Cloning of GRK2 cDNA from S49 murine lymphoma cells.
Beta-adrenergic receptor kinase is a member of the G protein-linked receptor kinase (GRK1) family that elicits receptor desensitization. We have cloned GRK2 from S49 mouse lymphoma cells. The nucleotide sequences of rat GRK2 and GRK3 were aligned and conserved primers chosen for use in reverse transcription-polymerase chain reaction (RT-PCR) of S49 mRNA. Direct sequencing of the PCR fragment provided a rapid means to identify the expression of the GRK2 but not the GRK3 transcript in these cells. Unique expression of GRK2 in S49 cells was confirmed by Western blotting. Three additional pairs of primers were chosen from the rat GRK2 sequence to amplify overlapping regions that together encompassed the entire coding sequence. After attempts to ligate the four fragments of S49 cell GRK2 cDNA by using PCR proved unsuccessful, the intact cDNA was assembled by digesting the PCR products in the region of the overlaps and ligating them in a single step into pBlue-script SK(+).
Hughes
R J
RJ
Department of Pharmacology, University of California at San Diego, La Jolla 92093-0636, USA.
Anderson
K L
KL
Kiel
D
D
Insel
P A
PA
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000096:biosynthesis*; D017931:DNA Primers; D018076:DNA, Complementary; D054770:G-Protein-Coupled Receptor Kinase 3; D008223:Lymphoma; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured; D051552:beta-Adrenergic Receptor Kinases
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D054770:G-Protein-Coupled Receptor Kinase 3; C517850:GRK3 protein, mouse; C517851:Grk3 protein, rat; D051552:beta-Adrenergic Receptor Kinases