CLONING OF GRK(2) CDNA FROM S49 MURINE LYMPHOMA-CELLS

beta-Adrenergic receptor kinase is a member of the G protein-linked re ceptor kinase (GRK(1)) family that elicits receptor desensitization. W e have cloned GRK(2) from S49 mouse lymphoma cells. The nucleotide seq uences of rat GRK(2) and GRK(3) were aligned and conserved primers cho sen 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 GRK(2) but not the GRK(3) tran script in these cells. Unique expression of GRK(2) in S49 cells was co nfirmed by Western blotting. Three additional pairs of primers were ch osen from the rat GRK(2) sequence to amplify overlapping regions that together encompassed the entire coding sequence. After attempts to lig ate the four fragments of S49 cell GRK(2) cDNA by using PCR proved uns uccessful, 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(+).