs phosphorylate serine/threonine residues in the intracellular loops and the carboxyl-termini of GPCRs. Phosphorylation promotes translocation of beta-arrestins to the receptors and inhibits further G protein activation by interrupting receptor-G protein coupling. The binding of beta-arrestins to the receptors also helps to promote receptor internalization by clathrin-coated pits. Thus, the GRK-catalyzed phosphorylation and subsequent binding of beta-arrestin to GPCRs are believed to be the common mechanism of GPCR desensitization and internalization. Recent studies have revealed that GRKs are also involved in the beta-arrestin-mediated signaling pathway. The GRK-mediated phosphorylation of the receptors plays opposite roles in conventional G protein- and beta-arrestin-mediated signaling. The GRK-catalyzed phosphorylation of the receptors results in decreased G protein-mediated signaling, but it is necessary for beta-arrestin-mediated signaling. Agonists that selectively activate GRK/beta-arrestin-dependent signaling without affecting G protein signaling are known as beta-arrestin-biased agonists. Biased agonists are expected to have potential therapeutic benefits for various diseases due to their selective activation of favorable physiological responses or avoidance of the side effects of drugs. Furthermore, GRKs are recognized as signaling mediators that are independent of either G protein- or beta-arrestin-mediated pathways. GRKs can phosphorylate non-GPCR substrates, and this is found to be involved in various physiological responses, such as cell motility, development, and inflammation. In addition to these effects, our group revealed that GRK6 expressed in macrophages mediates the removal of apoptotic cells (engulfment) in a kinase activity-dependent manner. These studies revealed that GRKs block excess stimulus and also induce cellular responses. Here, we summarized the involvement of GRKs in beta-arrestin-mediated and G protein-independent signaling pathways.
Journal of Molecular Signaling 03/2014; 9(1):1. DOI:10.1186/1750-2187-9-1
"SDF-1 Uses b-Arrestin1 and CXCR4 Structures in Neuroblastoma further support of our results, both b-arrestin1 and 2 were previously shown to conditionally associate with the CXCR4 tail region upon SDF-1 stimulation of HEK293 cells, and did so via a mechanism requiring a similar carboxyl-terminal region of CXCR4 (Busillo et al., 2010). In contrast, our results suggest that another b-arrestin binding site reportedly located within the third intracellular loop of CXCR4 (Roland et al., 2003), which was intact in all our CXCR4 mutants, either does not bind b-arrestin1 in Fig. 8. GRK2 participates in regulating CXCR4 cell-surface levels and internalization. "
[Show abstract][Hide abstract] ABSTRACT: CXCR4 is a G protein-coupled receptor (GPCR) located on the cell-surface that signals upon binding the chemokine SDF-1 (also called CXCL12). CXCR4 promotes neuroblastoma proliferation and chemotaxis. CXCR4 expression negatively correlates with prognosis and drives neuroblastoma growth and metastasis in mouse models. All functions of CXCR4 require its expression on the cell-surface, yet the molecular mechanisms that regulate CXCR4 cell-surface levels in neuroblastoma are poorly understood. We characterized CXCR4 cell-surface regulation in the related SH-SY5Y and SK-N-SH human neuroblastoma cell lines. SDF-1 treatment caused rapid down-modulation of CXCR4 in SH-SY5Y cells. Pharmacologic activation of Protein Kinase C (PKC) similarly reduced CXCR4, but via a distinct mechanism. Analysis of CXCR4 mutants delineated two CXCR4 regions required for SDF-1 treatment to decrease cell-surface CXCR4 in neuroblastoma cells: the IL motif at residues 328 and 329, and residues 343-352. In contrast, and unlike CXCR4 regulation in other cell types, serines 324, 325, 338 and 339 were not required. Arrestin proteins can bind and regulate GPCR cell-surface expression, often functioning together with kinases such as GRK2. Using SK-N-SH cells which are naturally deficient in β-arrestin1, we showed that β-arrestin1 is required for the CXCR4 343-352 region to modulate CXCR4 cell-surface expression following treatment with SDF-1. Moreover, GRK2 overexpression enhanced CXCR4 internalization, via a mechanism requiring both β-arrestin1 expression and the 343-352 region. Together, these results characterize CXCR4 structural domains and β-arrestin1 as critical regulators of CXCR4 cell-surface expression in neuroblastoma. β-arrestin1 levels may therefore influence the CXCR4-driven metastasis of neuroblastoma as well as prognosis.
"Specifically, GRK5 and 6 have been reported to compete with GRK2 and 3 for the phosphorylation of different receptors, with GRK5 and 6 promoting b-arrestin-dependent ERK activation whereas GRK2 and GRK3 inhibit the same pathway (Heitzler et al., 2012; Kim et al., 2005; Ren et al., 2005). Recent studies have provided direct evidence of the phosphorylation bar code for different GPCRs (Busillo et al., 2010; Butcher et al., 2011; Lau et al., 2011; Nobles et al., 2011). In particular, isoproterenol , a full agonist at the b2AR, was compared with carvedilol, a weak b-arrestin-biased ligand, in control cells or in cells depleted of either GRK6 or GRK2 (Nobles et al., 2011). "
[Show abstract][Hide abstract] ABSTRACT: Follicle-stimulating hormone (FSH) plays a crucial role in the control of reproduction by specifically binding to and activating a membrane receptor (FSHR) that belongs to the G protein-coupled receptor (GPCR) family. Similar to all GPCRs, FSHR activation mechanisms have generally been viewed as a two-state process connecting a unique FSH-bound active receptor to the Gs/cAMP pathway. Over the last decade, paralleling the breakthroughs that were made in the GPCR field, our understanding of FSH actions at the molecular level has dramatically changed. There are numerous facts indicating that the active FSHR is connected to a complex signalling network rather than the sole Gs/cAMP pathway. Consistently, the FSHR probably exists in equilibrium between multiple conformers, a subset of them being stabilized upon ligand binding. Importantly, the nature of the stabilized conformers of the receptor directly depends on the chemical structure of the ligand bound. This implies that it is possible to selectively control the intracellular signalling pathways activated by using biased ligands. Such biased ligands can be of different nature: small chemical molecules, glycosylation variants of the hormone or antibody/hormone complexes. Likewise, mutations or polymorphisms affecting the FSHR can also lead to stabilization of preferential conformers, hence to selective modulation of signalling pathways. These emerging notions offer a new conceptual framework that could potentially lead to the development of more specific drugs while also improving the way FSHR mutants/variants are functionally characterized.
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