Identification and chromosomal localization of a processed pseudogene of human GRK6.
G-protein-coupled receptor kinases (GRKs) phosphorylate agonist-occupied G-protein-coupled receptors, resulting in desensitization of receptor signaling. To date, 6 mammalian GRKs have been identified by molecular cloning. Several lines of evidence indicate that a homologue of GRK6, the most recently described GRK, is present in the human genome. Northern analysis identifies two transcripts which hybridize to GRK6, and genomic Southern analysis indicates that GRK6 is localized to chromosome 5, with a second GRK6-like locus on chromosome 13. To identify the GRK6 homologue on chromsome 13, several sets of closely-spaced primers were designed based on the GRK6 cDNA sequence and then used to amplify human genomic DNA by PCR. Two products were identified, the larger of which is a fragment of the GRK6 gene which contains introns, while the smaller fragment is 94% homologous to GRK6 and contains no introns. In order to further characterize this GRK6 homologue, primers from the 5' and 3' coding regions of GRK6 were used to amplify a product of 1458 base pairs from human genomic DNA. This 1458 base pair PCR fragment displays 94% homology to GRK6 and contains multiple nucleotide insertions and deletions compared to GRK6, including a C to T mutation at base pair 202 which creates a predicted in-frame stop codon. In an effort to determine whether this gene is transcriptionally active, primers designed to preferentially amplify either GRK6 or the homologue were used in reverse transcription PCR. In contrast to the GRK6-specific primers, primers which selectively amplify the GRK6 homologue fail to produce a PCR product in any RNA tested, indicating that this gene is most likely transcriptionally inactive. PCR amplification of rodent/human hybrid cell lines using these same primers confirms the previously established chromosome 5 localization of GRK6, and localizes this homologue to chromosome 13. Northern analysis indicates that the two GRK6-hybridizing species seen in RNA differ by approximately 500 base pairs in the 3' untranslated region, indicating that both transcripts likely arise from differential processing of a single gene. Taken together, these data indicate that the GRK6-hybridizing species on chromosome 13 is a transcriptionally inactive processed pseudogene of GRK6, while the two GRK6 transcripts differ in the 3' untranslated region.
Gagnon
A W
AW
Department of Pharmacology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Benovic
J L
JL
D001483:Base Sequence; D015152:Blotting, Northern; D002874:Chromosome Mapping; D002882:Chromosomes, Human, Pair 13 / Q000235:genetics; D003001:Cloning, Molecular; D017931:DNA Primers; D054768:G-Protein-Coupled Receptor Kinases; D019204:GTP-Binding Proteins / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D017346:Protein Serine-Threonine Kinases; D011544:Pseudogenes / Q000235:genetics*; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry* / Q000235:genetics; D017421:Sequence Analysis; D012689:Sequence Homology, Nucleic Acid
D017931:DNA Primers; D020794:Receptor Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D054768:G-Protein-Coupled Receptor Kinases; C089176:G-protein-coupled receptor kinase 6; D019204:GTP-Binding Proteins