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 ϳ3-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 ϳ3-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.
Stimulation of G protein-coupled receptors (GPCRs)1 in the plasma membrane triggers two events, activation of G proteinmediated signal transduction pathways and, in parallel, a deactivation or desensitization of signaling. One component of this desensitization, in particular homologous, receptor-specific desensitization, is agonist-dependent phosphorylation of the receptor by specific G protein-coupled receptor kinases (GRKs). This phosphorylation event leads to the recruitment of cytosolic proteins, -arrestins, to the receptor-signaling complex, the uncoupling of receptor from heterotrimeric G proteins, and loss of receptor responsiveness. Recent evidence indicates that GRKs and -arrestins not only promote receptor uncoupling but also may directly participate in GPCR sequestration and the initiation of events leading to clathrin-coated pit-mediated internalization of receptors (for recent reviews see Refs. 1-5).In addition, GRK activation may be required to initiate certain events unrelated to receptor desensitization (3, 4). At least six different isoforms of the GRK family have been isolated. GRK2, formerly termed ARK 1 , is a widely expressed member of this family and has been shown to phosphorylate various GPCRs (1, 2). GRK2 K220R, a dominant-negative GRK2 mutant (DN-GRK2) in which lysine at position 220 h...