Interaction of G protein-coupled receptor kinases and recoverin isoforms is determined by localization in zebrafish photoreceptors

Farnesylation of Zebrafish G-Protein-Coupled Receptor Kinase Using Bio-orthogonal Labeling

“…To identify the zGRK1a in immunoblots, we probed a parallel blot with an anti-zGRK1a antibody. 15 A clear signal of zGRK1a immunoreactivity at the same size was visible and is shown in Figure 1A (*). Additional bands in Figure 1B at the size of 75 kDa and higher in all samples indicated binding of streptavidin to possibly biotinylated endogenous proteins.…”

“…In photoreceptor outer segments, specific zGRKs colocalize with zebrafish recoverin isoforms in a distinct manner presumably by using the disk membrane system as an interacting platform. 15 Our bio-orthogonal labeling approach does not allow for the visualization of the presence of farnesyl moieties in zGRKs in native photoreceptor cells; nevertheless, the suitability of the approach can be tested in a cellular environment. Therefore, we further analyzed whether and where zGRK1a localized inside HEK293 cells by performing immunocytochemistry using a specific anti-zGRK1a antibody.…”

“…Therefore, we further analyzed whether and where zGRK1a localized inside HEK293 cells by performing immunocytochemistry using a specific anti-zGRK1a antibody. 15 For this purpose, zGRK1a was expressed in HEK293 cells like in the expression step described above. Then, HEK293 cells were fed with the alkyne derivative of farnesyldiphosphate 7 to incorporate it into zGRK1a.…”

“…Our findings are in agreement with our recent observation that zebrafish GRK isoforms and their calcium sensitive regulator proteins (recoverin isoforms) show differential expression profiles in the adult zebrafish retina, where they participate in shutting off the light resonse. 15 Although their protein−protein interaction properties seem redundant, their cellular and subcellular colocalization pattern exhibits a highly specific distribution profile.…”

“…The visual system of zebrafish ( Danio rerio ) has gained recent interest as a model organism to study cone vision, because the cone pigments in its cone cells span a similar range as that in human vision. , However, gene duplication during evolution has doubled (or tripled) the number of orthologous genes, which led to a complex picture concerning pairing of regulatory and target proteins. Four orthologs of zebrafish G-protein-coupled receptor kinase (zGRK) and four orthologs of the regulatory neuronal calcium sensor proteins named recoverin are suggested to form complexes. − Consensus sites for N-myristoyl transferases are present in recoverin variants, and the actual presence of myristoyl groups in purified recombinant proteins was demonstrated . GRKs in general contain consensus sites at their C-terminal end for isoprenoid modification, either farnesylation or geranylgeranylation .…”