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• W2094397762 abstract "Activation of the cyclic GMP phosphodiesterase (PDE6) by transducin is the central event of visual signal transduction. How the PDE6 inhibitory γ-subunit (Pγ) interacts with the catalytic subunits (Pαβ) and the transducin α-subunit (αt) in this process is not entirely clear. Here we have investigated this issue, taking advantage of site-specific label transfer from throughout the full-length Pγ molecule to both αt and Pαβ. The interaction profiling and pull-down experiments revealed that the Pγ C- terminal domain accounted for the major interaction with αt bound with guanosine 5′-3-O-(thio)triphosphate (αtGTPγS) in comparison with the central region, whereas an opposite pattern was observed for the Pγ-Pαβ interaction. This complementary feature was further exhibited when both αtGTPγS and Pαβ were present and competing for Pγ interaction, with the Pγ C-terminal domain favoring αt, whereas the central region demonstrated a preference for Pαβ. Furthermore, αtGTPγS co-immunoprecipitated with PDE6 and vice versa in a Pγ-dependent manner. Either Pαβ or αtGTPγS could be pulled down by the Btn-Pγ molecules on streptavidin beads that were saturated by the other partner, indicating simultaneous binding of these two partners to Pγ. These data together indicate that complementary Pγ interactions with its two targets facilitate the αt·PDE6 “transducisome” formation. Thus, our study provides new insights into the molecular mechanisms of PDE6 activation. Activation of the cyclic GMP phosphodiesterase (PDE6) by transducin is the central event of visual signal transduction. How the PDE6 inhibitory γ-subunit (Pγ) interacts with the catalytic subunits (Pαβ) and the transducin α-subunit (αt) in this process is not entirely clear. Here we have investigated this issue, taking advantage of site-specific label transfer from throughout the full-length Pγ molecule to both αt and Pαβ. The interaction profiling and pull-down experiments revealed that the Pγ C- terminal domain accounted for the major interaction with αt bound with guanosine 5′-3-O-(thio)triphosphate (αtGTPγS) in comparison with the central region, whereas an opposite pattern was observed for the Pγ-Pαβ interaction. This complementary feature was further exhibited when both αtGTPγS and Pαβ were present and competing for Pγ interaction, with the Pγ C-terminal domain favoring αt, whereas the central region demonstrated a preference for Pαβ. Furthermore, αtGTPγS co-immunoprecipitated with PDE6 and vice versa in a Pγ-dependent manner. Either Pαβ or αtGTPγS could be pulled down by the Btn-Pγ molecules on streptavidin beads that were saturated by the other partner, indicating simultaneous binding of these two partners to Pγ. These data together indicate that complementary Pγ interactions with its two targets facilitate the αt·PDE6 “transducisome” formation. Thus, our study provides new insights into the molecular mechanisms of PDE6 activation. IntroductionThe intricate visual transduction in rod photoreceptor cells provides a paradigm for G protein-coupled signaling. The outstanding visual sensitivity of the rod is largely due to the great signal amplification achieved by the cGMP 2The abbreviations used are: cGMPcyclic GMPαttransducin α-subunitPαβPDE6 catalytic heterodimerGAFa domain derived from cGMP phosphodiesterases, adenylyl cyclases, and the Escherichia coli protein Fh1APγPDE6 inhibitory subunitGAPGTPase-activating proteinACTPN-[3-iodo-4-azidophenylpropioamido-S-(2-thiopyridyl)]cysteinemBP4-(N-maleimido)benzophenoneHPLChigh performance liquid chromatographyROSrod outer segmentDTTdithiothreitolBSAbovine serum albuminGTPγSguanosine 5′-3-O-(thio)triphosphate. phosphodiesterase PDE6 (rod photoreceptor cGMP phosphodiesterase), the central effector enzyme (1Cote R.H. Photoreceptor Phosphodiesterase (PDE6): A G-protein-activated PDE Regulating Visual Excitation in Rod and Cone Photoreceptor Cells. CRC Press, Inc., Boca Raton, FL2006: 165-193Google Scholar). Upon absorption of a single photon, light-excited rhodopsin stimulates an exchange of GTP for GDP bound in the transducin α subunit (αt) (2Oldham W.M. Hamm H.E. Nat. Rev. Mol. Cell Biol. 2008; 9: 60-71Crossref PubMed Scopus (794) Google Scholar), which in turn relieves PDE6 from the inhibitory constraint exerted by its γ-subunit (Pγ). PDE6 activation causes rapid cGMP breakdown, which closes the cGMP-coupled ion channels, thus relaying visual signals to the brain in a form of electrical pulses (3Burns M.E. Arshavsky V.Y. Neuron. 2005; 48: 387-401Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). PDE6 in the rod is uniquely composed of a large catalytic heterodimer (Pαβ, ∼100 kDa each subunit) to which bind two small identical Pγ subunits (∼10 kDa) keeping the enzyme inactive in the dark (1Cote R.H. Photoreceptor Phosphodiesterase (PDE6): A G-protein-activated PDE Regulating Visual Excitation in Rod and Cone Photoreceptor Cells. CRC Press, Inc., Boca Raton, FL2006: 165-193Google Scholar, 4Artemyev N.O. Arshavsky V.Y. Cote R.H. Methods. 1998; 14: 93-104Crossref PubMed Scopus (30) Google Scholar). The PDE6 structure is less well understood compared with the other key players in phototransduction. This is primarily due to the fact that solving the atomic structure of PDE6 has been hindered by the lack of an expression system to produce active Pαβ heterodimers in large amounts (5Barren B. Gakhar L. Muradov H. Boyd K.K. Ramaswamy S. Artemyev N.O. EMBO J. 2009; 28: 3613-3622Crossref PubMed Scopus (46) Google Scholar). A low resolution electron microscopy image of Pαβ has revealed a linear alignment of three distinct domains of each subunit: the tandem GAFa and GAFb domains on the N-terminal side that host non-catalytic cGMP binding and the C-terminal catalytic domain that performs cGMP hydrolysis (6Kameni Tcheudji J.F. Lebeau L. Virmaux N. Maftei C.G. Cote R.H. Lugnier C. Schultz P. J. Mol. Biol. 2001; 310: 781-791Crossref PubMed Scopus (81) Google Scholar). Direct allosteric communication between GAF domains and the catalytic domain has been recently reported (7Zhang X.J. Cahill K.B. Elfenbein A. Arshavsky V.Y. Cote R.H. J. Biol. Chem. 2008; 283: 29699-29705Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar).The inhibitory Pγ subunit is an intrinsically disordered protein, yet structural elements important for its function are encoded in the free Pγ molecule (8Song J. Guo L.W. Muradov H. Artemyev N.O. Ruoho A.E. Markley J.L. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 1505-1510Crossref PubMed Scopus (79) Google Scholar). The Pγ sequence of 87 amino acids features a polycationic central domain (Gly19–Gly49) and a negatively charged C-terminal half that contains a linker region (Phe50–Gly61) and a hydrophobic C-terminal domain (Thr62–Ile87) (1Cote R.H. Photoreceptor Phosphodiesterase (PDE6): A G-protein-activated PDE Regulating Visual Excitation in Rod and Cone Photoreceptor Cells. CRC Press, Inc., Boca Raton, FL2006: 165-193Google Scholar, 9Guo L.W. Ruoho A.E. Curr. Protein Pept. Sci. 2008; 9: 611-625Crossref PubMed Scopus (14) Google Scholar). The last C-terminal dozen or so residues (herein termed the inhibitory region) are involved in the interaction with the Pαβ catalytic domain (8Song J. Guo L.W. Muradov H. Artemyev N.O. Ruoho A.E. Markley J.L. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 1505-1510Crossref PubMed Scopus (79) Google Scholar, 10Skiba N.P. Artemyev N.O. Hamm H.E. J. Biol. Chem. 1995; 270: 13210-13215Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 11Granovsky A.E. Artemyev N.O. Biochemistry. 2001; 40: 13209-13215Crossref PubMed Scopus (29) Google Scholar). The very recently reported crystal structure of the chimeric PDE5/6 catalytic domain complexed with the Pγ(70–87) inhibitory peptide (5Barren B. Gakhar L. Muradov H. Boyd K.K. Ramaswamy S. Artemyev N.O. EMBO J. 2009; 28: 3613-3622Crossref PubMed Scopus (46) Google Scholar) has confirmed the previous suggestion that the highly hydrophobic C terminus (Y84GII87) directly blocks the cGMP entry into the catalytic pocket (12Artemyev N.O. Natochin M. Busman M. Schey K.L. Hamm H.E. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 5407-5412Crossref PubMed Scopus (53) Google Scholar, 13Granovsky A.E. Natochin M. Artemyev N.O. J. Biol. Chem. 1997; 272: 11686-11689Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The other important Pαβ-interacting site on Pγ is the central domain, which has been shown to provide most of the binding strength for Pαβ (14Mou H. Cote R.H. J. Biol. Chem. 2001; 276: 27527-27534Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The central domain of Pγ binds to the Pαβ GAF domain (15Muradov K.G. Granovsky A.E. Schey K.L. Artemyev N.O. Biochemistry. 2002; 41: 3884-3890Crossref PubMed Scopus (40) Google Scholar, 16Guo L.W. Muradov H. Hajipour A.R. Sievert M.K. Artemyev N.O. Ruoho A.E. J. Biol. Chem. 2006; 281: 15412-15422Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) and couples non-catalytic cGMP binding in a positively cooperative manner, thus regulating the PDE-inhibiting function of Pγ (14Mou H. Cote R.H. J. Biol. Chem. 2001; 276: 27527-27534Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Remarkably, the C-terminal domain and the central domain also constitute αt-interacting sites (17Artemyev N.O. Rarick H.M. Mills J.S. Skiba N.P. Hamm H.E. J. Biol. Chem. 1992; 267: 25067-25072Abstract Full Text PDF PubMed Google Scholar, 18Skiba N.P. Bae H. Hamm H.E. J. Biol. Chem. 1996; 271: 413-424Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 19Granovsky A.E. McEntaffer R. Artemyev N.O. Cell Biochem. Biophys. 1998; 28: 115-133Crossref PubMed Scopus (9) Google Scholar).An overlap of the Pγ C-terminal αt-binding region (Thr62–Ile87) and the inhibitory region (Asn74–Ile87) forms the structural basis for transducin-mediated PDE6 activation (5Barren B. Gakhar L. Muradov H. Boyd K.K. Ramaswamy S. Artemyev N.O. EMBO J. 2009; 28: 3613-3622Crossref PubMed Scopus (46) Google Scholar, 8Song J. Guo L.W. Muradov H. Artemyev N.O. Ruoho A.E. Markley J.L. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 1505-1510Crossref PubMed Scopus (79) Google Scholar, 20Slep K.C. Kercher M.A. He W. Cowan C.W. Wensel T.G. Sigler P.B. Nature. 2001; 409: 1071-1077Crossref PubMed Scopus (222) Google Scholar). Various lines of evidence suggest that GTP-bound αt activates PDE6 by physically displacing the inhibitory region of Pγ from the Pαβ catalytic pocket, thus initiating the signaling state of phototransduction (5Barren B. Gakhar L. Muradov H. Boyd K.K. Ramaswamy S. Artemyev N.O. EMBO J. 2009; 28: 3613-3622Crossref PubMed Scopus (46) Google Scholar, 8Song J. Guo L.W. Muradov H. Artemyev N.O. Ruoho A.E. Markley J.L. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 1505-1510Crossref PubMed Scopus (79) Google Scholar, 10Skiba N.P. Artemyev N.O. Hamm H.E. J. Biol. Chem. 1995; 270: 13210-13215Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 11Granovsky A.E. Artemyev N.O. Biochemistry. 2001; 40: 13209-13215Crossref PubMed Scopus (29) Google Scholar, 12Artemyev N.O. Natochin M. Busman M. Schey K.L. Hamm H.E. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 5407-5412Crossref PubMed Scopus (53) Google Scholar, 20Slep K.C. Kercher M.A. He W. Cowan C.W. Wensel T.G. Sigler P.B. Nature. 2001; 409: 1071-1077Crossref PubMed Scopus (222) Google Scholar). In the ensuing transition state, αtGTP is converted back to the GDP-bound inactive structure, which has lowered affinity with Pγ, thus releasing it to reinhibit PDE6 and terminate signaling (3Burns M.E. Arshavsky V.Y. Neuron. 2005; 48: 387-401Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). Fast visual recovery is ensured by great acceleration of the αt GTPase activity, which is achieved by the GTPase-activating protein (GAP) complex composed of αt, Pγ, RGS9-1 (the ninth member of the regulators of G-protein signaling in photoreceptors), and its constitutive partner Gβ5 as well as the membrane anchoring protein R9AP (3Burns M.E. Arshavsky V.Y. Neuron. 2005; 48: 387-401Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 21Cheever M.L. Snyder J.T. Gershburg S. Siderovski D.P. Harden T.K. Sondek J. Nat. Struct. Mol. Biol. 2008; 15: 155-162Crossref PubMed Scopus (85) Google Scholar). Much of the molecular details of the Pγ-αtGTP interaction in the signaling state have been learned from the crystal structure of the partial transition state complex, which includes the GDP-AlF4−-bound αt/i1 chimera, the half-Pγ (Gly46–Ile87), and the catalytic core of RGS9-1 (20Slep K.C. Kercher M.A. He W. Cowan C.W. Wensel T.G. Sigler P.B. Nature. 2001; 409: 1071-1077Crossref PubMed Scopus (222) Google Scholar). As visualized by this structure, a stretch of Pγ residues around Trp70 forms a tight interaction with αt that is further reinforced by additional contacts provided by some residues in the Pγ inhibitory region. Recent NMR (8Song J. Guo L.W. Muradov H. Artemyev N.O. Ruoho A.E. Markley J.L. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 1505-1510Crossref PubMed Scopus (79) Google Scholar) and crystallography (5Barren B. Gakhar L. Muradov H. Boyd K.K. Ramaswamy S. Artemyev N.O. EMBO J. 2009; 28: 3613-3622Crossref PubMed Scopus (46) Google Scholar) studies indicated that when the Pγ inhibitory region was associated with the chimeric PDE5/6 catalytic domain, the critical αt-binding residues Trp70 and Leu76, however, were not involved. These studies lend further support to a model of PDE6 activation (5Barren B. Gakhar L. Muradov H. Boyd K.K. Ramaswamy S. Artemyev N.O. EMBO J. 2009; 28: 3613-3622Crossref PubMed Scopus (46) Google Scholar, 11Granovsky A.E. Artemyev N.O. Biochemistry. 2001; 40: 13209-13215Crossref PubMed Scopus (29) Google Scholar); i.e. an engagement of αtGTP with the Pγ residues Trp70 and Leu76 triggers a conformational change involving a hingelike rigid body movement of Pγ(78–87) away from the PDE6 catalytic pocket.Thus, Pγ plays a pivotal role, not only for turning on but also for turning off phototransduction and keeping the signaling system inactive in the dark (9Guo L.W. Ruoho A.E. Curr. Protein Pept. Sci. 2008; 9: 611-625Crossref PubMed Scopus (14) Google Scholar). Despite a wealth of information regarding phototransduction mechanisms, dynamic interactions of Pγ with αt and Pαβ, as well as RGS9-1, are not well understood. There has been controversy as to whether Pγ completely dissociates from Pαβ in the process of PDE6 activation. It is possible that whereas αt sequesters the Pγ C-terminal region from the Pαβ catalytic domain, the central domain of Pγ stays bound to the Pαβ GAF domain until the binding is allosterically reduced by the dissociation of cGMP from the GAF domain (1Cote R.H. Photoreceptor Phosphodiesterase (PDE6): A G-protein-activated PDE Regulating Visual Excitation in Rod and Cone Photoreceptor Cells. CRC Press, Inc., Boca Raton, FL2006: 165-193Google Scholar). This scenario of simultaneous Pγ interactions with both αt and Pαβ is consistent with the proposition of an intermediate αt·PDE6 complex during PDE6 activation (17Artemyev N.O. Rarick H.M. Mills J.S. Skiba N.P. Hamm H.E. J. Biol. Chem. 1992; 267: 25067-25072Abstract Full Text PDF PubMed Google Scholar, 22Navon S.E. Fung B.K. J. Biol. Chem. 1988; 263: 489-496Abstract Full Text PDF PubMed Google Scholar, 23Catty P. Pfister C. Bruckert F. Deterre P. J. Biol. Chem. 1992; 267: 19489-19493Abstract Full Text PDF PubMed Google Scholar, 24Clerc A. Bennett N. J. Biol. Chem. 1992; 267: 6620-6627Abstract Full Text PDF PubMed Google Scholar, 25Tsang S.H. Woodruff M.L. Chen C.K. Yamashita C.Y. Cilluffo M.C. Rao A.L. Farber D.B. Fain G.L. J. Neurosci. 2006; 26: 4472-4480Crossref PubMed Scopus (43) Google Scholar, 26Norton A.W. D'Amours M.R. Grazio H.J. Hebert T.L. Cote R.H. J. Biol. Chem. 2000; 275: 38611-38619Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Earlier studies suggested that direct αt-Pαβ contacts may be a driving force in forming the intermediate complex in the presence of disc membranes (24Clerc A. Bennett N. J. Biol. Chem. 1992; 267: 6620-6627Abstract Full Text PDF PubMed Google Scholar, 27Clerc A. Catty P. Bennett N. J. Biol. Chem. 1992; 267: 19948-19953Abstract Full Text PDF PubMed Google Scholar). However, it has not been determined whether the Pγ interactions with αt and Pαβ contribute important elements to the intermediate PDE6 activation complex.As presented in this study, the label transfer approach, which has proven to be powerful for systematically detecting interactions of full-length molecules (16Guo L.W. Muradov H. Hajipour A.R. Sievert M.K. Artemyev N.O. Ruoho A.E. J. Biol. Chem. 2006; 281: 15412-15422Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 28Guo L.W. Grant J.E. Hajipour A.R. Muradov H. Arbabian M. Artemyev N.O. Ruoho A.E. J. Biol. Chem. 2005; 280: 12585-12592Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 29Grant J.E. Guo L.W. Vestling M.M. Martemyanov K.A. Arshavsky V.Y. Ruoho A.E. J. Biol. Chem. 2006; 281: 6194-6202Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), offered us an opportunity to investigate this issue from a unique perspective. The data obtained through label transfer, immunoprecipitation, and pull-down suggest that complementary interactions, in which the Pγ C-terminal domain forms a strong interaction with αt while the central region binds tightly with Pαβ, assist the transducin·PDE6 complex formation, which elicits PDE6 activation.EXPERIMENTAL PROCEDURESThe chemicals and reagents used in this study were from the sources described previously (16Guo L.W. Muradov H. Hajipour A.R. Sievert M.K. Artemyev N.O. Ruoho A.E. J. Biol. Chem. 2006; 281: 15412-15422Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 28Guo L.W. Grant J.E. Hajipour A.R. Muradov H. Arbabian M. Artemyev N.O. Ruoho A.E. J. Biol. Chem. 2005; 280: 12585-12592Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) unless otherwise stated. The C-terminal Pγ peptide (Pγ(62–87)) was custom-synthesized at the Peptide Synthesis Facility of the Biotechnology Center, University of Wisconsin (Madison, WI).Transducin PreparationUsing frozen dark-adapted bovine retinas (J. A. & W. L. Lawson Co.), rod outer segment (ROS) membranes were isolated, from which holotransducin was prepared as described previously (29Grant J.E. Guo L.W. Vestling M.M. Martemyanov K.A. Arshavsky V.Y. Ruoho A.E. J. Biol. Chem. 2006; 281: 6194-6202Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 30Liu Y. Arshavsky V.Y. Ruoho A.E. J. Biol. Chem. 1996; 271: 26900-26907Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). αtGDP and βγt were then purified from holotransducin using a blue Sepharose CL-6B column. To prepare αtGTPγS, GTPγS was added to ROS membranes, and αtGTPγS was thus released and purified on the blue Sepharose CL-6B column. The purity of αt was determined to be >95% by SDS-PAGE and Coomassie staining. The purified proteins were stored at −80 °C.Preparation of PDE6The samples of bovine PDE6 were kindly provided by Dr. Nikolai O. Artemyev at the University of Iowa and prepared according to established methods (4Artemyev N.O. Arshavsky V.Y. Cote R.H. Methods. 1998; 14: 93-104Crossref PubMed Scopus (30) Google Scholar). Briefly, holo-PDE6 was extracted from bleached ROS membranes, and Pαβ was then obtained by removing Pγ through mild tryptic proteolysis of holo-PDE6. More vigorous tryptic treatment generated the Pαβ heterodimer with a nick at Lys146/Lys147 on Pβ. It has been reported that nicked Pαβ has unaltered functional properties (12Artemyev N.O. Natochin M. Busman M. Schey K.L. Hamm H.E. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 5407-5412Crossref PubMed Scopus (53) Google Scholar, 16Guo L.W. Muradov H. Hajipour A.R. Sievert M.K. Artemyev N.O. Ruoho A.E. J. Biol. Chem. 2006; 281: 15412-15422Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Unless otherwise stated, “Pαβ” refers to nicked Pαβ throughout this paper. The Pαβ preparations were purified to >95% by a Mono-Q column (Amersham Biosciences), as judged from Coomassie-stained SDS gels.Preparation of Pγ PhotoprobesThe constructs for expressing the full-length wild type Pγ with the single cysteine at position 68 (29Grant J.E. Guo L.W. Vestling M.M. Martemyanov K.A. Arshavsky V.Y. Ruoho A.E. J. Biol. Chem. 2006; 281: 6194-6202Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), and the single cysteine mutants were generated as described previously (28Guo L.W. Grant J.E. Hajipour A.R. Muradov H. Arbabian M. Artemyev N.O. Ruoho A.E. J. Biol. Chem. 2005; 280: 12585-12592Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). They were expressed in E. coli and purified by chitin beads, followed by reversed-phase HPLC using the POROS 20 R2 resin (31Guo L.W. Assadi-Porter F.M. Grant J.E. Wu H. Markley J.L. Ruoho A.E. Protein Expr. Purif. 2007; 51: 187-197Crossref PubMed Scopus (16) Google Scholar). The truncated Pγ variants (29Grant J.E. Guo L.W. Vestling M.M. Martemyanov K.A. Arshavsky V.Y. Ruoho A.E. J. Biol. Chem. 2006; 281: 6194-6202Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) with and without a His6 tag at the N terminus (HisPγ(1–61) and Pγ(1–61), respectively) were prepared using the same protocol. Full-length Pγ (>95% pure) was used for preparation of Pγ photoprobes. The radioactive [125I]ACTP-Pγ and non-radioactive [127I]ACTP-Pγ photoprobes were prepared as described earlier (28Guo L.W. Grant J.E. Hajipour A.R. Muradov H. Arbabian M. Artemyev N.O. Ruoho A.E. J. Biol. Chem. 2005; 280: 12585-12592Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar).The maleimido benzophenone (mBP)-Pγ photoprobes were prepared as described previously (16Guo L.W. Muradov H. Hajipour A.R. Sievert M.K. Artemyev N.O. Ruoho A.E. J. Biol. Chem. 2006; 281: 15412-15422Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Briefly, Pγ was derivatized with mBP in 10–20-fold molar excess, and mBP-Pγ was then separated from unreacted Pγ and free mBP through reversed phase HPLC. Correct molecular masses of the [127I]ACTP-Pγ and mBP-Pγ photoprobes have been confirmed by electrospray ionization mass spectrometry conducted at the Chemistry Department Mass Spectrometry Facility of the University of Wisconsin (Madison, WI).Functional Assay of the Pγ PhotoprobesThe transducin GTPase activity assay was kindly conducted by Dr. Kirill A. Martemyanov (now at the University of Minnesota) and Dr. Vadim Y. Arshavsky (now at Duke University), using a single turnover technique as described previously (32Martemyanov K.A. Arshavsky V.Y. J. Biol. Chem. 2002; 277: 32843-32848Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The assay was conducted at room temperature (22–24 °C) in a buffer containing 25 mm Tris-HCl (pH 8.0), 140 mm NaCl, and 8 mm MgCl2. The urea-treated ROS membranes, lacking endogenous activity of RGS9-1, were used as a source for the photoexcited rhodopsin required for transducin activation. The reactions were initiated by the addition of 10 μl of 0.6 μm [32P]GTP (∼105 dpm/sample) to 20 μl of urea-treated ROS membranes (20 μm final rhodopsin concentration) reconstituted with transducin heterotrimer (1 μm) and recombinant RGS9-1·Gβ5 complex (0.5 μm). The reactions were performed in either the absence or presence of Pγ derivatives (1 μm). The reaction was stopped by the addition of 100 μl of 6% perchloric acid. The 32P formation was measured with activated charcoal. All assays were conducted in the absence of reducing agent due to the presence of the disulfide linkage between the photoreactive group and Pγ.Photocross-linking/Label Transfer Using Pγ PhotoprobesA scheme is presented in supplemental Fig. S1A to explain the label transfer strategy. Unless otherwise described, photocross-linking reactions were performed in the HEPES buffer (10 mm HEPES, pH 7.5, 120 mm NaCl, 5 mm MgCl2). Samples were contained in ultraclear polypropylene microcentrifuge tubes (Axygen). The reactions using [125I]ACTP-Pγ photoprobes were exposed to the UV light generated by an AH-6 water-jacketed 1000-watt high pressure mercury lamp for 5 s at a distance of 10 cm (28Guo L.W. Grant J.E. Hajipour A.R. Muradov H. Arbabian M. Artemyev N.O. Ruoho A.E. J. Biol. Chem. 2005; 280: 12585-12592Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The reactions with mBP-Pγ were photolyzed at 5–10 °C for 2 × 15 min with a 5-min dark interval on ice in an RPR-100 Rayonet photochemical reactor equipped with 18 bulbs of 350 nm (Southern New England Ultraviolet Company). Immediately after photolysis, sample buffer was added to the reactions to final concentrations of 1% SDS and 50 mm DTT. The proteins were separated by SDS-PAGE and then subjected to Coomassie Blue staining and autoradiography. Autoradiography and protein quantitation were performed as described previously (28Guo L.W. Grant J.E. Hajipour A.R. Muradov H. Arbabian M. Artemyev N.O. Ruoho A.E. J. Biol. Chem. 2005; 280: 12585-12592Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar).Pull-down Assays of Pγ Interactions with αtGTPγS and Pαβ Using Affinity BeadsTo immobilize the full-length Pγ to the streptavidin beads, biotinylated Pγ was prepared by covalently attaching maleimide-PEO2-biotin (Pierce Biotechnology) to the single cysteine at position 3 of the Pγ mutant, L3C (28Guo L.W. Grant J.E. Hajipour A.R. Muradov H. Arbabian M. Artemyev N.O. Ruoho A.E. J. Biol. Chem. 2005; 280: 12585-12592Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The derivatization reaction and purification of the Btn-L3C derivative were performed following the protocol of mBP-Pγ preparation (16Guo L.W. Muradov H. Hajipour A.R. Sievert M.K. Artemyev N.O. Ruoho A.E. J. Biol. Chem. 2006; 281: 15412-15422Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). To prepare Btn-Pγ(46–87), the Pγ 87C mutant was first derivatized with maleimide-PEO2-biotin and then trypsinized, and Btn-Pγ(46–87) was purified by reversed phase HPLC using a C4 column (31Guo L.W. Assadi-Porter F.M. Grant J.E. Wu H. Markley J.L. Ruoho A.E. Protein Expr. Purif. 2007; 51: 187-197Crossref PubMed Scopus (16) Google Scholar).For each pull-down reaction, 0.4 μl of Ultra-Link Plus immobilized streptavidin gel (Pierce) was first equilibrated with the pull-down buffer, which contains 20 mm HEPES, pH 7.5, 120 mm NaCl, 5 mm MgCl2, 1 mm DTT, 0.1% n-dodecanoylsucrose (Calbiochem), and 50 μg/ml BSA, and then incubated with 4 μm Btn-Pγ by rotating the microcentrifuge tube for 10 min at room temperature. A high concentration (1 μg/μl) of BSA or soybean trypsin inhibitor was added at this step to block possible nonspecific protein-bead interactions. After this incubation, Btn-Pγ was found completely bound to the streptavidin beads (data not shown). To test if the Pγ peptides disrupt the Pγ-αtGTPγS or Pγ-Pαβ interaction, Pγ(62–87) or Pγ(1–61) in excess over Btn-Pγ was first incubated with αtGTPγS or Pαβ in the pull-down buffer for 1 h on ice and then added to the Btn-Pγ-streptavidin beads. After rotating the reactions at 4 °C for 1–2 h, the beads were washed twice with 400 μl of ice-cold pull-down buffer. Proteins were then eluted from the beads with SDS/DTT-containing sample buffer, run on a low cross-link 15% acrylamide gel (33Baehr W. Devlin M.J. Applebury M.L. J. Biol. Chem. 1979; 254: 11669-11677Abstract Full Text PDF PubMed Google Scholar), and visualized by staining with Coomassie Blue R-250, or SilverSNAP Stain Kit II (Pierce) when lower amounts of proteins were used.To study the interactions of the Pγ central region with Pαβ and αt, the Pγ construct HisPγ(1–61) was used. For each reaction, 0.5 μl of His-Select High-Flow nickel beads (Sigma) were first washed with 500 μl of H2O and then with 300 μl of pull-down buffer. HisPγ(1–61) of 5 μm was immobilized to the beads by incubation in the pull-down buffer (supplemented with 1 μg/μl trypsin inhibitor at this step) at room temperature for 10 min on rotating. Twenty mm imidazole was included in the pull-down buffer throughout the experimental procedures to prevent possible nonspecific binding of proteins to nickel beads. After the beads were washed with 2 × 500 μl of pull-down buffer to remove unbound HisPγ(1–61), Pαβ or/and αtGTPγS were added and incubated with the beads for 1–2 h at 4 °C. The beads were then washed twice with 200 μl of pull-down buffer. The proteins on the beads were eluted with the sample buffer and resolved by SDS-PAGE using a low cross-link 15% gel (33Baehr W. Devlin M.J. Applebury M.L. J. Biol. Chem. 1979; 254: 11669-11677Abstract Full Text PDF PubMed Google Scholar), which was then silver-stained using SilverSNAP Stain Kit II (Pierce).Immunoprecipitation Assay of the αtGTPγS-PDE6 InteractionCo-immunoprecipitation of αtGTPγS with holo-PDE6 was carried out using nProtein A Sepharose Fast-Flow beads (Amersham Biosciences) and the antibody against bovine rod Pα (Affinity Bioreagents). For each reaction, 0.5 μl of Protein A beads were first equilibrated with the HEPES buffer (10 mm HEPES, pH 7.5, 120 mm NaCl, 5 mm MgCl2) and then incubated with 0.5 μg of the anti-Pα antibody by rotating for 1 h at 4 °C. One μg/μl soybean trypsin inhibitor was included to block possible nonspecific protein binding sites on Protein A beads. The beads were washed three times with 300 μl of HEPES buffer prior to the immunoprecipitation reaction. Meanwhile, 0.5 μg of holo-PDE6 was incubated for 1–2 h on ice with 0.5 μg of αtGTPγS in the HEPES buffer containing 50 μg/ml trypsin inhibitor and 1 mm DTT. The reaction was then added to the washed Protein A beads with anti-Pα bound and incubated for 1 h by rotating at 4 °C. Pγ peptide Pγ(1–61) or Pγ(62–87) in a 200-fold molar excess ov" @default.
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• W2094397762 title "Complementary Interactions of the Rod PDE6 Inhibitory Subunit with the Catalytic Subunits and Transducin" @default.
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