G Protein-coupled Receptor Kinase GRK2 Is a Phospholipid-dependent Enzyme That Can Be Conditionally Activated by G Protein βγ Subunits

doi:10.1074/jbc.271.37.22552

G Protein-coupled Receptor Kinase GRK2 Is a Phospholipid-dependent Enzyme That Can Be Conditionally Activated by G Protein βγ Subunits*

From the Department of Molecular Pharmacology and Biological Chemistry, and Institute of Neuroscience, Northwestern University Medical School, Chicago, Illinois 60611 and ^§ Department of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

^∥ To whom correspondence should be addressed:
Dept. of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, 303 E. Chicago Ave., S215, Chicago, IL 60611.
Tel.: 312-503-3692; Fax: 312-503-5349; E-mail: mhosey{at}nwu.edu.

Abstract

G protein-coupled receptor kinases (GRKs) mediate agonist-dependent phosphorylation of G protein-coupled receptors (GPRs) and initiate homologous receptor desensitization. Previously, we reported that charged phospholipids directly interacted with the two GRK isoforms, GRK2 and GKR3, via a pleckstrin homology (PH) domain to regulate GRK activity (DebBurman, S. K., Ptasienski, J., Boetticher, E., Lomasney, J. W., Benovic, J. L., and Hosey, M. M. (1995) J. Biol. Chem. 270: 5742-5747). Here, evidence is provided to support the hypothesis that charged phospholipids are required for agonist-dependent phosphorylation of receptors by GRK2. In the absence of charged phospholipids, the purified human m2 muscarinic acetylcholine receptor (hm2mAChR) reconstituted in pure phosphatidylcholine vesicles or in a noninhibitory detergent was not a substrate for GRK2. However, these receptor preparations were stoichiometrically phosphorylated in an agonist-dependent manner upon addition of charged phospholipids. The known ability of G protein βγ subunits to stimulate mAChR phosphorylation also was found to be absolutely dependent on the presence of charged phospholipids, including phosphatidylinositol 4,5-bisphosphate (PIP₂). Phospholipids also regulated GRK-mediated phosphorylation of casein, a nonreceptor-soluble substrate. Among lipids tested, lipid inositol phosphates, PIP₂ and phosphatidylinositol 4-monophosphate, were found to be the most potent activators of GRK2 and were the only lipids that regulated GRK2 in a complex biphasic manner. At low μM concentrations, PIP₂ activated GRK2 via an interaction with the GRK pleckstrin homology domain; however, at high μM concentrations, PIP₂ inhibited GRK2, apparently via another mechanism. PIP₂-mediated inhibition could be partly relieved by increasing ATP. The results support the hypothesis that GRK2 is a lipid-dependent protein kinase that requires charged phospholipids for enzyme activation, for regulation by G_βγ subunits, and potentially for membrane association.

Footnotes

↵^‡ Supported by an Advanced Predoctoral Fellowship from the Pharmaceutical Research and Manufacturers of America Foundation.
↵^¶ An Established Investigator of the American Heart Association.
↵* This work was supported in part by National Institutes of Health Grants HL50121 (to M. M. H.) and GM 44944 (to J. L. B.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

GPR

G protein-coupled receptor

β₂AR

β₂-adrenergic receptor

βARK

β-adrenergic receptor kinase

PH

pleckstrin homology

G_βγ

βγ subunits of G proteins

G protein

guanine nucleotide-binding protein

GRK

G protein-coupled receptor kinase

GST

glutathione S-transferase

hm2 mAChR

human m2 muscarinic acetylcholine receptor

IP₃

inositol 1,4,5-trisphosphate

PC

phosphatidylcholine

PIP₂

phosphatidylinositol 4,5-bisphosphate

PIP

phosphatidylinositol 4-monophosphate

PI

phosphatidylinositol

PE

phosphatidylethanolamine

PS

phosphatidylserine

PG

phosphatidylglycerol

Sf9

Spodoptera frugiperda

βγ_t

transducin βγ

βγ_b

brain βγ

CHO

Chinese hamster ovary.
↵²R. Anderson, personal communication.
- Received April 15, 1996.
- Revision received June 27, 1996.