INHIBITION OF G-PROTEIN-COUPLED RECEPTOR KINASE SUBTYPES BY CA2+ CALMODULIN/

G protein-coupled receptor kinases (GRKs) are implicated in the homolo gous 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 ubiqu itous beta ARK1 (GRK2), can be inhibited by the photoreceptor-specific Ca2+-binding protein recoverin through direct binding. The present st udy was aimed to investigate regulation of other GRKs by alternative C a2+-binding proteins such as calmodulin (CaM). We found that G beta ga mma-activated GRK2 and GRK3 were inhibited by CaM to similar extents ( IC50 similar to 2 mu M), while a 50-fold more potent inhibitory effect was observed on GRK5 (IC50 = 40 nM). Inhibition by CaM was strictly d ependent on Ca2+ and was prevented by the CaM inhibitor CaMBd. Since G beta gamma, which is a binding target of Ca2+/CaM, is critical for th e activation of GRK2 and GRK3, it provides a possible site of interact ion between these proteins. However, since GRK5 is G beta gamma-indepe ndent, an alternative mechanism is conceivable. A direct interaction b etween GRK5 and Ca2+/CaM was revealed using CaM-conjugated Sepharose 4 B. This binding does not influence the catalytic activity as demonstra ted using the soluble GRK substrate casein. Instead, Ca2+/CaM signific antly reduced GRK5 binding to the membrane. The mechanism of GRK5 inhi bition 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 effec t of Ca2+/CaM on some GRK subtypes, thus expanding the range of differ ent mechanisms regulating the functional states of these kinases.