©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Lipid-mediated Regulation of G Protein-coupled Receptor Kinases 2 and 3 (*)

(Received for publication, October 12, 1994; and in revised form, December 8, 1994)

Shubhik K. DebBurman (1) (2)(§) Judy Ptasienski (1) Evan Boetticher (1) (2) Jon W. Lomasney (1) (2) (3) Jeffrey L. Benovic (4)(¶) M. Marlene Hosey (1) (2)(**)

From the  (1)Department of Molecular Pharmacology and Biological Chemistry, the (2)Institute of Neuroscience, and the (3)Department of Pathology and Feinberg Cardiovascular Research Institute, Northwestern University Medical School, Chicago, Illinois 60611 and the (4)Department of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

G protein-coupled receptor-mediated signaling is attenuated by a process referred to as desensitization, wherein agonist-dependent phosphorylation of receptors by G protein-coupled receptor kinases (GRKs) is proposed to be a key initial event. However, mechanisms that activate GRKs are not fully understood. In one scenario, beta-subunits of G proteins (G) activate certain GRKs (beta-adrenergic receptor kinases 1 and 2, or GRK2 and GRK3), via a pleckstrin homology domain in the COOH terminus. This interaction has been proposed to translocate cytosolic beta-adrenergic receptor kinases (betaARKs) to the plasma membrane and facilitate interaction with receptor substrates. Here, we report a novel finding that membrane lipids modulate betaARK activity in vitro in a manner that is analogous and competitive with G. Several lipids, including phosphatidylserine (PS), stimulated, whereas phosphatidylinositol 4,5-bisphosphate inhibited, the ability of these GRKs to phosphorylate agonist-occupied m2 muscarinic acetylcholine receptors. Furthermore, both PS and phosphatidylinositol 4,5-bisphosphate specifically bound to betaARK1, whereas phosphatidylcholine, a lipid that did not modulate betaARK activity, did not bind to betaARK1. The lipid regulation of betaARKs did not occur via a modulation of its autophosphorylation state. PS- and G-mediated stimulation of betaARK1 was compared and found strikingly similar; moreover, their effects together were not additive (except at initial stages of reaction), which suggests that PS and G employed a common interaction and activation mechanism with the kinase. The effects of these lipids were prevented by two well known G-binding proteins, phosducin and GST-betaARK-(466-689) fusion protein, suggesting that the G-binding domain (possibly the pleckstrin homology domain) of the GRKs is also a site for lipid:protein interaction. We submit the intriguing possibility that both lipids and G proteins co-regulate the function of GRKs.


INTRODUCTION

G protein-coupled receptors (GPRs) (^1)represent a superfamily of plasma membrane receptors that recognize an eclectic variety of environmental stimuli and transduce their signals to the internal milieu of cells(1) . GPR-activated cellular signaling rapidly wanes due to regulatory processes collectively known as desensitization. An important component to desensitization, especially underlying its rapid phase, is an uncoupling of the GPR from its G protein by phosphorylation. Two classes of protein kinases mediate this phosphorylation: second messenger-dependent kinases (e.g. PKC and PKA) mediate agonist-independent phosphorylation of GPRs and initiate heterologous desensitization, whereas a unique class of serine-threonine protein kinases, namely G protein-coupled receptor kinases (GRKs), mediate agonist-dependent phosphorylation of GPRs and initiate homologous desensitization(2, 3, 4) .

Until now, six GRKs have been identified by molecular cloning (GRK1 to GRK6)(5, 6, 7, 8, 9, 10, 11) . The molecular mechanisms that govern the activity of these kinases, both in their interaction with receptor substrates and in regulation of their function, are not completely understood. An important step in GRK activation is translocation, whereby the cytosolic enzyme is targeted to its substrate (the agonist occupied form of the GPR) in the plasma membrane. It appears that different GRKs have evolved distinct mechanisms of membrane localization. Rhodopsin kinase (GRK1) is unique among GRKs in that it is modified at its carboxyl terminus by a geranylgeranyl isoprenoid moiety, which allows the kinase to be directly anchored to the plasma membrane(12, 13) . GRK5 nonspecifically binds lipids which stimulate its autophosphorylation and may help target it to cell membranes(14) . In contrast, GRK2 and GRK3 (betaARK1 and betaARK2) are not modified by isoprenylation. Instead, in vitro studies suggest that membrane-associated beta subunits of heterotrimeric G proteins (G) aid both in the membrane targeting and activation of betaARKs(15, 16, 17, 18) . G binds betaARK1 and betaARK2 in vitro(16) , and the site of this interaction has been mapped to a region partly within the pleckstrin homology (PH) domain in the carboxyl terminus of betaARK1 and betaARK2(18) . Recent studies in which the solution structure of the PH domain of pleckstrin was solved suggested that PH domains may contain lipid-binding domains(19) . Subsequently, Harlan et al.(20) reported that phosphatidylinositol-4,5-bisphosphate (PIP(2)) directly binds to the PH domains of several proteins, including betaARK1 (GRK2); however, the functional consequence of lipid binding was not assessed.

In the present effort, we evaluated the role of various classes of membrane lipids in regulating betaARK1 and betaARK2 activity, by measuring their ability to mediate agonist-dependent phosphorylation of human m2 (hm2) muscarinic acetylcholine receptors (mAChRs) in vitro. We report here novel results demonstrating that these GRKs bound to and were dually regulated either in a positive or negative manner by charged phospholipids. Lipid-mediated regulation of betaARK1 and 2 activity did not appear to occur through a mechanism that involves autophosphorylation, in contrast to what was recently reported for GRK5(14) . Furthermore, analogous to G, the lipids appeared to interact with betaARKs via the carboxyl-terminal region which contains the G-binding domain and PH domain.


EXPERIMENTAL PROCEDURES

Materials

Spodoptera frugiperda (Sf9) insect cells were obtained from Invitrogen. Sf-900 II insect cell media and gentamicin were purchased from Life Technologies, Inc. Recombinant baculoviruses encoding the human m2 muscarinic acetylcholine receptors were kindly provided by Drs. Elliot Ross and E. M. Parker, University of Texas (Dallas). G purified from bovine brain was a generous gift from Dr. Pat Casey, Duke University (Durham, NC). Phosducin purified from bovine retina was kindly provided by Dr. Y. K. Ho, University of Illinois (Chicago). [-P]ATP and 1-quinuclidinyl[phenyl-4-^3H]benzilate were purchased from Amersham Corp. Lipids were either purchased from Sigma or Avanti Polar Lipids.

Cell Culture

Sf9 cells were cultured as described (21) , either in monolayers or in suspension using Sf-900 II serum-free media supplemented with gentamicin (50 µg/ml).

Purification and Reconstitution of hm2 mAChRs

Sf9 cells (at a density of 2 times 10^6 cells/ml for suspension culture) were infected with recombinant baculovirus expressing the hm2 mAChRs at a multiplicity of infection of 5. At 65-72 h post-infection, cells were harvested and membranes prepared essentially as described in Richardson et al.(16) . The hm2 mAChRs were purified from Sf9 cell membranes and reconstituted into chick heart lipids (16) . The specific activity of the purified receptors was determined by [^3H]1-quinuclidinyl[phenyl-4-[^3H]benzilate ligand binding assays(22) .

Phosphorylation of the Purified and Reconstituted hm2 mAChRs

The GRKs, betaARK1, and betaARK2 were expressed in Sf9 cells using recombinant baculovirus encoding the various GRKs and purified (>95%) as described previously(23) . The GST-betaARK-(466-689) fusion protein was prepared as reported(18) . All phosphorylation reactions were carried out with purified and reconstituted hm2 mAChRs (0.2-0.4 pmol) and betaARKs (30 nM) in 20 mM Tris-HCl, pH 7.4, 5 mM MgCl(2), 2 mM EDTA, in the presence of 50 µM [--P]ATP (specific activity of 1000 cpm/pmol), for 1 h at 37 °C in a total reaction volume of 100 µl. Reactions were terminated with SDS sample buffer, and samples were resolved on 8% polyacrylamide gels(24) . Phosphorylated receptors were visualized and the amount of incorporated P quantified by phosphorimaging with a Bas2000 Fuji-bioimager. The extent of phosphorylation of the receptors by betaARK1 observed in the presence of carbachol, and the absence of added lipid, was taken as 100% (4-5 mol of phosphate/mol of receptor). Purified lipids were lyophilized and sonicated and used at the concentrations indicated. In reactions that contained lipids, lipids were preincubated with the receptors and betaARKs for 5-10 min at room temperature before reactions were started with [--P]ATP.

Peptide Mapping of Phosphorylated hm2 mAChRs

Phosphorylated receptors were incubated in 0.05% digitonin and centrifuged in a Centricon device to remove excess [-P]ATP. Subsequently, the receptors were electrophoresed on an 8% polyacrylamide gel and then electroblotted onto an Immobilon-CD filter. The phosphorylated receptors were visualized by phosphorimaging, and the corresponding band was excised and minced into small pieces. The receptors were digested with Staphylococcus aureus V8 protease (50 µg/ml) at 37 °C for 16 h in buffer containing 50 mM NH(4)CO(3), 7 mM EDTA, pH 8.0. The filters were washed and phosphopeptides were eluted by adding 50 µl of 4 M guanidine HCl, 0.1% Triton X-100 and sonicating for 10 min(25) . This process was repeated twice and resulted in the recovery of 60-80% of applied phosphoprotein. The peptides were lyophilized, resuspended in 125 mM Tris-HCl, pH 6.8, and electrophoresed on 25% acrylamide gels prepared as in (26) . The gels were dried, and peptide maps were analyzed by phosphorimaging.

betaARK Binding to Phospholipid Vesicles

Lipids of desired composition were mixed and dried together under high purity N(2) and then redissolved in phosphate-buffered saline buffer. The lipid mixtures (25 mg/ml) were sonicated in short bursts for 1-2 min, chilled on ice for 1 min, and re-sonicated. Lipid vesicles (concentrations as shown in the legend to Fig. 1, C and D) were added to purified betaARK1 (100 ng) in a total volume of 50 µl in a Beckman TLA-100 centrifuge tube and incubated for 5-15 min (or until equilibrium was attained) at room temperature. The tubes were spun at 100,000 times g for 30 min. The vesicles pelleted with this treatment effectively separated lipid-bound betaARK1 from the unbound betaARK in the supernatant. The betaARK1 content in both the pellet and supernatant fractions was determined using Western blot analysis using an anti-betaARK polyclonal antibody. Visualization was performed using the Renaissance Chemiluminescence Kit and Reflection Autoradiography film (DuPont NEN). NIH Image 1.52 software was used to quantitate the films.


Figure 1: A, lipid regulation of agonist-dependent phosphorylation of mAChRs by betaARK1. The figure is a graphic representation of phosphorylation of purified reconstituted hm2 mAChRs by betaARK1, in the presence of muscarinic agonist carbachol (1 mM), with (open bar) or without (closed bar; control at 100%) the addition of various lipids. The results are means from two to five experiments with similar results. The maximal effects of lipids (observed with 50 µg for all lipids, except for phosphatidic acid, whose effects were maximal with 0.5-5 µg), whether stimulatory or inhibitory, are shown. B, concentration-dependent stimulation by phosphatidylserine and inhibition by PIP(2) of betaARK1 activity. The graph shows the percent change in receptor phosphorylation by betaARK1 with increasing amounts of PS or PIP(2) (1.0 ng to 100 µg/100-µl reaction), when compared with receptor phosphorylation in the absence of lipid (100%). Experiments were performed two to three times with similar results. The insets are representative phosphorimages. Concentration-dependent effects of the other lipids were also assessed (data not shown). C and D, direct binding of betaARK1 to phospholipids. Centrifugation assays to study betaARK1 binding to phospholipids were performed as described under ``Experimental Procedures.'' Binding reactions were performed with betaARK1 (100 ng) and phospholipid vesicles that were made of varying concentrations of either PC alone (C, D), PC and PS (C), or PC and PIP(2) (D). The amount of betaARK1 that bound to lipid vesicles was assessed by quantifying betaARK1 immunoreactivity in pellet fractions and is expressed as percent of the total betaARK1 in the supernatant and pellet combined. In control experiments with PC vesicles, betaARK immunoreactivity was negligible at 0.05% (500 µM PC; Fig. 1C), and 0.0-0.1% (180-900 µM PC; Fig. 1D).




RESULTS AND DISCUSSION

Various membrane lipids were found to modulate the ability of betaARK1 to phosphorylate agonist-activated hm2 mAChRs (Fig. 1A), which are known substrates for betaARK1 and betaARK2(15, 16, 27) . Of the lipids tested, phosphatidic acid, phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) enhanced betaARK1-mediated receptor phosphorylation by 2-3-fold, compared with reactions in the absence of added lipid (control; 100%). Phosphatidylcholine (PC) had no significant effect on betaARK1 activity. In direct contrast, a key phospholipid, PIP(2), markedly inhibited receptor phosphorylation by betaARK1 by as much as 90%. No effects were observed with three other classes of membrane lipids: cholesterol, sphingolipids (sphingosine and sphingomyelin), and glycolipids (cerebroside). The effects of PS and PIP(2) were further characterized and shown to occur in a concentration-dependent manner (Fig. 1B). Parallel experiments with betaARK2 and lipids produced similar results (data not shown). These results are the first demonstration that agonist-dependent phosphorylation of a G protein-coupled receptor by GRKs may be governed by membrane lipids.

In order to assess whether regulation of betaARK activity by lipids was a consequence of direct interaction between lipid and kinase, we employed a phospholipid binding assay utilizing ultracentrifugation, similar to that developed by Harlan et al.(20) , and measured the appearance of betaARK immunoreactivity in pellets consisting of various mixtures of phospholipids. Phospholipid vesicles consisting of 500 µM PC alone bound betaARK1 poorly (<1% of betaARK1 bound to these vesicles) and were used as controls (Fig. 1, C and D). In studies with PS, 37% of the total added betaARK1 was detected in pellets that contained phospholipid vesicles of 60 µM PS and 500 µM PC (Fig. 1C). betaARK1 immunoreactivity in these vesicles increased to 55% when the PS content in PC/PS vesicles was increased to 100 µM (Fig. 1C). Similarly, in studies with PC and PIP(2) we observed specific binding of betaARK1 to PIP(2) (Fig. 1D). Increased PIP(2) content in PC/PIP(2) vesicles resulted in higher amounts of betaARK1 immunoreactivity in pellet fractions (Fig. 1D). These results demonstrated that lipids which regulated betaARK activity toward the hm2 mAChRs (PS and PIP(2)) specifically bound betaARK1; in contrast, PC, which did not regulate betaARK-mediated phosphorylation of hm2 mAChRs, bound betaARK1 minimally. Although the exact mechanism by which lipids regulate betaARKs is unknown, our data strongly suggest that lipids directly interact with betaARKs to modulate their function.

We wondered if PS and PIP(2) interacted with GRKs at independent or similar sites. When both PIP(2) or PS were added, PS effectively alleviated the PIP(2)-mediated inhibition of betaARK1 phosphorylation of agonist-occupied hm2 receptor in a concentration-dependent manner (Fig. 2). Similarly, PIP(2) inhibited PS stimulation of betaARK1 in a concentration-dependent fashion (Fig. 2). These results suggest that PS and PIP(2) regulate betaARK1 in a competitive manner and, despite their opposing actions on the kinase, might share common sites of interaction with betaARK1.


Figure 2: Competitive interactions of PS and PIP(2) with betaARK1. The figure depicts agonist-dependent phosphorylation of the hm2 mAChRs by betaARK1 either in the presence of different amounts of PS and PIP(2) or control reactions in the absence of either lipid (100%; solid bar). PIP(2) strikingly inhibited PS stimulation of BARK1 to levels of receptor phosphorylation observed in presence of atropine (10-20%). PS effectively alleviate PIP(2) inhibition of betaARK1 to 80% of control. The inset is a representative phosphorimage. Reactions were performed twice with similar results.



PS increased the initial rate and extent (Fig. 3A) of receptor phosphorylation by betaARK1. These results resembled the effects of G to increase both the rate and extent of betaARK1-mediated phosphorylation of hm2 mAChRs ( (15) and (16) and Fig. 3A), suggesting that PS might activate betaARK1 in a manner analogous to G. To address this possibility, we first asked if the stimulatory effects of PS and G were additive. When PS and G were tested together, their effects on receptor phosphorylation by betaARK1 were additive during the initial stages of the reactions (Fig. 3B), but nonadditive at 60 min (Fig. 3C). Moreover, addition of G alleviated PIP(2) inhibition of betaARK1 in a concentration-dependent manner (data not shown). To further assess whether PS and G activated betaARKs in a similar fashion, peptide maps of betaARK1-phosphorylated hm2 mAChRs (phosphorylated either in the presence of either PS or G or both) were compared. The results revealed that both PS and G enhanced the phosphate content of a set of similar peptides in a non-additive manner, when compared with peptides obtained in the absence of either lipid or G (carbachol control, Fig. 3D). Collectively, these results suggest that lipids and G either share common sites of interaction with betaARK or that their sites are proximally located and binding of one allosterically modulates the binding of the other.


Figure 3: Comparison of PS and G protein beta subunit stimulation of betaARK1. A compares the time course (0-90 min) of agonistdependent phosphorylation of hm2 mAChRs by betaARK1 in the presence of either PS (50 µg) or G (subunits) purified from bovine brain (100 nM), with control reactions carried out in the absence of added lipid and G (carbachol). B shows that the PS and G effects on receptor phosphorylation by betaARK1 were additive within the first 2 min of the reaction, while C shows that PS and G were nonadditive at 60 min. The data shown are means from two to three independent experiments with similar results. D compares phosphopeptide maps obtained from hm2 mAChRs that were phosphorylated with betaARK1 and subsequently proteolyzed with S. aureus V8 protease. Reactions were carried out in the presence of either atropine (1 mM) (lane 1) or carbachol (1 mM) (lanes 2-5) and contained PS (lane 3), G (lane 4), or both (lane 5).



G is known to bind betaARK1 in vitro and the G-binding domain is localized within the carboxyl terminus of betaARK1-(467-689)(17, 18) . A glutathione S-transferase (GST)-betaARK1-(466-689) fusion protein, which is known to bind G and thus prevent its ability to stimulate betaARK(17, 18, 28, 29) , strikingly reduced the ability of PS to stimulate betaARK1-mediated phosphorylation of the hm2 mAChRs by 80% (Fig. 4). GST-betaARK(466-689) contains the entire PH domain of betaARK1, which has been shown to directly bind PIP(2)(20) . The present result suggested to us that the GST-betaARK fusion protein served as a sink for lipids, and therefore bound PS, and prevented its ability to stimulate receptor phosphorylation by betaARK1. In support of this hypothesis, the effects of the GST-betaARK fusion protein were competitively eliminated in a dose-dependent fashion when increasing amounts of PS were added to the phosphorylation reaction (Fig. 4B).


Figure 4: A, modulation of lipid regulation by GST-betaARK by GST-betaARK-(466-689) fusion protein and phosducin. Agonist-dependent phosphorylation of the hm2 mAChRs by betaARK1 was carried out in the presence of added lipid (50 µg of either PS or PIP(2)), with or without GST-betaARK-(466-689) fusion protein (7 µM) or phosducin purified from bovine retina (3 µM). Receptor phosphorylation by betaARK1 in the absence of added lipid was taken as 100% (solid bar). GST-betaARK-(466-689) fusion protein and phosducin inhibited PS stimulation of betaARK1 by 80 and 75%, respectively; furthermore, phosducin alleviated PIP(2) inhibition of betaARK1 to 90% of control. The data shown are means from two to three independent experiments. B, concentration-dependent elimination of the effects of the GST-betaARK fusion protein by PS. Increasing amounts of PS (50-250 µg) were added to phosphorylation reactions that contained purified reconstituted hm2 mAChRs (0.3 pmol), betaARK1, PS (25 µg), carbachol, and GST-betaARK (7 µM). Control reactions (solid bar) did not contain either GST-betaARK or PS and receptor phosphorylation observed under these conditions were taken as 100%.



It is unclear whether the effect of the GST-betaARK fusion protein to prevent the effect of lipids on betaARK occurred as a result of lipid binding to the PH domain or G domain or both. To further determine whether other proteins with G-binding domains could modify lipid regulation of betaARK1, the ability of retinal phosducin, a G-binding protein(30, 31) , to prevent PS stimulation of betaARK1 was assessed. Here, in studies with mAChR and betaARK1, phosducin markedly inhibited PS stimulation of betaARK1 by 75% (Fig. 4); the effects of phosducin were concentration-dependent (data not shown). Furthermore, phosducin also alleviated PIP(2) inhibition of betaARK1 (Fig. 4). These results support the concept that G and lipids interact with betaARK1 in a common region. The results also suggest that phosducin contains lipid-binding sites that allow it to compete with betaARK1 for both PIP(2) and PS. The results observed with phosducin are interesting. Hekman et al.(32) recently demonstrated that phosducin inhibits phosphorylation of purified and reconstituted beta(2)ARs by betaARK1 only in the presence of G proteins (or G); in the absence of G proteins, phosducin had no effect on betaARK activity. It is thought that phosducin has no direct interaction with betaARKs, but rather it regulates the ability of betaARKs to bind G(32) . The results we observed with phosducin and lipids were strikingly similar and suggest to us that the effect of phosducin was to regulate the ability of betaARKs to interact with lipids.

The current theory that membrane-anchored G subunits aid the targeting of betaARKs to their membrane-bound substrates is both attractive and well supported(2, 17, 18) . Based on our observations, we propose that both G proteins and lipids participate in the translocation and activation of betaARKs. A critical question regarding GRK function has been whether GRKs are constitutively active or are normally inhibited or activated by other unidentified regulatory molecules in the cell. The observation that different lipids may either enhance or suppress kinase activity is revealing, since it provides a candidate molecule (PIP(2)) that may directly inhibit GRKs and others that may stimulate GRKs. Furthermore, the apparent specificity of charged phospholipids to produce these effects complements the fact that the intracellular face of the plasma membrane is enriched with such lipids. Binding of GRKs to G and/or charged lipids in vivo might direct these enzymes to the plasma membrane, where they may phosphorylate agonist-activated GPRs and initiate receptor desensitization. In support of this speculation, recent reports have suggested that a small, but significant, population of betaARK is normally localized to the plasma membrane in cells and that other pools of betaARKs also exist in intracellular membranes(33, 34) . However, the relative contributions of G and lipids in regulation of betaARKs need to be dissected. It also remains unclear why some lipids stimulate betaARKs, whereas other lipids inhibit their activity. Furthermore, we cannot preclude the possibility of a supplementary effect of lipids in directly modulating the reconstituted receptors. Further studies will address these questions and elucidate the exact domains in GRKs involved in their regulation by lipids.

The present results support a mechanism of modulation of betaARK1 and betaARK2 that is quite distinct from a previous finding concerning effects of lipids on another member of the GRK family. Kunapuli et al.(34) demonstrated that lipids increased autophosphorylation of GRK5 and suggested a role for autophosphorylation in regulating GRK5 by demonstrating that an autophosphorylation-deficient mutant had decreased GRK5 activity. It is to be noted however that the previous study did not demonstrate that the lipid-mediated increase in autophosphorylation had a functional effect on the ability of GRK5 to phosphorylate receptor substrates. The lipid effects on betaARK reported here occur via a mechanism that does not involve autophosphorylation. In contrast to what may be the case for GRK5, autophosphorylation appears to be unimportant, or less important, in regulating betaARK1 and betaARK2. In the present study, we observed autophosphorylation of both betaARKs, but at very low substoichiometric levels. In studies with betaARKs and mAChR, the stoichiometry of betaARK1 autophosphorylation was 0.03 mol of phosphate/mol of betaARK or less; this increased to no more than 0.1 mol phosphate/mol of protein in the presence of PS and decreased with PIP(2). Thus, these substoichiometric levels of autophosphorylation do not support a role for autophosphorylation of betaARK in the lipid-mediated regulation of activity. A second important difference that should be noted in terms of the regulation of the betaARK isozymes and other members of the GRK family, including GRK5, is that the other GRKs (rhodopsin kinase, GRK4, GRK5, and GRK6) lack a G-binding domain and do not possess PH domains.

The G-binding domain of betaARK1 and betaARK2 is partially contained within a PH domain(28, 35, 36) . PH domains have only recently been recognized as sites for interaction between proteins (28, 35, 36) and have been subsequently found in an increasing number of diverse molecules(35, 37, 38, 39, 40, 41, 42) ; however, their function(s) remain uncertain. In one proposal, proteins with PH domains have recently been suggested to be either effectors of G or molecules similar to G(28). An alternate suggestion has been made that PH domains are interaction sites for phosphate groups(42) . However, neither phosphoserine nor phosphothreonine appeared to regulate the ability of betaARK1 to phosphorylate the hm2 mAChRs (data not shown). In contrast, our results indicate that, at least in betaARK1 and betaARK2, the G-binding domain and/or PH domain can also interact with phospholipids.

The resolution of the solution structure of the PH domain of pleckstrin by nuclear magnetic resonance spectroscopy has suggested that PH domains may be lipid interaction sites in vivo(19) . In this regard, PIP(2) has been shown recently to directly bind the PH domain in betaARK1 and various other PH domain-containing proteins (20) . It is proposed that the NH(2) terminus of PH domains contain the lipid-binding motif, and this has been specifically demonstrated in the PH domain in pleckstrin(20) . In betaARK1 and betaARK2, the G-binding domain overlaps with the COOH-terminal end of the PH domain. Therefore, it is possible that the lipid binding (presumably NH(2)-terminal) and G-binding regions are separate modules within the PH domain in betaARK. Yet, the close proximity of these sites in betaARK may allow for allosteric regulation between lipids and G in their interactions with betaARK. Our data provide initial evidence to support this hypothesis.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants HL 50121 (to M. M. H.) and GM 44944 (to J. L. B.) and by grants from the American Heart Association (to M. M. H.) and the Northwestern Memorial Foundation (to J. W. 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.

§
Supported by an advanced predoctoral fellowship from the Pharmaceutical Research and Manufacturer's of America Foundation.

Established investigator of the American Heart Association.

**
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; mhosey{at}nwu.edu.

(^1)
The abbreviations used are: GPR, G protein-coupled receptor; betaAR, beta-adrenergic receptor; betaARK, beta-adrenergic receptor kinase; G, beta subunits of G proteins; G protein, guanine nucleotide-binding protein; GRK, G protein-coupled receptor kinase; GST, glutathione S-transferase; mAChR, muscarinic acetylcholine receptors; PC, phosphatidylcholine; PG, phosphatidylglycerol; PS, phosphatidylserine; PIP(2), phosphatidylinositol 4,5-bisphosphate; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PH, pleckstrin homology.


ACKNOWLEDGEMENTS

We thank Roger Bodine for excellent technical assistance and Robin Rylaarsdam for helpful discussions.


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