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 CRK6, 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 wildtype GRK6, GRK6-pal(-) had a similar to 5-fold higher K-m and similar to 2-fold lower V-max for phosphorylating rhodopsin, whereas GRRG-GG exhibited a similar to 2-fold lower K, and similar to 14-fold higher V-max for rhodopsin, In contrast, wildtype GRR6 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 beta(2)-adrenergic receptor, while sequestration was significantly increased in cells expressing either wild-type GRKG 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.