Inhibition of G protein-coupled receptor kinases (GRKs) by EF-hand Ca2+-sensor proteins appears to be a universal mechanism regulating receptor phosphorylation When Ca2+ level rises in the dark-adapted photoreceptors, recoverin (Rv) inhibits rhodopsin kinase (RK or GRK1) and extends the lifetime of activated state of rhodopsin (Rh*). Calmodulin (CaM) has been shown to inhibit GRKs such as GRK2, GRK4-and GRK5, but not RK We found that RK can interact with CaM in vitro. Our goal is to investigate the specificity of interaction between Ca2+-binding proteins and RK by kinetic and structure-function analysis. The interaction of RK with Rv and CaM was analyzed by surface plasmon resonance and by effects on Rh* phosphorylation. The apparent KD for RK-Rv binding was determined at 1 uM and for RK-CaM at 10 nM. The interactions were Ca2+-dependent with EC50 for Ca2+ about 0.5 uM (RK-Rv) and 2 uM (RK-CaM). Autophosphorylation of RK reduced its binding to Rv ten-fold but had no effect on the RK-CaM binding. Analysis of RK fragments expressed as GST-fusion proteins has located a single binding site for Rv within the first 25 amino acid residues of RK. In addition, two distinct CaM binding sites were identified in the N- and C-terminal parts of RK molecule. The sites for Rv and CaM on the N-terminal part of RK do not overlap. The nanomolar binding affinity between RK and CaM suggests that they should interact in vivo. The functional significance of this interaction is currently under investigation and will be discussed.
|Original language||English (US)|
|State||Published - Dec 1 1998|
ASJC Scopus subject areas
- Molecular Biology