Development of a Yeast Bioassay to Characterize G Protein-coupled Receptor Kinases

Self Cite

G protein-coupled receptor kinases (GRKs) specifically phosphorylate agonist-occupied G protein-coupled receptors at the inner surface of the plasma membrane (PM), leading to receptor desensitization. GRKs utilize a variety of mechanisms to bind tightly, and sometimes reversibly, to cellular membranes. Previous studies demonstrated the presence of a membrane binding domain in the C terminus of GRK5. Here we define a mechanism by which this short C-terminal stretch of amino acids of GRK5 mediates PM localization. Secondary structure predictions suggest that a region contained within amino acids 546 -565 of GRK5 forms an amphipathic helix, with the key features of the predicted helix being a hydrophobic patch of amino acids on one face of the helix, hydrophilic amino acids on the opposite face, and a number of basic amino acids surrounding the hydrophobic patch. We show that amino acids 546 -565 of GRK5 are sufficient to target the cytoplasmic green fluorescent protein (GFP) to the PM, and the hydrophobic amino acids are necessary for PM targeting of GFP-546 -565. Moreover, full-length GRK5-GFP is localized to the PM, but mutation of the hydrophobic patch or the surrounding basic amino acids prevents PM localization of GRK5-GFP. Last, we show that mutation of the hydrophobic residues severely diminishes phospholipid-dependent autophosphorylation of GRK5 and phosphorylation of membrane-bound rhodopsin by GRK5. The findings in this report thus suggest the presence of a membrane binding motif in GRK5 and define the importance of a group of hydrophobic amino acids within this motif in mediating its PM localization.Cell surface localized G protein-coupled receptors (GPCRs) 1 detect a large variety of extracellular stimuli and initiate numerous intracellular signaling pathways. Proper regulation of GPCR signaling is maintained in part by a process known as desensitization (1), which refers to the turning off of a GPCRinitiated signaling event in the continuous presence of agonist. A key mechanism of GPCR desensitization is phosphorylation of agonist-occupied receptor by the G protein-coupled receptor kinases (GRKs) (2, 3). This phosphorylation promotes binding of arrestin proteins to the GPCR and results in subsequent uncoupling of the GPCR from G protein.To function properly GRKs need to be localized to the cytoplasmic face of the plasma membrane (PM) where their GPCR substrates are located. Different members of the GRK family utilize a variety of mechanisms to bind to cellular membranes (2, 3). GRK2 and GRK3 contain well characterized C-terminal pleckstrin homology domains (4) that mediate translocation of the kinases from the cytoplasm to the PM in response to GPCR activation (5, 6). The pleckstrin homology domains allow PM binding by interacting with both phospholipids and free G protein ␤␥ subunits. In contrast to GRK2/3, other GRK family members exhibit a more constitutive association with cellular membranes. GRK1 and GRK7 contain a C-terminal CAAX motif, which directs covalent modification by a farnesyl or gerany...

Section: Discussionsupporting

confidence: 55%

A Predicted Amphipathic Helix Mediates Plasma Membrane Localization of GRK5

Thiyagarajan

Stracquatanio

Pronin³

et al. 2004

Self Cite

“…3a). This region comprises many of the residues known to be important for receptor phosphorylation by GRKs (19,41,42). The N terminus makes extensive contacts with both RH subdomains, burying ϳ1,200 Å 2 of accessible surface area.…”

Section: Resultsmentioning

confidence: 99%

Structures of Rhodopsin Kinase in Different Ligand States Reveal Key Elements Involved in G Protein-coupled Receptor Kinase Activation

Singh

Wang

Maeda

et al. 2008

154

G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate activated heptahelical receptors, leading to their uncoupling from G proteins. Here we report six crystal structures of rhodopsin kinase (GRK1), revealing not only three distinct nucleotide-binding states of a GRK but also two key structural elements believed to be involved in the recognition of activated GPCRs. The first is the C-terminal extension of the kinase domain, which was observed in all nucleotide-bound GRK1 structures. The second is residues 5-30 of the N terminus, observed in one of the GRK1⅐(Mg 2؉ ) 2 ⅐ATP structures. The N terminus was also clearly phosphorylated, leading to the identification of two novel phosphorylation sites by mass spectral analysis. Co-localization of the N terminus and the C-terminal extension near the hinge of the kinase domain suggests that activated GPCRs stimulate kinase activity by binding to this region to facilitate full closure of the kinase domain.

“…Ser 21 is part of an ␣-helical domain in the amino terminus that is structurally conserved across the GRK family of proteins. Biochemical and structural studies indicate that this region generally plays a role in receptor interaction, leading to allosteric activation of the kinase (22,24,34), possibly through interaction with the hinge region of the RGS homology domain and a region of the kinase domain of the carboxyl terminus (23,35). Although Ser 21 is visualized in the crystal structure of GRK1, its potential interactions with rhodopsin or regions of the kinase have not been defined.…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation of G Protein-coupled Receptor Kinase 1 (GRK1) Is Regulated by Light but Independent of Phototransduction in Rod Photoreceptors

Osawa

Xiong

et al. 2011

Phosphorylation of rhodopsin by G protein-coupled receptor kinase 1 (GRK1, or rhodopsin kinase) is critical for the deactivation of the phototransduction cascade in vertebrate photoreceptors. Based on our previous studies in vitro, we predicted that Ser 21 in GRK1 would be phosphorylated by cAMP-dependent protein kinase (PKA) in vivo. Here, we report that dark-adapted, wild-type mice demonstrate significantly elevated levels of phosphorylated GRK1 compared with light-adapted animals. Based on comparatively slow half-times for phosphorylation and dephosphorylation, phosphorylation of GRK1 by PKA is likely to be involved in light and dark adaptation. In mice missing the gene for adenylyl cyclase type 1, levels of phosphorylated GRK1 were low in retinas from both dark-and light-adapted animals. These data are consistent with reports that cAMP levels are high in the dark and low in the light and also indicate that cAMP generated by adenylyl cyclase type 1 is required for phosphorylation of GRK1 on Ser 21 . Surprisingly, dephosphorylation was induced by light in mice missing the rod transducin ␣-subunit. This result indicates that phototransduction does not play a direct role in the light-dependent dephosphorylation of GRK1.