Mutational Analysis of the Pleckstrin Homology Domain of the β-Adrenergic Receptor Kinase.
1995; Elsevier BV; Volume: 270; Issue: 28 Linguagem: Inglês
10.1074/jbc.270.28.17000
ISSN1083-351X
AutoresKazushige Touhara, Walter J. Koch, Brian E. Hawes, Robert J. Lefkowitz,
Tópico(s)Pharmacogenetics and Drug Metabolism
ResumoThe βγ subunits of heterotrimeric G proteins (Gβγ) play a variety of roles in cellular signaling, one of which is membrane targeting of the β-adrenergic receptor kinase (βARK). This is accomplished via a physical interaction of Gβγ and a domain within the carboxyl terminus of βARK which overlaps with a pleckstrin homology (PH) domain. The PH domain of βARK not only binds Gβγ but also interacts with phosphatidylinositol 4,5-bisphosphate (PIP2). Based on previous mapping of the Gβγ binding region of βARK, and conserved residues within the PH domain, we have constructed a series of mutants in the carboxyl terminus of βARK in order to determine important residues involved in Gβγ and PIP2 binding. To examine the effects of mutations on Gβγ binding, we employed three different methodologies: direct Gβγ binding to GST fusion proteins; the ability of GST fusion proteins to inhibit Gβγ-mediated βARK translocation to rhodopsin-enriched rod outer segments; and the ability of mutant peptides expressed in cells to inhibit Gβγ-mediated inositol phosphate accumulation. Direct PIP2 binding was also assessed on mutant GST fusion proteins. Ala residue insertion following Trp643 completely abolished the ability of βARK to bind Gβγ, suggesting that a proper α-helical conformation is necessary for the Gβγ·βARK interaction. In contrast, this insertional mutation had no effect on PIP2 binding. Both Gβγ binding and PIP2 binding were abolished following Ala replacement of Trp643, 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 βARK carboxyl terminus with Gβγ and PIP2. These results suggest that the last PH subdomain and its neighboring sequences within the carboxyl terminus of βARK, including Trp643, Leu647, and residues Lys663-Arg669, are critical for Gβγ binding while Trp643 and residues Asp635-Glu639 are important for the PH domain to form the correct structure for binding to PIP2. The βγ subunits of heterotrimeric G proteins (Gβγ) play a variety of roles in cellular signaling, one of which is membrane targeting of the β-adrenergic receptor kinase (βARK). This is accomplished via a physical interaction of Gβγ and a domain within the carboxyl terminus of βARK which overlaps with a pleckstrin homology (PH) domain. The PH domain of βARK not only binds Gβγ but also interacts with phosphatidylinositol 4,5-bisphosphate (PIP2). Based on previous mapping of the Gβγ binding region of βARK, and conserved residues within the PH domain, we have constructed a series of mutants in the carboxyl terminus of βARK in order to determine important residues involved in Gβγ and PIP2 binding. To examine the effects of mutations on Gβγ binding, we employed three different methodologies: direct Gβγ binding to GST fusion proteins; the ability of GST fusion proteins to inhibit Gβγ-mediated βARK translocation to rhodopsin-enriched rod outer segments; and the ability of mutant peptides expressed in cells to inhibit Gβγ-mediated inositol phosphate accumulation. Direct PIP2 binding was also assessed on mutant GST fusion proteins. Ala residue insertion following Trp643 completely abolished the ability of βARK to bind Gβγ, suggesting that a proper α-helical conformation is necessary for the Gβγ·βARK interaction. In contrast, this insertional mutation had no effect on PIP2 binding. Both Gβγ binding and PIP2 binding were abolished following Ala replacement of Trp643, 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 βARK carboxyl terminus with Gβγ and PIP2. These results suggest that the last PH subdomain and its neighboring sequences within the carboxyl terminus of βARK, including Trp643, Leu647, and residues Lys663-Arg669, are critical for Gβγ binding while Trp643 and residues Asp635-Glu639 are important for the PH domain to form the correct structure for binding to PIP2. The βγ subunits of heterotrimeric G proteins (Gβγ)1 1The abbreviations used are: Gβγβγ subunits of heterotrimeric G proteinsPHpleckstrin homologyARadrenergic receptorAChRmuscarinic cholinergic receptorβARKβ-adrenergic receptor kinasePIP2phosphatidylinositol 4,5-bisphosphatePCphosphatidylcholineGSTglutathione S-transferaseIPinositol phosphatePIphosphoinositideβARKctcarboxyl terminus of βARK. 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 β 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 β-adrenergic receptor kinase (βARK)(1Clapham D.E. Neer E.J. Nature. 1993; 365: 403-406Crossref PubMed Scopus (590) Google Scholar, 2Lefkowitz R.J. Cell. 1993; 74: 409-412Abstract Full Text PDF PubMed Scopus (403) Google Scholar, 3Inglese J. Freedman N.J. Koch W.J. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 23735-23739Abstract Full Text PDF PubMed Google Scholar). Most recently, Gβγ has been shown to activate the MAP kinase cascade mediated through serpentine Gi protein-coupled receptors such as that for lysophosphatidic acid, the M2-muscarinic acetylcholine (AChR) and α2-adrenergic (AR) receptors (4-6). This activation is p21ras-dependent(5Crespo P. Xu N. Simonds W.F. Gutkind J.S. Nature. 1994; 369: 418-420Crossref PubMed Scopus (766) Google Scholar, 6Koch W.J. Hawes B.E. Allen L.F. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12706-12710Crossref PubMed Scopus (409) Google Scholar). It is now clear that both Gα and Gβγ subunits interact with various effectors and receptors to regulate cellular signaling. βγ subunits of heterotrimeric G proteins pleckstrin homology adrenergic receptor muscarinic cholinergic receptor β-adrenergic receptor kinase phosphatidylinositol 4,5-bisphosphate phosphatidylcholine glutathione S-transferase inositol phosphate phosphoinositide carboxyl terminus of βARK. Although the role of Gβγ in various signal transduction pathways has been established, the structural and molecular basis of the interaction between Gβγ and its effectors has yet to be fully understood(7Inglese J. Koch W.J. Touhara K. Lefkowitz R.J. Trends Biol. Sci. 1995; 20: 151-156Abstract Full Text PDF PubMed Scopus (165) Google Scholar). The region of phospholipase Cβ interacting with Gβγ has been broadly mapped to the NH2-terminal two-thirds of the protein(8Wu D. Katz A. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5297-5301Crossref PubMed Scopus (174) Google Scholar). The K+ channel has its Gβγ-sensitive region in the cytoplasmic carboxyl-terminal region (9). The Gβγ-binding domain of βARK, however, has been most intensively studied(10Haga K. Haga T. FEBS Lett. 1990; 268: 43-47Crossref PubMed Scopus (57) Google Scholar, 11Haga K. Haga T. J. Biol. Chem. 1992; 267: 2222-2227Abstract Full Text PDF PubMed Google Scholar, 12Pitcher J.A. Inglese J. Higgins J.B. Arriza J.L. Casey P.J. Kim C. Benovic J.L. Kwatra M.M. Caron M.G. Lefkowitz R.J. Science. 1992; 257: 1264-1267Crossref PubMed Scopus (573) Google Scholar, 13Koch W.J. Inglese J. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 8256-8260Abstract Full Text PDF PubMed Google Scholar). The translocation and activation of the cytosolic enzyme βARK is mediated by the prenylated membrane-anchored Gβγ(14Inglese J. Koch W.J. Caron M.G. Lefkowitz R.J. Nature. 1992; 359: 147-150Crossref PubMed Scopus (234) Google Scholar). The specific region of βARK which directly interacts with and binds Gβγ is located within the carboxyl 125-amino-acid residue stretch(13Koch W.J. Inglese J. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 8256-8260Abstract Full Text PDF PubMed Google Scholar), and this Gβγ-binding domain peptide inhibits Gβγ-mediated phosphoinositide (PI) hydrolysis and type II adenylyl cyclase stimulation in both a transient transfection (15Koch W.J. Hawes B.E. Inglese J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 6193-6197Abstract Full Text PDF PubMed Google Scholar) and a cell permeabilized system(16Inglese J. Luttrell L.M. Iniguez-Lluhi J. Touhara K. Koch W.J. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3637-3641Crossref PubMed Scopus (71) Google Scholar). It has been demonstrated that this domain can be utilized to probe and dissect a broad range of Gβγ-mediated signaling pathways. The Gβγ-binding domain of βARK includes a pleckstrin homology domain (PH domain) that has been found in a variety of proteins involved in cellular signaling(17Haslam R.J. Koide H.B. Hemmings B.A. Nature. 1993; 363: 310-311Crossref Scopus (389) Google Scholar, 18Mayer B.J. Ren R. Clark K.L. Baltimore D. Cell. 1993; 73: 629-630Abstract Full Text PDF PubMed Scopus (383) Google Scholar, 19Musacchio A. Gibson T. Rice P. Thompson J. Saraste M. Trends Biol. Sci. 1993; 18: 343-348Abstract Full Text PDF PubMed Scopus (487) Google Scholar, 20Shaw G. Biochem. Biophys. Res. Commun. 1993; 195: 1145-1151Crossref PubMed Scopus (83) Google Scholar, 21Gibson T.J. Hyvonen M. Birney E. Musacchio A. Saraste M. Trends Biol. Sci. 1994; 19: 349-353Abstract Full Text PDF PubMed Scopus (297) Google Scholar). 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 Gβγ to varying extents(22Touhara K. Inglese J. Pitcher J.A. Shaw G. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 10217-10220Abstract Full Text PDF PubMed Google Scholar). Some of these PH domain peptides have been shown to behave as antagonists of Gβγ-mediated signaling in intact cells, such as Gβγ-mediated inositol phosphate (IP) production and Gβγ-mediated p21ras-GTP exchange and MAP kinase activation (23). The region responsible for binding Gβγ, 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 (Trp643 to Ser670) containing the carboxyl portion of the PH domain of βARK (see Fig. 1) inhibited Gβγ-mediated activation of βARK1(13Koch W.J. Inglese J. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 8256-8260Abstract Full Text PDF PubMed Google Scholar), and desensitization of the cAMP response elicited via odorant activation of olfactory receptors in permeabilized rat olfactory cilia(24Boekhoff I. Inglese J. Schleicher S. Koch W.J. Lefkowitz R.J. Breer H. J. Biol. Chem. 1994; 269: 37-40Abstract Full Text PDF PubMed Google Scholar). In addition, the Gβγ binding region of retinal phosducin contains sequences homologous with the carboxyl half of Gβγ-binding domain of βARK(25Hawes B.E. Touhara K. Kurose H. Lefkowitz R.J. Inglese J. J. Biol. Chem. 1994; 269: 29825-29830Abstract Full Text PDF PubMed Google Scholar). Most recently, three-dimensional structures of PH domains from pleckstrin, spectrin, and dynamin have been determined(26Yoon H.S. Hajduk P.J. Petros A.M. Olejniczak E.T. Meadows R.P. Fesnik S.W. Nature. 1994; 369: 672-675Crossref PubMed Scopus (189) Google Scholar, 27Macias M.J. Musacchio A. Ponstingl H. Nilges M. Saraste M. Oschkinat H. Nature. 1994; 369: 675-677Crossref PubMed Scopus (207) Google Scholar, 28Ferguson K. Lemmon M.A. Schlessinger J. Sigler P.B. Cell. 1994; 79: 199-209Abstract Full Text PDF PubMed Scopus (243) Google Scholar, 29Timm D. Salim K. Gout I. Guruprasad L. Waterfield M. Blundell T. Nature Struct. Biol. 1994; 1: 782-788Crossref PubMed Scopus (107) Google Scholar). The core structures are almost superimposable, consisting of a β-barrel of seven antiparallel β-sheets and a carboxyl-terminal amphiphilic α-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 (PIP2) in the cleft of the β-barrel(30Harlan J.E. Hajduk P.J. Yoon H.S. Fesik S.W. Nature. 1994; 371: 168-170Crossref PubMed Scopus (676) Google Scholar). The PH domain of spectrin has been shown to be the site of membrane interaction(31Davis L.H. Bennett V.J. J. Biol. Chem. 1994; 269: 4409-4416Abstract Full Text PDF PubMed Google Scholar). The PH domain of the B-cell tyrosine kinase, however, binds protein kinase C (32Yao L. Kawakami Y Kawakami T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9175-9179Crossref PubMed Scopus (310) Google Scholar) as well as Gβγ subunits(33Tsukada S. Simon M.I. Witte O.N. Katz A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11256-11260Crossref PubMed Scopus (238) Google Scholar), suggesting that the function of PH domains is more complex. The structures of PH domains suggest that the carboxyl-terminal α-helix including the most conserved Trp residue may mediate Gβγ association. Simonds et al.(34Simonds W.F. Manji H.K. Lupas A.N. Garritsen A. Trends Biol. Chem. 1993; 18: 315-317Abstract Full Text PDF PubMed Scopus (29) Google Scholar) suggested that the G protein α, β, and γ subunits form a triple coiled-coil structure through the amino termini and that the dissociation of Gα from the Gβγ complex allows βARK 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 Gβ subunit, in which the putative NH2-terminal coiled-coil domain of Gβ is missing, still binds to βARK(35Wang D.S. Shaw R. Winkelmann J.C. Shaw G. Biochem. Biophys. Res. Commun. 1994; 203: 29-35Crossref PubMed Scopus (88) Google Scholar). In order to determine more precisely which residues in the Gβγ-binding domain of βARK are crucial for this specific protein-protein interaction and to test the triple coiled-coil hypothesis, this report studies the Gβγ binding abilities of a series of βARK1 constructs containing mutations in the PH domain and adjacent sequences. Effects of these mutations on the ability of the βARK PH domain to bind to PIP2 were also examined. Bovine brain Gβγ was purified in our laboratory. The cDNA for the human α2-C10 AR was cloned in our laboratory. The cDNA for the human M1 AChR was kindly provided by Dr. Ernest Peralta. The cDNAs encoding Gβ1 and Gγ2 were kindly provided by Dr. Mel Simon. Sources of other reagents were as described previously(13Koch W.J. Inglese J. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 8256-8260Abstract Full Text PDF PubMed Google Scholar, 22Touhara K. Inglese J. Pitcher J.A. Shaw G. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 10217-10220Abstract Full Text PDF PubMed Google Scholar, 23Luttrell L.M. Hawes B.E. Touhara K. van Biesen T. Koch W.J. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 12984-12989Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 25Hawes B.E. Touhara K. Kurose H. Lefkowitz R.J. Inglese J. J. Biol. Chem. 1994; 269: 29825-29830Abstract Full Text PDF PubMed Google Scholar). All mutants of the βARK1 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 βARK1 as the template as described(13Koch W.J. Inglese J. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 8256-8260Abstract Full Text PDF PubMed Google Scholar, 22Touhara K. Inglese J. Pitcher J.A. Shaw G. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 10217-10220Abstract Full Text PDF PubMed Google Scholar). The truncated βARK1ct fusion protein construct (δ671-689) (Fig. 1) was used as the template for the KKKR→EEEE (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(22Touhara K. Inglese J. Pitcher J.A. Shaw G. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 10217-10220Abstract Full Text PDF PubMed Google Scholar). 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(15Koch W.J. Hawes B.E. Inglese J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 6193-6197Abstract Full Text PDF PubMed Google Scholar). These DNAs were used for transfection of COS-7 cells using LipofectAMINE (Life Technologies, Inc.) as described(23Luttrell L.M. Hawes B.E. Touhara K. van Biesen T. Koch W.J. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 12984-12989Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Expression of the mutant peptides was determined by Western blot of whole cell lysates as described using anti-βARKct serum(15Koch W.J. Hawes B.E. Inglese J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 6193-6197Abstract Full Text PDF PubMed Google Scholar). The binding of bovine brain Gβγ to the fusion proteins and the detection of bound Gβγ were accomplished essentially as described previously(22Touhara K. Inglese J. Pitcher J.A. Shaw G. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 10217-10220Abstract Full Text PDF PubMed Google Scholar). Briefly, 500 nM GST fusion protein and 73 nM purified bovine brain Gβγ were incubated in phosphate-buffered saline, 0.01% Lubrol, and the bound Gβγ on the immobilized beads was detected by using antibodies to the Gβ subunit (DuPont NEN). Laser densitometry was used to quantitate the relative amounts of bound Gβγ. Translocation of βARK1 to rod outer segment membranes and its inhibition by the fusion proteins were carried out as described previously(12Pitcher J.A. Inglese J. Higgins J.B. Arriza J.L. Casey P.J. Kim C. Benovic J.L. Kwatra M.M. Caron M.G. Lefkowitz R.J. Science. 1992; 257: 1264-1267Crossref PubMed Scopus (573) Google Scholar, 22Touhara K. Inglese J. Pitcher J.A. Shaw G. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 10217-10220Abstract Full Text PDF PubMed Google Scholar). Briefly, purified recombinant βARK1 and urea-stripped rod outer segment membranes were incubated at 30°C for 5 min in either the presence or absence of bovine Gβγ (185 nM). Incubations containing Gβγ 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 βARK1 activity. Quantitation of the band corresponding to phosphorylated rhodopsin was accomplished using a Molecular Dynamics PhosphorImager. COS-7 cells were cotransfected with receptor cDNA and either empty pRK5 vector DNA or pRK-mutant βARK1 DNA. In some experiments, cells were cotransfected with Gβ1 and Gγ2 cDNAs rather than receptor DNA. After 24 h of incubation, cells were prelabeled with 2 μCi/ml myo-[3H]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(15Koch W.J. Hawes B.E. Inglese J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 6193-6197Abstract Full Text PDF PubMed Google Scholar). 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% PIP2 (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. We focused on subdomain 6 of the PH domain and its adjacent sequences to construct various mutants of the βARK carboxyl-terminal peptide as shown in Fig. 1. The Trp643 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 Gβγ binding region of PH domains. Thus, we made a series of point mutations around the most conserved Trp643 in βARK1 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 α-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 βARK1ct fusion protein mutant KKKR→EEEE co-migrates with the truncated wild type fusion protein (δ671-689) since the mutant is derived from the δ671-689. Other mutant fusion proteins derived from the wild type fusion protein (GST-βARKct; Pro467-Leu689) 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 δ671-689, it is likely that sequences up to Ser670 are still intact. The Gβγ 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 δ671-689, still significantly binds Gβγ (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 (KKKR→EEEE), the Gβγ binding activity was completely lost, suggesting that these basic residues are crucial for the Gβγ binding ability of βARK. All other mutations altered Gβγ binding to varying extents, with the most deleterious mutations being W643A and L647G. K645E, E646K, and DSD→KKK 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(36Dayhoff M.O. Atlas of Protein Sequence and Structure. Vol. 5. National Biochemical Research Foundation, Georgetown University Medical Center, Washington D. C.1978Google Scholar), 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 Trp643 (WA and WAA, respectively) was assessed. These insertions completely block binding activity, suggesting that the proper orientation of residues in the α-helix is disrupted. 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 Gβγ-mediated translocation of βARK1 to rod outer segment membranes. As shown in Fig. 3, the wild type βARK1ct fusion protein significantly inhibits Gβγ-mediated translocation of βARK1. The truncated mutant, δ671-689, also inhibits the translocation, but less effectively than the wild type, consistent with the direct Gβγ 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 Gβγ than WAA in the direct binding assay (Fig. 2B), slightly inhibited Gβγ-mediated βARK translocation. Thus, the relative inhibitory effects of these fusion proteins correlate well with the relative binding activities observed in the direct Gβγ-binding assay. The DSD→KKK mutant, which shows moderate Gβγ binding in the direct binding assay, did not inhibit translocation of βARK1. However, the inability of this mutant to bind PIP2, as described later, probably is the reason for this discrepancy. Since bacterially expressed proteins are utilized in the above two assays, we constructed plasmid minigenes encoding the same mutant βARKct domains and expressed them in mammalian cells. In COS-7 cells, agonist stimulation of transiently expressed α2-ARs produces pertussis toxin-sensitive, Gβγ-mediated PI hydrolysis. In contrast, agonist stimulation of M1-AChRs produces pertussis toxin-insensitive, Gβγ-independent PI hydrolysis mediated via Gqα subunits. We utilized these two pathways to examine the ability of co-expressed mutant βARKct peptides to selectively antagonize Giβγ-mediated PI hydrolysis. Expression of each mutant peptide was confirmed by Western blot as described previously(15Koch W.J. Hawes B.E. Inglese J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 6193-6197Abstract Full Text PDF PubMed Google Scholar), demonstrating that the level of cellular expression of all mutant peptides was similar (data not shown). Co-expression of wild type βARKct peptide resulted in ∼70% attenuation of α2-AR-mediated IP production, while the mutant βARKct peptides WAA, L647G, and W643A had no significant effect (Fig. 4A). These mutants were shown to have the lowest Gβγ binding activities according to the direct Gβγ binding assay and the translocation assay (Fig. 2 and 3). Other mutant peptides, K645E, Q642G, E646K, and K644E, inhibited α2-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 PIP2, 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, PIP2, under these conditions. The effect of the mutant peptides on IP production provoked by co-expression of Gβ and Gγ was also determined. The results are consistent with the α2-AR data (Fig. 4C), demonstrating that the inhibitory effects are dependent on the ability to sequester Gβγ subunits specifically in intact cells. Some PH domains including that of βARK have been shown to bind PIP2 in the cleft of the NH2-terminal β-barrel(30Harlan J.E. Hajduk P.J. Yoon H.S. Fesik S.W. Nature. 1994; 371: 168-170Crossref PubMed Scopus (676) Google Scholar). The ability of the mutants to bind PIP2 was assessed and compared to the Gβγ binding ability. Binding of mutant GST fusion proteins to PC vesicles containing 5% PIP2 was examined using a centrifugation assay. The nonspecific binding of GST-βARKct wild type fusion protein to PC vesicles is relatively high (45-60% in pellet after centrifugation) (Fig. 5) in comparison to native βARK. Nonetheless, the binding of the wild type fusion protein to PIP2-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, δ671-689, and KKKR→EEEE (Fig. 5). Thus, the carboxyl end of the fusion protein appears to be lipophilic. The W643A mutant and the DSD→KKK mutant bind significantly less to PIP2-containing PC vesicles (Fig. 5), suggesting that the β-barrel structure necessary for the PIP2 binding was impaired by these mutations. The DSD→KKK mutant did not inhibit the translocation of βARK (Fig. 3) perhaps due to its weak PIP2 binding. Other mutants exhibited significant translocation to PIP2-containing vesicles (Fig. 5). Interestingly, the Ala insertion, WA, and the KKKR→EEEE mutants, which did not bind to Gβγ, still contained PIP2 binding activity. On the other hand, a mutant which still had some Gβγ-binding activity (DSD→KKK) showed less PIP2 binding. These data suggest that crucial residues for the Gβγ binding activity are quite different from those for PIP2 binding. The Gβγ binding region of the PH domain of βARK is in a 125-amino-acid residue stretch located within the carboxyl terminus (13). This region includes the 28-mer peptide (Trp643-Ser670) that shows inhibitory activity on the Gβγ-mediated activation of βARK(13Koch W.J. Inglese J. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 8256-8260Abstract Full Text PDF PubMed Google Scholar). 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(34Simonds W.F. Manji H.K. Lupas A.N. Garritsen A. Trends Biol. Chem. 1993; 18: 315-317Abstract Full Text PDF PubMed Scopus (29) Google Scholar). The homology between two Gβγ-binding proteins, βARK and phosducin, starts at subdomain 4 of the PH domain of βARK and continues to the adjacent sequences of the βARK PH domain(25Hawes B.E. Touhara K. Kurose H. Lefkowitz R.J. Inglese J. J. Biol. Chem. 1994; 269: 29825-29830Abstract Full Text PDF PubMed Google Scholar). Based on these observations, we made a series of mutants from the region encompassing the last subdomain of the PH domain. Mutations at Trp643, Leu647, or within the cluster of basic residues located more distally most effectively reduced the ability of βARK to bind to Gβγ and were less capable of inhibiting Gβγ-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 Gβγ binding such as Trp643, Leu637, Lys664, and Lys648 are 100% conserved among members of the βARK family (from human to bovine) and the Drosophila GPRK1 that bind Gβγ (Fig. 1). Based on the triple coiled-coil hypothesis, Gα, Gβ, and Gγ subunits form a coiled-coil structure through their amino termini, and the dissociation of Gα from Gβγ subunits allows βARK to form a new triple coiled-coil(34Simonds W.F. Manji H.K. Lupas A.N. Garritsen A. Trends Biol. Chem. 1993; 18: 315-317Abstract Full Text PDF PubMed Scopus (29) Google Scholar). The hydrophobic and ionic interactions of the residues within the 28-mer peptide region were thought to explain the mechanism of binding of βARK and Gβγ subunits. Based on this model, Lys644, Lys645, and Glu646 provide crucial hydrogen bonds, and Trp643 and Leu647 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(26Yoon H.S. Hajduk P.J. Petros A.M. Olejniczak E.T. Meadows R.P. Fesnik S.W. Nature. 1994; 369: 672-675Crossref PubMed Scopus (189) Google Scholar, 27Macias M.J. Musacchio A. Ponstingl H. Nilges M. Saraste M. Oschkinat H. Nature. 1994; 369: 675-677Crossref PubMed Scopus (207) Google Scholar, 28Ferguson K. Lemmon M.A. Schlessinger J. Sigler P.B. Cell. 1994; 79: 199-209Abstract Full Text PDF PubMed Scopus (243) Google Scholar, 29Timm D. Salim K. Gout I. Guruprasad L. Waterfield M. Blundell T. Nature Struct. Biol. 1994; 1: 782-788Crossref PubMed Scopus (107) Google Scholar). Moreover, the trypsin-digested Gβ subunit, which lacks the coiled-coil sequence of Gβ, still binds to the PH domain of βARK and spectrin(35Wang D.S. Shaw R. Winkelmann J.C. Shaw G. Biochem. Biophys. Res. Commun. 1994; 203: 29-35Crossref PubMed Scopus (88) Google Scholar). Together with our results that the Lys644, Lys645, and Glu646 mutants still bind to Gβγ and inhibit Gβγ-mediated IP production, these observations suggest that the interaction between Gβγ and βARK cannot simply be explained by the tripled coiled-coil model. Nonetheless, the fact that the alanine insertions after Trp643 impair Gβγ binding activity suggests that this region could form an α-helical structure like other PH domains, thereby playing a crucial role in interacting with Gβγ. The binding of PIP2 to the PH domain of βARK 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 β-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 α-helix are important to stabilize the domain structure.2 2K. Touhara, R. J. Lefkowitz, unpublished results. Therefore, the most conserved Trp residue is critical for both Gβγ binding and stablization of the PIP2-binding cleft in the NH2 terminus of the βARK PH domain. Mutations in the cluster of anionic residues located in the beginning of the carboxyl α-helix (DSD→KKK mutation) attenuated binding to PIP2. Although the DSD→KKK mutant binds Gβγ as well as K645E or E646K, the ability of the DSD→KKK mutant to inhibit Gβγ-mediated βARK translocation to rod outer segment membranes was less than that of K645E or E646K, consistent with the evidence that both PIP2 and Gβγ are required for PH domain-mediated membrane translocation of βARK(37Pitcher J.A. Touhara K. Payne E.S. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 11707-11710Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). Other mutants with markedly impaired Gβγ binding still possess PIP2 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 Gβγ has been mapped using guanine nucleotide releasing factor and phospholipase Cγ, demonstrating that the NH2-terminal half of PH domain is not necessary for Gβγ binding activity(22Touhara K. Inglese J. Pitcher J.A. Shaw G. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 10217-10220Abstract Full Text PDF PubMed Google Scholar). Later, this was confirmed by using the PH domain of Bruton tyrosine kinase(33Tsukada S. Simon M.I. Witte O.N. Katz A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11256-11260Crossref PubMed Scopus (238) Google Scholar). Apparently, the carboxyl α-helix of PH domain mediates the interaction with Gβγ. One function of the NH2-terminal region of the PH domain, in contrast, is binding to PIP2 (30). Moreover, the PH domain of Bruton tyrosine kinase has been shown to bind protein kinase C at the NH2-terminal region of the PH domain, based on the evidence that the mutation of Arg28 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 Gβγ and PIP2 to the PH domain of βARK appears to be cooperative, since binding of the COOH terminus of the PH domain to Gβγ potentially affects PIP2 binding(37Pitcher J.A. Touhara K. Payne E.S. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 11707-11710Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). The following novel conclusions emerge from our studies. First, we have identified the critical residues involved in Gβγ binding to the βARK PH domain and localized these within subdomain 6 of the PH domain and the adjacent region. Second, although the region can form an α-helical structure which is crucial for the Gβγ interaction, the Gβγ-βARK interaction cannot simply be explained by a triple coiled-coil model. Third, the effects of mutations in the βARK PH domain on the ability to bind Gβγ are distinct from those on PIP2 binding. Finally, the most highly conserved Trp in subdomain 6 of the PH domain is absolutely required for both Gβγ and PIP2 binding. We thank W. Carl Stone for assisting in mutant DNA construction and sequencing, W. Darrel Capel for purified βARK, rod outer segment membranes, and Gβγ, 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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