Pleckstrin homology domain-mediated membrane association and activation of the beta-adrenergic receptor kinase requires coordinate interaction with G beta gamma subunits and lipid.
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 G beta gamma subunits of heterotrimeric G proteins (G beta gamma), which bind to the amino and carboxyl termini of the PH domain, respectively. Here we report that the binding of G beta gamma and lipid to the PH domain of the beta-adrenergic receptor kinase (beta ARK) 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 G beta gamma, of enhancing beta ARK activity. In contrast, the Km and Vmax for phosphorylation of a soluble beta ARK substrate (casein) was not altered in either the presence or absence of G beta gamma and/or PIP2. A fusion protein of the beta ARK containing an intact PH domain inhibits G beta gamma/PIP2-dependent beta ARK 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.
Pitcher
J A
JA
Howard Hughes Medical Research Institute, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
Touhara
K
K
Payne
E S
ES
Lefkowitz
R J
RJ
D000818:Animals; D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry*; D002364:Caseins / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000502:physiology*; D008024:Ligands; D008081:Liposomes / Q000378:metabolism; D008563:Membrane Lipids / Q000502:physiology; D008565:Membrane Proteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010712:Phosphatidic Acids / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000494:pharmacology; D010743:Phospholipids / Q000494:pharmacology / Q000502:physiology*; D010750:Phosphoproteins; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D051552:beta-Adrenergic Receptor Kinases
D001798:Blood Proteins; D002364:Caseins; D008024:Ligands; D008081:Liposomes; D008563:Membrane Lipids; D008565:Membrane Proteins; D010712:Phosphatidic Acids; D010716:Phosphatidylinositols; D010743:Phospholipids; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C039077:platelet protein P47; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D019204:GTP-Binding Proteins