The G protein-coupled receptor regulatory kinase GPRK2 participates in Hedgehog signaling in Drosophila.

Signaling by Smoothened (Smo) plays fundamental roles during animal development and is deregulated in a variety of human cancers. Smo is a transmembrane protein with a heptahelical topology characteristic of G protein-coupled receptors. Despite such similarity, the mechanisms regulating Smo signaling are not fully understood. We show that Gprk2, a Drosophila member of the G protein-coupled receptor kinases, plays a key role in the Smo signal transduction pathway. Lowering Gprk2 levels in the wing disc reduces the expression of Smo targets and causes a phenotype reminiscent of loss of Smo function. We found that Gprk2 function is required for transducing the Smo signal and that when Gprk2 levels are lowered, Smo still accumulates at the cell membrane, but its activation is reduced. Interestingly, the expression of Gprk2 in the wing disc is regulated in part by Smo, generating a positive feedback loop that maintains high Smo activity close to the anterior-posterior compartment boundary.

The cell biology of Smo signalling and its relationships with GPCRs.

The Smoothened (Smo) signalling pathway participates in many developmental processes, contributing to the regulation of gene expression by controlling the activity of transcription factors belonging to the Gli family. The key elements of the pathway were identified by means of genetic screens carried out in Drosophila, and subsequent analysis in other model organisms revealed a high degree of conservation in both the proteins involved and in their molecular interactions. Recent analysis of the pathway, using a combination of biochemical and cell biological approaches, is uncovering the intricacies of Smo signalling, placing its elements in particular cellular compartments and qualifying the molecular processes involved. These include the synthesis, secretion and diffusion of the ligand, the activation of the receptor and the modifications in the activity of nuclear effectors. In this review we discuss recent advances in understanding biochemical and cellular aspects of Smo signalling, with particular focus in the similarities in the mechanism of signal transduction between Smo and other transmembrane proteins belonging to the G-Protein coupled receptors superfamily (GPCR).

G protein-coupled receptor kinase 2-mediated phosphorylation of downstream regulatory element antagonist modulator regulates membrane trafficking of Kv4.2 potassium channel.

Downstream regulatory element antagonist modulator (DREAM)/potassium channel interacting protein (KChIP3) is a multifunctional protein of the neuronal calcium sensor subfamily of Ca2+-binding proteins with specific roles in different cell compartments. In the nucleus, DREAM acts as a Ca2+-dependent transcriptional repressor, and outside the nucleus DREAM interacts with Kv4 potassium channels, regulating their trafficking to the cell membrane and their gating properties. In this study we characterized the interaction of DREAM with GRK6 and GRK2, members of the G protein-coupled receptor kinase family of proteins, and their phosphorylation of DREAM. Ser-95 was identified as the site phosphorylated by GRK2. This phosphorylation did not modify the repressor activity of DREAM. Mutation of Ser-95 to aspartic acid, however, blocked DREAM-mediated membrane expression of the Kv4.2 potassium channel without affecting channel tetramerization. Treatment with the calcineurin inhibitors FK506 and cyclosporin A also blocked DREAM-mediated Kv4.2 channel trafficking and calcineurin de-phosphorylated GRK2-phosphorylated DREAM in vitro. Our results indicate that these two Ca2+-dependent posttranslational events regulate the activity of DREAM on Kv4.2 channel function.