Expression and characterization of the G protein-coupled receptor kinase GRK6

Robert Peter Loudon, Thomas Jefferson University


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. ^

Subject Area

Biology, Cell|Chemistry, Biochemistry|Health Sciences, Immunology

Recommended Citation

Loudon, Robert Peter, "Expression and characterization of the G protein-coupled receptor kinase GRK6" (1997). ETD Collection for Thomas Jefferson University. AAI9836610.