Chuang T T, Paolucci L, De Blasi A
Consorzio Mario Negri Sud, Istituto di Ricerche Farmacologiche "Mario Negri," 66030 Santa Maria Imbaro, Italy.
J Biol Chem. 1996 Nov 8;271(45):28691-6. doi: 10.1074/jbc.271.45.28691.
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.
G蛋白偶联受体激酶（GRKs）与G蛋白偶联受体的同源脱敏有关。迄今为止，已鉴定出六种GRK亚型，命名为GRK1至GRK6。GRKs的功能状态可以通过不同方式进行积极调节。特别是，人们发现视网膜视紫红质激酶（GRK1），而不是普遍存在的βARK1（GRK2），可被光感受器特异性钙结合蛋白恢复蛋白通过直接结合而抑制。本研究旨在探讨其他钙结合蛋白如钙调蛋白（CaM）对其他GRKs的调节作用。我们发现，Gβγ激活的GRK2和GRK3受到CaM的抑制程度相似（IC50约为2μM），而对GRK5的抑制作用则强50倍（IC50 = 40 nM）。CaM的抑制作用严格依赖于Ca2+，并被CaM抑制剂CaMBd所阻断。由于Ca2+/CaM的结合靶点Gβγ对GRK2和GRK3的激活至关重要，它提供了这些蛋白之间可能的相互作用位点。然而，由于GRK5不依赖Gβγ，因此可以设想一种替代机制。使用CaM偶联的琼脂糖4B揭示了GRK5与Ca2+/CaM之间的直接相互作用。如使用可溶性GRK底物酪蛋白所证明的，这种结合不会影响催化活性。相反，Ca2+/CaM显著降低了GRK5与膜的结合。GRK5的抑制机制似乎是通过直接与Ca2+/CaM结合，导致膜结合的抑制，从而抑制受体磷酸化。本研究为Ca2+/CaM对某些GRK亚型的调节作用提供了首个证据，从而扩展了调节这些激酶功能状态的不同机制的范围。
