Cloning and expression of GRK5: a member of the G protein-coupled receptor kinase family.
Guanine nucleotide binding protein (G-protein)-coupled receptor kinases (GRKs) specifically phosphorylate the agonist-occupied form of G-protein-coupled receptors such as the beta 2-adrenergic receptor and rhodopsin. The best characterized members of this family include the beta-adrenergic receptor kinase (beta ARK) and rhodopsin kinase. To identify additional members of the GRK family, the polymerase chain reaction was used to amplify human heart cDNA using degenerate oligonucleotide primers from highly conserved regions unique to the GRK family. Here we report the isolation of a cDNA that encodes a 590-amino acid protein kinase, termed GRK5, which has 34.8% and 47.2% amino acid identities with beta ARK and rhodopsin kinase, respectively. Interestingly, GRK5 has an even higher homology with Drosophila GPRK-2 (71.0% identity) and the recently identified human IT11 (69.1% identity). Northern blot analysis of GRK5 with selected human tissues reveals a message of approximately 3 kilobases with highest levels in heart, placenta, lung > skeletal muscle > brain, liver, pancreas > kidney. GRK5, overexpressed in Sf9 insect cells using the baculovirus system, was able to phosphorylate rhodopsin in a light-dependent manner. In addition, GRK5 neither contains a consensus sequence for isoprenylation like rhodopsin kinase nor is activated by G-protein beta gamma subunits like beta ARK1. Thus, GRK5 represents a member of the GRK family that likely has a unique physiological role.
Kunapuli
P
P
Department of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107.
Benovic
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D054774:G-Protein-Coupled Receptor Kinase 5; D019204:GTP-Binding Proteins / Q000096:biosynthesis*; D015870:Gene Expression; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009036:Moths; D005810:Multigene Family; D009838:Oligodeoxyribonucleotides; D009928:Organ Specificity; D011061:Poly A / Q000032:analysis / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000096:biosynthesis* / Q000235:genetics; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis*; D012313:RNA / Q000032:analysis / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000096:biosynthesis*; D012374:Rod Cell Outer Segment / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D005136:Eye Proteins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D011061:Poly A; D012313:RNA; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; C496764:GRK1 protein, human; D054774:G-Protein-Coupled Receptor Kinase 5; C517898:GRK5 protein, human; D019204:GTP-Binding Proteins