Retrovirally Mediated Transfer of a G Protein-coupled Receptor Kinase (GRK) Dominant-negative Mutant Enhances Endogenous Calcitonin Receptor Signaling in Chinese Hamster Ovary Cells

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 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...

Serine 447 in the Carboxyl Tail of Human VPAC1 Receptor Is Crucial for Agonist-Induced Desensitization but Not Internalization of the Receptor

“…This serine, which is a likely GRK-mediated phosphorylation site, further extends the previous data, suggesting that GRKs are involved in desensitizing VPAC 1 and certain other related class II G protein-coupled receptors, such as secretin and calcitonin receptors (Shetzline et al, 1998(Shetzline et al, , 2002Horie and Insel, 2000). In sharp contrast to the above data, serine 447 is not involved in VIP-induced down-regulation or the internalization of VPAC 1 receptor, which occurred concomitantly with its desensitization.…”

“…GRK2, GRK5, and, to a lesser extent, GRK3 seem to be involved in the desensitization of the rat secretin receptor expressed in HEK 293 cells (Shetzline et al, 1998). The use of dominant-negative GRK mutant has revealed the clear involvement of GRK2 in calcitonin receptor desensitization (Horie and Insel, 2000). Truncation of a C-terminal domain of the human GRF receptor has also been suggested to decrease receptor desensitization (Gaylinn, 2002).…”

Expression of a G Protein-coupled Receptor (GPCR) Leads to Attenuation of Signaling by Other GPCRs

“…To do this, we confirmed the presence and the functionality of CT and PGE 2 receptors that were previously described on the CHO-K1 cell line (22,23) (Fig. 2).…”

Arrestins and two receptor kinases are upregulated in Parkinson's disease with dementia

“…If the compensation for DA depletion was aimed at restoring a normal level of G protein-mediated signaling via DA receptors, a down-regulation of arrestins and GRKs in the PD brain should be expected. It is well established that reduction of the arrestin and/or GRK concentration invariably reduces GPCR desensitization and internalization and enhances G protein-mediated signaling [3,14,27,41,58,75,103]. Therefore, decreased concentrations of arrestin/GRKs in the DA-depleted basal ganglia would impede desensitization of DA receptors and help sustain the G protein-mediated signaling.…”