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Volume 271, Number 45, Issue of November 8, 1996 pp. 28691-28696
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Inhibition of G Protein-coupled Receptor Kinase Subtypes by Ca²⁺/Calmodulin

(Received for publication, July 15, 1996, and in revised form, August 26, 1996)

From the Consorzio Mario Negri Sud, Istituto di Ricerche Farmacologiche ``Mario Negri,'' 66030 Santa Maria Imbaro, Italy

G protein-coupled receptor kinases (GRKs) are implicated in the homologous desensitization of G protein-coupled receptors. Six GRK subtypes have so far been identified, named GRK1 to GRK6. The functional state of the GRKs can be actively regulated in different ways. In particular, it was found that retinal rhodopsin kinase (GRK1), but not the ubiquitous ARK1 (GRK2), can be inhibited by the photoreceptor-specific Ca²⁺-binding protein recoverin through direct binding. The present study was aimed to investigate regulation of other GRKs by alternative Ca²⁺-binding proteins such as calmodulin (CaM). We found that G-activated GRK2 and GRK3 were inhibited by CaM to similar extents (IC₅₀ ~ 2 µM), while a 50-fold more potent inhibitory effect was observed on GRK5 (IC₅₀ = 40 nM). Inhibition by CaM was strictly dependent on Ca²⁺ and was prevented by the CaM inhibitor CaMBd. Since G, which is a binding target of Ca²⁺/CaM, is critical for the activation of GRK2 and GRK3, it provides a possible site of interaction between these proteins. However, since GRK5 is G-independent, an alternative mechanism is conceivable. A direct interaction between GRK5 and Ca²⁺/CaM was revealed using CaM-conjugated Sepharose 4B. This binding does not influence the catalytic activity as demonstrated using the soluble GRK substrate casein. Instead, Ca²⁺/CaM significantly reduced GRK5 binding to the membrane. The mechanism of GRK5 inhibition appeared to be through direct binding to Ca²⁺/CaM, resulting in inhibition of membrane association and hence receptor phosphorylation. The present study provides the first evidence for a regulatory effect of Ca²⁺/CaM on some GRK subtypes, thus expanding the range of different mechanisms regulating the functional states of these kinases.

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