ANNUAL REVIEWS
1991. 60:349-400
Copyright © 1991 by Annual
Annu. Rev. Biochem. 1991.60:349-400. Downloaded from www.annualreviews.org by Moscow State University - Scientific Library of Lomonosov on 08/12/13. For personal use only.
Annu. Rev. Biochem.
Further
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Reviews Inc. All rights reserved
STRlJCTURE AND FUNCTION OF
SIGNAL-TRANSDUCING
GTP-BINDING PROTEINS
Yoshito Kaziro / ,2 Hiroshi Itoh,3 Tohru Kozasa,4 Masato Nakafukll,2 and Takaya Satoh2 Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108, Japan KEY WORDS:
elongation factor, Ras proteins, G proteins, GTPase cycle, confonnational switch.
CONTENTS 1. PERSPECTIVES AND SUMMARy . . . . . . . . . .
350
II. INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
35 1
III. GENERAL CHARACTERISTICS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tra nslati onal Fa ctors . . . . . . . . . . . . .. . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... . . .. . . .. ... . . Heterotrimeric G Protein s . . .. . . .. . . . . ... . . ... .. ... .. .. .. .... . .... . . .. . . . .. . .. . . . .. . . .. . .. . . . . .. Ra s Protein s . .. . . . . . . .. . . . . . . . . . . .. . . . . . . . . . . . . . . .. ... . . . . . . . . . . . . . . . .. ... . . . . . .. . . ... . . . . . . . ... . .. Structu ral Simi /aritie s Be w t een Vari ou s G TP-Binding Protein s. . . . . . . . . . . . . . . . .
354 354 356 357 359
IV . MAMMALIAN G PRO TEINS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M olecular Entitie s and Gene Organizations of G P rotein s . . . . . . . . . . .... . ... .. . . . . . . .. ... O verall St ru ctu re of G P rotein a-Su bunits .. . . .. . . . . . . . . .. . . . ... . . . . . . . . . . . . . . . . . ... . . . . . . . . Structu re -Fun ction Relation s hip of G P rotein a-Subunits . . . . . . . . . . . . . . . ... . .... . .. . .. . . . Structure of fly-Subunits of G Protein . . . .. . . . .. . . . .. . . ..... ..... . ... . . . . . . ... . . ..... . . . . . . . .
363 368 363
368 376
ITo whom correspondence should be addressed. 2Present address: DNAX Research Institute of Molecular & Cellular Biology, 901 California Avenue, Palo Alto, California 94304-1 104 3Present address: Department of Pharmacology, The University of Texas, SouthWestern Medical Center, Dallas, Texas 75235-9041 4Present address: Department of Phannacology, The University of JIIinois at Chicago, Chica go, Illinois 606 12
0066-4154/9110701-0349$02.00
349
350
KAZIRO ET AL
Annu. Rev. Biochem. 1991.60:349-400. Downloaded from www.annualreviews.org by Moscow State University - Scientific Library of Lomonosov on 08/12/13. For personal use only.
V. G PROTEINS IN YEAST SACCHAROMYCES CEREVISIAE . . . . . . . . . . . .. . . . . . . . . . . . . . . Comparison of Structures of Yeast and Mammalian GQ Proteins . , . . . . . . . . . . . . . . . . . . . . The Role of G Proteins in Yeast Signal Transduction . . . . .. . . . . . .. . . . .. . . . . . . . .. . . . . . . . . . .
376 376 377
V I . G PROTEINS FROM O THER ORGANISMS . . . . . . . . . .. . . . .. . . . .. . . .. . . . . . . . . . . . . . . . . . . . . G Protein of Drosophila melanogaster.... . . . . . . . .. . . . . . . . . .. . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . G Proteins from Dictyostelium discoideum. . . . . .. . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . Plant G Protein . . . . . . . . . . . . . . . . . .. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G Protein from Schizosaccharomyces pombe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
383 383 384 385 385
VII. MAMMALIAN RAS PROTEINS . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of Ras Activity by a Bound Guanine Nucleotide . . . . . . . . . . . . . . . . .. . . .. . . . . . . . GAP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
385 385 388
VIII. RAS PROTEINS IN yEAS T .. . . . . . .. . . . .. . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. S . cerevisiae Ras and Cyclic AMP Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Downstream Effector of Yeast Ras . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of Ras Proteins in yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .. . . . . . . . . . . . . . RAS Proteins in Schizosaccharomyces pombe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
390 390 391 392 394
I. PERSPECTIVES AND SUMMARY Extracellular signals are recognized by receptors on the surface of the cytoplasmic membrane and transmitted through transducers to amplifiers, which synthesize catalytically many molecules of second messengers that reach final targets through many additional steps. Transducers, which regu late the opening and closing of the signalling pathways, are GTP-binding proteins, which we discuss in detail in this review article. Signal-transducing GTP-binding proteins, or GTPases, are classified large ly into two groups. One is the high-molecular-weight or heterotrimeric GTP binding proteins (G proteins), consisting of three subunits, ct, {3, and y. Another group is the low-molecular-weight, monomeric GTP-binding pro teins (often abbreviated as "small Os"). The two groups, both growing in size, contain a number of subgroups, each of which in tum contains several members. GTP-binding proteins are widely distributed in various tissues and among organisms, and the structures of individual proteins are highly con served among distant organisms. The basic mechanism of the reactions catalyzed by GTP-binding proteins is essentially analogous to that proposed for translational factors ( 1 ) . The GTP bound form is an active conformation that turns on the transmission of signals, and the hydrolysis of bound GTP to bound ODP is required to shift the conformation to an inactive form, i.e. to tum off the signal transduction. Interconversion of the GTP-bound form (E'OTP) and the GDP-bound form (E'GDP) is regulated by two mechanisms. The conversion of E'ODP to E'GTP by nucleotide exchange is stimulated by an exchange-promoting pro tein (EP), which increases the off-rate (Kdiss.GDP) of GDP from E·GDP. On the other hand, the conversion of E·GTP to E'ODP is accelerated by a protein that stimulates the intrinsic GTPase activity. A GTPase-activating protein (GAP) functions by increasing the first-order rate constant of GTP hydrolysis (kcat.GTP), often by several orders of magnitude.
Annu. Rev. Biochem. 1991.60:349-400. Downloaded from www.annualreviews.org by Moscow State University - Scientific Library of Lomonosov on 08/12/13. For personal use only.
GTP-BINDING PROTEINS
351
The above regulatory mechanism, of course, varies with individual GTP binding proteins. In the classical example of E. coli elongation factors , the exchange reaction, EF-Tu'GDP ;;:::: EF-Tu'GTP is stimulated by EF-Ts, and the hydrolysis of GTP bound to EF-Tu by ribosomes (1). In the case of signal-transducing GTP-binding proteins , heterotrimeric G proteins are acti vated through the ligand·receptor-induced stimulation of the exchange reac tion . However, the hydrolysis of bound GTP appears to be determined by the intrinsic (built-in) GTPase activity of G proteins ( 3-5 min -I). On the other hand, in ras p2 1 proteins, where the rates of GDP release and GTP hydrolysis are exceedingly small «0.01 min- I) , both EP and GAP are required for the GTPase cycle to proceed. We discuss later the detailed reaction mechanisms of individual GTP-binding proteins as well as the regulation of the GTPase cycle by signals coming from outside the cells. Finally, we emphasize that no matter how complex the diverse reactions �
appear, the basic principle of the function of GTP-binding proteins as molecu
lar switches is universal. All GTP-binding proteins utilize the same cycle of reactions, i .e. activation by GTP binding and relaxation by GTP hydrolysis. They unde:rgo sequential conformational alterations depending on the phos phate potential of their ligand, and their abilities to interact with their cognate macromolecules are altered qualitatively. This cycle is irreversible and unidi rectional by virtue of GTP hydrolysis . The most important problem still remaining i s the identification o f the downstream targets of GTP-binding proteins. Except for a few cases, the assignment of functions for each individual GTP-binding protein is still under way. For example, in spite of much effort, the immediate upstream and downstream molecules for mammalian Ras proteins are still unknown . The precise role of individual GTP-binding proteins in switching the signalling for cellular growth and differentiation as well as the response to hormones , neurotransmitters , and sensory stimuli remains for further investigation. II. INTRODUCTION
The superfamily of GTP-binding proteins consists of several families includ ing (a) translational factors , (b) heterotrimeric GTP-binding proteins involved in transmembrane signalling processes (abbreviated as G proteins), (c) proto oncogenic ras proteins (Ras proteins), (d) other low-molecular-weight GTP binding proteins, including the products of rab, rap, rho, rae, smg21, smg25, YPT, SEC4, ARF genes (abbreviated as "small Gs") , and (e) tubulins . There are also other metabolic enzymes that interact with GTP (such as succinate thiokinase, and phosphoenolpyruvate carboxykinase) , but they are not dis cussed here . In this review article, we limit our discussions mainly to the proteins that
352
KAZIRO ET AL
(b) and (c), i.e. G proteins and Ras proteins. We describe (2) and Barbacid (3) in the Annual Review of Biochemistry. This review is not belong to Groups
mainly progress achieved since the previous reviews by Gilman
comprehensive; rather, we emphasize our special interest, which is how GTP-binding proteins function as a "molecular switch" that regulates the
Annu. Rev. Biochem. 1991.60:349-400. Downloaded from www.annualreviews.org by Moscow State University - Scientific Library of Lomonosov on 08/12/13. For personal use only.
opening and closing of signal transmission. For a broader view, we recom
& Clapham (4), Freissmuth et al (5), (6), Gibbs & Marshall (7), Kaziro (8), Hall (9) , and Bourne et al ( 10) .
mend the more recent reviews by Neer Ross
Several monographs have also been published recently; they include those edited by Bosch, Kraal, and Parmeggiani Houslay and Milligan
(13),
(11), Iyenger and Bimbaumer ( 1 2), (14).
and Moss and Vaughan
All signal-transducing GTP-binding proteins bind and hydrolyze GTP, properties that are crucial to their function as a molecular switch for diverse cellular functions. Each GTP-binding protein, however, has its own dissocia
tion constant for guanine nucleotides, Kd value for GTP and GOP, and rate constant for GTP hydrolysis
(kcat.GTP)'
GTP-binding proteins undergo two alternate conformations (and reactivi ties) depending on the phosphate potentials of the ligand (Figure lA). The
GTP-bound form is an active conformation; in this form the protein can recognize and interact with its target molecules. On hydrolysis of the bound GTP to GOP and inorganic phosphate, the conformation as well as the reactivity of the protein is shifted to the GOP-bound form (an inactive form). The conversion of the GOP-bound form to the GTP-bound form is achieved by the exchange of the bound GOP with an external GTP. This is the step in which the energy from outside is fed into this system in the form of GTP. On the other hand, in the relaxation process, the bound GTP is hydrolyzed to
GDP and inorganic phosphate. Depending on the kinetic parameters of each GTP-binding protein, factors that stimulak (he exchange reaction (EP or exchange-promoting proteins), or factors that increase the rate of hydrolysis of bound GTP (GAP or GTPase-activating proteins) are required. The above basic mechanism, originally proposed from studies on trans lational factors (see the next section), has been found in a variety of systems to be dependent on either GTP or ATP. It has been known that replication, recombination, and repair of ONA are dependent on ATP hydrolysis, and a number of DNA-dependent ATPases are involved in these processes
( 15).
These ATPases function in a manner analogous to translation GTPases; i.e. in the presence of ATP, the proteins are bound to DNA, inducing structural changes that are required for the sequential events in the processes of DNA replication, recombination, and repair. Furthermore, we can find an analogous mechanism in protein phosphoryla tion and dephosphorylation reactions. Proteins can be activated or inactivated by a covalent phosphorylation. In this case, a protein can undergo, again, two alternate conformational changes, i.e. between phosphorylated and nonphos-
GTP-BINDING PROTEINS
�
(ATP) G
A Annu. Rev. Biochem. 1991.60:349-400. Downloaded from www.annualreviews.org by Moscow State University - Scientific Library of Lomonosov on 08/12/13. For personal use only.
353
(AOP) GOP·
(ADP) GDP (ATP) . GTP
B -P
Figure
1
kinases (8). For details, see text.
General reaction mechanism of GIP- (or AIP-) binding proteins (A) and protein
phorylated fonus (Figure IB). As described below, intracellular signals are mostly transmitted via protein-protein interactions. The interactions are mod ulated by conformational change induced by ligands covalently or nonco valently as.sociated with the proteins. Recently, it was found that in growth regulation, many protein kinases as well as protein phosphatases play an essential role in response to growth factor stimuli.
354
KAZIRO ET AL
III. GENERAL CHARACTERISTICS In this section, we first outline the classical GTP-utilizing reaction, i.e. the reaction catalyzed by the translational elongation factors Tu and G. Then, we compare this reaction with newly developed systems.
Annu. Rev. Biochem. 1991.60:349-400. Downloaded from www.annualreviews.org by Moscow State University - Scientific Library of Lomonosov on 08/12/13. For personal use only.
Translational Factors Several GTP-binding proteins are known to be involved in protein biosynthe sis. These include initiation factor 2 (IF-2), elongation factor Tu (EF-Tu), and elongation factor G (EF-G) for prokaryotes, and initiation factor 2 (eIF-2),
elongation factor I a (EF-I a), and elongation factor 2 (EF-2) for eukaryotes.
Below we discuss some general characteristics of the polypeptide chain (1). Figure 2 summarizes the reaction mechanism of
elongation cycle
GTP
B
EF-Tu GOP •
Pi
~ •. Figure 2
GOP
�t�_F
\
(EF-Ts)
n8
GOP
@ Ammoacyl-
� �
GTP
.
,,�VD tRNA � 67
Tu ·GTP
1991. 60:349-400
Copyright © 1991 by Annual
Annu. Rev. Biochem. 1991.60:349-400. Downloaded from www.annualreviews.org by Moscow State University - Scientific Library of Lomonosov on 08/12/13. For personal use only.
Annu. Rev. Biochem.
Further
Quick links to online content
Reviews Inc. All rights reserved
STRlJCTURE AND FUNCTION OF
SIGNAL-TRANSDUCING
GTP-BINDING PROTEINS
Yoshito Kaziro / ,2 Hiroshi Itoh,3 Tohru Kozasa,4 Masato Nakafukll,2 and Takaya Satoh2 Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108, Japan KEY WORDS:
elongation factor, Ras proteins, G proteins, GTPase cycle, confonnational switch.
CONTENTS 1. PERSPECTIVES AND SUMMARy . . . . . . . . . .
350
II. INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
35 1
III. GENERAL CHARACTERISTICS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tra nslati onal Fa ctors . . . . . . . . . . . . .. . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... . . .. . . .. ... . . Heterotrimeric G Protein s . . .. . . .. . . . . ... . . ... .. ... .. .. .. .... . .... . . .. . . . .. . .. . . . .. . . .. . .. . . . . .. Ra s Protein s . .. . . . . . . .. . . . . . . . . . . .. . . . . . . . . . . . . . . .. ... . . . . . . . . . . . . . . . .. ... . . . . . .. . . ... . . . . . . . ... . .. Structu ral Simi /aritie s Be w t een Vari ou s G TP-Binding Protein s. . . . . . . . . . . . . . . . .
354 354 356 357 359
IV . MAMMALIAN G PRO TEINS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M olecular Entitie s and Gene Organizations of G P rotein s . . . . . . . . . . .... . ... .. . . . . . . .. ... O verall St ru ctu re of G P rotein a-Su bunits .. . . .. . . . . . . . . .. . . . ... . . . . . . . . . . . . . . . . . ... . . . . . . . . Structu re -Fun ction Relation s hip of G P rotein a-Subunits . . . . . . . . . . . . . . . ... . .... . .. . .. . . . Structure of fly-Subunits of G Protein . . . .. . . . .. . . . .. . . ..... ..... . ... . . . . . . ... . . ..... . . . . . . . .
363 368 363
368 376
ITo whom correspondence should be addressed. 2Present address: DNAX Research Institute of Molecular & Cellular Biology, 901 California Avenue, Palo Alto, California 94304-1 104 3Present address: Department of Pharmacology, The University of Texas, SouthWestern Medical Center, Dallas, Texas 75235-9041 4Present address: Department of Phannacology, The University of JIIinois at Chicago, Chica go, Illinois 606 12
0066-4154/9110701-0349$02.00
349
350
KAZIRO ET AL
Annu. Rev. Biochem. 1991.60:349-400. Downloaded from www.annualreviews.org by Moscow State University - Scientific Library of Lomonosov on 08/12/13. For personal use only.
V. G PROTEINS IN YEAST SACCHAROMYCES CEREVISIAE . . . . . . . . . . . .. . . . . . . . . . . . . . . Comparison of Structures of Yeast and Mammalian GQ Proteins . , . . . . . . . . . . . . . . . . . . . . The Role of G Proteins in Yeast Signal Transduction . . . . .. . . . . . .. . . . .. . . . . . . . .. . . . . . . . . . .
376 376 377
V I . G PROTEINS FROM O THER ORGANISMS . . . . . . . . . .. . . . .. . . . .. . . .. . . . . . . . . . . . . . . . . . . . . G Protein of Drosophila melanogaster.... . . . . . . . .. . . . . . . . . .. . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . G Proteins from Dictyostelium discoideum. . . . . .. . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . Plant G Protein . . . . . . . . . . . . . . . . . .. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G Protein from Schizosaccharomyces pombe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
383 383 384 385 385
VII. MAMMALIAN RAS PROTEINS . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of Ras Activity by a Bound Guanine Nucleotide . . . . . . . . . . . . . . . . .. . . .. . . . . . . . GAP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
385 385 388
VIII. RAS PROTEINS IN yEAS T .. . . . . . .. . . . .. . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. S . cerevisiae Ras and Cyclic AMP Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Downstream Effector of Yeast Ras . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of Ras Proteins in yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .. . . . . . . . . . . . . . RAS Proteins in Schizosaccharomyces pombe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
390 390 391 392 394
I. PERSPECTIVES AND SUMMARY Extracellular signals are recognized by receptors on the surface of the cytoplasmic membrane and transmitted through transducers to amplifiers, which synthesize catalytically many molecules of second messengers that reach final targets through many additional steps. Transducers, which regu late the opening and closing of the signalling pathways, are GTP-binding proteins, which we discuss in detail in this review article. Signal-transducing GTP-binding proteins, or GTPases, are classified large ly into two groups. One is the high-molecular-weight or heterotrimeric GTP binding proteins (G proteins), consisting of three subunits, ct, {3, and y. Another group is the low-molecular-weight, monomeric GTP-binding pro teins (often abbreviated as "small Os"). The two groups, both growing in size, contain a number of subgroups, each of which in tum contains several members. GTP-binding proteins are widely distributed in various tissues and among organisms, and the structures of individual proteins are highly con served among distant organisms. The basic mechanism of the reactions catalyzed by GTP-binding proteins is essentially analogous to that proposed for translational factors ( 1 ) . The GTP bound form is an active conformation that turns on the transmission of signals, and the hydrolysis of bound GTP to bound ODP is required to shift the conformation to an inactive form, i.e. to tum off the signal transduction. Interconversion of the GTP-bound form (E'OTP) and the GDP-bound form (E'GDP) is regulated by two mechanisms. The conversion of E'ODP to E'GTP by nucleotide exchange is stimulated by an exchange-promoting pro tein (EP), which increases the off-rate (Kdiss.GDP) of GDP from E·GDP. On the other hand, the conversion of E·GTP to E'ODP is accelerated by a protein that stimulates the intrinsic GTPase activity. A GTPase-activating protein (GAP) functions by increasing the first-order rate constant of GTP hydrolysis (kcat.GTP), often by several orders of magnitude.
Annu. Rev. Biochem. 1991.60:349-400. Downloaded from www.annualreviews.org by Moscow State University - Scientific Library of Lomonosov on 08/12/13. For personal use only.
GTP-BINDING PROTEINS
351
The above regulatory mechanism, of course, varies with individual GTP binding proteins. In the classical example of E. coli elongation factors , the exchange reaction, EF-Tu'GDP ;;:::: EF-Tu'GTP is stimulated by EF-Ts, and the hydrolysis of GTP bound to EF-Tu by ribosomes (1). In the case of signal-transducing GTP-binding proteins , heterotrimeric G proteins are acti vated through the ligand·receptor-induced stimulation of the exchange reac tion . However, the hydrolysis of bound GTP appears to be determined by the intrinsic (built-in) GTPase activity of G proteins ( 3-5 min -I). On the other hand, in ras p2 1 proteins, where the rates of GDP release and GTP hydrolysis are exceedingly small «0.01 min- I) , both EP and GAP are required for the GTPase cycle to proceed. We discuss later the detailed reaction mechanisms of individual GTP-binding proteins as well as the regulation of the GTPase cycle by signals coming from outside the cells. Finally, we emphasize that no matter how complex the diverse reactions �
appear, the basic principle of the function of GTP-binding proteins as molecu
lar switches is universal. All GTP-binding proteins utilize the same cycle of reactions, i .e. activation by GTP binding and relaxation by GTP hydrolysis. They unde:rgo sequential conformational alterations depending on the phos phate potential of their ligand, and their abilities to interact with their cognate macromolecules are altered qualitatively. This cycle is irreversible and unidi rectional by virtue of GTP hydrolysis . The most important problem still remaining i s the identification o f the downstream targets of GTP-binding proteins. Except for a few cases, the assignment of functions for each individual GTP-binding protein is still under way. For example, in spite of much effort, the immediate upstream and downstream molecules for mammalian Ras proteins are still unknown . The precise role of individual GTP-binding proteins in switching the signalling for cellular growth and differentiation as well as the response to hormones , neurotransmitters , and sensory stimuli remains for further investigation. II. INTRODUCTION
The superfamily of GTP-binding proteins consists of several families includ ing (a) translational factors , (b) heterotrimeric GTP-binding proteins involved in transmembrane signalling processes (abbreviated as G proteins), (c) proto oncogenic ras proteins (Ras proteins), (d) other low-molecular-weight GTP binding proteins, including the products of rab, rap, rho, rae, smg21, smg25, YPT, SEC4, ARF genes (abbreviated as "small Gs") , and (e) tubulins . There are also other metabolic enzymes that interact with GTP (such as succinate thiokinase, and phosphoenolpyruvate carboxykinase) , but they are not dis cussed here . In this review article, we limit our discussions mainly to the proteins that
352
KAZIRO ET AL
(b) and (c), i.e. G proteins and Ras proteins. We describe (2) and Barbacid (3) in the Annual Review of Biochemistry. This review is not belong to Groups
mainly progress achieved since the previous reviews by Gilman
comprehensive; rather, we emphasize our special interest, which is how GTP-binding proteins function as a "molecular switch" that regulates the
Annu. Rev. Biochem. 1991.60:349-400. Downloaded from www.annualreviews.org by Moscow State University - Scientific Library of Lomonosov on 08/12/13. For personal use only.
opening and closing of signal transmission. For a broader view, we recom
& Clapham (4), Freissmuth et al (5), (6), Gibbs & Marshall (7), Kaziro (8), Hall (9) , and Bourne et al ( 10) .
mend the more recent reviews by Neer Ross
Several monographs have also been published recently; they include those edited by Bosch, Kraal, and Parmeggiani Houslay and Milligan
(13),
(11), Iyenger and Bimbaumer ( 1 2), (14).
and Moss and Vaughan
All signal-transducing GTP-binding proteins bind and hydrolyze GTP, properties that are crucial to their function as a molecular switch for diverse cellular functions. Each GTP-binding protein, however, has its own dissocia
tion constant for guanine nucleotides, Kd value for GTP and GOP, and rate constant for GTP hydrolysis
(kcat.GTP)'
GTP-binding proteins undergo two alternate conformations (and reactivi ties) depending on the phosphate potentials of the ligand (Figure lA). The
GTP-bound form is an active conformation; in this form the protein can recognize and interact with its target molecules. On hydrolysis of the bound GTP to GOP and inorganic phosphate, the conformation as well as the reactivity of the protein is shifted to the GOP-bound form (an inactive form). The conversion of the GOP-bound form to the GTP-bound form is achieved by the exchange of the bound GOP with an external GTP. This is the step in which the energy from outside is fed into this system in the form of GTP. On the other hand, in the relaxation process, the bound GTP is hydrolyzed to
GDP and inorganic phosphate. Depending on the kinetic parameters of each GTP-binding protein, factors that stimulak (he exchange reaction (EP or exchange-promoting proteins), or factors that increase the rate of hydrolysis of bound GTP (GAP or GTPase-activating proteins) are required. The above basic mechanism, originally proposed from studies on trans lational factors (see the next section), has been found in a variety of systems to be dependent on either GTP or ATP. It has been known that replication, recombination, and repair of ONA are dependent on ATP hydrolysis, and a number of DNA-dependent ATPases are involved in these processes
( 15).
These ATPases function in a manner analogous to translation GTPases; i.e. in the presence of ATP, the proteins are bound to DNA, inducing structural changes that are required for the sequential events in the processes of DNA replication, recombination, and repair. Furthermore, we can find an analogous mechanism in protein phosphoryla tion and dephosphorylation reactions. Proteins can be activated or inactivated by a covalent phosphorylation. In this case, a protein can undergo, again, two alternate conformational changes, i.e. between phosphorylated and nonphos-
GTP-BINDING PROTEINS
�
(ATP) G
A Annu. Rev. Biochem. 1991.60:349-400. Downloaded from www.annualreviews.org by Moscow State University - Scientific Library of Lomonosov on 08/12/13. For personal use only.
353
(AOP) GOP·
(ADP) GDP (ATP) . GTP
B -P
Figure
1
kinases (8). For details, see text.
General reaction mechanism of GIP- (or AIP-) binding proteins (A) and protein
phorylated fonus (Figure IB). As described below, intracellular signals are mostly transmitted via protein-protein interactions. The interactions are mod ulated by conformational change induced by ligands covalently or nonco valently as.sociated with the proteins. Recently, it was found that in growth regulation, many protein kinases as well as protein phosphatases play an essential role in response to growth factor stimuli.
354
KAZIRO ET AL
III. GENERAL CHARACTERISTICS In this section, we first outline the classical GTP-utilizing reaction, i.e. the reaction catalyzed by the translational elongation factors Tu and G. Then, we compare this reaction with newly developed systems.
Annu. Rev. Biochem. 1991.60:349-400. Downloaded from www.annualreviews.org by Moscow State University - Scientific Library of Lomonosov on 08/12/13. For personal use only.
Translational Factors Several GTP-binding proteins are known to be involved in protein biosynthe sis. These include initiation factor 2 (IF-2), elongation factor Tu (EF-Tu), and elongation factor G (EF-G) for prokaryotes, and initiation factor 2 (eIF-2),
elongation factor I a (EF-I a), and elongation factor 2 (EF-2) for eukaryotes.
Below we discuss some general characteristics of the polypeptide chain (1). Figure 2 summarizes the reaction mechanism of
elongation cycle
GTP
B
EF-Tu GOP •
Pi
~ •. Figure 2
GOP
�t�_F
\
(EF-Ts)
n8
GOP
@ Ammoacyl-
� �
GTP
.
,,�VD tRNA � 67
Tu ·GTP
