©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Pleckstrin Homology Domain-mediated Membrane Association and Activation of the -Adrenergic Receptor Kinase Requires Coordinate Interaction with G Subunits and Lipid(*)

Julie A. Pitcher , Kazushige Touhara , E. Sturgis Payne , Robert J. Lefkowitz (§)

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

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 (PIP) 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 -adrenergic receptor kinase (ARK) synergistically enhances agonist-dependent receptor phosphorylation and that both PH domain-binding ligands are required for membrane association of the kinase. PIP 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 ARK activity. In contrast, the Kand V for phosphorylation of a soluble ARK substrate (casein) was not altered in either the presence or absence of G and/or PIP. A fusion protein of the ARK containing an intact PH domain inhibits G/PIP-dependent 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.


INTRODUCTION

The pleckstrin homology (PH)() domain, named for the protein in which it was first identified, is an approximately 100-amino-acid region of sequence homology (1, 2) . To date more than 70 PH domain-containing proteins have been identified including serine/threonine and tyrosine kinases, several isoforms of phospholipase C, GTPase-activating proteins, nucleotide exchange factors, and cytoskeletal elements (1, 2, 3, 4, 5, 6) . Whether the PH domains of these proteins share common functions and what these functions are remain to be elucidated. Despite limited sequence identity the structural similarities recently reported for the PH domains of pleckstrin (7) , spectrin (8) , and dynamin (9, 10) support the classification of these regions as discrete domains.

Several lines of evidence suggest that PH domains may function to target proteins to membranes, potentially facilitating appropriate interactions with other components of the signal transduction pathway. The PH domain of spectrin has been implicated as a probable site for membrane binding (11) , and the PH domain of PLC-1 has been shown to be involved in binding this enzyme to lipid bilayers containing phosphatidylinositol 4,5-bisphosphate (PIP) (12) . Furthermore, the carboxyl terminus of the -adrenergic receptor kinase-1 (ARK) has been demonstrated to bind the G subunits of heterotrimeric G proteins (G) (13, 14) . This interaction leads to the membrane localization of the kinase and subsequent increased phosphorylation of agonist-occupied G protein-coupled receptor substrates. Fusion proteins encompassing the PH domains of several other proteins have additionally been demonstrated to bind G, although with varying affinities (15) . Importantly the G binding site is limited to and extends slightly beyond the carboxyl terminus of the PH domain (15) , raising speculation as to the function of the amino terminus of this domain.

Following the observation that PH domains and lipid binding molecules share structural similarities Harlan et al.(16) demonstrated that fusion proteins encompassing the PH domains from several proteins including the ARK bound specifically to PIP. Furthermore, the amino terminus of the PH domain was implicated as being the important site of lipid interaction. The PH domain of the ARK has thus been shown to interact with both G and PIP at the carboxyl and amino termini, respectively, of the PH domain. Here we demonstrate that interaction of these two ligands with the intact enzyme is required for its membrane association and activation.


EXPERIMENTAL PROCEDURES

Materials

Bovine ARK was overexpressed and purified from baculovirus-infected Sf9 cells (17) , and G subunits were purified from bovine brain (18) according to previously published procedures. Purified lipids, soybean phosphatidylcholine (20% phosphatidylcholine (PC)), and partially dephosphorylated casein were from Sigma. All other reagents were of the highest grade commercially available.

Purification and Reconstitution of the -Adrenergic Receptor

The human -adrenergic receptor (AR) was expressed and purified from baculovirus-infected Sf9 cells. Briefly, Sf9 cells were harvested 48 h postinfection with recombinant virus. The cells were lysed and a membrane fraction prepared by centrifugation at 33,000 g. The membranes (5 mg/ml) were subsequently solubilized with 0.25% w/v n-dodecyl -D-maltoside, and the AR was purified by affinity chromatography on an alprenolol-Sepharose column as described in Ref. 19.

Purified receptor was reconstituted into either soybean phosphatidylcholine vesicles (20% PC) or into vesicles of defined lipid composition. To form vesicles the required amounts of lipid in chloro-form were dried under a stream of nitrogen, resuspended in 10 mM Tris-HCl, pH 7.2, 100 mM NaCl by vortexing, and sonicated with a microtip sonicator. Purified AR was reconstituted into these vesicles as described previously (20) . The AR-containing vesicles were resuspended in 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, and the receptor concentration was determined by radioligand binding using I-labeled (-)-iodocyanopindolol.

ARK-mediated AR Phosphorylation

The AR (40 nM) reconstituted in various lipid environments (described in the text and figure legends) was incubated with ARK (10 nM) in 20 mM Tris-HCl (pH 7.5), 2.0 mM EDTA, 10 mM MgCl, 1 mM dithiothreitol containing 60 µM ATP (6000 cpm/pmol) in a total volume of 25 µl. All assays were performed in the presence of 50 µM(-)-isoproterenol unless otherwise indicated. Purified G subunits (20-200 nM) were also included in the phosphorylation reactions where indicated. Reactions were incubated at 30 °C and stopped by addition of an equal volume of SDS sample-loading buffer (8% SDS, 25 mM Tris-HCl, pH 6.5, 10% glycerol, 5% mercaptoethanol, 0.003% bromphenol blue) and electrophoresed on 10% SDS-polyacrylamide gels. The dried gels were subjected to autoradiography and PhosphorImager analysis to determine the picomoles of phosphate transferred to the receptor substrate.

Construction and Purification of Glutathione S-transferase Fusion Proteins

Glutathione S-transferase fusion proteins derived from the ARK carboxyl terminus (ct) were expressed in the Escherichia coli strain NM522 or BL21 and were purified as described previously (14) .()

Binding of the ARK to Lipid Vesicles

The AR (3 pmol) reconstituted in vesicles composed of either 100% PC or 95% PC, 5% PIP was incubated in the presence or absence of G subunits (0.2 µg) for 1 h on ice. After incubation the receptor was added to polycarbonate tubes (7 20 mm) (Beckman) containing purified ARK (0.5 µg), and the reaction mixture was diluted to a final volume of 30 µl with PBS. The final lipid concentration was 1.7 mg/ml in all assays. After incubation at room temperature for 10 min and on ice for 5 min, the tubes were centrifuged at 100,000 rpm (TL-100 rotor) for 15 min at 4 °C. The supernatant was removed and the pellet rinsed once with PBS. The pellet was subsequently resuspended in 15 µl of PBS and transferred to a clean tube. SDS sample-loading buffer was added to the supernatant, and pellet fractions and the samples were electrophoresed on 4-20% gradient polyacrylamide gels (Novex) and subjected to Western blot analysis (ECL, Amersham Corp.) using anti-ARKct antibodies (22) . The distribution of the ARK between the pellet and the supernatant was determined by densitometric analysis of the Western blot.

RESULTS AND DISCUSSION

Previous studies have demonstrated that the binding of G to the PH domain of the ARK leads to its membrane association and enhanced receptor phosphorylation (13, 14, 23, 24) . However, these experiments all utilized receptor substrates presented in heterogeneous lipid environments such as the AR reconstituted in crude lipid preparations or rhodopsin in rod outer segment membranes. To investigate potential interactions between the PH domain of ARK and lipids, ARs reconstituted into vesicles of defined lipid composition were utilized. Addition of G to the ARK significantly enhanced the rate and extent of receptor phosphorylation when the substrate was presented in a heterogeneous lipid environment (20% PC) (closed and opentriangles) but not when the receptor was reconstituted in a homogeneous lipid environment (100% PC) (closed and opensquares) (Fig. 1A). Thus, vesicles composed of 100% PC lack an essential cofactor required for G-mediated enhancement of ARK activity. Could this missing component be PIP, a lipid previously documented to bind to the amino terminus of the PH domain of ARK (16) ? Indeed, although AR reconstituted into PC vesicles containing PIP (3 or 10%) was a poor substrate for the ARK (closedsymbols), addition of G led to a dramatic enhancement of receptor phosphorylation (opensymbols) (Fig. 1B). In the presence of 100 nM G subunits and 3 or 10% PIP the initial rate of receptor phosphorylation increased approximately 25- and 65-fold, and the maximal extent of phosphorylation increased approximately 12- and 20-fold, respectively. Thus the ARK-mediated phosphorylation of purified reconstituted AR is dependent upon the presence of two ligands, each previously shown to bind PH domains.


Figure 1: PIP2 is required for G-mediated activation of ARK. A, phosphorylation of AR reconstituted in vesicles composed of an impure lipid preparation (20% PC) or purified PC. Reactions were performed in the presence of 50 µM (-)-isoproterenol in either the presence or absence of 100 nM G subunits. , AR in 20% PC; , AR in 20% PC + G; , AR in 100% PC; , AR in 100% PC + G. B, phosphorylation of AR reconstituted in vesicles composed of purified PC containing 3 or 10% PIP. Reactions were performed in either the presence or absence of 100 nM G and in the presence of 50 µM (-)-isoproterenol. , AR in 3% PIP; , AR in 3% PIP + G; , AR in 10% PIP; , AR in 10% PIP + G. Phosphorylation reactions were performed as described under ``Experimental Procedures'' and were stopped at the times indicated. The data represent the mean values obtained from three separate determinations.



Is the simultaneous presence of G and lipid required for the membrane association of ARK or does PIP interact with and directly activate a membrane-associated G/ARK complex? To distinguish between these two possibilities the kinetics of phosphorylation of a soluble ARK substrate (casein) were investigated. The Kand V for ARK-catalyzed casein phosphorylation were unaffected when assayed in the presence of vesicles composed of either 100% PC or 95% PC, 5% PIP in either the presence or absence of G (data not shown). These results support the hypothesis that both PH domain ligands are required for the membrane association of the ARK since addition of G and lipid did not directly activate the kinase. Indeed, when incubated with AR-containing vesicles, enhanced ARK activity (Fig. 2A) and membrane association of the kinase (Fig. 2B) was observed only when both PIP and G were present. Under the conditions utilized in this study neither PH domain ligand alone was sufficient to cause association of the ARK with lipid vesicles (Fig. 2B). Interestingly, the same pattern of membrane association of the ARK was observed in either the absence or presence (data not shown) of AR agonist. Agonist occupancy of the receptor was, however, required for ARK-mediated AR phosphorylation (data not shown).


Figure 2: Membrane association of the ARK requires the presence of both G and PIP. A, autoradiograph showing ARK-mediated AR phosphorylation. The AR was reconstituted in lipid vesicles composed of 100% PC (PC) or 95% PC, 5% PIP (PIP). G subunits were included where indicated. Phosphorylation reactions were performed in the presence of 50 µM (-)-isoproterenol for 10 min. The concentrations of all components were as for B (see ``Experimental Procedures''). B, ARK binding to AR-containing vesicles composed of 100% PC (PC) or 95% PC, 5% PIP (PIP). G subunits were included where indicated, and assays were performed as described under ``Experimental Procedures.'' The distribution of the ARK between the supernatant (S, whitebars) and pellet (P, blackbars) fractions is shown ±S.E. for three separate determinations. A representative Western blot is also displayed.



The membrane association of ARK in the presence of AR requires both 5% PIP and G and would appear to account for the enhanced rate and extent of receptor phosphorylation observed under these conditions. Increasing the concentration of PIP and G enhances the initial rates of ARK phosphorylation of AR in a dose-dependent fashion (Fig. 3). As the concentration of G increases from 20 nM to an apparently saturating concentration of 120 nM a dramatic increase in the V of the enzyme and a modest increase in the apparent affinity for PIP is observed (Fig. 3). Analysis of this data using the ALLFIT program (25) reveals slope factors greater than 1, an observation that may suggest positive cooperativity.


Figure 3: Dose-dependent activation of the ARK by PIP and G. Purified AR was reconstituted in vesicles composed of PC containing various concentrations of PIP. The initial rate of phosphorylation of these receptor substrates by the ARK was subsequently determined in the absence of any additions () or in the presence of 20 nM (), 40 nM (), 120 nM (), or 200 nM () G subunits. The results shown represent the mean values from two separate determinations. The EC values for PIP binding are 5.7% PIP (20 nM G), 4.35%PIP (40 nM G), 4.0% PIP (120 nM G), and 3.0% PIP (200 nM G). These values were determined using the ALLFIT program. Constraining parameters such that all curves shared the same EC value had a significant deleterious effect on the quality of the fit (p < 0.01).



Which lipids in the presence of G are capable of mediating membrane association of the ARK? To address this question AR was reconstituted in a lipid background of PC in the presence of an EC concentration of PIP (3%) or 3% of various other lipids. At low G subunit concentrations (20 nM) the only lipids capable of promoting ARK phosphorylation of AR were PIP and to a much lesser extent phosphatidylinositol 4-phosphate (PIP) (Fig. 4, left panel). This pattern of lipid specificity is similar to that observed by Harlan et al. (16), who looked directly at the binding of PH domain-containing fusion proteins to lipid vesicles. PIP and (with a lower affinity) PIP were the only lipids capable of promoting membrane association of these proteins. Raising the concentration of G (200 nM) increases the extent of AR phosphorylation and appears to increase the apparent affinity of the ARK for lipid. Under these conditions other precursors (phosphatidylinositol and PIP) or metabolites (phosphatidic acid) of PIP become effective promoters of ARK-mediated AR phosphorylation (Fig. 4, right panel). Thus, although at 20 nM G PIP is approximately 4-fold more effective at promoting ARK-mediated AR phosphorylation than PIP (Fig. 4, left panel), at a higher concentration of G (200 nM), PIP and PIP were equally effective (Fig. 4, right panel). Of the lipids tested only PIP, PIP, phosphatidylinositol, and phosphatidic acid were capable of promoting G activation of the ARK (Fig. 4, right panel).


Figure 4: PIP most effectively mediates lipid/G enhancement of ARK activity. Purified AR reconstituted in 97% PC, 3% of the indicated lipids was phosphorylated for 10 min in the absence of G (whitebars) or alternatively in the presence of either 20 nM G (leftpanel, black bars) or 200 nM G (right panel, black bars). PA, phosphatidic acid; PI, phosphatidylinositol, PE, phosphatidylethanolamine; MAG, monoacylglycerol (1-monopalmitoyl-rac-glycerol (C16:0)); DAG, diacylglycerol (1,2-dioleoyl-rac-glycerol (C18:1, [cis]-9)); ceramides (type III); type III); GC, galactocerebrosides (type II). The results shown represent the mean values obtained from at least three separate determinations.



That the PH domain of the ARK is the region of the enzyme that interacts with both G and PIP is suggested by previous studies demonstrating direct interactions between these ligands and fusion proteins encompassing the PH domain of this enzyme (15, 16) . Furthermore, here we show that a carboxyl-terminal fusion protein of the ARK, which contains the PH domain, inhibits G/PIP-dependent ARK activity (Fig. 5, ARKct). However, a fusion protein in which a tryptophan residue invariant in all PH domain sequences is mutated to alanine (Trp in the ARK) and which shows no binding to either G or PIP has no inhibitory activity (Fig. 5, Trpmutant).


Figure 5: Inhibition of G/PIP-mediated ARK activation by the PH domain of ARK. Purified AR reconstituted in 97% PC, 3% PIP was phosphorylated in the presence of 20 nM G for 10 min. The glutathione S-transferase fusion proteins Pro-Leu of the ARK (ARKct) and a mutant fusion protein in which Trp of the ARK is mutated to alanine (Trpmutant) were included in the phosphorylation reactions at the concentrations indicated. The results represent the means ±S.E. for three separate determinations.



The data presented demonstrate that effective membrane localization of the ARK, which enhances both the rate and extent of phosphorylation of receptor substrates, requires the simultaneous presence of two PH domain ligands (PIP and G). Neither PH domain binding ligand alone is sufficient to affect this functional activation of the enzyme. The requirement for a specific lipid to effect G-mediated activation of the ARK was previously unappreciated since purified receptor preparations reconstituted in heterogeneous lipid environments have been exclusively utilized as substrates for this kinase. Increasing the concentration of G appears to increase the affinity of the ARK for lipid. Thus, binding of a ligand to the carboxyl terminus may affect binding of a ligand to the amino terminus of the PH domain. The precise mechanism and potentially cooperative nature of multiple ligand binding to the PH domain of the ARK are currently under investigation.

Comparison of the recently elucidated structural features of several PH domains (7, 8, 9, 10) reveals a degree of polarity of structure such that the highly conserved carboxyl-terminal -helix and three loops connecting the -strands, /, /, and /, fall on opposite faces of the domain. These three loops represent the most variable region of PH domains and provide the potential for differences in ligand specificity. Thus, although in this and previous studies the PH domain of the ARK has been shown to interact with PIP and G(15, 16) not all PH domains have been demonstrated to bind to these specific ligands, and various other molecules have been implicated as ligands for these domains. The PH domain of spectrin has been implicated as binding to specific sites in bovine brain membranes (11) , and the PH domain of Bruton tyrosine kinase in addition to binding G(26) has been shown to bind protein kinase C (21) . The number and variety of ligands for PH domains remain to be elucidated; however, the synergistic activation of the ARK by G and lipid suggests that reexamination of the binding of these particular ligands to the PH domains of other proteins may be fruitful. In particular, proteins that have been shown to bind weakly to G or PIP might display a higher affinity for these ligands and potentially altered functional characteristics when assayed in the presence of both. The potentially cooperative binding of multiple ligands to PH domains may also in part explain the failure of glutathione S-transferase-PH domain fusion proteins to interact with protein ligands in either the two hybrid yeast system or when utilized to screen T7 promoter-based bacterial expression libraries (9) . It remains to be determined if the membrane localization of proteins via multiple ligand binding is a function common to all PH domains.


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.

§
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.

The abbreviations used are: PH, pleckstrin homology; G, -subunits of heterotrimeric G proteins; AR, -adrenergic receptor; ARK, -adrenergic receptor kinase; PC, phosphatidylcholine; PIP, phosphatidylinositol 4,5-bisphosphate; PIP, phosphatidylinositol 4-phosphate; PBS, phosphate-buffered saline; ct, carboxyl terminus.

K. Touhara, W. J. Koch, B. E. Hawes, and R. J. Lefkowitz, manuscript submitted.


ACKNOWLEDGEMENTS

We thank Darrell Capel for purified ARK and G subunits, W. Carl Stone for DNA sequencing and purification of the ARKct fusion protein, W. Carl Stone and Dr. Jeffrey L. Arizza for generation of the anti-ARK polyclonal sera, Grace P. Irons for virus and cell culture, and Dr. Mario Tiberi for analysis of data using the ALLFIT program.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.