Altered activity of palmitoylation-deficient and isoprenylated forms of the G protein-coupled receptor kinase GRK6.

G protein-coupled receptor kinases (GRKs) utilize diverse mechanisms to associate with the plasma membrane and mediate phosphorylation of agonist-occupied receptors. For example, two members of this family, GRK4 and GRK6, contain C-terminal cysteine residues that are palmitoylated. To address whether the activity and membrane association of GRK6 is regulated by palmitoylation, we overexpressed and characterized wild-type GRK6 and two GRK6 mutants, one with the palmitoylation sites mutated to serines (GRK6-pal-) and one containing a C-terminal CAAX motif to promote geranylgeranylation (GRK6-GG). Compared with wild-type GRK6, GRK6-pal- had a approximately 5-fold higher Km and approximately 2-fold lower Vmax for phosphorylating rhodopsin, whereas GRK6-GG exhibited a approximately 2-fold lower Km and approximately 14-fold higher Vmax for rhodopsin. In contrast, wild-type GRK6 and GRK6-pal- displayed similar activity toward the nonreceptor substrate phosvitin, indicating that nonpalmitoylated GRK6 is catalytically active. Wild-type GRK6 and GRK6-GG, but not GRK6-pal-, also bound significantly to phosphatidylcholine vesicles (36 +/- 3, 79 +/- 4, and 4 +/- 2%, respectively) suggesting that GRK6 activity is dependent upon its ability to interact with the plasma membrane. When assayed in COS-1 cells GRK6-pal- promoted minimal agonist-dependent sequestration of the beta2-adrenergic receptor, while sequestration was significantly increased in cells expressing either wild-type GRK6 or GRK6-GG. These data demonstrate an important functional link between the ability of GRK6 to bind to the plasma membrane, a process that appears to be regulated by palmitoylation, and its activity toward receptor substrates.