Molecular cloning and expression of GRK6. A new member of the G protein-coupled receptor kinase family.
G protein-coupled receptor kinases (GRK), such as the beta-adrenergic receptor kinase (beta ARK) and rhodopsin kinase, specifically phosphorylate the activated form of G protein-coupled receptors. To identify additional members of the GRK family, we screened a human heart cDNA library by low stringency hybridization using the catalytic domains of two beta ARK isoforms. Here we report the isolation of a cDNA that encodes a 576-amino-acid protein kinase, termed GRK6, that has significant homology with GRK5 (70.1% amino acid identity), IT11 (68.5%), rhodopsin kinase (47.1%), and beta ARK (37.4%). RNA blot analysis of GRK6 with selected human tissues reveals two distinct mRNAs of 3 and 2.4 kilobases with a distribution very similar to that of beta ARK (i.e. brain, skeletal muscle > pancreas > heart, lung, kidney, placenta > liver). GRK6, overexpressed in Sf9 insect cells using the baculovirus system, was able to phosphorylate both the beta 2-adrenergic receptor and rhodopsin in a stimulus-dependent fashion, although it was significantly less active then beta ARK on these substrates. These data extend the family of GRKs and suggest that GRK6 may have a substrate specificity quite distinct from beta ARK and rhodopsin kinase.
Benovic
J L
JL
Department of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
Gomez
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D001921:Brain / Q000201:enzymology*; D002460:Cell Line; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases; D005136:Eye Proteins; D005333:Fetus; D051606:G-Protein-Coupled Receptor Kinase 1; D054768:G-Protein-Coupled Receptor Kinases; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D009036:Moths; D005810:Multigene Family; D009206:Myocardium / Q000201:enzymology*; D009928:Organ Specificity; D011494:Protein Kinases / Q000096:biosynthesis* / Q000235:genetics; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D051552:beta-Adrenergic Receptor Kinases
D005136:Eye Proteins; D012333:RNA, Messenger; D011494:Protein Kinases; D020794:Receptor Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; C496764:GRK1 protein, human; D051552:beta-Adrenergic Receptor Kinases; D054768:G-Protein-Coupled Receptor Kinases; C089176:G-protein-coupled receptor kinase 6; D019204:GTP-Binding Proteins