Pleckstrin homology domain-mediated membrane association and activation of the β-adrenergic receptor kinase requires coordinate interaction with Gβγ subunits and lipid

Abstract

The pleckstrin homology (PH) domain is an approximately 100-amino-acid region of sequence homology present in numerous proteins of diverse functions, which forms a discrete structural module. Several ligands capable of binding to PH domain-containing proteins have been identified including phosphatidylinositol 4,5-bisphosphate (PIP2) and the Gβγ subunits of heterotrimeric G proteins (Gβγ), which bind to the amino and carboxyl termini of the PH domain, respectively. Here we report that the binding of Gβγ and lipid to the PH domain of the β-adrenergic receptor kinase (βARK) synergistically enhances agonist-dependent receptor phosphorylation and that both PH domain-binding ligands are required for membrane association of the kinase. PIP2 and to a lesser extent phosphatidylinositol 4-phosphate, phosphatidylinositol, and phosphatidic acid were the only lipids tested capable, in the presence of Gβγ, of enhancing βARK activity. In contrast, the Km and Vmax for phosphorylation of a soluble βARK substrate (casein) was not altered in either the presence or absence of Gβγ and/or PIP2. A fusion protein of the βARK containing an intact PH domain inhibits Gβγ/PIP2-dependent βARK activity. In contrast, a mutant fusion protein in which a tryptophan residue, invariant in all PH domain sequences, is mutated to alanine shows no inhibitory activity. The requirement for the simultaneous presence of two PH domain binding ligands represents a previously unappreciated mechanism for effecting membrane localization of a protein and may have relevance to other PH domain-containing proteins.