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 t o associate with the plasma membrane and mediate phosphorylation of ag onist-occupied receptors, For example, two members of this family, GRK 4 and CRK6, contain C-terminal cysteine residues that are palmitoylate d, 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 muta ted to serines (GRK6-pal(-)) and one containing a C-terminal CAAX moti f to promote geranylgeranylation (GRK6-GG). Compared with wildtype GRK 6, GRK6-pal(-) had a similar to 5-fold higher K-m and similar to 2-fol d 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 simila r activity toward the nonreceptor substrate phosvitin, indicating that nonpalmitoylated GRK6 is catalytically active, Wild-type GRK6 and GRK 6-GG, but not GRK6-pal(-), also bound significantly to phosphatidylcho line vesicles (36 +/- 3, 79 +/- 4, and 4 +/- 2%, respectively) suggest ing 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 rece ptor, while sequestration was significantly increased in cells express ing either wild-type GRKG or GRK6-GG. These data demonstrate an import ant functional Link between the ability of GRK6 to bind to the plasma membrane, a process that appears to be regulated by palmitoylation, an d its activity toward receptor substrates.