Plasma membrane and nuclear localization of G protein coupled receptor kinase 6A.
G protein-coupled receptor (GPCR) kinases (GRKs) specifically phosphorylate agonist-occupied GPCRs at the inner surface of the plasma membrane (PM), leading to receptor desensitization. Here we show that the C-terminal 30 amino acids of GRK6A contain multiple elements that either promote or inhibit PM localization. Disruption of palmitoylation by individual mutation of cysteine 561, 562, or 565 or treatment of cells with 2-bromopalmitate shifts GRK6A from the PM to both the cytoplasm and nucleus. Likewise, disruption of the hydrophobic nature of a predicted amphipathic helix by mutation of two leucines to alanines at positions 551 and 552 causes a loss of PM localization. Moreover, acidic amino acids in the C-terminus appear to negatively regulate PM localization; mutational replacement of several acidic residues with neutral or basic residues rescues PM localization of a palmitoylation-defective GRK6A. Last, we characterize the novel nuclear localization, showing that nuclear export of nonpalmitoylated GRK6A is sensitive to leptomycin B and that GRK6A contains a potential nuclear localization signal. Our results suggest that the C-terminus of GRK6A contains a novel electrostatic palmitoyl switch in which acidic residues weaken the membrane-binding strength of the amphipathic helix, thus allowing changes in palmitoylation to regulate PM versus cytoplasmic/nuclear localization.
Jiang
Xiaoshan
X
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Benovic
Jeffrey L
JL
Wedegaertner
Philip B
PB
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology*; D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology*; D002522:Chlorocebus aethiops; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D054768:G-Protein-Coupled Receptor Kinases; D006801:Humans; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010168:Palmitates / Q000494:pharmacology; D019308:Palmitic Acid / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D021381:Protein Transport / Q000187:drug effects; D013347:Subcellular Fractions / Q000187:drug effects / Q000201:enzymology
D005231:Fatty Acids, Unsaturated; D010168:Palmitates; C022776:2-bromopalmitate; D019308:Palmitic Acid; D017346:Protein Serine-Threonine Kinases; D054768:G-Protein-Coupled Receptor Kinases; C089176:G-protein-coupled receptor kinase 6; C038753:leptomycin B