A Predicted Amphipathic Helix Mediates Plasma Membrane Localization of GRK5

Abstract

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...

Role of the Amphipathic Peptide of Semliki Forest Virus Replicase Protein nsP1 in Membrane Association and Virus Replication

“…The N-terminal helices of regulator of G protein signaling 2 and Rho guanine nucleotide dissociation inhibitor 3 can direct these proteins to the plasma membrane and Golgi complex, respectively (7,13). A C-terminal helix mediates the plasma membrane localization of G protein-coupled receptor kinase 5 (46). All these helices are also able to mediate the targeting of GFP, although the relocalization can be only partial.…”

G Protein-coupled Receptor Kinases of the GRK4 Protein Subfamily Phosphorylate Inactive G Protein-coupled Receptors (GPCRs)

“…GRK5 possesses an amphipathic helix in its C terminus (44), whereas the GRK6A splice variant in addition to this helix also has palmitoylation sites (45)(46)(47). In contrast, the recruitment of GRK2 and GRK3 to the membrane is believed to depend on the interaction of their pleckstrin homology domain with G␤␥ released upon G protein activation (48,49).…”

Mutational Analysis of the Cytoplasmic Tail of Jaagsiekte Sheep Retrovirus Envelope Protein

“…This was somewhat unexpected since alanines would not be expected to disrupt the hydrophobicity of this face. In fact, others have also reported that alanine substitutions in the hydrophobic faces of other amphipathic helices can change their properties (25). Another possibility is that the hydrophobic face of the JSRV amphipathic helix could interact with hydrophobic regions of the same or other proteins within the lipid bilayer.…”