Binding of multiple ligands to pleckstrin homology domain regulates membrane translocation and enzyme activity of β-adrenergic receptor kinase

AbstractPleckstrin homology (PH) domains are discrete structural modules present in numerous proteins involved in signal transduction processes. In the case of the β-adrenergic receptor kinase (βARK), PH domain-mediated binding of two ligands, the βγ subunits of heterotrimeric G proteins (Gβγ) and phosphatidylinositol 4,5-bisphosphate (PIP2), has been shown to be required for the kinase function. In this study, the ability of Gβγ and PIP2 to affect membrane localization of βARK is used to define the ligand binding characteristics of the βARK PH domain. The binding of these ligands to the PH domain of the intact kinase is shown to be cooperative, Gβγ increasing the affinity of the PH domain for PIP2. Notably, although PIP2-dependent membrane association of βARK is observed at high concentrations of this lipid, in the absence of Gβγ, no receptor phosphorylation is observed. Peptides derived from the receptor intracellular loop inhibit the receptor phosphorylation without affecting the membrane translocation of the kinase complex, suggesting that βARK activity does not necessarily correlate with the amount of βARK associated with the membrane. These results point to a distinct role for each PH domain ligand in βARK-mediated receptor phosphorylation. Strikingly, the ligand binding characteristics of the isolated βARK PH domain fused to glutathione S-transferase are very different from those of the PH domain of the intact kinase. Thus, in contrast to the native protein, the isolated PH domain binds Gβγ and PIP2 independently and with no apparent cooperativity. That protein environment plays an important role in determining the ligand binding characteristics of a particular PH domain highlights the potential risks of inferring mechanisms from studies of isolated PH domains