title: Structure-function analysis of G protein-coupled receptor kinase-5. Role of the carboxyl terminus in kinase regulation.
abstract: Many G protein-coupled receptors are phosphorylated and regulated by a distinct family of G protein-coupled receptor kinases (GRKs) that specifically target the activated form of the receptor. Recent studies have revealed that the GRKs are also subject to post-translational regulation. For example, GRK5 activity is strongly inhibited by protein kinase C phosphorylation and by Ca2+-calmodulin binding. Ca2+-calmodulin binding also promotes GRK5 autophosphorylation, which further contributes to kinase inhibition. In this study we identify two important structural domains in GRK5, a phospholipid binding domain (residues 552-562) and an autoinhibitory domain (residues 563-590), that significantly contribute to GRK5 localization and function. We demonstrate that the C-terminal region of GRK5 (residues 563-590) contains residues autophosphorylated in the presence of calmodulin as well as the residues phosphorylated by protein kinase C. Deletion of this domain increases the apparent affinity of GRK5 for receptor substrates 3-4-fold but has no effect on nonreceptor substrates. These findings define residues 563-590 of GRK5 as an autoinhibitory domain with efficacy that is regulated by phosphorylation. Another C-terminal domain in GRK5 that appears to be functionally important is found between residues 552 and 562. Deletion of this region significantly inhibits kinase phosphorylation of membrane-bound receptor substrates but has no effect on soluble substrates. Additional studies reveal that this domain is critical for GRK5 interaction with phospholipids and for the intracellular localization of the kinase. Interestingly, similar regions in GRK4 and GRK6 appear to be palmitoylated (and involved in membrane interaction), suggesting evolutionary conservation of the function of this domain.
authors.affiliation: Departments of Biochemistry and Molecular Pharmacology and Microbiology and Immunology, Kimmel Cancer Cancer, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
authors.forename: A N
authors.initials: AN
authors.lastname: Pronin
authors.forename: C V
authors.initials: CV
authors.lastname: Carman
authors.forename: J L
authors.initials: JL
authors.lastname: Benovic
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002147:Calmodulin / Q000494:pharmacology; D002451:Cell Compartmentation; D054774:G-Protein-Coupled Receptor Kinase 5; D019204:GTP-Binding Proteins / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008081:Liposomes / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
chemical_list: D002147:Calmodulin; D008081:Liposomes; D010446:Peptide Fragments; D010743:Phospholipids; D011994:Recombinant Proteins; D020794:Receptor Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D054774:G-Protein-Coupled Receptor Kinase 5; C517898:GRK5 protein, human; D019204:GTP-Binding Proteins