G protein-coupled receptors were considered to be the only natural substrates of G protein-coupled receptor kinases (GRKs). However, it was recently demonstrated that GRKs can also bind to other signal molecules. The purpose of this study was to investigate new molecules that might interact with the GRK5 using a yeast two-hybrid system to screen the cDNA library.

For the yeast two-hybrid system, the "bait" was constructed to generate a LexA-GRK5 fusion protein in the EGY48 yeast strain. Rat library cDNA was inserted into the "prey". The first step in the library screening was performed by a galactose dependent leucine orthotrophism. For the second step screening, a beta-galactosidase dependent discoloration of colonies was used. Sequencing and searching of the gene bank was undertaken to characterize the clones.

We screened a total of 1.3X10 6 clones from the cDNA library. On the first screening, 162 clones were identified by leucine orhotrophism. Another 54 clones were identified on the second screening by beta-galactosidase activation. Seven clones were selected by PCR and restriction patterns. Sequencing of seven molecules revealed that four of the clones were emerin fragments, with 2 of the remaining 3 clones being: an ID2 protein and a mitochondrial cytochrome c oxidase subunit II, with the last one remaining an unknown molecule. For the emerin fragments, their interactions with the GRK5 were confirmed by immunoprecipitation.

We describe the novel protein-protein interactions of the GRK5, specifically, with three molecules. At first, these proteins may modulate the activation of the GRK5 through this specific protein-protein interaction desensitizing the beta-adrenergic receptors. Conversely, the localization of these molecules inside the cell indicates a potential new physiological role for the GRK5.