Identification by polymerase chain reaction of the G protein-coupled receptor kinases expressed in the mouse gonadotropic alpha T3-1 cell line.
G protein-coupled receptor kinases (GRK 1-6) stimulate short-term desensitization (< 5 min) by phosphorylating G-protein coupled receptors, and also participate in receptor sequestration, which may relate to intermediate-term desensitization (30-60 min). The existence of such kinases and hence a potential role for them in gonadotrope/GnRH receptor desensitization was investigated using the PCR to identify GRKs in messenger RNA (mRNA) from the mouse alpha T3-1 gonadotrope cell line. The 150-bp complementary DNAs amplified by PCR from the kinase catalytic domain were cloned and sequenced. Seventeen of 42 clones were receptor kinases based on high nucleotide identities of 85-100% and amino acid identities of 97-100% with rat GRK2 and 3, and with human GRK6. Among the eight GRK3 clones was one differing from rat GRK3 by a single nucleotide and seven differing by six; no amino acid difference resulted from the nucleotide differences. Of the five GRK2 clones, one sequence was identical with rat GRK2, but four sequences differed by three nucleotides and one amino acid. Among four GRK6 sequences, one showed 15 nucleotide differences from human GRK6 (with no amino acid differences), and three had 16 nucleotide and one amino acid differences. For each of the three GRKs found, the most closely related isoform is assumed to be the mouse homolog of rat GRK2 and GRK3, and human GRK6, whereas the others are assumed to be previously undescribed isoforms or subtypes of GRK2, 3, and 6. Immunocytochemical staining using antibodies to GRK2, 3, and 6 confirmed their presence in alpha T3-1 cells. The function of these GRKs in alpha T3-1 cells is unknown, but they may be involved in short-term desensitization of the gonadotrope/GnRH receptor or perhaps, more likely, the sequestration of this receptor during intermediate-term desensitization.
Neill
J D
JD
Department of Physiology and Biophysics, Schools of Medicine and Dentistry, University of Alabama at Birmingham 35294, USA. Neill@Phybio.bhs.uab.edu
Musgrove
L C
LC
Sellers
J C
JC
Duck
L W
LW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis* / Q000235:genetics; D054770:G-Protein-Coupled Receptor Kinase 3; D054768:G-Protein-Coupled Receptor Kinases; D019204:GTP-Binding Proteins / Q000032:analysis; D006065:Gonadotropins, Pituitary / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D010903:Pituitary Gland, Anterior / Q000737:chemistry* / Q000166:cytology; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000032:analysis* / Q000235:genetics; D014158:Transcription, Genetic; D051552:beta-Adrenergic Receptor Kinases
D006065:Gonadotropins, Pituitary; D015345:Oligonucleotide Probes; D020794:Receptor Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D054770:G-Protein-Coupled Receptor Kinase 3; C517848:GRK3 protein, human; C517850:GRK3 protein, mouse; C517851:Grk3 protein, rat; D051552:beta-Adrenergic Receptor Kinases; D054768:G-Protein-Coupled Receptor Kinases; C089176:G-protein-coupled receptor kinase 6; D019204:GTP-Binding Proteins