h-89 and Retinoblastoma

Potential G protein-coupled receptor kinase (GRK) and protein kinase A (PKA) mediation of homologous desensitization of corticotropin-releasing factor type 1 (CRF1) receptors was investigated in human retinoblastoma Y-79 cells. Inhibition of PKA activity by PKI(5-22) or H-89 failed to attenuate homologous desensitization of CRF1 receptors, and direct activation of PKA by forskolin or dibutyryl cAMP failed to desensitize CRF-induced cAMP accumulation. However, treatment of permeabilized Y-79 cells with heparin, a nonselective GRK inhibitor, reduced homologous desensitization of CRF1 receptors by approximately 35%. Furthermore, Y-79 cell uptake of a GRK3 antisense oligonucleotide (ODN), but not of a random or mismatched ODN, reduced GRK3 mRNA expression by approximately 50% without altering GRK2 mRNA expression and inhibited homologous desensitization of CRF1 receptors by approximately 55%. Finally, Y-79 cells transfected with a GRK3 antisense cDNA construct exhibited an approximately 50% reduction in GRK3 protein expression and an ~65% reduction in homologous desensitization of CRF1 receptors. We conclude that GRK3 contributes importantly to the homologous desensitization of CRF1 receptors in Y-79 cells, a brain-derived cell line.

Topics: beta-Adrenergic Receptor Kinases; Colforsin; Cyclic AMP-Dependent Protein Kinases; DNA, Antisense; Enzyme Inhibitors; Eye Neoplasms; G-Protein-Coupled Receptor Kinase 3; Gene Expression Regulation, Enzymologic; Humans; Isoquinolines; Oligodeoxyribonucleotides, Antisense; Peptide Fragments; Protein Serine-Threonine Kinases; Receptors, Corticotropin-Releasing Hormone; Recombinant Proteins; Retinoblastoma; RNA, Messenger; Sulfonamides; Transcription, Genetic; Transfection; Tumor Cells, Cultured