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- W2019915186 abstract "Phospholipase C (PLC)-γ is unique among the PLC enzymes because each PLC-γ isozyme contains a split pleckstrin homology (PH) domain with an SH2SH2SH3 tandem repeat insertion (where SH indicates Src homology domain) in the middle of its sequence. Split PH domains exist in a number of other proteins that play crucial signaling roles. However, little is known about the structure and function of split PH domains. The C-terminal half of the PLC-γ split PH domain has been implicated to interact directly with the TRPC3 calcium channel, thereby providing a direct coupling mechanism between PLC-γ and agonist-induced calcium entry. However, this interaction has not been proved by direct biochemical or structural studies. Here we determined the three-dimensional structure of the split PH domain of PLC-γ1, and we found that the split PH domain of the enzyme folds into a canonical PH domain fold with high thermostability. The SH2SH2SH3 insertion between the β3 and β4 strands does not change the structure of the split PH domain. In contrast to the majority of phospholipid-binding PH domains, the PLC-γ1 split PH domain lacks the signature lipid-binding motif located between the β1 and β2 strands. Consistent with this structural feature, the split PH domain of PLC-γ1 does not bind to phospholipids. Multiple biochemical and biophysical experiments have argued against a direct interaction between TRPC3 and the C-terminal half of the PLC-γ1 split PH domain. Our data pointed to the existence of a yet to be elucidated interaction mechanism between TRPC3 and PLC-γ1. Phospholipase C (PLC)-γ is unique among the PLC enzymes because each PLC-γ isozyme contains a split pleckstrin homology (PH) domain with an SH2SH2SH3 tandem repeat insertion (where SH indicates Src homology domain) in the middle of its sequence. Split PH domains exist in a number of other proteins that play crucial signaling roles. However, little is known about the structure and function of split PH domains. The C-terminal half of the PLC-γ split PH domain has been implicated to interact directly with the TRPC3 calcium channel, thereby providing a direct coupling mechanism between PLC-γ and agonist-induced calcium entry. However, this interaction has not been proved by direct biochemical or structural studies. Here we determined the three-dimensional structure of the split PH domain of PLC-γ1, and we found that the split PH domain of the enzyme folds into a canonical PH domain fold with high thermostability. The SH2SH2SH3 insertion between the β3 and β4 strands does not change the structure of the split PH domain. In contrast to the majority of phospholipid-binding PH domains, the PLC-γ1 split PH domain lacks the signature lipid-binding motif located between the β1 and β2 strands. Consistent with this structural feature, the split PH domain of PLC-γ1 does not bind to phospholipids. Multiple biochemical and biophysical experiments have argued against a direct interaction between TRPC3 and the C-terminal half of the PLC-γ1 split PH domain. Our data pointed to the existence of a yet to be elucidated interaction mechanism between TRPC3 and PLC-γ1. PH 3The abbreviations used are: PH, pleckstrin homology; PLC, phospholipase C; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; GST, glutathione S-transferase; TOCSY, total correlation spectroscopy; HSQC, heteronuclear single quantum coherence; SH, Src homology. domains are abundant protein modules that play critical roles in cellular signaling and cytoskeletal organization (1Lemmon M.A. Ferguson K.M. Biochem. J. 2000; 350: 1-18Crossref PubMed Scopus (617) Google Scholar). All PH domains with known structures contain a conserved core structure composed of a partially open, two-sheeted β-barrel with one end of the barrel capped with a C-terminal α-helix (1Lemmon M.A. Ferguson K.M. Biochem. J. 2000; 350: 1-18Crossref PubMed Scopus (617) Google Scholar, 2Yoon H.S. Hajduk P.J. Petros A.M. Olejniczak E.T. Meadows R.P. Fesik S.W. Nature. 1994; 369: 672-675Crossref PubMed Scopus (189) Google Scholar, 3Ferguson K.M. Lemmon M.A. Schlessinger J. Sigler P.B. Cell. 1994; 79: 199-209Abstract Full Text PDF PubMed Scopus (243) Google Scholar, 4Fushman D. Cahill S. Lemmon M.A. Schlessinger J. Cowburn D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 816-820Crossref PubMed Scopus (82) Google Scholar, 5Macias M.J. Musacchio A. Ponstingl H. Nilges M. Saraste M. Oschkinat H. Nature. 1994; 369: 675-677Crossref PubMed Scopus (207) Google Scholar). The best characterized function of PH domains is binding to inositol phospholipids (1Lemmon M.A. Ferguson K.M. Biochem. J. 2000; 350: 1-18Crossref PubMed Scopus (617) Google Scholar). Only a minority of PH domains are capable of binding to lipids with high affinity and specificity. Some PH domains are known to be weak, nonspecific membrane phosphoinositide binders (6Yu J.W. Mendrola J.M. Audhya A. Singh S. Keleti D. DeWald D.B. Murray D. Emr S.D. Lemmon M.A. Mol. Cell. 2004; 13: 677-688Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar), whereas others interact with proteins (e.g. the PH domain of the β-adrenergic receptor) (7Touhara K. Inglese J. Pitcher J. Shaw G. Lefkowitz R. J. Biol. Chem. 1994; 269: 10217-10220Abstract Full Text PDF PubMed Google Scholar). However, the functions of the majority of PH domains are unknown (6Yu J.W. Mendrola J.M. Audhya A. Singh S. Keleti D. DeWald D.B. Murray D. Emr S.D. Lemmon M.A. Mol. Cell. 2004; 13: 677-688Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). Split PH domains represent a unique subclass of PH domains that are characterized by insertions of one or several autonomously folded protein modules in the middle of PH domain sequences. Split PH domains are also found in various proteins, including the second messenger generating enzymes phospholipase C-γ (PLC-γ), the syntrophin scaffold proteins (8Yan J. Wen W. Xu W. Long J.F. Adams M.E. Froehner S.C. Zhang M. EMBO J. 2005; 24: 3985-3995Crossref PubMed Scopus (56) Google Scholar), the Rock1 family Ser/Thr kinases, and the actin filament-based molecular motor myosin X (9Berg J.S. Derfler B.H. Pennisi C.M. Corey D.P. Cheney R.E. J. Cell Sci. 2000; 113: 3439-3451Crossref PubMed Google Scholar). Recent biochemical and structural studies showed that the split PH domain of α-syntrophin folds into a canonical PH domain fold with or without the PDZ domain insertion. It was further demonstrated that the PDZ domain insertion functions synergistically with the split PH domain in binding to phosphoinositol lipids (8Yan J. Wen W. Xu W. Long J.F. Adams M.E. Froehner S.C. Zhang M. EMBO J. 2005; 24: 3985-3995Crossref PubMed Scopus (56) Google Scholar). Little is known about the structural and biochemical properties of the split PH domains other than what has been demonstrated in α-syntrophin. Phospholipase C (PLC) hydrolyzes phosphatidylinositol 4,5-biphosphate to produce the second messengers inositol 1,4,5-triphosphate and diacylglycerol. There are as many as 12 different PLC gene products found in mammalian cells that can be grouped into five subfamilies: β (β1-β4), γ (γ1 and γ2), δ (δ1-δ4), ϵ, and ζ (10Patterson R.L. van Rossum D.B. Nikolaidis N. Gill D.L. Snyder S.H. Trends Biochem. Sci. 2005; 30: 688-697Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 11Rhee S.G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1220) Google Scholar, 12Carpenter G. Ji Q. Exp. Cell Res. 1999; 253: 15-24Crossref PubMed Scopus (209) Google Scholar). All PLC isoforms are modular proteins invariably containing from their N-terminal to C-terminal ends a PH domain, catalytic X and Y domains, and a C2 domain. Among the various isozymes of PLC, members of the γ subfamily are structurally distinct in that the catalytic X and Y domains are separated by an ∼450-residue insertion. The center of the insertion sequences in each PLC-γ isozyme consists of two Src homology 2 (SH2) domains and an SH3 domain, and the two ends of the SH2SH2SH3 supramodule are flanked by the split halves of a PH domain (Fig. 1A). Extensive studies in the past demonstrated that in addition to docking the enzymes to various receptors and adaptor proteins, the SH2 and SH3 domains also directly regulate the catalytic activities of PLC-γ (11Rhee S.G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1220) Google Scholar, 12Carpenter G. Ji Q. Exp. Cell Res. 1999; 253: 15-24Crossref PubMed Scopus (209) Google Scholar, 13Poulin B. Sekiya F. Rhee S.G. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 4276-4281Crossref PubMed Scopus (88) Google Scholar). An emerging feature of the PLC-γ family isozyme is that many PLC-γ cellular functions are not dependent on lipase activity. For example, the mitogenic activity of PLC-γ1 was not affected by a lipase-inactive mutation (14Smith M.R. Liu Y.L. Matthews N.T. Rhee S.G. Sung W.K. Kung H.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6554-6558Crossref PubMed Scopus (105) Google Scholar) but could be inhibited by the SH3 domain of the enzyme (15Smith M.R. Liu Y.L. Kim S.R. Bae Y.S. Kim C.G. Kwon K.S. Rhee S.G. Kung H.F. Biochem. Biophys. Res. Commun. 1996; 222: 186-193Crossref PubMed Scopus (33) Google Scholar). The SH3 domain of PLC-γ1 was found to contain guanine nucleotide exchange factor activity specifically for the phosphatidylinositol 3-kinase enhancer small GTPase PIKE (16Ye K. Aghdasi B. Luo H.R. Moriarity J.L. Wu F.Y. Hong J.J. Hurt K.J. Bae S.S. Suh P.G. Snyder S.H. Nature. 2002; 415: 541-544Crossref PubMed Scopus (146) Google Scholar), and it was suggested that the guanine nucleotide exchange factor activity of the SH3 domain may be associated with the lipase-independent mitogenic activity of the enzyme. Another example of lipase-independent activity of PLC-γ is the regulation of agonist-induced Ca2+ entry via the TRPC3 calcium channel (17Patterson R.L. van Rossum D.B. Ford D.L. Hurt K.J. Bae S.S. Suh P.G. Kurosaki T. Snyder S.H. Gill D.L. Cell. 2002; 111: 529-541Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). In that study, the authors showed that the lipase-inactive mutant of PLC-γ1 functions as effectively as the wild-type enzyme in augmenting agonist-induced Ca2+ entry in PC12 cells. It was further found that a fragment of the enzyme containing the SH3 domain and the C-terminal half of the split PH domain of PLC-γ1 (PLCγ1-PHC) can directly associate with TRPC3. Very recently, the same research group showed that PLCγ1-PHC is solely responsible for direct binding to a short fragment of TRPC3 located at its N-terminal end (18van Rossum D.B. Patterson R.L. Sharma S. Barrow R.K. Kornberg M. Gill D.L. Snyder S.H. Nature. 2005; 434: 99-104Crossref PubMed Scopus (164) Google Scholar). More significantly, the authors suggested that the PLCγ1-PHC-binding segment of TRPC3 represents a complementary partial PH domain “hidden” in the ion channel. They demonstrated that binding of the two partial PH domain fragments from PLC-γ1 and TRPC3 forms a functional “PH domain” capable of binding to specific lipids and regulating the surface expression of the TRPC3 ion channel. Given the potentially wide distribution of split PH domains in diverse proteins and enzymes, the work presented by van Rossum et al. (18van Rossum D.B. Patterson R.L. Sharma S. Barrow R.K. Kornberg M. Gill D.L. Snyder S.H. Nature. 2005; 434: 99-104Crossref PubMed Scopus (164) Google Scholar) suggests a novel mode of function of many PH domains (19Lemmon M.A. Cell. 2005; 120: 574-576Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). To advance this important hypothesis, it is critical to know whether the two halves of the split PH domain in PLCγ1 can fold into a canonical PH domain structure; whether PLCγ1-PHC alone can stably exist in solution for binding to a complementing partial PH domain from another protein such as TRPC3; and whether the complex formed by two fragments from PLCγ1 and TRPC3 (or PLCγ1 and translational elongation factor 1α (20Chang J.-S. Seok H. Kwon T.-K. Min D.S. Ahn B.-H. Lee Y.H. Suh J.-W. Kim J.-W. Iwashita S. Omori A. Ichinose S. Numata O. Seo J.-K. Oh Y.-S. Suh P.-G. J. Biol. Chem. 2002; 277: 19697-19702Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar)) or from other proteins with split PH domains can indeed assume a PH domain-like fold. In this study, we determined the solution structure of the split PH domain of PLC-γ1. We further showed that the insertion of the SH2SH2SH3 domain does not affect the structure of the split PH domain of PLC-γ1. Finally, we characterized potential interactions of PLCγ1-PHC with the hypothetical hidden PHN fragment from TRPC3. Protein Expression and Purification—The joined PHN-PHC domain (residues 489-547 and 851-933), PHC fragment (residues 851-933), and the PHN-SH2SH2SH3-PHC (residues 489-933) of rat PLC-γ1 were cloned into a modified version of the pET32a vector (21Feng W. Fan J.S. Jiang M. Shi Y.W. Zhang M. J. Biol. Chem. 2002; 277: 41140-41146Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The joined PHN-PHC domain contains an 8-residue protease 3C recognition sequence (“LEVLFQGP”) at the joint site of the two halves of the PH domain. The human TRPC3 fragment (residues 1-52) and the PLC-γ1 PHN fragment (residues 489-547) were cloned into the pET32a vector. GST-fused PHN-PHC, the PHC fragment, and the SH3-PHC fragment (residues 794-933) were cloned into pGEX4T-1 plasmid (Amersham Biosciences). Bacterial cells harboring each fusion protein expression plasmid were grown at 37 °C, and protein expression was induced by isopropyl β-d-thiogalactoside at 16 °C overnight. Uniformly 15N- and 15N/13C-labeled proteins were prepared by growing bacteria in M9 medium containing NH4Cl with or without 13C6-glucose. The Histagged fusion proteins were purified under native conditions using an Ni2+-nitrilotriacetic acid-agarose (Qiagen) affinity chromatography. The remaining small amount of contaminant proteins was removed by size-exclusion chromatography. The GST-fused proteins were purified using GSH-Sepharose affinity chromatography followed by size-exclusion chromatography. Lipid Binding Assay—Liposomes consisting of total bovine brain lipids were prepared by resuspending brain lipid extracts (Folch fraction I, Sigma B1502, which contains ∼10% phosphatidylinositol lipids) at 2 mg/ml in a buffer containing 20 mm HEPES, pH 7.4, 150 mm NaCl, 1 mm dithiothreitol. The protein sample (5-10 μm) was incubated with 0.6 mg/ml liposomes in 40 μl of buffer for 15 min at room temperature and then spun at 65,000 × g for 15 min at 4 °C in a Beckman TLA100.1 rotor. The supernatants were removed for determination of proteins not bound to liposomes. The pellets were washed twice with the same buffer and brought up to the same volume as the supernatant. The supernatant and the pellet proteins were subjected to SDS-PAGE and visualized by Coomassie Blue staining. Pull-down Experiments—Purified GST-PHC, GST-PHN-PHC, or GST-SH3-PHC (10 μg) was mixed with purified Trx-TRPC3-(1-52) (100 μg) with or without the presence of 0.5 mg/ml of brain liposome. Then the complexes were pelleted with 30 μl of fresh GSH-Sepharose beads (Amersham Biosciences). The pelleted beads were washed extensively with phosphate-buffered saline buffer and subsequently boiled with 2× SDS-PAGE sample buffer. The proteins were resolved by SDS-PAGE and visualized by Coomassie Blue staining. NMR Spectroscopy—NMR samples contained ∼1.0 mm of the PHN-PHC tandem in 50 mm potassium phosphate, pH 6.5, in 90% H2O, 10% D2O or 99.9% D2O. NMR spectra were acquired at 35 °C on Varian Inova 500- and 750-MHz spectrometers each equipped with an actively z-gradient shielded triple resonance probe. Backbone and side chain resonance assignments of the protein were obtained by standard heteronuclear correlation experiments (22Bax A. Grzesiek S. Acc. Chem. Res. 1993; 26: 131-138Crossref Scopus (795) Google Scholar, 23Kay L.E. Gardner K.H. Curr. Opin. Struct. Biol. 1997; 7: 722-731Crossref PubMed Scopus (156) Google Scholar). Nonaromatic, nonexchangeable side chain resonances were assigned using HCCH-TOCSY experiments. The side chains of aromatics were assigned by standard 1H two-dimensional TOCSY/NOESY experiments. Structure Calculations—Approximate interproton distance restraints were derived from the NOESY spectra (a 1H two-dimensional homonuclear NOESY, a 15N-separated NOESY, and a 13C-separated NOESY). The NOEs were grouped into three distance ranges as follows: 1.8-2.7 Å (1.8-2.9 Å for NOEs involving NH protons), 1.8-3.3 Å (1.8-3.5 Å for NOEs involving NH protons), and 1.8-5.0 Å, corresponding to strong, medium, and weak NOEs, respectively. Hydrogen bonding restraints were generated from the standard secondary structure of the protein based on the NOE patterns and backbone secondary chemical shifts. The backbone dihedral angle restraints (φ and ψ angles) were derived from the chemical shift analysis program TALOS (24Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2738) Google Scholar). Structures were calculated using the program CNS (25Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D. Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16967) Google Scholar). Figures were generated using MOLMOL (26Koradi R. Billeter M. Wuthrich K. J. Mol. Graphics. 1996; 14: 51-55Crossref PubMed Scopus (6489) Google Scholar), MOLSCRIPT (27Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), and Raster3D (28Merritt E. Murphy M. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 869-873Crossref PubMed Scopus (2857) Google Scholar). NMR Titration—NMR-based interaction studies were by recording 1H-15N HSQC spectra of performed 15N-labeled protein samples (∼0.2 mm) with or without addition of their respective binding partners at natural abundance. The N-terminal SH2 domain-binding phosphotyrosine peptide (D{pY}IIPLPDP) was commercially synthesized (GenScript Corp., Piscataway, NJ). The buffer condition was identical to that used in the samples for the structural determination of the split PH proteins. The Split PH Domain of PLC-γ1 Adopts a Stable Fold—Several approaches were used to assess whether the two split halves of the PH domain of PLC-γ1 (referred to as PHN and PHC) can directly interact with each other to form a stable structure. First, we deleted the SH2SH2SH3 insert (residues 548-850 in rat PLC-γ1) from the PHN-SH2SH2SH3-PHC supramodule, resulting in a fusion protein with the two halves of the split PH domain connected directly (i.e. PHN-PHC). The recombinant PHN-PHC was eluted at a molecular mass indicative of a stable monomer when analyzed by analytical gel filtration chromatography (data not shown). The well dispersed 1H, 15N HSQC spectrum indicates that the joined PHN-PHC is well folded (Fig. 1B, black dots). It is possible that the covalent linkage of PHN and PHC may artificially induce folding of the linked protein. To address this possibility, in the middle of the linking sequence of PHN-PHC, we inserted an 8-residue peptide fragment that can be cleaved by protease 3C. Digestion of PHN-PHC with protease 3C produces two fragments with molecular masses corresponding to PHN and PHC, respectively (Fig. 1C). The NMR spectrum of the protease 3C-cleaved PHN-PHC is essentially identical to that of the uncleaved protein (Fig. 1B), indicating that the covalent linkage between PHN and PHC is dispensable to the folding of the split PH domain in PLC-γ1. Furthermore, both the joined and cleaved PHN-PHC showed excellent thermostability, as the proteins remained well folded in the NMR tubes at temperature as high as 50 °C (supplemental Fig. 1). Structure of the Split PH Domain of PLC-γ1—To determine whether the split PH domain of PLC-γ1 folds into a canonical PH domain structure, we solved the three-dimensional structures of the joined PHN-PHC by NMR spectroscopy (Fig. 2 and Table 1). The PHN and PHC fragments fold together to form a canonical PH domain structure containing seven β-strands and one C-terminal α-helix. As in the split PH domain of α-syntrophin (8Yan J. Wen W. Xu W. Long J.F. Adams M.E. Froehner S.C. Zhang M. EMBO J. 2005; 24: 3985-3995Crossref PubMed Scopus (56) Google Scholar), the PHN half is composed of three β-strands (β1-β3), and the PHC half contains the remaining four β-strands (β4-β7) and the C-terminal α-helix. Inserted at the β3/β4-loop of the PH domain is a 56-residue flexible linker. The flexibility of this 56-residue linker is confirmed by a lack of any detectable medium, long range NOEs and negative backbone amide 1H, 15N NOE values (data not shown). In the native PLC-γ1, the β3/β4-loop of the split PH domain also contains a 300-residue SH2SH2SH3 tandem insertion in the middle of the loop.TABLE 1Structural statistics for the family of 15 structures of the joined PHN-PHC domainDistance restraintsIntraresidue (i - j = 0)837Sequential (|i - j| = 1)501Medium range (2 ≤ |i - j| ≤4)220Long range (|i - j| >5)465Hydrogen bonds62Total2085Dihedral angle restraintsΦ31Ψ29Total60Mean r.m.s. deviations from the experimental restraintsDistance (Å)0.008 ± 0.000Dihedral angle (°)0.003 ± 0.005Mean r.m.s. deviations from idealized covalent geometryBond (Å)0.001 ± 0.000Angle (°)0.291 ± 0.004Improper (°)0.125 ± 0.004Mean energies (kcal mol−1)ENOEaThe final values of the square-well NOE and dihedral angle potentials were calculated with force constants of 50 kcal mol−1 Å−2 and 200 kcal mol−1 rad−2, respectively.10.26 ± 0.44EcdihaThe final values of the square-well NOE and dihedral angle potentials were calculated with force constants of 50 kcal mol−1 Å−2 and 200 kcal mol−1 rad−2, respectively.0.00 ± 0.00EL-J−368 ± 19Ramachandran plotbThe program Procheck (40) was used to assess the overall quality of the structures.Residues 1-33 and 90-150% residues in the most favorable regions74.6Additional allowed regions20.0Generously allowed regions4.6Disallowed regions0.8Atomic r.m.s. difference (Å)cThe precision of the atomic coordinates is defined as the average r.m.s. difference between 15 final structures and the mean coordinates of the protein.Residues 1-11, 16-31, 91-118, and 125-147Backbone heavy atoms (N, Cα, and C′)0.45Heavy atoms0.93a The final values of the square-well NOE and dihedral angle potentials were calculated with force constants of 50 kcal mol−1 Å−2 and 200 kcal mol−1 rad−2, respectively.b The program Procheck (40Laskowski R.A. Rullmannn J.A. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4428) Google Scholar) was used to assess the overall quality of the structures.c The precision of the atomic coordinates is defined as the average r.m.s. difference between 15 final structures and the mean coordinates of the protein. Open table in a new tab Sequence alignment analysis showed that the split PH domains of PLCγ1 are highly conserved throughout evolution (Fig. 3A). When compared with a number of PH domains that bind to phosphoinositide head groups with high affinities (29Ferguson K.M. Lemmon M.A. Schlessinger J. Sigler P.B. Cell. 1995; 83: 1037-1046Abstract Full Text PDF PubMed Scopus (532) Google Scholar, 30Rameh L.E. Arvidsson A.-K. Carraway III, K.L. Couvillon A.D. Rathbun G. Crompton A. VanRenterghem B. Czech M.P. Ravichandran K.S. Burakoff S.J. Wang D.-S. Chen C.-S. Cantley L.C. J. Biol. Chem. 1997; 272: 22059-22066Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar, 31Kavran J.M. Klein D.E. Lee A. Falasca M. Isakoff S.J. Skolnik E.Y. Lemmon M.A. J. Biol. Chem. 1998; 273: 30497-30508Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar, 32Lietzke S.E. Bose S. Cronin T. Klarlund J. Chawla A. Czech M.P. Lambright D.G. Mol. Cell. 2000; 6: 385-394Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 33Thomas C.C. Deak M. Alessi D.R. van Aalten D.M. Curr. Biol. 2002; 12: 1256-1262Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar), the split PH domain of PLCγ1 lacks a number of critical residues necessary for binding to phosphoinositides. For example, the phosphoinositide-binding PH domains share a signature motif with conserved positively charged amino acid residues, “KXn(K/R)XR,” where the first Lys locates at the penultimate position of the β1 strand, and the “(K/R)XR” sequence corresponds to residues 2-4 of the β2 strand (Fig. 3A) (32Lietzke S.E. Bose S. Cronin T. Klarlund J. Chawla A. Czech M.P. Lambright D.G. Mol. Cell. 2000; 6: 385-394Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 33Thomas C.C. Deak M. Alessi D.R. van Aalten D.M. Curr. Biol. 2002; 12: 1256-1262Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 34Cronin T.C. DiNitto J.P. Czech M.P. Lambright D.G. EMBO J. 2004; 23: 3711-3720Crossref PubMed Scopus (80) Google Scholar, 35Ferguson K.M. Kavran J.M. Sankaran V.G. Fournier E. Isakoff S.J. Skolnik E.Y. Lemmon M.A. Mol. Cell. 2000; 6: 373-384Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). These conserved basic residues play critical roles in binding to negatively charged phosphate groups from the head groups of phosphoinositides (see Fig. 3B for an example). In contrast, the penultimate residue in the β1 strand is a Leu instead of a Lys, and the second and the fourth residues in the β2 strand are Tyr and His, respectively, in the split PH domains of PLC-γ1. Because all three positively charged residues in the otherwise phosphoinositol lipid-binding signature motif are absent, we predicted that the split PH domain of PLC-γ1 is not likely to function as a lipid binding module. To test this hypothesis, we assayed the binding of the joined PHN-PHC domain of PLCγ1 to liposomes prepared from total bovine brain lipids. As predicted, the split PH domain of PLC-γ1 showed no detectable binding to brain liposomes (Fig. 3C). We further demonstrated that the PHN-SH2SH2SH3-PHC supramodule of PLCγ1 does not bind to brain liposomes either, indicating that the SH2SH2SH3 insertion does not alter the lipid binding property of the split PH domain (Fig. 3C). The SH2SH2SH3 insertion is known to play important roles in regulating the enzyme activities of PLC-γ1. It has been suggested, based on indirect experimental evidence, that the insertion may change the structure of the split PH domain, thereby influencing the assembly of the catalytic X and Y boxes (10Patterson R.L. van Rossum D.B. Nikolaidis N. Gill D.L. Snyder S.H. Trends Biochem. Sci. 2005; 30: 688-697Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 11Rhee S.G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1220) Google Scholar). We used NMR spectroscopy to investigate potential structural changes that the SH2SH2SH3 insertion might exert on the split PH domain. As shown in Fig. 4A and supplemental Fig. 3, the HSQC spectrum of the joined PHN-PHC domain overlaps well with a subset of peaks from the HSQC spectrum of the PHN-SH2SH2SH3-PHC supramodule, indicating that the insertion of the SH2SH2SH3 tandem domains in the β3/β4-loop does not alter the structure of the split PH domain. We further tested whether ligand binding to the SH2SH2SH3 insertion might result in structural changes of the split PH domain. We chose a peptide ligand that is specific to the C-terminal SH2 domain to test potential ligand binding-induced structural changes to the split PH domain, as the two SH2 domains have been shown to play critical roles in enzyme activity regulation (11Rhee S.G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1220) Google Scholar, 12Carpenter G. Ji Q. Exp. Cell Res. 1999; 253: 15-24Crossref PubMed Scopus (209) Google Scholar). Binding of a phospho-Tyr-containing peptide encompassing the Tyr(P)-1021 site of platelet-derived growth factor receptor (36Pascal S.M. Singer A.U. Gish G. Yamazaki T. Shoelson S.E. Pawson T. Kay L.E. Forman-Kay J.D. Cell. 1994; 77: 461-472Abstract Full Text PDF PubMed Scopus (229) Google Scholar) to the PHN-SH2SH2SH3-PHC supramodule induced minimal chemical shift changes in the entire split PH domain (Fig. 4, B and C), indicating that the binding of the C-terminal SH2 ligand to PLC-γ1 does not change the conformation and therefore the assembly of the split PH domain. As expected, binding of the C-terminal SH2 ligand peptide to the PHN-SH2SH2SH3-PHC supramodule induced significant chemical shift changes in a number of residues other than those from the split PH domain, and these residues presumably belong to the ligand-binding SH2 domain (Fig. 4C). Residue-specific chemical shift assignments of the PHN-SH2SH2SH3-PHC supramodule are required for correlating the peptide-induced shift changes to the individual residues within the SH2SH2SH3 domains. Characterization of the Interaction between the Split PH Domain of PLC-γ1 and TRPC3—Having characterized the structure of the split PH domain of PLC-γ1 in detail, we went on to study the earlier reported interaction between PLC-γ1 and TRPC3 (17Patterson R.L. van Rossum D.B. Ford D.L. Hurt K.J. Bae S.S. Suh P.G. Kurosaki T. Snyder S.H. Gill D.L. Cell. 2002; 111: 529-541Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 18van Rossum D.B. Patterson R.L. Sharma S. Barrow R.K. Kornberg M. Gill D.L. Snyder S.H. Nature. 2005; 434: 99-104Crossref PubMed Scopus (164) Google Scholar), hoping to lay a foundation for structural characterization of the PLCγ1-TRPC3 complex. We were able to obtain large quantities of recombinant proteins encompassing the N-terminal 52 residues of TRPC3 (Fig. 5A), as well as a fragment containing the N-terminal 48 residues (data not shown). The authenticity of the TRPC3 fragments was verified using mass" @default.
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- W2019915186 title "Structural Characterization of the Split Pleckstrin Homology Domain in Phospholipase C-γ1 and Its Interaction with TRPC3" @default.
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