Inhibition of G protein-coupled receptor kinase subtypes by Ca2+/calmodulin.
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 betaARK1 (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 Gbetagamma-activated GRK2 and GRK3 were inhibited by CaM to similar extents (IC50 approximately 2 microM), 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 Gbetagamma, 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 Gbetagamma-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.
Consorzio Mario Negri Sud, Istituto di Ricerche Farmacologiche "Mario Negri," 66030 Santa Maria Imbaro, Italy. deblasi@cmns.mnegri.it
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Paolucci
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De Blasi
D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology*; D002364:Caseins / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D054770:G-Protein-Coupled Receptor Kinase 3; D054771:G-Protein-Coupled Receptor Kinase 4; D054774:G-Protein-Coupled Receptor Kinase 5; D054768:G-Protein-Coupled Receptor Kinases; D006801:Humans; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D012243:Rhodopsin / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism; D051552:beta-Adrenergic Receptor Kinases
D002147:Calmodulin; D002364:Caseins; D004791:Enzyme Inhibitors; D012243:Rhodopsin; D020794:Receptor Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D054770:G-Protein-Coupled Receptor Kinase 3; C517848:GRK3 protein, human; D051552:beta-Adrenergic Receptor Kinases; D054771:G-Protein-Coupled Receptor Kinase 4; D054774:G-Protein-Coupled Receptor Kinase 5; D054768:G-Protein-Coupled Receptor Kinases; C089176:G-protein-coupled receptor kinase 6; C517855:GRK4 protein, human; C517898:GRK5 protein, human; D002118:Calcium