G protein-coupled receptor kinases (GRKs) are a family of serine/ threonine kinases that phosphorylate and desensitize G protein-coupled receptors. GRK6 is the most recently cloned member of this family. Since little was known about GRK6 we undertook studies that would enhance our understanding of this kinase.
To characterize GRK6, it was overexpressed in Sf9 cells and purified to homogeneity. GRK6 shares a number of in vitro characteristics with GRK5 including: potent inhibition by polyanionic compounds, hyperstimulation by polycations, and preference for phosphorylation of non-acidic peptides. Rhodopsin, the $\beta\sb2$-adrenergic and m2 muscarinic cholinergic receptors serve as stimulus-dependent substrates for GRK6, but with stoichiometries significantly lower than achieved by GRK5 and $\beta$ARK. Additionally, GRK6 does not undergo significant autophosphorylation. These data suggest that GRK6 has a substrate specificity distinct from $\beta$ARK, rhodopsin kinase, and GRK5.
We analyzed the regulation of $\beta$-adrenergic receptor kinase ($\beta$ARK) and GRK6 expression and activity in myelomonocytic and lymphoid cells. In the promyelocytic cell line HL-60, GRK6 protein levels and activity rose 2-4-fold over the course of treatment with agents that induce differentiation toward either the myeloid or monocytic lineage, while $\beta$ARK protein levels and activity were only slightly altered. Treatment of human lymphocytes with several polyclonal activators resulted in enhanced expression and activity of both $\beta$ARK and GRK6. These data demonstrate that $\beta$ARK and GRK6 expression are differentially regulated during myelomonocytic cell development and lymphocyte activation.
GRK6 contains 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$\sp-$) and GRK6-pal$\sp-$ containing a C-terminal CAAX motif to promote geranylgeranylation (GRK6-GG). Compared to wild-type GRK6, GRK6-pal$\sp-$ had an $\sim$8-fold reduced ability to phosphorylate rhodopsin, while GRK6-GG exhibited an $\sim$10-fold higher activity. Wild-type GRK6 and GRK6-GG, but not GRK6-pal$\sp-$, also bound to phosphatidylcholine vesicles. In COS-1 cells, GRK6-pal$\sp-$ was ineffective in promoting agonist-dependent sequestration of the $\beta\sb2$-adrenergic receptor, while sequestration was enhanced significantly in cells expressing either wild-type GRK6 or GRK6-GG versus control cells. These data demonstrate that palmitoylation of GRK6 is necessary for its membrane association and activity.
