Korean Circ J.  2002 Jul;32(7):613-622. 10.4070/kcj.2002.32.7.613.

Novel Protein Interactions of G Protein-Coupled Receptor Kinase 5 (GRK5) Searched with Yeast Two-Hybrid System

• Jin BC
• Park TJ
• Kim EJ
• Lee JE
• Lee JH
• Mun IK
• Park JR
• Choi DJ
• Seo BG

• KMID: 2093861
• DOI: http://doi.org/10.4070/kcj.2002.32.7.613

Abstract

BACKGROUND AND OBJECTIVES: 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. MATERIALS AND MEDTHODS: 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.
RESULTS
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.
CONCLUSION
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.

Keyword

GTP-binding proteins; GRK5; Yeasts; Two-hybrid system techniques; Rats; Heart

MeSH Terms

Animals
beta-Galactosidase
Clone Cells
DNA, Complementary
Electron Transport Complex IV
Galactose
Gene Library
GTP-Binding Proteins
Heart
Immunoprecipitation
Leucine
Mass Screening
Phosphotransferases*
Polymerase Chain Reaction
Rats
Receptors, Adrenergic, beta
Two-Hybrid System Techniques*
Yeasts*
DNA, Complementary
Electron Transport Complex IV
GTP-Binding Proteins
Galactose
Leucine
Phosphotransferases
Receptors, Adrenergic, beta
beta-Galactosidase