NEUROMETHODS [7 3 1 G Protein Methods and Protocols NEUROMETHODS Program Editors: Alan A. Boulton and Glen B. Baker 31 G Protein Methods and Protocols: 16 Molecular Neurobiological Techniques, Role of G Protems m Psuchlatnc and edltedb yA lan A. Boulton, Glen 9. Baker, Neuroioglcal Disorders, e&ted by Ram and Anthony T. Campagnoni, 1990 K. Mishra. Glen 9. Baker. and Alan 15 Neurophvsioloaical Techniaues: Aotka- A. Boulton, 1997 tlons ‘to- Neural Systems, edltkh by 30. Regulatory Protein Modification: Alan A. Boulton, Glen 9. Baker, and Technques and Protocols, edlted by Case H. Vanderwolf, 1990 Hugh C. Hemmings, Jr., 1997 14. Neurophysiological Techniques: Basic 29. Apoptosis Techniques and Protocols, Methods and Concepts, edltedb y Alan edited by Judes Poirier, 1997 A. Boulton, Glen 9. Baker, and Case 28 Transgenic Xenopus: Mlcromjechon H. Vanderwolf. 1990 Methods and Developmental Neuro- 13 Psychopharmkology, editedb y Alan biology, by Shlomo Seidman and A. Boulton, Glen B. Baker. and Andrew Hermona Soreq, 1997 J. Greenshaw, 1989 27. Voltammetric Methods in Brain Sys- 12 Drugs as Tools in Neurotransmitter tems, edlted by Alan A. Boulton, Research, edltedb y Alan A. Boulton, Glen 9. Baker, and Ralph N. Adams, Glen B Baker, and August0 V. 1995 Juorio, 1989 26. Patch-Clamp Applications and Pro- 11. Carbohydrates and Energy Metabo- tocols, edrted by Alan A. Boulton, lism, edrtedb y AlanA. Boulton, Glen Glen 9. Baker, and Wolfgang Walz, 9. Baker, and Roger F. Butterworth, 1_99-5- - 1989 25 Neurotrophic Factors, edltedb y Alan 10 Analysis of Psychiatric Drugs, edlted A. Boulton, Glen 9. Baker, and Franz by Alan A. Boulton, Glen 9. Baker, Hefti. 1993 and Ronald T. Coutts, 1988 24 An&al Models of Drug Addiction, 9 The Neuronal Microenvironment, edlted edlted bv Alan A. Boulton, Glen 9. by Alan A. Boulton, Glen 9. Baker, Baker, and Peter Wu, 1992 and Wolfgang Walz, 1988 23 Practical Cell Culture Techniques, 8 Imaging and Correlative Physico- edlted by Alan A. Boulton, Glen B. chemical Techniques, edrtedb y Alan Baker, and Wolfgang Walz, 1992 A. Boulton, Glen 9. Baker, and 22 Animal Models of Neurological Disease, Donald P. Bolsvert, 1988 II: Metabok Enceohalooathles and the Lipids and Related Compounds, edlted Eprlepsres, edltedb y Aian A. Boulton, by Alan A. Boulton, Glen 9. Baker, Glen 9. Baker. and Roaer F. and Lloyd A. Horrocks, 1988 Butterworth, 1992’ Peptides, edlted by Alan A. Boulton, 21. Animal Models of Neurological Dis- Glen 9. Baker, and Quentin Plttman, ease. I: Neurodeaeneratwe Disease, 1987 edlted by Alan A: Boulton, Glen 9’ Neurotransmitter Enzymes, edltedb y Baker, and Roger F. Butterworth, Alan A. Boulton, Glen 9. Baker, and 1992 Peter H. Yu, 1986 20. Intracellular Messengers, edlted by Receptor Binding Techniques, edlted Alan A. Boulton, Glen 9. Baker, and by Alan A. Boulton, Glen 9. Baker, Colin W. Taylor, 1992 and Pave1 D. Hrdma, 1986 19 Animal Models in Psychiatry, II, edlted Amino Acids, edltedb y Alan A. Boulton, by Alan A. Boulton, Glen 9. Baker, Glen 9. Baker, and James D. Wood, and Mathew T. Martin-lverson, 1991 1985 18 Animal Models in Psychiatry, I, edrted Amines and Their Metabolites, edlted by Alan A. Boulton, Glen 9. Baker, by Alan A. Boulton, Glen 9. Baker, and Mathew T. Martin-lverson, 199 1 and Judith M. Baker, 1985 17 Neuropsychology, edlted by Alan A. General Neurochemical Techniques, Boulton, Glen 9. Baker, and Merrill edited by Alan A. Boulton and Glen Hlscock, 1990 I3 Baker, 1985 NEUROMETHODS 0 31 G Protein Methods and Protocols Role of G Proteins in Psychiatric and lYeurologica1 Disorders Edited by Ram K. Mishra McMaster University, Hamilton, Canada Glen B. Baker University of Alberta, Edmonton, Canada and Alan A. Boulton University of Saskatchewan, Saskatoon, Canada Humana Press Totowa, New Jersey 0 1997 Humana Press Inc 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 All rights reserved No part of this book may be reproduced, stored m a retrieval system, or transmitted m any form or by any means, electronic, mechamcal, photocopymg, microfilmmg, recordmg, or otherwise without written permtssion from the Publisher All authored papers, comments, opmtons, conclustons, or recommendations are those of the author(s) and do not necessarily reflect the views of the pubhsher This publication is printed on acid-free paper @ ANSI 239 48-1984 (American National Standards Institute) Permanence of Paper for Prmted Library Materials For additional copies, pricmg for bulk purchases, and/or mformatlon about other Humana titles, contact Humana at the above address or at any of the followmg numbers Tel 973-256- 1699, Fax 973-256-8341, E-mall humana@mmdsprmg corn, or visit our Webstte http / / humanapress corn Cover illustration Fig 1 m “G Protem-Coupled Melatonm Receptors” by Lennard P Niles Cover destgn by Patricia F Cleary Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, IS granted by Humana Press Inc , provided that the base fee of US $8 00 per copy, plus US $00.25 per page, IS paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923 For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and IS acceptable to Humana Press Inc The fee code for users of the Transactional Reporting Service IS [O-89603-490-9/97 $8 00 + 500 251 ISBN o-89603-490-9 ISSN 0893-2336 Printed m the Umted States of America 10 9 8 7 6 5 4 3 2 1 Preface The G proteins are a family of structurally homologous, plasma membrane-associated guanine-nucleotide-binding proteins. These proteins play an integral role in the trans- duction of extracellular signals through second messenger systems. As such, G proteins affect a wide variety of intra cellular biochemical reactions by regulating the concentra- tion of second messengers in cells. G proteins are heterotrimeric, consisting of a, p, and y polypeptide chains, with G protein specificity largely deter- mined by the a-subunit, Molecular cloning of G protein sub- units has revealed 23 distinct a-subunits, encoded by 17 different genes. Based on functional measures, G proteins are generally classified into three major categories: the G, family, which is stimulatory for adenylyl cyclase; the G, fam- ily, which is inhibitory for adenylyl cyclase; and the G, fam- ily, which stimulates phospholipases (Birnbaumer and Birnbaumer, 1995). Alternatively, on the basis of sequence homology, G proteins can be subdivided into four catego- ries: G,, G,, G,, and G12. Although the a-subunits are specific for a given G pro- tein, the p- and y-subunits share considerable homology with each other. As previously mentioned, it has generally been assumed that the diversity of the a-subunits provides the essential specificity for G protein receptor interactions. How- ever, there is also evidence suggesting that the structural diversity of the p- and y-subunits may also contribute to the specificity of these interactions. On the basis of molecular cloning studies, 5 P-subunits and 10 y-subunits have been described. These two subunits can be regarded as one func- tional subunit, considering their tight association. The five P-subunits are highly homologous, and display a character- istic feature of repetitive tryptophan-aspartate (WD) motif, V vi Preface outside Fig. 1. A hypothetical model of G protein a-subunit (modified from Gordeladze et al., 1994). The figure displays various functions and the spatial interrelationships within the a-subunit. The C-terminus is involved in receptor binding, whereas the N-terminal and probably some other stretch of the subunit are involved in its anchoring to the p y-sub- unit complex. Various other regions, G-l, G-2, G-3, and G-4, are impor- tant for the binding of GTP. The GTPase activity and the GDP/GTP exchange reside in G-2, G-3 regions. NTP stands for nucleotide triphos- phate. Amino acids denoted with a number represent their correspond- ing position in the sequence. Mutations in these positions have led to the identification of their functional role. whereas the y-subunits display significantly more diverse primary structures (Simon et al., 1991; Helper and Gilman, 1992; Ray et al., 1995; Guderman et al., 1996). G proteins cycle between a GDP-bound inactive form (which maintains the high-affinity agonist-binding state of the receptor) and a GTP-bound active form (low-affinity ago- nist binding state of the receptor). On agonist binding, the cell surface receptor catalyzes the GDP/GTP exchange at the a-subunit, and this active form goes on to stimulate effector pathways in transmembrane signaling. The salient features of a-subunit signaling are displayed in Fig. 1. al , al a et nt e 8 g m 8 n g References Horn et al, 1988 Horn et al., 1995 Bohm et al., 1990 Neumann et al, 1988 Longabaugh et al, 19Bohm et al, 1995 Okada et al, 1991 Manji et al, 1995 Lesch et al., 1992, You1994 Self et al, 1994 Manjr, 1992 Parolaro et al, 1993 Van Vliet et al., 1993 mbens, VTA, ventral te u c c a C s A u N cle D D D D D D C, C Nu es N- NNNNN N- N - C, Stat s AC, m, NA Table 1 in Disease ND - ND ND ND ND 5 1 JNAC Low levelC, VTA, N t & clostruNE - ontal cortex, G Proteins Subtypes ?37% t K!!% 4 NC ?FC WAC Low levels VTA, NALc NC k16% hange, FC, fr al ns) no corm Gsa -180% L ND ND J50% ND NC - T30-70% (hype) ?>50% (&natneuro ned, NC, endoprrif mis u ere etucl dn Disease states Congestive heart failure Dilated cardromyopathy Heart failure Left ventricle failure Heart-sepnc shock Schizophrenia Depression Cocaine addiction Morphine tolerance “Abbreviatrons ND, not LC, locus ceruleus, NE, c r=: Changes in G Protein Subunits in Diseases In view of the critical and important role carried out by G proteins in signal transduction, it is very likely that quali- tative or quantitative changes in G proteins could have sig- nificant effects on intracellular effecters. It has been recently recognized by many investigators that the simple estimation of receptor numbers in disease states may not provide a com- plete perspective of receptor activity (Brodde and Michel, 1989). Indeed, the modification of signal transduction path- ways could provide important clues to the pathophysiology of many disease conditions. For example, it has been dem- onstrated that in experimentally induced diabetes, the gene expression of the G,,-subunit is significantly reduced (Gawler et al., 1987). Similarly, in patients with type IA pseudo- hypoparathyroidism, reduced levels of G, protein and mRNA levels have been reported (Carter et al., 1987). In congestive heart failure, the levels of G, in lymphocyte membranes are significantly reduced, and this reduction can be fully restored by therapeutic agents, such as angiotensin-converting enzyme inhibitor (Horn et al., 1988). A summary of alter- ations in G protein levels in various diseases is presented in Table 1. Although in the past decade significant advances have been made toward unraveling the role of neurotrans- mitter receptors in various psychiatric and neurological dis- orders, the role of second messenger systems in these disease states is still poorly understood. The purpose of G Protein Methods and Protocols is to provide novel information and techniques pertaining to the study of G proteins and their role in CNS functions. References Birnbaumer, L. and Birnbaumer, M. (1995) Signal transduction by G-pro- teins. J Recept Szgnal Tram Res. 15,213-252. Bohm, M., Gierschik, P , Pleske, B., Schnabel, P , Ungerer, M., and Erdmann, E (1990) Increase of Gi alpha m human hearts with dilated but not ischemic cardiomyopathy. Cmulation 82,1249-1265. Brodde, 0 E. and Michel, M. C (1989) Disease states can modify both receptor number and signal transduction pathways. TlPS 10,383,384. Carter, A., Bardin, C., Collins, R., Simons, C., Bray, I?., and Spiegel, A (1987) Reduced expression of multiple forms of the alpha subunit of the stimulatory GTP binding protein in pseudohypopara- thyroidism type Ia. Proc Nafl. Acad. Sci. USA 84,7266-7269. Gawler, D. J., Milligan, G., Spiegel, A M., Unson, C. G., and Houslay, M. D. (1987) Abolition of the expression of inhibitory guanine nucle- otide regulatory protein Gi activity in diabetes Nature 327, 229- 232 Gordeladze, J. O., Johansen, P W., Paulson, R. H., Paulssen, E J., and Gautvik, K M. (1994) G-proteins. implications for pathophysiol- ogy and disease. Eur J Endocrznol. 131,557-574. Guderman, T , Kalbrenner, F., and Schultz, G. (1996) Diversity and selectivity of receptor G-protein interaction. Ann Rev Pharmacol Tox~col. 36,429-459. Helper, J, R. and Gilman, A G. (1992) G-proteins. Trends Biochem Scz. 17, 383-387. Horn, E. M., Corwm, S. J., Steinberg, S F., Chow, Y K., Neuberg, G. W., Cannon, P. J., Powers, E. R., and Bilezikian, J. P. (1988) Reduced lymphocyte stimulatory guanine nucleotide regulatory protem and beta-adrenergic receptors in congestive heart failure and reversal with angiotensin converting enzyme inhibitor therapy Cwculafion 78,1373-1379. Horn, E. M., Kukin, M. L., Neuberg, G W., Goldsmith, R. L , McCarty, M , Gratch, M , Medina, N., Yushak, M., and Packer, M. (1995) Lym- phocyte G-proteins reflect response to treatment in congestive heart failure. Am. Heart J 129,98-106. Lesch, K. I’., Hough, C J , Aulakh, C. S , Wolozin, B L., Tolliver, T J , Hill, J L., Akiyoshi, J., Chuang, D., and Murphy, D L. (1992) Fluoxetine modulates G-protein alpha-s, alpha-q and alpha-12 sub- unit mRNA expression m rat brain. Eur J Pharmacol Mol Pharmacol. 227,233-237. Longabaugh, J. P., Batner, D. E., Batner, S. F., and Homey, C. J. (1988) Decreased stimulatory guanosine triphosphate bindmg protein in dogs with pressure overload left ventricular failure. J Clan Invest 81,420-424. Manji, H K. (1992) G-proteins: implications for psychiatry. Am. J. Psy- chzatry 149,746-760. Manji, H. K , Chen, G., and Shimon, H (1995) Guanine nucleotide bind- ing proteins in bipolar affective disorders: effect of long-term lithium treatment. Arch Gen. Psychiatry 52,135-144. Neumann, J., Schmitz, W., Scholz, H., von Meyermck, L., Dormg, V., and Kalmar, P. (1988) Increase in myocardial Gi-proteins m heart fail- ure. Lancet 2,936,937. Okada, F., Crow, T. J., and Roberts, G. W. (1990) G proteins (Gi, Go) in the basal ganglia of control and schizophrenic brain. J. Neural Transm. [GenSectI 79,227-234. Parolaro, D., Rubino, T., Gori, E., Massi, P., Bendotti, C., Patrini, G., Marcozzi, C., and Parenti, M. (1993) In situ hybridization reveals specific increases in G-alpha-s and G-alpha-o mRNA in discrete brain regions of morphine-tolerant rats Eur J. Pharmacol. MoZ Pharmacol 244,211-222. Ray, K , Kunsch, C., Bonnes, L. M , and Robishaw, J D. (1995) Isolation of cDNA clones encoding eight different human G-protein g sub- units, including three novel forms designated the 84, g10 and gll sunbumts. 1 Brol Chem 270,21,765-21,771 Self, D. W., Terwilliger, R. Z., Nestler, E. J , and Stein, L. (1994) Inactiva- tion of G, and G, proteins in nucleus accumbens reduces both cocaine and heroin reinforcment. J. Neuroscz 14,6239-6247 Simon, M. I, Strathmann, M. I’., and Gautam, N. (1991) Diversity of G- proteins u-t signal transduction. Science 252,802-808. Young, L. T., Li, P. P., Kamble, A., SIU, K. P., and Warsh, J J. (1994) Mono- nuclear levels of G-proteins in depressed patients with bipolar dis- order or major depressive disorders. Am 1 Psychiatry 151,594-596.