Pleckstrin Homology Domain-mediated Membrane Association and Activation of the -Adrenergic Receptor Kinase Requires Coordinate Interaction with G Subunits and Lipid

doi:10.1074/jbc.270.20.11707

Pleckstrin Homology Domain-mediated Membrane Association and Activation of the -Adrenergic Receptor Kinase Requires Coordinate Interaction with G Subunits and Lipid(*)

From the (1) Howard Hughes Medical Research Institute, Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

^§To whom correspondence should be addressed:
Howard Hughes Medical Research Inst., Depts. of Medicine and Biochemistry, Duke University Medical Center, P. O. Box 3821, Durham, NC 27710.

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 subunits of heterotrimeric G proteins (G), which bind to the amino and carboxyl termini of the PH domain, respectively. Here we report that the binding of G 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 G, of enhancing βARK activity. In contrast, the Kand V_max for phosphorylation of a soluble βARK substrate (casein) was not altered in either the presence or absence of G and/or PIP₂. A fusion protein of the βARK containing an intact PH domain inhibits G/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.

Footnotes

↵* This work was supported by National Institutes of Health Grant HL16037 (to R. J. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PH

pleckstrin homology

G

β-subunits of heterotrimeric G proteins

βAR

β₂-adrenergic receptor

βARK

β-adrenergic receptor kinase

PC

phosphatidylcholine

PIP₂

phosphatidylinositol 4,5-bisphosphate

PIP

phosphatidylinositol 4-phosphate

PBS

phosphate-buffered saline

ct

carboxyl terminus.
↵²K. Touhara, W. J. Koch, B. E. Hawes, and R. J. Lefkowitz, manuscript submitted.