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Publication Detail
Pleckstrin homology domain-mediated membrane association and activation of the beta-adrenergic receptor kinase requires coordinate interaction with G beta gamma subunits and lipid.
  • Publication Type:
    Journal article
  • Publication Sub Type:
    Comparative Study
  • Authors:
    Pitcher JA, Touhara K, Payne ES, Lefkowitz RJ
  • Publication date:
    19/05/1995
  • Pagination:
    11707, 11710
  • Journal:
    J Biol Chem
  • Volume:
    270
  • Issue:
    20
  • Country:
    UNITED STATES
  • Print ISSN:
    0021-9258
  • Language:
    eng
  • Keywords:
    Animals, Binding Sites, Blood Proteins, Caseins, Cattle, Cell Membrane, Cyclic AMP-Dependent Protein Kinases, Enzyme Activation, GTP-Binding Proteins, Ligands, Liposomes, Membrane Lipids, Membrane Proteins, Mutagenesis, Site-Directed, Phosphatidic Acids, Phosphatidylinositols, Phospholipids, Phosphoproteins, Phosphorylation, Protein Processing, Post-Translational, Protein Structure, Tertiary, Recombinant Fusion Proteins, Sequence Homology, Amino Acid, Structure-Activity Relationship, beta-Adrenergic Receptor Kinases
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 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.
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