title: Retrovirally mediated transfer of a G protein-coupled receptor kinase (GRK) dominant-negative mutant enhances endogenous calcitonin receptor signaling in Chinese hamster ovary cells. GRK inhibition enhances expression of receptors and receptor mRNA.
abstract: G protein-coupled receptor kinases (GRKs) initiate pathways leading to agonist-dependent phosphorylation and desensitization of G protein-coupled receptors. However, the role of GRKs in modulation of signaling properties of native receptors has not been clearly defined. Here we addressed this question by generating Chinese hamster ovary (CHO) cells stably expressing a dominant-negative mutant of GRK2 (DN-GRK2), K220R, using retrovirally mediated gene transfer, and we assessed function of the endogenously expressed calcitonin (CT) receptors. We found that CT-mediated responses were prominently enhanced in CHO cells expressing DN-GRK2 compared with mock-infected control CHO cells with approximately 3-fold increases in CT-promoted cAMP production in whole cells and adenylyl cyclase activity in membrane fractions. CT-promoted phosphoinositide hydrolysis was also enhanced in DN-GRK2 cells. The number of CT receptors was increased approximately 3-fold in DN-GRK2 cells, as assessed by (125)I-salmon CT-specific binding, and this was associated with increased CT receptor mRNA levels. These results indicate that DN-GRK2 has multiple consequences for CT receptor signaling, but a primary effect is an increase in CT receptor mRNA and receptor number and, in turn, enhanced CT receptor signaling. As such, our findings provide a mechanistic basis for previous observations regarding agonist-promoted down-regulation of CT receptors and for resistance and escape from response to CT in vitro and in vivo. Moreover, the data suggest that blunting of receptor desensitization by DN-GRK2 blocks a GRK-mediated tonic inhibition of CT receptor expression and response. We speculate that GRKs play a similar role for other G protein-coupled receptors as well.
authors.affiliation: Department of Pharmacology, University of California, San Diego, La Jolla, California 92093, USA.
authors.forename: K
authors.initials: K
authors.lastname: Horie
authors.forename: P A
authors.initials: PA
authors.lastname: Insel
mesh_terms: D000818:Animals; D016466:CHO Cells; D002116:Calcitonin / Q000235:genetics* / Q000378:metabolism*; D006224:Cricetinae; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism*; D012190:Retroviridae; D015398:Signal Transduction / Q000235:genetics*
chemical_list: D007527:Isoenzymes; D002116:Calcitonin; D017346:Protein Serine-Threonine Kinases; D019204:GTP-Binding Proteins