G protein-coupled receptor-mediated signaling is attenuated by a process referred to as desensitization, wherein agonist-dependent phosphorylation of receptors by G protein-coupled receptor kinases (GRKs) is proposed to be a key initial event. However, mechanisms that activate GRKs are not fully understood. In one scenario, -subunits of G proteins (G) activate certain GRKs (-adrenergic receptor kinases 1 and 2, or GRK2 and GRK3), via a pleckstrin homology domain in the COOH terminus. This interaction has been proposed to translocate cytosolic -adrenergic receptor kinases (ARKs) to the plasma membrane and facilitate interaction with receptor substrates. Here, we report a novel finding that membrane lipids modulate ARK activity in vitro in a manner that is analogous and competitive with G. Several lipids, including phosphatidylserine (PS), stimulated, whereas phosphatidylinositol 4,5-bisphosphate inhibited, the ability of these GRKs to phosphorylate agonist-occupied m2 muscarinic acetylcholine receptors. Furthermore, both PS and phosphatidylinositol 4,5-bisphosphate specifically bound to ARK1, whereas phosphatidylcholine, a lipid that did not modulate ARK activity, did not bind to ARK1. The lipid regulation of ARKs did not occur via a modulation of its autophosphorylation state. PS- and G-mediated stimulation of ARK1 was compared and found strikingly similar; moreover, their effects together were not additive (except at initial stages of reaction), which suggests that PS and G employed a common interaction and activation mechanism with the kinase. The effects of these lipids were prevented by two well known G-binding proteins, phosducin and GST-ARK-(466-689) fusion protein, suggesting that the G-binding domain (possibly the pleckstrin homology domain) of the GRKs is also a site for lipid:protein interaction. We submit the intriguing possibility that both lipids and G proteins co-regulate the function of GRKs.

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