©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutational Analysis of the Pleckstrin Homology Domain of the -Adrenergic Receptor Kinase
DIFFERENTIAL EFFECTS ON G AND PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE BINDING (*)

Kazushige Touhara , Walter J. Koch , Brian E. Hawes , Robert J. Lefkowitz (§)

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The subunits of heterotrimeric G proteins (G) play a variety of roles in cellular signaling, one of which is membrane targeting of the -adrenergic receptor kinase (ARK). This is accomplished via a physical interaction of G and a domain within the carboxyl terminus of ARK which overlaps with a pleckstrin homology (PH) domain. The PH domain of ARK not only binds G but also interacts with phosphatidylinositol 4,5-bisphosphate (PIP). Based on previous mapping of the G binding region of ARK, and conserved residues within the PH domain, we have constructed a series of mutants in the carboxyl terminus of ARK in order to determine important residues involved in G and PIP binding. To examine the effects of mutations on G binding, we employed three different methodologies: direct G binding to GST fusion proteins; the ability of GST fusion proteins to inhibit G-mediated ARK translocation to rhodopsin-enriched rod outer segments; and the ability of mutant peptides expressed in cells to inhibit G-mediated inositol phosphate accumulation. Direct PIP binding was also assessed on mutant GST fusion proteins. Ala residue insertion following Trp completely abolished the ability of ARK to bind G, suggesting that a proper -helical conformation is necessary for the GARK interaction. In contrast, this insertional mutation had no effect on PIP binding. Both G binding and PIP binding were abolished following Ala replacement of Trp, suggesting that this conserved residue within the last subdomain of the PH domain is crucial for both interactions. Other mutations also produced differential effects on the physical interactions of the ARK carboxyl terminus with G and PIP. These results suggest that the last PH subdomain and its neighboring sequences within the carboxyl terminus of ARK, including Trp, Leu, and residues Lys-Arg, are critical for G binding while Trp and residues Asp-Glu are important for the PH domain to form the correct structure for binding to PIP.


INTRODUCTION

The subunits of heterotrimeric G proteins (G)()play a variety of roles in modulating various cellular signaling cascades. These include regulation of certain isoforms of adenylyl cyclases, activation of some phospholipase C isoforms and phospholipase A2, modulation of muscarinic K channels, mediation of the pheromone-induced mating response in yeast, binding to retinal phosducin, and stimulation of G protein-coupled receptor-specific kinases such as muscarinic receptor kinase and -adrenergic receptor kinase (ARK)(1, 2, 3) . Most recently, G has been shown to activate the MAP kinase cascade mediated through serpentine G protein-coupled receptors such as that for lysophosphatidic acid, the M2-muscarinic acetylcholine (AChR) and -adrenergic (AR) receptors (4-6). This activation is p21-dependent(5, 6) . It is now clear that both G and G subunits interact with various effectors and receptors to regulate cellular signaling.

Although the role of G in various signal transduction pathways has been established, the structural and molecular basis of the interaction between G and its effectors has yet to be fully understood(7) . The region of phospholipase C interacting with G has been broadly mapped to the NH-terminal two-thirds of the protein(8) . The K channel has its G-sensitive region in the cytoplasmic carboxyl-terminal region (9). The G-binding domain of ARK, however, has been most intensively studied(10, 11, 12, 13) . The translocation and activation of the cytosolic enzyme ARK is mediated by the prenylated membrane-anchored G(14) . The specific region of ARK which directly interacts with and binds G is located within the carboxyl 125-amino-acid residue stretch(13) , and this G-binding domain peptide inhibits G-mediated phosphoinositide (PI) hydrolysis and type II adenylyl cyclase stimulation in both a transient transfection (15) and a cell permeabilized system(16) . It has been demonstrated that this domain can be utilized to probe and dissect a broad range of G-mediated signaling pathways.

The G-binding domain of ARK includes a pleckstrin homology domain (PH domain) that has been found in a variety of proteins involved in cellular signaling(17, 18, 19, 20, 21) . Although the function of the PH domain is not clear, several hypotheses have been raised and tested. The PH domains of numerous proteins appear to bind G to varying extents(22) . Some of these PH domain peptides have been shown to behave as antagonists of G-mediated signaling in intact cells, such as G-mediated inositol phosphate (IP) production and G-mediated p21-GTP exchange and MAP kinase activation (23). The region responsible for binding G, however, is not identical to the PH domain, but rather encompasses the carboxyl portion of the PH domain plus immediately distal sequences. A 28-mer peptide (Trp to Ser) containing the carboxyl portion of the PH domain of ARK (see Fig. 1) inhibited G-mediated activation of ARK1(13) , and desensitization of the cAMP response elicited via odorant activation of olfactory receptors in permeabilized rat olfactory cilia(24) . In addition, the G binding region of retinal phosducin contains sequences homologous with the carboxyl half of G-binding domain of ARK(25) .


Figure 1: Schematic representation of the G-binding domain and the PH domain of ARK and the location of mutations constructed in this study. Both the G-binding domain (Pro-Ser) and PH domain (Tyr-Gln) are located in the COOH terminus of ARK. The PH domain is divided into six subdomains according to Musacchio et al. (19). Shown are subdomain 6 of the PH domain and the adjacent sequences from ARK1. The bold amino acids indicate the perfectly conserved amino acids among the G-binding active ARK family (bovine ARK1 and 2, human ARK1 and 2, and Drosophila GPRK1) (38, 39). The PH domain consensus sequences (, hydrophobic residue) are based on previous publications.



Most recently, three-dimensional structures of PH domains from pleckstrin, spectrin, and dynamin have been determined(26, 27, 28, 29) . The core structures are almost superimposable, consisting of a -barrel of seven antiparallel -sheets and a carboxyl-terminal amphiphilic -helix. The structural similarity to other proteins immediately suggests that one function of PH domains is to bind small lipophilic molecules or peptides. Indeed, some PH domains can apparently bind phosphatidylinositol 4,5-bisphosphate (PIP) in the cleft of the -barrel(30) . The PH domain of spectrin has been shown to be the site of membrane interaction(31) . The PH domain of the B-cell tyrosine kinase, however, binds protein kinase C (32) as well as G subunits(33) , suggesting that the function of PH domains is more complex.

The structures of PH domains suggest that the carboxyl-terminal -helix including the most conserved Trp residue may mediate G association. Simonds et al.(34) suggested that the G protein , , and subunits form a triple coiled-coil structure through the amino termini and that the dissociation of G from the G complex allows ARK to form a new triple coiled-coil structure through the 28-mer peptide region encompassing the PH domains. Recent evidence, however, demonstrates that the trypsin-digested G subunit, in which the putative NH-terminal coiled-coil domain of G is missing, still binds to ARK(35) . In order to determine more precisely which residues in the G-binding domain of ARK are crucial for this specific protein-protein interaction and to test the triple coiled-coil hypothesis, this report studies the G binding abilities of a series of ARK1 constructs containing mutations in the PH domain and adjacent sequences. Effects of these mutations on the ability of the ARK PH domain to bind to PIP were also examined.


EXPERIMENTAL PROCEDURES

Materials

Bovine brain G was purified in our laboratory. The cDNA for the human 2-C10 AR was cloned in our laboratory. The cDNA for the human M1 AChR was kindly provided by Dr. Ernest Peralta. The cDNAs encoding G1 and G2 were kindly provided by Dr. Mel Simon. Sources of other reagents were as described previously(13, 22, 23, 25) .

Construction and Isolation of GST Fusion Proteins

All mutants of the ARK1 carboxyl terminus (ct) were constructed with the GST fusion vector pGEX-2T (Pharmacia) by standard techniques utilizing the polymerase chain reaction and wild type bovine ARK1 as the template as described(13, 22) . The truncated ARK1ct fusion protein construct (671-689) (Fig. 1) was used as the template for the KKKREEEE (K663E, K665E, K667E, R669E) mutant. All mutations were verified by dideoxy sequencing using Sequenase (United States Biochemical Corp.). Mutant constructs were introduced into the Escherichia coli strain NM522 or BL21. The fusion proteins were expressed and purified as described previously using glutathione-agarose(22) .

Construction and Expression of Minigenes

The cDNAs encoding various mutant proteins were amplified from the pGEX plasmid cDNAs encoding the corresponding mutant GST fusion protein to construct EcoRI-BclI minigene cassettes and inserted into the mammalian expression vector pRK5 as described(15) . These DNAs were used for transfection of COS-7 cells using LipofectAMINE (Life Technologies, Inc.) as described(23) . Expression of the mutant peptides was determined by Western blot of whole cell lysates as described using anti-ARKct serum(15) .

Binding of G to Fusion Proteins

The binding of bovine brain G to the fusion proteins and the detection of bound G were accomplished essentially as described previously(22) . Briefly, 500 nM GST fusion protein and 73 nM purified bovine brain G were incubated in phosphate-buffered saline, 0.01% Lubrol, and the bound G on the immobilized beads was detected by using antibodies to the G subunit (DuPont NEN). Laser densitometry was used to quantitate the relative amounts of bound G.

Translocation Assay of ARK1 to Rod Outer Segment Membranes

Translocation of ARK1 to rod outer segment membranes and its inhibition by the fusion proteins were carried out as described previously(12, 22) . Briefly, purified recombinant ARK1 and urea-stripped rod outer segment membranes were incubated at 30 °C for 5 min in either the presence or absence of bovine G (185 nM). Incubations containing G additionally contained one of the fusion proteins (1 µM). Following incubation, samples were subjected to centrifugation at 350,000 g for 5 min, and the supernatant and pellet obtained were assayed for ARK1 activity. Quantitation of the band corresponding to phosphorylated rhodopsin was accomplished using a Molecular Dynamics PhosphorImager.

Inositol Phosphate Assays

COS-7 cells were cotransfected with receptor cDNA and either empty pRK5 vector DNA or pRK-mutant ARK1 DNA. In some experiments, cells were cotransfected with G1 and G2 cDNAs rather than receptor DNA. After 24 h of incubation, cells were prelabeled with 2 µCi/ml myo-[H]inositol for 24 h. Cells were then stimulated for 45 min with or without agonist, and IP accumulation was determined by Dowex anion-exchange chromatography as described(15) .

Binding of PIP to Fusion Proteins

Fusion proteins (0.5-1 µg) in phosphate-buffered saline were incubated in polycarbonate centrifuge tubes (7 20 mm) for 10 min at room temperature. Phosphatidylcholine (PC) vesicles or PC vesicles containing 5% PIP (Sigma) were added for a final lipid concentration of 0.8 mg/ml in 30 µl. After a 10-min incubation at room temperature and 5 min on ice, the tubes were then centrifuged at 100,000 revolutions/minute (TL-100 rotor) for 15 min at 4 °C. The supernatant was removed, and the pellet was washed once with phosphate-buffered saline and transferred to a new tube. The percentage of fusion protein in the supernatant and pellet was determined by using Western blot analysis (ECL, Amersham Corp.) and densitometry.


RESULTS

Design and Isolation of Mutant Fusion Proteins

We focused on subdomain 6 of the PH domain and its adjacent sequences to construct various mutants of the ARK carboxyl-terminal peptide as shown in Fig. 1. The Trp residue in subdomain 6 is perfectly conserved in all PH domains as are several hydrophobic residues. In addition, clusters of acidic (Asp or Glu) and basic (Lys or Arg) residues are found in the G binding region of PH domains. Thus, we made a series of point mutations around the most conserved Trp in ARK1 and also reversed the ionic charge of the acidic and basic regions. In addition, since this region is predicted, by computer analysis, to form an -helix we inserted 1 or 2 Ala residues to disrupt the proper orientation of the helical structure.

Fig. 2A shows the purified GST fusion proteins. The ARK1ct fusion protein mutant KKKREEEE co-migrates with the truncated wild type fusion protein (671-689) since the mutant is derived from the 671-689. Other mutant fusion proteins derived from the wild type fusion protein (GST-ARKct; Pro-Leu) migrate as expected except the E646K mutant. This mutant migrates faster than the wild type due to either a change in the ionic strength or some degradation. Because the mutant peptide migrates close to the 671-689, it is likely that sequences up to Ser are still intact.


Figure 2: Isolation of mutant GST-PH domain fusion proteins and binding to bovine brain G. A, Coomassie Blue-stained SDS-polyacrylamide gel of various GST fusion proteins used in this study. B, Western blot assessing G binding ability of the various GST fusion proteins. The position of the subunit of G is indicated by the arrowhead. GST and wild type GST-ARK-ct were used as negative and positive controls, respectively. C, the relative amounts of bound G as the mean percentage ± S.D. or range of the band intensity of wild type GST-ARK-ct.



G Binding to Mutant Fusion Proteins

The G binding abilities of these mutant GST fusion proteins (Fig. 2A) were assessed by the direct binding assay shown in Fig. 2B. The truncated wild type 671-689, still significantly binds G (although weaker than the wild type), consistent with our previous data (13). When the basic residues in the truncated protein were changed to acidic residues (KKKREEEE), the G binding activity was completely lost, suggesting that these basic residues are crucial for the G binding ability of ARK. All other mutations altered G binding to varying extents, with the most deleterious mutations being W643A and L647G. K645E, E646K, and DSDKKK still display some binding, although significantly weaker than the wild type (Fig. 2, B and C). Since Arg and Ser are most frequently substituted with Trp according to the Dayhoff's table(36) , W643S and W643R were also tested. The binding of these mutants is similar to that of W643A. Finally, the effect of 1 or 2 Ala residue insertions after Trp (WA and WAA, respectively) was assessed. These insertions completely block binding activity, suggesting that the proper orientation of residues in the -helix is disrupted.

Inhibition of G-mediated Translocation of ARK1 to Membranes by Mutant Fusion Proteins

In order to confirm the relative binding affinity of the mutant fusion proteins, a fixed concentration (1 µM) of each was tested for the ability to inhibit G-mediated translocation of ARK1 to rod outer segment membranes. As shown in Fig. 3, the wild type ARK1ct fusion protein significantly inhibits G-mediated translocation of ARK1. The truncated mutant, 671-689, also inhibits the translocation, but less effectively than the wild type, consistent with the direct G binding data. Other mutants are less effective inhibitors, but the relative efficacy among the mutants is consistent with the direct binding data. For example, the mutations with two Ala insertions (WAA) had no inhibitory effect, but K645E and E646K, which bind more G than WAA in the direct binding assay (Fig. 2B), slightly inhibited G-mediated ARK translocation. Thus, the relative inhibitory effects of these fusion proteins correlate well with the relative binding activities observed in the direct G-binding assay. The DSDKKK mutant, which shows moderate G binding in the direct binding assay, did not inhibit translocation of ARK1. However, the inability of this mutant to bind PIP, as described later, probably is the reason for this discrepancy.


Figure 3: Inhibition of G-dependent translocation of ARK1 to rod outer segment membranes by various mutant fusion proteins. Each fusion protein was included in the G-mediated ARK-translocation assays described under ``Experimental Procedures'' at 1 µM. In the absence of G, 5.1 ± 3.0% (n = 5) of the total ARK1 activity was associated with the membrane fraction. In contrast, when assays were performed in the presence of G, 59 ± 5.0% (n = 5) of the total ARK1 activity was membrane-associated. *, less than the G control, p < 0.05.



Effects of Mutant ARKct Peptides on G-mediated IP Production

Since bacterially expressed proteins are utilized in the above two assays, we constructed plasmid minigenes encoding the same mutant ARKct domains and expressed them in mammalian cells. In COS-7 cells, agonist stimulation of transiently expressed 2-ARs produces pertussis toxin-sensitive, G-mediated PI hydrolysis. In contrast, agonist stimulation of M1-AChRs produces pertussis toxin-insensitive, G-independent PI hydrolysis mediated via Gq subunits. We utilized these two pathways to examine the ability of co-expressed mutant ARKct peptides to selectively antagonize Gi-mediated PI hydrolysis. Expression of each mutant peptide was confirmed by Western blot as described previously(15) , demonstrating that the level of cellular expression of all mutant peptides was similar (data not shown). Co-expression of wild type ARKct peptide resulted in 70% attenuation of 2-AR-mediated IP production, while the mutant ARKct peptides WAA, L647G, and W643A had no significant effect (Fig. 4A). These mutants were shown to have the lowest G binding activities according to the direct G binding assay and the translocation assay ( Fig. 2and 3). Other mutant peptides, K645E, Q642G, E646K, and K644E, inhibited 2-AR-stimulated IP production. When assayed for the ability to antagonize M1-AChR-mediated IP production, none of the peptides exhibited significant activity (Fig. 4B). It is of note that the wild type peptide, which binds PIP, did not attenuate the M1-AChR-mediated PI hydrolysis by phospholipase C, suggesting that the PH domain peptide does not sequester the phospholipase C substrate, PIP, under these conditions. The effect of the mutant peptides on IP production provoked by co-expression of G and G was also determined. The results are consistent with the 2-AR data (Fig. 4C), demonstrating that the inhibitory effects are dependent on the ability to sequester G subunits specifically in intact cells.


Figure 4: Comparison of the effects of mutant PH domain peptides on IP production mediated by 2-C10 AR, M1 AChR, and tranfected G. COS-7 cells were transiently transfected with plasmid DNA encoding the a2-C10 AR (A), M1 AChR (B) (0.2 µg/well), or G1 and G2 (C) (1 µg each/well) plus empty pRK5 vector or the indicated mutant PH domain peptide minigene (2 µg/well). Cells were stimulated with the appropriate agonists (2 AR, 1 µM UK14304; M1-AChR, 1 mM carbachol) IP production is represented as a percentage of the maximal agonist-induced stimulation (A and C), or fold stimulation (B) observed in vector cotransfected cells. Each column represents mean ± S.E. values for three to five separate experiments. * signifies value less than control, p < 0.01.



Binding of Mutant Fusion Proteins to PIP

Some PH domains including that of ARK have been shown to bind PIP in the cleft of the NH-terminal -barrel(30) . The ability of the mutants to bind PIP was assessed and compared to the G binding ability. Binding of mutant GST fusion proteins to PC vesicles containing 5% PIP was examined using a centrifugation assay. The nonspecific binding of GST-ARKct wild type fusion protein to PC vesicles is relatively high (45-60% in pellet after centrifugation) (Fig. 5) in comparison to native ARK. Nonetheless, the binding of the wild type fusion protein to PIP-containing PC vesicles is clearly significantly higher, since no fusion protein is detected in the supernatant. Interestingly, much less nonspecific binding to PC vesicles was observed for some mutant fusion proteins such as G642G, 671-689, and KKKREEEE (Fig. 5). Thus, the carboxyl end of the fusion protein appears to be lipophilic. The W643A mutant and the DSDKKK mutant bind significantly less to PIP-containing PC vesicles (Fig. 5), suggesting that the -barrel structure necessary for the PIP binding was impaired by these mutations. The DSDKKK mutant did not inhibit the translocation of ARK (Fig. 3) perhaps due to its weak PIP binding. Other mutants exhibited significant translocation to PIP-containing vesicles (Fig. 5). Interestingly, the Ala insertion, WA, and the KKKREEEE mutants, which did not bind to G, still contained PIP binding activity. On the other hand, a mutant which still had some G-binding activity (DSDKKK) showed less PIP binding. These data suggest that crucial residues for the G binding activity are quite different from those for PIP binding.


Figure 5: PIP-mediated translocation of various GST-PH domain fusion proteins. The percentage of the fusion protein in PC vesicles (open bars) or 5% PIP/95% PC vesicles (shaded bars) are presented. The results shown represent the mean values obtained from three separate determinations. * signifies a fusion protein that does not show significant binding to the PIP-containing vesicles in comparison to PC vesicles.




DISCUSSION

The G binding region of the PH domain of ARK is in a 125-amino-acid residue stretch located within the carboxyl terminus (13). This region includes the 28-mer peptide (Trp-Ser) that shows inhibitory activity on the G-mediated activation of ARK(13) . The region of this 28-mer peptide is the putative triple coiled-coil domain and encompasses the most conserved last subdomain of the PH domain(34) . The homology between two G-binding proteins, ARK and phosducin, starts at subdomain 4 of the PH domain of ARK and continues to the adjacent sequences of the ARK PH domain(25) . Based on these observations, we made a series of mutants from the region encompassing the last subdomain of the PH domain. Mutations at Trp, Leu, or within the cluster of basic residues located more distally most effectively reduced the ability of ARK to bind to G and were less capable of inhibiting G-mediated IP production. These residues are fairly conserved among the PH domain-containing proteins (Fig. 1). Notably, the Trp residue in subdomain 6 is 100% conserved in the PH domain sequences. Moreover, the crucial residues for G binding such as Trp, Leu, Lys, and Lys are 100% conserved among members of the ARK family (from human to bovine) and the Drosophila GPRK1 that bind G (Fig. 1).

Based on the triple coiled-coil hypothesis, G, G, and G subunits form a coiled-coil structure through their amino termini, and the dissociation of G from G subunits allows ARK to form a new triple coiled-coil(34) . The hydrophobic and ionic interactions of the residues within the 28-mer peptide region were thought to explain the mechanism of binding of ARK and G subunits. Based on this model, Lys, Lys, and Glu provide crucial hydrogen bonds, and Trp and Leu participate in the hydrophobic interactions. However, the three-dimensional structures of the PH domains of pleckstrin, spectrin, and dynamin suggest that the Trp residue faces the inside of the protein rather than localizing to the surface of the helix(26, 27, 28, 29) . Moreover, the trypsin-digested G subunit, which lacks the coiled-coil sequence of G, still binds to the PH domain of ARK and spectrin(35) . Together with our results that the Lys, Lys, and Glu mutants still bind to G and inhibit G-mediated IP production, these observations suggest that the interaction between G and ARK cannot simply be explained by the tripled coiled-coil model. Nonetheless, the fact that the alanine insertions after Trp impair G binding activity suggests that this region could form an -helical structure like other PH domains, thereby playing a crucial role in interacting with G.

The binding of PIP to the PH domain of ARK was impaired by the W643A mutation. According to the three-dimensional structure from other PH domains, the Trp residue interacts with the core of the -barrel, most likely contributing to domain stability. We have made several Trp mutants from other PH domain-containing proteins, and bacterial expression of these mutants was lower, confirming that the Trp residue and perhaps the carboxyl -helix are important to stabilize the domain structure.()Therefore, the most conserved Trp residue is critical for both G binding and stablization of the PIP-binding cleft in the NH terminus of the ARK PH domain. Mutations in the cluster of anionic residues located in the beginning of the carboxyl -helix (DSDKKK mutation) attenuated binding to PIP. Although the DSDKKK mutant binds G as well as K645E or E646K, the ability of the DSDKKK mutant to inhibit G-mediated ARK translocation to rod outer segment membranes was less than that of K645E or E646K, consistent with the evidence that both PIP and G are required for PH domain-mediated membrane translocation of ARK(37) . Other mutants with markedly impaired G binding still possess PIP binding activity, suggesting that these mutations do not result in global misfolding of the domain peptides.

The region of the PH domain that interacts with G has been mapped using guanine nucleotide releasing factor and phospholipase C, demonstrating that the NH-terminal half of PH domain is not necessary for G binding activity(22) . Later, this was confirmed by using the PH domain of Bruton tyrosine kinase(33) . Apparently, the carboxyl -helix of PH domain mediates the interaction with G. One function of the NH-terminal region of the PH domain, in contrast, is binding to PIP (30). Moreover, the PH domain of Bruton tyrosine kinase has been shown to bind protein kinase C at the NH-terminal region of the PH domain, based on the evidence that the mutation of Arg in the PH domain resulted in lower protein kinase C binding capacity (32). Considering the heterogeneity in sequences of PH domains, various other molecules have been implicated as ligands for the PH domain. Since the molecules which bind PH domains seem to be quite diverse ranging from lipids to macromolecules, the function of the PH domain and the mechanisms of PH domain action may be quite complex and delicately regulated. In at least one instance, binding of G and PIP to the PH domain of ARK appears to be cooperative, since binding of the COOH terminus of the PH domain to G potentially affects PIP binding(37) .

The following novel conclusions emerge from our studies. First, we have identified the critical residues involved in G binding to the ARK PH domain and localized these within subdomain 6 of the PH domain and the adjacent region. Second, although the region can form an -helical structure which is crucial for the G interaction, the G-ARK interaction cannot simply be explained by a triple coiled-coil model. Third, the effects of mutations in the ARK PH domain on the ability to bind G are distinct from those on PIP binding. Finally, the most highly conserved Trp in subdomain 6 of the PH domain is absolutely required for both G and PIP binding.


FOOTNOTES

*
This work was supported in part 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 and reprint requests should be addressed: Duke University Medical Center, P. O. Box 3821, Durham, NC 27710. Tel.: 919-684-2974; Fax: 919-684-8875.

The abbreviations used are: G, subunits of heterotrimeric G proteins; PH, pleckstrin homology; AR, adrenergic receptor; AChR, muscarinic cholinergic receptor; ARK, -adrenergic receptor kinase; PIP, phosphatidylinositol 4,5-bisphosphate; PC, phosphatidylcholine; GST, glutathione S-transferase; IP, inositol phosphate; PI, phosphoinositide; ARKct, carboxyl terminus of ARK.

K. Touhara, R. J. Lefkowitz, unpublished results.


ACKNOWLEDGEMENTS

We thank W. Carl Stone for assisting in mutant DNA construction and sequencing, W. Darrel Capel for purified ARK, rod outer segment membranes, and G, Sabrina T. Exum for assisting tissue culture, and Drs. J. A. Pitcher and J. Inglese for helpful discussions throughout the course of this work. We also thank Donna Addison and Mary Holben for excellent secretarial assistance.


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