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

G protein-coupled receptor kinases (GRKs) initiate pathways leading to agon ist-dependent phosphorylation and desensitization of G protein-coupled rece ptors. However, the role of GRKs in modulation of signaling properties of n ative receptors has not been clearly defined. Here we addressed this questi on by generating Chinese hamster ovary (CHO) cells stably expressing a domi nant-negative mutant of GRK2 (DN-GRK2), K220R, using retrovirally mediated gene transfer, and we assessed function of the endogenously expressed calci tonin (CT) receptors. We found that CT-mediated responses were prominently enhanced in CHO cells expressing DN-GRK2 compared with mock-infected contro l CHO cells with similar to 3-fold increases in CT-promoted cAMP production in whole cells and adenylyl cyclase activity in membrane fractions. CT-pro moted phosphoinositide hydrolysis was also enhanced in DN-GRK2 cells. The n umber of CT receptors was increased similar to 3-fold in DN-GRK2 cells, as assessed by I-125-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 rece ptors and for resistance and escape from response to CT in vitro and in viv o. Moreover, the data suggest that blunting of receptor desensitization by DN-GRK2 blocks a GRK-mediated tonic inhibition of CT receptor expression an d response. We speculate that GRKs play a similar role for other G protein- coupled receptors as well.