heterotrimeric g-protein effectors

Preface Transduction of signals through G-protein pathways is achieved largely by production of intracellular messengers that either directly or through protein kinases regulate cellular biochemical and physiological processes Several enzymes, including adenylyl cyclase, phospholipase C, and the cGMP phosphodiesterase, are well established as effectors directly regulated by G proteins These enzymes are covered in this volume Others such as phospholipase A2 and phospholipase D are often described as G-protein effectors Though receptors that couple to G proteins undoubtedly regulate the activity of these enzymes, there is no compelling evidence at this time to indicate these enzymes are directly regulated by G-protein subunits Hence these enzymes are not included Over the past two years it has become increasingly obvious that there is considerable molecular and functional diversity of the effector enzymes as well Eight mammalian Gs-sensitive adenylyl cyclases and four Gqstimulated phospholipases C-fl have been cloned, and many of these have been characterized as having distinct capabilities for signal input The varied functional characteristics and the tissue-specific distribution of the effector isoforms allow the various cell types and tissues to develop customized response systems by altering the mix of the effector isoforms Several approaches to characterize the molecular and functional identities of the effector isoforms are presented Several mitogens use G-protein pathways to communicate proliferative signals At this time it is not clear if the immediate effectors in Gprotein signaling pathways are those that we already know of or are as yet unidentified ones Hence techniques currently used to study G-protein regulation of cell proliferation measure the activity of downstream elements These techniques are nevertheless useful in tracing G-protein pathways and are covered in this volume G proteins can also modulate cellular functions by presumably direct regulation of channels to alter the flow of ions through the plasma membranes To date there have been no descriptions of reconstitution experiments in which purified G-protein subunits can alter the function of purified ion channels The circumstantial evidence for direct regulation is substantial, so it is reasonable to assume that some channels will be Gprotein effectors A number of imaging and electrophysiological techniques relevant to G-protein regulation of channels are included In selecting the chapters for this volume an attempt was made to restrict areas covered to those likely to be of direct interest to researchers xiii xiv PREFACE working in cell surface signal transducing systems Such selections are undoutedly subjective, and I am certain that there are some areas that are not as thoroughly covered as they could be However, G-protein effector research is a very active area in many laboratories including my own, and undoubtedly techniques emerging from these studies will have to be covered at later dates I would like to thank the authors for their contributions I am especially thankful to those involved in phospholipase C research, a field that moved at a brisk pace during the year these chapters were being compiled, for providing chapters documenting the very latest advances I am also grateful to Ms Lina Mazzella for her unfailingly cheerful help in organizing the chapters RAVI IYENGAR Contributors to V o l u m e Article numbers are in parenthesesfollowingthe names of contributors Affiliationslisted are current ROBERT ALVAREZ (3), Institute of Pharma- M P CAULFIELD (30), Department of Phar- cology, Syntex Research, Paid Alto, California 94304 JEFF AMUNDSON (27), Department of Pharmacology, Mayo Foundation, Rochester, Minnesota 55905 RODRIGO ANDRADE (29), Department of Pharmacological and Physiological Sciences, St Louis University School of Medicine, St Louis, Missouri 63130 NIKOLAI O ARTEMYEV (2), Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612 ANDREW BALL (12), Department of Physiology, University College London, London WC1E 6JJ, United Kingdom JONATHAN L BLANK (19), Division of Basic Sciences, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206 ROBERT D BLITZER (11, 32), Departments of Psychiatry and Pharmacology, Bronx Veterans Admnistrations Medical Center, and Mount Sinai School of Medicine, New York, New York 10029 ANTHONY A BOMINAAR (16), Department of Biochemistry, University of Groningen, 9747 AG Groningen, The Netherlands D A BROWN (30), Department of Pharmacology, University College London, London WC1E 6BT, United Kingdom KEVlN P CAMPBELL (28), Howard Hughes Medical Institute, College of Medicine, University of Iowa, Iowa City, Iowa 52242 MONTSERRAT CAMPS (14), Molecular Pharmacology Division, German Cancer Research Center, 69120 Heidelberg, Germany macology, University College London, London WCIE 6BT, United Kingdom JIANQIANG CHEN (8), Department of Phar- macology, Mount Sinai School of Medicine, City University of New York, Ne~t York, New York 10029 DAVID E CLAPHAM (27), Department of Pharmacology, Mayo Foundation, Rochester, Minnesota 55905 SHAMSHAD COCKCROFT (12, 13), Department of Physiology, University College London, London WCIE 6JJ, United Kingdom DERMOT M F COOPER (5), Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262 ADRIENNE D C o x (23, 24), Departments of Radiation Oncology and Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 EMER CUNNINGHAM , 13), Department of Physiology, University College London, London WCIE 6JJ, United Kingdom MICHAEL DE VIVO (10), Department of Pharmacology, Mount Sinai School of Medicine, City University of New York, New York, New York 10029 NICOLAS DEMAUREX (26), Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1)(8 CHANNING J DER (23, 24), Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 MICHEL DE WAARD (28), Howard Hughes Medical Institute, College of Medicine, University of lowa, Iowa City, Iowa 52242 ix X CONTRIBUTORS TO VOLUME 238 J H EXTON (19), Department of Molecular DEOK-YOUNGJHON (17), Laboratory of BioPhysiology and Biophysics, Howard chemistry, National Heart, Lung, and Hughes Medical Institute, Vanderbilt Blood Institute, National Institutes of University School of Medicine, Nashville, Health, Bethesda, Maryland 20892 Tennessee 37232 GARY L JOHNSON (22), Division of Basic ANNE M GARDNER (22), Division of Basic Sciences, National Jewish Center for ImSciences, National Jewish Center for Immunology and Respiratory Medicine, munology and Respiratory Medicine, Denver, Colorado 80206, and Department Denver, Colorado 80206 of Pharmacology, University of Colorado Medical School, Denver, Colorado 80262 PETER GIERSCmK (14), Department of Pharmacology and Toxicology, University of ROGER A JOHNSON (3, 4), Department of Ulm, 89069 Ulm, Germany Physiology and Biophysics, School of ALFRED G GILMAN (7), Department of Medicine, Health Sciences Center, State Pharmacology, University of Texas University of New York at Stony Brook, Southwestern Medical Center, Dallas, Stony Brook, New York 11794 Texas 75235 STEVEN D KAHL (28), Howard Hughes HEIDI E HAMM (2), Department of PhysiolMedical Institute, College of Medicine, ogy and Biophysics, University of lllinois University of lowa, Iowa City, Iowa 52242 at Chicago, Chicago, Illinois 60612 KARL-HEINZ KRAUSE (26), Infectious DisT KENDALL HARDEN (15), Department of eases Division, University Hospital, CHPharmacology, School of Medicine, Uni1211 Geneva 4, Switzerland versity of North Carolina at Chapel Hill, YOSHIHISA KURACHI (34), Division of CarChapel Hill, North Carolina 27599 diovascular Diseases, Departments of lnternal Medicine and Pharmacology, ANNE E HARWOOD (21), Division of Basic Sciences, National Jewish Center for ImMayo Foundation, Rochester, Minnesota munology and Respiratory Medicine, 55905, and Department of Pharmacology Denver, Colorado 80206, and Department H, Faculty of Medicine, Osaka University of Pharmacology, University of Colorado Medical School, Suita, Osaka 565, Japan Medical School, Denver, Colorado 80262 EMMANUEL M LANDAU (] ], 32), DepartCRAIG A HAUSER (23), Cancer Research ments of Psychiatry and Pharmacology, Center, La Jolla Cancer Research FounBronx Veterans Administration Medical dation, La Jolla, California 92037 Center, and Mount Sinai School of Medicine, New York, New York 10029 JORGEN HESCHELER(31), lnstitut far Pharmakologie, Freie Universitdt Berlin, CAROL A LANGE-CARTER(22), Division of 14195 Berlin, Germany Basic Sciences, National Jewish Center for Immunology and Respiratory MediYEE-KIN HO (1), Departments of Biochemcine, Denver, Colorado 80206 istry and Ophthalmology, University of Illinois at Chicago, Chicago, Illinois 60612 CHANG-WON LEE (18), Laboratory of Biochemistry, National Heart, Lung, and RAVI IYENGAR(8, 20), Department of PharBlood Institute, National Institutes of macology, Mount Sinai Medical Center, Health, Bethesda, Maryland 20892 New York, New York 10029 VEER JACOBOWITZ(8), Department of Phar- KWEON-HAENG LEE (18), Laboratory of Biochemistry, National Heart, Lung, and macology, Mount Sinai School of MediBlood Institute, National Institutes of cine, City University of New York, New Health, Bethesda, Maryland 20892 York, New York 10029 MARISA E E JACONI (26), Department of P DANIEL LET (26), Infectious Diseases Division, University Hospital, CH-1211 Pharmacology, Mayo Foundation, RochGeneva 4, Switzerland ester, Minnesota 55905 CONTRIBUTORS TO VOLUME 238 HAI-WEN MA (20), Department of Pharmacology, Mount Sinai Medical Center, New York, New York 10029 I MCFADZEAN (30), Pharmacology Group, King's College London, London SW3 6LX, United Kingdom JOHN S MILLS (2), Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612 ANDREW J MORRIS (15), Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 DONGEUN PARK 07), Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 RICHARD T PREMONT (9, 20), Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710 HELEN M RARICK(2), Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612 STEPHEN R RAWLINGS (25, 26), Foundation for Medical Research, Department of Medicine, University of Geneva, CH-1211 Geneva 4, Switzerland SUE Goo RULE (17, 18), Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 YORAM SALOMON (3), Weizmann Institute of Science, Department of Hormone Research, Rehovot 76100, Israel WERNER SCHLEGEL(25, 26), Foundation for Medical Research, Department of Medicine, University of Geneva, CH-1211 Geneva 4, Switzerland 1LANA SHOSHANI(4), Department of Physiology and Biophysics, School of Medicine, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, New York 11794 N1KOLAI P SKIBA (2), Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612 xi LISA STEHNO-BITTEL (27), Department of Pharmacology, Mayo Foundation, Rochester, Minnesota 55905 WEI-JEN TANG (7), Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235 ANDREATAR (1), Department of Biochemistry, University of Illinois at Chicago, Chicago, Illinois 60612 RONALD TAUSSIG(7), Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235 ANDRl~ TERZIC (34), Departments of Internal Medicine and Pharmacology, Mayo Foundation, Rochester, Minnesota 55905 JEAN-MARC THELER (25), Division of Clinical Biochemistry, Department of Medicine, University of Geneva, CH-1211 Geneva 4, Switzerland GERAINT M H THOMAS (12, 13), Department of Physiology, University College London, London WCIE 6JJ, United Kingdom T u o w D TING (1), Department of Biochemistry, University of Illinois at Chicago Chicago, Illinois 60612 RICHARD R VA1LLANCOURT(21, 22), Division of Basic Sciences, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206 Y VALLIS (30), Department of Pharmacology, University College London, London WCIE 6BT, United Kingdom PETER J M VAN HAASTERT(16), Department of Biochemistry, University of Groningen, 9747 AG Groningen, The Netherlands GARY L WALDO (15), Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 SIM WINITZ (21), Division of Basic Sciences, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206, and Department of Pharmacology, University of Colorado Medical School, Denver, Colorado 80262 xii CONTRIBUTORS TO VOLUME 238 DERRICK R WITCHER (28), Howard Hughes MITSUHIKO YAMADA (34), Pharmacology Medical Institute, College of Medicine, University of lowa, Iowa City, Iowa 52242 YUNG H WONG (6), Department of Biology, Hong Kong University of Science and Technology, Kowloon, Hong Kong H, Osaka University Medical School, Suita, Osaka 565, Japan ATSUKO YATANI (33), Departments of Pharmacology and Cell Biophysics, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267 [1] PURIFYING RETINAL cGMP PHOSPHODIESTERASE [1] P u r i f i c a t i o n of B o v i n e R e t i n a l c G M P P h o s p h o d i e s t e r a s e By ANDREA TAR, TUOW D TING, a n d YEE-KIN H o Introduction Visual excitation in vertebrate rod photoreceptor cells involves a light-activated cGMP enzyme cascade in the rod outer segment (ROS) Absorption of a photon by the receptor molecule, rhodopsin (R*), leads to the activation of a latent cGMP phosphodiesterase (PDE) which rapidly hydrolyzes cytosolic cGMP The transient decrease in cGMP concentration causes the closure of cGMP-sensitive cation channels in the plasma membrane and results in hyperpolarization of the cell 1,2 The PDE can either be bound on ROS disk membranes or exist in a soluble form in the cytosol In both forms, PDE is a latent enzyme complex composed of three types of polypeptides, P~ (88 kDa), P~ (84 kDa), and P~ (14 kDa), with a ratio of : : Polypeptides P~ and P~ contain separate catalytic sites which are inhibited by the binding of the inhibitory P~ subunits Signal coupling between photolyzed rhodopsin (R*) and PDE is mediated by a signal-transducing G protein called transducin (T) via a GTP-binding and hydrolysis cycle Transducin is a trimeric protein composed of three polypeptides: T~ (39 kDa), T¢ (37 kDa), and T~ (8.5 kDa) In the dark-adapted state, T~ contains a bound GDP (T~-GDP) and interacts with T~ The T~-GDP • T~ complex tightly associates with rhodopsin On photoexcitation, R* catalyzes a GTP/GDP exchange reaction converting T~-GDP to the active form of T~-GTP The T~-GTP complex activates PDE by relieving the restraint exerted by the P~ inhibitory subunit on the P~ and Po catalytic sites After the hydrolysis of the tightly bound GTP, T~-GDP releases P~ and recombines with T~ In turn, P~ shuts off the cGMP hydrolysis, and the cascade is ready for another cycle of activation This chapter delineates methods for the purification of retinal cGMP PDE from bovine retinas and also describes biochemical asssays for the enzymatic activities i M L Applebury and P A Hargrave, Vision Res 26, 1881 (1986) -' L Stryer, J Biol Chem 266, 10711 (1991) Y.-K Ho, V N Hingorani, S E Navon, and B K.-K Fung, Curr Top Cell Regul 30, 171 (1989) METHODS IN ENZYMOLOGY, VOL 238 Copyright © 1994 by Academic Press, Inc All rights of reproduction in any form reserved cGMP PHOSPHODIESTERASE [1] Purification of Retinal cGMP Phosphodiesterase The purification procedure was originally developed by Hurley and Stryer and was modified for large-scale preparation from 400 retinas 4-6 The y subunit of PDE can be isolated from bovine retina according to the procedure of Hurley Phosphodiesterase can be isolated from Rana catesbiana according to a procedure of Yamazaki et al This chapter describes the purification of bovine PDE from crude ROS membranes prepared from frozen retinas As a peripheral membrane protein, PDE can be extracted from photolyzed ROS membranes by repeatedly washing with low ionic strength buffer Transducin, the other major peripheral membrane protein, remains tightly associated with R* in the absence of GTP, and transducin subunits are retained on the ROS membrane during low ionic extraction The extracted PDE is further purified by column chromatography.9 An isolation process using 400 retinas typically yields more than mg of pure PDE Based on the convenience of the purification scheme, it is recommended that a minimum of 200 retinas be used to obtain optimal yields Preparation of Crude Rod Outer Segment Membranes for Phosphodiesterase Extraction Frozen dark-adapted bovine retinas can be purchased from supply houses (G A Hormel Co., Austin, MN, or J A Lawson Co., Lincoln, NE) Fresh bovine eyes are collected from local packing companies and kept in the dark for several hours prior to dissection of the retinas under dim red light (Kodak, Rochester, NY, red No safety light filter) These retinas can be stored in the dark at - ° for over a year without loss of activity of the enzymes involved in the cGMP cascade The isolation procedure is carried out under dim red light to keep rhodopsin in the darkadapted state Two liters of ice-cold isolation buffer is required, which consists of 10 mM MOPS [3-(N-morpholino)propanesulfonic acid], 60 mM KCI, 30 mM NaCI, mM MgCI2, 0.1 mM PMSF (phenylmethylsulfonyl fluoride), and mM DTT (dithiothreitol) at pH 7.5 Two 500 ml sucrose solutions are prepared using the isolation buffer, namely, a 50% (w/v) solution and a 38% (w/v) solution B K.-K Fung, J Biol Chem 258, 10495 (1983) B K.-K Fung, J B Hurley, and L Stryer, Proc Natl Acad Sci U,S.A 78, 152 (1981) Y.-K Ho and B K.-K Fung, J Biol Chem 259, 6694 0984) j B Hurley, this series, Vol 81, p 542 s A Yamazald, N Mild,and M W Bitensky, this series, Vol 81, p 526 j B Hurley and L Stryer, J Biol Chem 257, 11094 (1982) [1] PURIFYING RETINAL cGMP PHOSPHODIESTERASE For the preparation of crude ROS membranes, 400 frozen retinas are thawed in 200-350 ml of the 50% sucrose buffer The suspension is transferred to a beaker and stirred with a magnetic stirrer for approximately 20 The retinal suspension is forced twice through a 50-ml syringe without a needle to break up the tissue, then divided equally among sixteen 50-ml centrifuge tubes used in superspeed centrifuges (Du Pont Sorval RC5 centrifuges with SS-34 rotors, Du Pont Company, Wilmington, DE; or Beckman Instruments J2-21 centrifuges with J-21 rotors, Beckman Instruments Inc., Palo Alto, CA) The volume of each tube is adjusted to 40 ml with 50% sucrose buffer After the contents are mixed by shaking, the suspension is centrifuged at 15,000 rpm (27,000 g) for 15 at 4° Under these conditions, the ROS membranes float to the top of the tubes, while the remainder of the retinal membranes pellet to the bottom With a rubber policeman, the floating ROS membranes are scraped off the walls of the tubes and resuspended in the supernatant The supernatant is gently poured off and distributed equally among 28 clean 50-ml centrifuge tubes The volume in each tube is increased to 40 ml with isolation buffer without sucrose to dilute the sucrose concentration to approximately 28% The tubes are shaken and centrifuged at 18,000 rpm (38,000 g) for 15 at 4° The ROS membranes sediment to the bottom of the tubes After discarding the supernatant, the ROS membrane pellets are resuspended in 150 ml of 38% sucrose in isolation buffer and divided equally among 16 tubes The volume of each tube is adjusted to 30 ml with the 38% sucrose buffer, and the tubes are centrifuged at 18,000 rpm for 45 The ROS membranes float to the top of the tubes and are resuspended in the supernatant The 38% sucrose membrane suspension is divided among 16 clean centrifuge tubes To reduce the sucrose concentration, the volume of each tube is increased to 45 ml with isolation buffer After mixing, the tubes are centrifuged at 19,000 rpm (43,000 g) for 20 at 4° to pellet the ROS membranes The supernatants are discarded, and the pellets are resuspended with 40 ml of isolation buffer The samples are centrifuged again at 19,000 rpm for 20 at 4° Residual sucrose is removed by washing the ROS membranes twice more with isolation buffer The final ROS membranes are resuspended with 40 ml of isolation buffer and divided equally among four tubes The crude ROS membranes can be kept in the dark overnight on ice prior to PDE extraction Extraction of PDE from crude ROS membranes is carried out under room light Two liters of low ionic strength extraction buffer containing mM Tris, 0.5 mM MgClz, 0.1 mM PMSF, and 1.0 mM DTT at pH 7.5, a 120-ml Teflon-glass homogenizer, and two 30-ml syringes fitted with cm of Tygon tubing are required The crude ROS membranes are resus- cGMP PHOSPHODIESTERASE [1] pended in 100 ml extraction buffer and homogenized with the Teflon-glass homogenizer by five up and down strokes The homogenate is divided equally among four 50-ml centrifuge tubes, and the volume of each tube is increased to 45 ml with extraction buffer The samples are photolyzed on ice under a lamp for 15 until the red color of the suspension changes to bright orange, indicating the conversion of rhodopsin to the meta-II state (R*) Under these conditions, transducin remains tightly bound to the R*-containing membrane, whereas most of the peripheral proteins, including PDE, can be extracted by the low ionic strength buffer After photolyzing the ROS membranes, the suspensions are centrifuged at 19,000 rpm for 30 rain The supernatants are carefully removed using a 30-ml syringe fitted with Tygon tubing and transferred to four clean 50-ml centrifuge tubes The supernatants containing the extracted proteins are centrifuged again at 19,000 rpm for 30 to remove residual ROS membranes Supernatant from the second centrifugation is removed with a clean 30-ml syringe and stored on ice for subsequent chromatographic separation of PDE The ROS membrane pellets from the two centrifugation steps are pooled and homogenized again with 100 ml buffer for the second extraction The repetitive extraction of PDE with double centrifugation steps is carried out at 0°-4 ° for a total of six times The protein concentration of each extract is monitored In general, supernatants from the first four extractions (-480 ml) are pooled for PDE purification by column chromatography steps, and supernatants from the last two extractions are discarded because of the low protein content.The final ROS membrane pellets are pooled and saved for extraction of transducin with GTP The purification of transducin is described elsewhere in this series ~° and is not elaborated here, Purification of Phosphodiesterase by Column Chromatographies DEAE-Sephadex Chromatography The low ionic strength extract of the ROS membrane is applied to an 18 x 2.5 cm DEAE-Sephadex (Sigma Chemical Co., St Louis, MO) column equilibrated with buffer A (10 mM MOPS, mM MgC12, mM DTT, pH 7.5) at ° After washing the column with 50 ml buffer A, the bound proteins are eluted with a 500 ml linear gradient of NaC1 from 0.1 to 0.6 M in buffer A Fractions of ml are collected The protein content in each fraction is determined by the Bradford assay, ~t and the PDE activity in each fraction is assayed using a pH electrode method after trypsin activation as described in a later section ~0j Bigay and M Chabre, this series, Vol 237 [11] ii M M Bradford, Anal Biochem 72, 248 (1986) 442 AUTHOR INDEX Woon, C W., 14 Wootton, J F., 352, 356, 371 Wray, V P., 106 Wray, W., 106 Wu, D., 90, 182, 183(23), 237, 244-245 Wu, J., 259 Wuestehube, L J., 356 Wulfern, M., 365, 368, 369(15), 370(15) Wurtman, R J., 86(k), 87 Wynalda, M A., 413-414 X Xu, K., 280 Yeager, R E., 95 Yee, D., 90 Yee, R., Yeung, S.-M.H., 56-57, 57(4), 58(6), 60(6), 65(6), 66(6), 67(6) Yokota, Y., 147 Yokoyama, C., 416(7), 417 Yoo, O J., 182, 219, 220(3), 229, 243, 244(18), 245, 246(4) Yoon, J., 246 Yoshimasa, T., 126 Yoshimoto, T., 416(7), 417 Yoshimura, M., 95, 96(11), 117, 120(6), 124(14), 125(6, 14), 126(6), 127(14) Yoshioka, T., 227 Yuen, P.S.T., 40 Y Yadagiri, P., 416(4), 417 Yajima, M., 368, 369(15), 370(15) Yajnik, V., 255 Yamada, M., 394, 406 Yamamoto, K., 184 Yamamoto, S., 416(7), 417 Yamamoto, T., 256 Yamazaki, A., Yancopoulos, G D., 258 Yang-Feng, T L., 86 Yatani, A., 183,337, 357, 365, 373,385-386, 388, 391,391(6, 11, 12), 392-393,393(6, 12, 19), 395,406, 421 Yates, J., 288 Yau, K W., 228 Yazawa, K., 386 Z Zachary, I., 82-83, 83(1), 86(1), 87(1), 91(1), 94(1) Zenser, T V., 41, 45(33) Zhang, L., 376 Zhang, X.-F., 256, 260 Zhang, Y., 79, 80(20), 86 Zho, W., 147, 151(33) Zhou, Q Y., 86 Zhou, W., 352 Zijlstra, F J., 416(9), 417 Zubiaur, M., 83 Zwaagstra, J C., 117, 120(11), 124(11), 125(11), 126(11) Zwiers, H., 200 SUBJECT INt~EX 443 Subject Index A Acetylcholine receptors, muscarinic, s e e Muscarinic acetylcholine receptors Adenine, radioassay for cAMP, 91-92 Adenosine deaminase, ATP-regeneration system, 35 Adenylyl cyclase assay ATP-regeneration systems, 34-35 chromatography on alumina column, 45-47, 49-52, 93 on Dowex 50, 45, 48-49, 92-93 contaminating enzymes, inhibition, 33-34 data analysis blank, 53-54, 93-94 calculations, 54-55 sample recovery, 54 value reporting, 55-56, 93 enzyme concentration, 36 incubation temperature, 37 inhibition assays, 90-94 radioimmunoassay, 31 reaction stopping ATP/sodium dodecyl sulfate/cAMP, 43 hydrochloric acid, 43-45, 50-51 zinc acetate/sodium carbonate/ cAMP, 41-42, 45, 52-53 reaction time, 36-37 substrate radiolabel, s e e ATP transfected cells, 1t 1-112 ATP requirements, 32 calcium-sensitive, s e e Calcium-sensitive adenylyl cyclase cloning, 108, 116-117, 124-126 degenerative primers, 118-121 divalent metal cation requirements, 32 expression systems, s e e COS cells; HEK-293 cells; S p o d o p t e r a frugiperda G protein regulation, 37-38, 81, 116 isoforms cloning, 124-126 distribution, 96 forskolin response, 114 functions, 126-127 Gs response, 127 immunoblot analysis, 99-101 regulation, 96 sizes, 96, 106-107 types, 95-96, 116-117, 124-126 membrane topology, 118-119 oxidation, 68 phosphoenolpyruvate effects, 34-35 polymerase chain reaction, 121-124 "P"-site, s e e "P"-site purification, recombinant histidinetagged enzyme, 102-108 sequence homology between species, 117-121, 124-126 substrate affinity, 32 ultraviolet irradiation inactivating effects, 68 protection from, 69 fl-Adrenergic receptor, adenylyl cyclase stimulation, 115 Affinity chromatography adenylyl cyclase, 102-105 cGMP phosphodiesterase, inositol phosphates, 197-200 N-type calcium channel, 338-340 phosphatidylinositol transfer protein, 172-173, 175-176 phospholipase C-/3, 204,223,225,233, 235, 239-241 Albumin, arachidonic acid solubilization, 413-414 Alumina column care, 49, 52 preparation, 46-47, 93 nucleotide binding, 45-46 444 SUBJECT I N D E X Aluminum fluoride, G protein activation, 31 Angiotensin II receptor, superfrog expression, 150 Angrelide, cAMP phosphodiesterase inhibition, 34 Antibodies cell loading cell permeation, 358 controls, 358-359 microelectrode injection, 360-363 patch pipette, 359, 369-370 scrape loading, 363-364 visualization, 357-358 phospholipase C-fl isoform generation, 221-222 Antisense oligonucleotides, G protein, 145, 329-331,373-374 Arachidonic acid albumin binding, 413-414 compatible perfusion systems, 414-415 inhibitors, 414, 416 metabolism, 409 patch clamp configurations, 415, 418-419 pK, 413 potassium channel regulation, 409, 411 solubilization, 41 I-413 stability, 411 Armyworm, see Spodoptera frugiperda 3'-Arylazidoiodo-2',5'-dideoxyadenosine photocoupling, 61 structure, 62 synthesis, 61-62 ATP 32p-labeled detection, 40, 48, 50-51 disposal, 53 half-life, 40 a-phosphate labeling specificity, 39-40 quality of preparations, 40 regeneration systems, 34-35 stability, 352-354 tritiated assay advantages, 38 detection, 39, 48, 50-51 disposal, 52-53 half-life, 38 stability of label, 39 3'-(p-Azido-m-iodophenylacetyl)-2' ,5'dideoxyadenosine adenylyl cyclase inactivation, 68-71 photocoupling, 67-68 "P"-site modification, 71 radioactive labeling, 68 solubility, 68 synthesis, 65 3'-(p-Azido-m-iodophenylbutyryl)-2',5'dideoxyadenosine adenylyl cyclase inactivation, 68-71 photocoupling, 67-68 "P"-site modification, 71 radioactive labeling, 68 solubility, 68 synthesis, 65 B Baculovirus, Spodoptera frugiperda expression system using, see Spodoptera frugiperda Barium, nucleotide precipitation, 41 Brain, whole cell recording, see Whole cell recording C Caged compounds confocal microscopy, 333 GTP analogs, 371 Calcium chelation, 74, 76, 297 chloride current dependent on, 140, 143, 146, 321 fluorescence imaging, 303-304, 322-325, 334-335 homeostasis mechanisms, 80 hormonal elevation, 79 inositol phosphate effect on release, 207 levels cytosolic, 73 determination computer methods, 73-75 with fura-2, 73, 146 with indo-1,298, 316-317 with quin-2, 297 hormone effects, 79 ionomycin effects, 79 membrane depolarization effects, 80 thapsigargin effects, 79-80 light adaptation mediation, 228 SUBJECT INDEX in phospholipase C assay calcium-45, 153-154 free ion determination, 139-140 Calcium channels dihydropyridine-sensitive function, 335-336 G protein stimulation, 337 hormonal effects in PC-12 cells, 371-372 N-type blockers, 336-337 function, 336 immunoblotting, 341-342 labeling, 337 purification from rabbit brain heparin-agarose chromatography, 339-340 labeling, 340 membrane isolation, 337-338 solubilization, 338, 340 sucrose density gradient centrifugation, 341 wheat germ agglutinin chromatography, 338-339 subunits, 341 reconstitution bilayer formation, 345-346 channel insertion, 346-348 monolayer formation, 344 technique selection, 343-344 tip-dip method, 344-345 subunit composition, 336, 341,343 types, 335-336 Calcium Green cell loading, 324-325 photobleaching, 324 Calcium phosphate, coprecipitation transfection technique, 89-90, 274,282 Calcium-sensitive adenylyl cyclase assay, 74, 101-102 calcium concentration effects, 72-77, 80 inhibition, 72, 76-77 stimulation, 72 calmodulin effects, 77-78, 95 expression system, see Spodoptera frugiperda hormone effects, 79 immunoblot analysis, 99-101 ionomycin effects, 79 magnesium effects, 76 manganese effects, 76 445 Calmodulin contamination in creatine phosphokinase, 76 endogenous, removal, 77-78 regulation of adenylyl cyclase, detection, 77-78 Camera, fluorescence detection, 302-303 cAMP assays protein binding, 90 radioimmunoassay, 90 with tritiated adenine, 91-92 chromatography, 45-47, 49-52, 92-94 phospholipase C stimulation, 212-213 cAMP phosphodiesterases adenylyl cyclase preparation contamination, 33 inhibitors, 33-34 Cell lines, see COS cells; HEK-293 ceils cGMP phosphodiesterase activation by rhodopsin, 3, 11-13 by transducin, 3, 11-13, 21-23 by trypsin, 9, 11, 13, 19-21 bovine retina assay, 9, 11-12, 19 chromatography, 6-9 extraction from rod outer segments, 4-6, 19 structure, 3, 13 inhibitory subunit, 3, 13, 19, 23-27 factor Xa fusion protein, 23 fluorescence labeling, 24 transducin c~ subunit binding affinity, 24-26 assay, 24 site, 23, 25-28 Chloramphenicol acetyltransferase, assay, 274-275 Chloride current calcium dependence, 140, 143, 146, 321 inositol 1,4,5-trisphosphate effect, 145 Xenopus oocyte, 143, 145, 325-331 Chloroform, lipid extraction, 167 Cholera toxin, G protein sensitivity, 373 Collagenase, defolliculation of Xenopus oocytes, 150 Confocal microscopy caged compound activation, 333 cost, 308 446 SUBJECT INDEX detectors, 333 light source, 307-308, 331 muscarinic receptors, 322-325, 331, 333-334 optics, 331-332 resolution, 307, 333 scanning, 307 scan speed, 334 voxel, 307 co-Conotoxin GVIA blocking of N-type calcium channel, 336 labeling of N-type calcium channel, 340 radiolabel, 337, 339 COS cells, expression systems adenylyl cyclase assay, 111 cDNA preparation, 109-110 cell culture, 109, 124 transfection, 110-111, 116 phospholipase C-~ cell culture, 186 expression vector construction, 186 immunochemical analysis, 187 transfection, 186-187 Creatine kinase ATP-regeneration system, 34-35 calmodulin contamination, 76 D DEAE-dextran transfection technique, 8889 Degenerative primers adenylyl cyclase design, 118-121 sequences, 119-121 phospholipase C-/3 design, 246-248 sequences, 248, 250, 252 Dictyostelium discoideum, phospholipase C assay, 208-212 calcium effects, 213 cAMP stimulation, 212-213 2' ,5'-Dideoxyadenosine adenylyl cyclase affinity, 57 synthesis, 57-58 2',5'-Dideoxy-3'-p-fluorosulfonylbenzoyladenosine adenylyl cyclase inactivation, 66-67 "P"-site modification, 65-67 purification, 59-60 reactivity, 65 structure, 60 synthesis, 58-59 tritiation, 59-60, 67 E Eicosanoids metabolism, 409 potassium channel regulation, 409, 411 solubilization, 411-413 stability, 411 Electroporation transfection technique, 90 Elongation factor-TU, effector region, 15 Epidermal growth factor receptor Ras activation, 255 stimulation of phospholipase C, 196 Exocytosis patch clamp monitoring, 320 regulation by Ge, 168 F Fast atom bombardment mass spectrometry, peptides, 16 Fast protein liquid chromatography phosphatidylinositol transfer protein, 173 phospholipase C-fl, 206-207 Fibroblasts NIH 3T3 focus formation assay, 277-281 growth in nude mouse, 292-293 maintenance, 281-282 oncogene response element assays, 272, 274-275 transfection assay, 283-284 DNA carrier, 282 Rat- focus formation assay, 277-281 maintenance, 284 transfection, 285 REF52 maintenance, 286-287 oncogene cooperation assay, 285-286 transfection, 287 SUBJECT INDEX transformation assays anchorage-independent growth, 290291 focus-formation method, 277-281 growth rate acceleration, 288, 290 oncogene cooperation method, 285287 saturation density, 288, 290 serum growth factor requirements, 289 stable cell line establishment, 289-290 Fluorescence, s e e a l s o Confocal microscopy assay, 24 cell loading of probes, 299, 315-316 cGMP phosphodiesterase, inhibitory subunit labeling, 24 data acquisition with patch clamp measurements, 312-313,318-320 detection camera, 302-303, 304, 306 photomultiplier tube, 302, 304, 306 dual emission advantages, 306-307 dichroic mirror, 304, 310 dye calibration, 316-317 excitation source, 309-310 imaging, 304 dual excitation detector synchronization, 303 disadvantages, 306-307 image processing ratio calculation, 303 video mixing technique, 303-304 light source, 300 quantitation principles, 297-298 wavelength selection filters, 302 monochromators, 300, 302 imaging, 302-304, 306 microscope requirements, 299-300, 309 perfusion chamber, 313-314 probes, 72, 146, 297, 325 Focus-formation assays cell lines for, 277 maintenance, 281-282, 284 morphological changes, 277-279 selection, 280 transfection, 282-285 disadvantages, 279 447 time course, 284 transfection assay, 283-284 Forskolin, in analysis of adenylyl cyclase activation, 95-96 affinity chromatography, 102-105 isoform response, 114 protection against thermal inactivation, 37 Frog identification, 150 maintenance, 148 oocyte collection, 148-150 superfrog types, 150 Fura-2 cell loading, 299 in intracellular calcium determination 73, 146, 298 ratio fluorescence application, 298 G GDP/~S G protein binding, 352-353 patch pipette perfusion, 369 Ge, exocytosis regulation, 168 Gel filtration cGMP phosphodiesterase, phosphatidylinositol transfer protein 173-174, 176-177 phospholipase C-fl, 204 Genes, oncogenic, s e e Oncogenes Gi adenylyl cyclase regulation, 37-38, 81, 116 a-subunit mutation, 81-82 cell expression systems, 82-83 COS cells, 85, 87 HEK-293 cells, 85-87 PAl2 cells, 84-85 retroviral infection of mammalian cells cell culture, 84-85 DNA preparation, 83-84 cloning, 81 detection, 94 pertussis toxin sensitivity, 145, 155, 196, 237 signal transduction role, 81 subunit sequence homology, 81 transfection, 82, 88-90 types, 81 448 SUBJECT I N D E X Gk a subunit, 393 arachidonic acid modulation, 420-421 effect on potassium channels, 392-393, 420-421 Go antisense oligonucleotide, 145, 374 pertussis toxin sensitivity, 145, 155, 196, 237 G proteins, s e e a l s o Ge; Gi; Gk; Go; Gq; Gs; Transducin activation in X e n o p u s oocyte, 144 activators, 31 ADP ribosylation, 31, 94, 371-373 c~ subunit diversity, 365-366 immunoblotting, 399 patch pipette perfusion, 369 antisense oligonucleotides, 145, 329-331, 373-374 BY subunit denaturation, 407-408 detergent solubilization, 405-406 phospholipase C stimulation, 238-239 purification, bovine brain, 238 classes, 196 expression in HEK-293 cells, 85-87 membrane association, 155, 196 oncogene response element, transactivation assay, 275-276 regulation adenylyl cyclase, 37-38, 81, 116 ion channels, 308, 349, 357, 365, 371372, 385, 394-396 phospholipase A2, 422 phospholipase C, 131-132, 144-146, 155, 182, 208 structure, 196 G~, pertussis toxin insensitivity, 155, 196 Growth factor receptor-bound protein, Ras protein regulation, 255 Gs adenylyl cyclase isoform response, 127 ADP ribosylation, 373 regulation of adenylyl cyclase, 37-38, 81 Gt, s e e Transducin GTP analogs caged compounds, 371 types, 37-38, 350-353,369, 390 in sharp microelectrode recording, 354356 in single-channel recording, 354 stability, 352-354 thin-layer chromatography, 258 in whole-cell recording, 348-349 GTPase-activating protein, Ras protein regulation, 255 GTP-binding proteins, s e e G proteins GTPyS adenylyl cyclase activation, 38 complex with G protein a subunit, stability, 407 G protein binding, 356 patch pipette perfusion, 369 phospholipase C-/3 response, 163-164, 169 potassium channel activation, 390 receptor response, 350-352 Guanylyl imidodiphosphate patch pipette perfusion, 369 receptor response, 350-351 H Heart, perfusion, 396-397 HEK-293 cells adenylyl cyclase expression activity, 112-113 assay, 111 cDNA preparation for, 109-110 forskolin response, 114-115 hormonal stimulation, 115 cell culture, 88, 109, 124 G protein expression, 85-87 transfection calcium phosphate coprecipitation, 89-90 DEAE-dextran technique, 88-89, 110111, 116 electroporation, 90 lipofection, 90 High-performance liquid chromatography IP3, 216-217 phospholipase C, 225-226, 230-231, 235-236 synthetic peptides, 16 Histidine, tagging of recombinant proteins, 102-105,267 HL-60 cells, s e e Neutrophils SUBJECT INDEX HPLC, s e e High-performance liquid chromatography Human chorionic gonadotropin, adenylyl cyclase stimulation, 115 Human chorionic gonadotropin receptor, COS cell expression, 116 Hydrochloric acid, assay stopping, 43-44 I IBMX, s e e 3-Isobutyl-l-methylxanthine Indo-1 calibration, 316-317 cell loading, 316 confocal microscopy application, 334 detection, 310 excitation, 310 in intracellular calcium determination, 298,316-317 lnositol phosphates calcium-releasing effects, 207 Dowex separation, 165-166, 190, 215 fluorescent probes, 335 neomycin affinity chromatography, 197200 radiolabel, 159-160, 197-198 synthesis by turkey erythrocytes, 196197 Inositol 1,4,5-trisphosphate effect on chloride current amplitude, 145 high-performance liquid chromatography, 216-217 identification, 213,215-217 isotope dilution assay, 213,215-216 light-induced regulation in retina, 227 quantitation, 210-213 Inositol 1,4,5-trisphosphate-binding protein, isolation, 217-218 Inside-out patch recording G protein criteria for defining active subunits, 400-402 diffusion, 399-400 potassium channels apparatus, 387, 397-398 buffers, 391 cell preparation, 386, 396-397 concentration clamp, 388-389 data acquisition, 387 analysis, 387-388 449 GTP analogs, 390-391,398 method suitability, 385-386, 397 patch solution, 387, 398 pertussis toxin uncoupling, 391-393 pipette glass, 387 time course, 389 lodoazidodideoxyadenosine, precursor preparation p-azido-m-iodophenylacetic acid, 62-64 p-azido-m-iodophenylacetic anhydride 64 p-azido-m-iodophenylbutyric anhydride 64-65 Ion channels, s e e Calcium channels: Potassium channels Ion-exchange chromatography adenyly[ cyclase assay, reaction stopping, 45-49 cGMP phosphodiesterase, 6-7 inositol phosphates, 165-166, 190 phosphatidylinositol transfer protein, 175, 179 phospholipase C assay, 133, 165-166 purification, 203-204, 206-207 226227,236-237 Ionomycin, effect on adenylyl cyclase, 79 3-1sobutyl-l-methylxanthine, cAMP phosphodiesterase inhibition, 33-34 Isoelectric point, peptide solubility effect 17 K Kidney, s e e HEK-293 cells L Lipofection, transfection technique, 90 Liposomes, preparation, 172 Lucifer Yellow vinyl sulfone, cGMP phosphodiesterase subunit labeling, 24 Luteinizing hormone receptor, adenylyl cyclase stimulation, 115 M Magnesium effect on adenylyl cyclase, 76 role in GTP activation of ion channels, 389, 403 450 SUBJECT INDEX MAPK, s e e Mitogen-activated protein kinase Mass spectrometry, fast atom bombardment, peptides, 16 MEK-I assay coupled, 263 separations, 262-264 mitogen-activated protein kinase specificity, 259-260 phosphorylation, 260 recombinant, preparation cell growth, 266-267 expression plasmid, 265 histidine tagging, 267 induction, 266 purification, 237 solubility, 265 MEK kinase assay, 270 MEK-1 activation, 260 purification, 270 Membranes, reconstitution bilayer, 344-346 monolayer, 344 Microfluorimetry, s e e Fluorescence Microsomes, preparation, 171-172 Mitogen-activated protein kinase activation phosphorylation, 258-260 receptor, 258-259 regulation, 259 assay separations, 260-261 substrates, 260-262 functions, 258-259 recombinant, preparation cell growth, 266-267 expression plasmid, 265 histidine tagging, 267 induction, 266 MEK-1 assay substrate, 262-264 purification, 237 solubility, 265 Mitogen-activated protein kinase kinase, s e e MEK-1 Muscarinic acetylcholine receptors confocal microscopy, 322-325, 331,333334 tissue ditribution, 321 types, 321-322 oocyte chloride current, 322, 325-330 expression, 322 receptor expression stability, 327-328 signal transduction, 322 Myelin basic protein, mitogen-activated protein kinase substrate, 262 Myocytes; cardiac, preparation, 396-397 Myokinase, s e e Adenylyl cyclase Xenopus N Nerve growth factor receptor, Ras activation, 255 Neutrophils, HL-60 cells calcium influx, 317-319 intracellular pH, measurement, 319-320 membranes, preparation, 186 phospholipase C, preparation, 184-186 Nickel, histidine affinity, 102-105, 267 O Oligonucleotides, antisense, G protein, 145, 329-331,373-374 Oncogene-responsive elements activation assay chloramphenicol acetyltransferase, 274-275 cotransfection analysis, 272 G protein, 275-276 reporter gene expression, 273 transactivation, 275-276 mechanism, 271 transcription increase, 271 Oncogenes cooperation assay, 285-287 cell line for, 286-287 transfection, 287 transcription activation of other genes, 271-272 Oocytes, X e n o p u s calcium oscillations, 147, 323 chloride current acetylcholine response, 325-330 calcium dependence, 140, 143, 146, 321 SUBJECT INDEX recording amplifier, 153,324 data storage, 153 micropipettes, 153 tissue bath medium, 142, 152-153 response with different receptors, 143-144, 322 values, 143, 326 collection, 148-150 defolliculation, 149-150, 323 development, stages, 141 glutamate receptor expression, 141 G proteins activation, 144 c~ subunit cloning, 321 antisense oligonucleotides, 329-331 ion permeability, 142-143 layers, 141-142 muscarinic acetylcholine receptors chloride current, 322, 325-330 expression, 322 receptor expression stability, 327-328 signal transduction, 322 phospholipase C assay calcium-45, 153-154 chloride current, 152-153 G protein sensitivity, 144-146 receptor expression system, 146-147 resting potential, 142 RNA injection, 150-151,324 size, 141, 147 small molecule injection, 151-152 P p21ra~, effector region, 14-15 Papaverine, cAMP phosphodiesterase inhibition, 33-34 Patch clamp, s e e a l s o Chloride current, X e n o p u s oocyte amplifiers, 153,310 arachidonic acid techniques, 415,418419 cell loading antisense oligonucleotides, 374 microelectrode injection, 360-363 patch pipette antibodies, 359, 369-370 451 dyes, 315-316 G protein, 369 guanine nucleotides, 348-350 scrape loading, 363-364 trituration method, 374 data acquisition with fluorescence measurements, 312-313, 318-320 exocytosis monitoring, 320 Faraday cage, 310-311 GTP supplementation sharp microelectrode, 354-356 single-channel, 354 whole-cell, 348-349 inside-out, s e e Inside-out patch recording micromanipulator, 310 oscilloscope, 312 perfusion chamber, 313-314 solutions pipette, 314-315 recording, 142, 152-153 stimulator, 311 temperature control, 314 tip-dip method, 344-348 whole-cell, s e e Whole-cell recording Peptides fast atom bombardment mass spectrometry, 16 purification, 16 quantitation, 18 solubility determination, 18 factors affecting, 17 isoelectric point effect, 17 solvent, 17 synthesis, 16-17 Pertussis toxin ADP ribosylation of G proteins, 237, 371-373 G protein sensitivity, 144-145, 155, 196, 237,357,371-372 potassium channel blocking, 391-393 structure, 371 Phosphatidylinositol, s e e Inositol phosphates; Phosphatidylinositol transfer protein Phosphatidylinositol 4,5-bisphosphate, s e e Inositol phosphates Phosphatidylinositol transfer protein activity, 169 452 SUBJECT INDEX assay isoelectric focusing, 180 microsome preparation, 171-172 quantitation, 172 reconstitution, 173, 181 bovine brain, purification cytosol preparation, 175 gel filtration, 176-177 heparin-Sepharose chromatography, 175-176 ion exchange chromatography, 175, 179 Phenyl-Superose chromatography, 177-179 phosphatidylcholine and phosphatidylinositol forms, 169 conversion, 179-180 separation, 179 phospholipase C-/3 reconstitution assay, 166-167, 169 rat brain phospholipase C contamination, 172 purification cytosol preparation, 170-171 gel filtration, 173-174 heparin-Sepharose chromatography, 171-173 size, 173 substrate specificity, 169 Phosphoinositidase, see Phospholipase C Phospholipase A2, G protein regulation, 422 Phospholipase C activation by hormone receptors, 196, 244-245 cellular distribution, 154 cloning, 131, 195 Dictyostelium discoideum assay, 208-212, 218 calcium effects, 213 cAMP stimulation, 212-213 G protein regulatory effects, assays applications, 133-134, 137 calcium effects, 139-140 data interpretation, 134-135, 140 detergent interference, 138 Dowex chromatography, 133 enzyme concentration, 138-139 quantitation, 135, 137, 140 reaction stopping, 132-134 substrates endogenous, 132-134 exogenous, 135-137 temperature, 138 G protein sensitivity, 131-132, 144-146, 155, 182, 208, 237 high-performance liquid chromatography, 230-231 isozymes bovine retina, 229-233 families, 131, 154, 168, 182, 195, 219220, 244 sequence homology, 181,220, 245-248 sizes, 220 nomenclature, 131 phosphorylation, 155, 196 phototransduction role, 227-228 reconstitution, 164-167, 169 second messenger generation, 131, 168, 181, 195, 207, 219, 237 substrate specificity, 131, 159 temperature sensitivity, 138 in Xenopus oocytes, chloride current assay, 140-154 Phospholipase C-/3 assays calculation of data, 166-167 with phosphatidylinositol 4,5-bisphosphate substrates, 160-161, 187, 197-200, 228-229 with phosphatidylinositol substrates, 159-160, 197-200, 228-229 degenerative primers, see Degenerative primers G protein/3y subunit-stimulated assay, 238 /3y response, 243-244 bovine brain family, 243-244 purification, 238-241 size, 241 G protein stimulation a subunit, 182-183, 245 fly subunit, 182-183, 188-191,220, 245 calcium effects, 190-191 detergent effects, 191 salt effects, 191 SUBJECT INDEX isoforms, 182,220 antibodies, 221-222 sequence homology, 221,246-248 tissue distribution, 221-222 membrane-associated activity, 162 particulate, assay, 192-193 phospholipase C-/33 bovine brain, truncated form, 243244 rat brain antibody generation, 221 assay, 222-223 cloning, 220 immunoblot analysis, 227 purification, 223,225-227 sequence, 221 phospholipase C-/34, bovine retina purification heparin-Sepharose chromatography, 233,235 high-performance liquid chromatography, 231-233,235-236 ion-exchange chromatography, 236237 salt extract, 233 tryptic peptides, 232-233 polymerase chain reaction, s e e Polymerase chain reaction recombinant, expression assay, 193-195 cell culture, 186 immunochemical analysis, 187 transfection, 186-187 vector construction, 186 reconstitution system assay, 164-167, 169 cytosol depletion, 155-157 GTP3,S, loss of responsiveness, 163164, 169 immunoblot monitoring of enzyme release, 161-162 protein efflux, time course, 157-158 solubilization bovine tissue, 185-186 HL-60 cells, 184-185 solubilized assay, 187-192 substrate preparation, 187-188 substrate specificity, 159 453 turkey erythrocyte assay, 200-201 properties, 206-207 purification ammonium sulfate precipitation, 202-203 cytosolic fraction, 202 erythrocyte preparation, 201 gel filtration, 204 heparin-Sepharose chromatography, 204 hydroxylapatite chromatography, 204 ion-exchange chromatography, 203204, 206-207 Phospholipase C-8 bovine retina, 231-232 high-performance liquid chromatography, 231-232 Phospholipase C-3, high-performance liquid chromatography, 230-231 immunoblotting, 230-231 phosphorylation, 220, 245 Phospholipase D assay reconstitution, 167-168 substrate labeling, 167 GTP-binding protein requirement, 167 Photomultiplier tube, fluorescence detection, 302, 304, 306 pI, s e e Isoelectric point Platelet-derived growth factor receptor Ras activation, 255 stimulation of phospholipase C, 196 Polymerase chain reaction, s e e also Degenerative primers adenylyl cyclase amplification, 122-123 cDNA synthesis, 122 RNA isolation, 121-122 subcloning, 123-124 type enzyme cloning, 124 nested screening, 126 phospholipase C-/3 amplification, 249, 252 cDNA synthesis, 249 product characterization, 250-251 RNA isolation, 249 454 SUBJECT INDEX Potassium channels ATP-activated, G protein subunit activation, 408-409 inside-out patch recording, G protein effects buffers, 391,398 GTP analog studies, 390-391 method suitability, 385-386, 397 pertussis toxin uncoupling, 391-393 time course, 389 muscarinic, cardiac arachidonic acid activation direct action, 420 Gk effects, 422 indirect action, 420-421 G protein subunit activation, 402-409 patch clamp studies, 397-399 perfusion system, 397 regulation arachidonic acid metabolites, 409, 411, 419-422 G protein subunit activation, 365, 385, 391-395,402-409 c~ subunit, 392-396, 404 fly subunit, 395-396, 402-404 concentration dependence, 404-405 detergent solubilization, 405-406 reproducibility, 404 Protein kinase, mitogen-activated, see Mitogen-activated protein kinase "P"-site adenylyl cyclase inhibition, 34, 37 structural requirements, 56 homology with catalytic domain, 57 Pyruvate kinase, ATP-regeneration system, 34-35 Q Quin-2, calcium quantitation, 297 R R0-20-1724, cAMP phosphodiesterase inhibition, 33-34 Raf-1 assay, 269 immunoprecipitation, 267-268 MEK-I activation, 260 Ras protein assay, in guanine nucleotide analysis, 256-258 GTP binding, 255 mitogen-activated protein kinase activation, 262 Raf protein binding, 256 receptor activation, 255 size, 255 Retina, phototransduction, 227-228 Rhodopsin, cGMP phosphodiesterase activation, 3, 11-13 Rod outer segment cGMP enzyme cascade, 3, 13 cGMP phosphodiesterase extraction from membranes, 4-6, 19 light adaptation, mediation by calcium, 228 purification, 4, 18 transducin extraction, 183-184 $ Sf9 cells, see Spodoptera frugiperda SNARF- detection, 310 excitation, 310 intracellular pH determination, 298, 319320 Sodium dodecyl sulfate, enzyme assay, reaction stopping, 43 Spodoptera frugiperda adenylyl cyclase expression, baculovirus system assay, 101-102 cell culture, 97-98 enzyme purification affinity chromatography, 102-105 detergent extracts, 103 expression level, 108 G protein activation, 107-108 histidine tagging, 97, 102 immunoblot analysis, 99-10l plasmid construction, 96-97 recombinant baculovirus production, 98 time course of expression, 99-100 membrane isolation, 98-99 SUBJECT INDEX Streptolysin O cytosol depletion, 155-157, 163, 169 membrane lesion size, 158, 167 455 cGMP phosphodiesterase activation, 3, 11-13 GTP binding, 13-14 heterotrimeric, purification, 183-184 subunit structure, 3, 12-13 Trituration method, intracellular applicaT tion of proteins, 374 Thapsigargin, effect on adenylyl cyclase, Trypsin, cGMP phosphodiesterase activa79 tion, 9, 11, 13, 19-21 Thin-layer chromatography Tumor cells chloramphenicol acetyltransferase assay, growth potential assay, 291-293 274-275 transformation 2',5'-dideoxy-3'-p-fluorosulfonylbenzoylassays adenosine, 59 anchorage-independent growth, GTP, 258 290-291 phosphatidylinositol transfer protein focus-formation method, 277-281 assay, 172 growth rate acceleration, 288, 290 Tip-dip method, see Patch clamp oncogene cooperation method, 285Transducin 287 a subunit saturation density, 288 cGMP phosphodiesterase activation, serum growth factor requirement, 13 289 effector region, 14-15 stable cell line establishment, 289peptides 290 activation constants, 23 types, 277,294 affinity assay, 24 growth characteristics, 277,287-290 cGMP phosphodiesterase activation, morphological changes, 277-279 21-23 competition assays, 19-21 inhibitory subunit binding site, 25W 28 mechanism of action, 18 Whole-cell recording purification, 18-19 average current values, 375 synthesis, 16-17 brain slices phospholipase C activation, 182-183 apparatus, 377-378 purification, 184, 399 blind technique, 376-377 size, 13 discontinuous single-electrode voltage clamping, 382 three-dimensional structure, 14 gigaseal, 380-381 fly subunit junction potentials, 383-384 phospholipase C stimulation pipette assay, 188-192 diameter, 375 calcium effects, 190-191 detergent effects, 191 fabrication, 378 linearity with time, 192 solutions, 378-379 particulate enzyme, 192-193 protein injection, 384 recombinant enzyme, 193-195 recording, 379-382 salt effects, 191 response rundown, 382-383 purification, 183-184, 399 series resistance, 381-382 solubility, 406 sharp electrode technique, 375-376 456 SUBJECT I N D E X slice preparation, 378 thin slice technique, 376-377 GTP depletion of cells, 348-350 patch pipette intracellular perfusion, 368-370, 384 sealing, 367 voltage pulse, 367-368 X Xenopus, see Frog Z Zinc, nucleotide precipitation, 41-42 ... [2] P E P T I D E PROBES FOR G PROTEINS AND EFFECTORS 13 [2] Specific P e p t i d e P r o b e s for G - P r o t e i n I n t e r a c t i o n with Effectors B y HELEN M RARICK, NIKOLAI O ARTEMYEV,... certain that there are some areas that are not as thoroughly covered as they could be However, G-protein effector research is a very active area in many laboratories including my own, and undoubtedly... and P/3 (84 kDa), and two identical inhibitory subunits, P3'' (11 kDa) The catalytic activity of heterotrimeric PDE is kept low in the dark by the inhibitory subunits In the light Gta-GTP activates