Interaction of rhodopsin kinase with recoverin and calmodulin

D. K. Satpaev, K. Levay, Vladlen Z Slepak

Research output: Contribution to journalArticle

Abstract

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 languageEnglish
JournalFASEB Journal
Volume12
Issue number8
StatePublished - Dec 1 1998

Fingerprint

G-Protein-Coupled Receptor Kinase 1
Recoverin
rhodopsin
calmodulin
Calmodulin
phosphotransferases (kinases)
calcium
G-Protein-Coupled Receptor Kinases
Phosphorylation
Rhodopsin
binding sites
phosphorylation
Binding Sites
EF Hand Motifs
surface plasmon resonance
Surface Plasmon Resonance
protein phosphorylation
Surface plasmon resonance
photoreceptors
binding proteins

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

Interaction of rhodopsin kinase with recoverin and calmodulin. / Satpaev, D. K.; Levay, K.; Slepak, Vladlen Z.

In: FASEB Journal, Vol. 12, No. 8, 01.12.1998.

Research output: Contribution to journalArticle

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