SIGNAL TRANSDUCTION: EVOLUTION
OF AN IDEA

Nobel Lecture, December 8, 1994

`>Y

National Institute of Environmental Health Scienes, P.O. Box 12233,
Research Triangle Park, North Carolina, USA

In general there is no set of observations conceivable which can
e've enough information about the past of a system to give com-
@de information as to its future.. .
                               Norbert Wiener

Think simplicity; then discard it.. . .

Alfred North Whitehead

INTRODUCTION

I was born in 1925, a time when there were no talking movies, radio was just
emerging as a popular listening device, when newspapers printed important
information, and libraries were sources of both pleasure and learning. My
father's grocery store (above which we lived) was a community center where
people from blocks away would come for their groceries and to gossip. We
knew or knew about everyone in our neighborhood. In that atmosphere I
grew up as a young man feeling the warmth of this community.
Retrospcclively, I have come to realize how important this long-gone com-
munity and the intense human relationships have been to my development
as a scientist. My scientific neighborhood encompasses a place where cultu-
ral and language differences have been melded seamlessly and with synergy
to promote communication, to expand knowledge with a kinship of purpo-
se, and to create new thought. Nature, which we often equate with our gene-
tic make-up, and Nurture, which symbolizes our environment, interact mutu-
ally and synergistically in this community. These are the forces that have
given meaning to life; i.e. the parable ofwhich comes first, the chicken or the
egg, is not of biological importance.

My lecture symbolizes my interest in societal/cellular relationships and
concerns the broad issues of biological communication. The first half will
deal with the development of the concept of transducers and their role in
cell signaling. Since this concepl is still at an evolulionary phase, I will con-

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elude with an hypothesis which in its simplest message argues that biological
communication consists of a complex meshwork of structures in which G
proteins, surface receptors, the extracellular matrix, and the vast cytoskeletal
network within cells are joined in a community of effort, for which my life
and those of my colleagues is a metaphor.

RECEPTORS, ALLOSTERY AND THE SECOND MESSENGER THEORY

The concept of receptors as sensory elements in biology has a long history.
Early in this century Paul Ehrlich realized the importance of surface recep-
tors and postulated a "lock and key" theory to explain their interactions with
antigenic materials and drugs. Today, it is understood that receptors are pro-
teins with the patterns of design and malleability of structure required for
discriminating between an extraordinary variety of chemical signals. My
interest in receptors began in the early 60's, when I uncorked the means of
freeing adipocytes from their tissue matrix by collagenase treatment and
found that insulin at physiological concentrations stimulated glucose upta-
ke(1). Searching for the possible site of action of the hormone, I tested the
effects of treating adipocytes with phospholipases and proteases on the
assumption that, if the surface or plasma membrane contains insulin recep-
tors, these digestive enzymes might prevent insulin action. Surprisingly,
phospholipases mimicked the known actions of insulin on glucose utiliza-
tion and protein synthesis (2,3), Based on such observations I postulated that
insulin might act by stimulating phospholipases (4)) not a bad hypothesis in
view of the accumulated evidence of the importance of phospholipases in
mediating the actions of a variety of hormones (5) . .

During the 60's two major theories influenced the course of my research
on hormone receptors. One was the "Second Messenger" theory (6,7). This
theory suggested that extracellular or primary messengers in the form of
hormones or neurotransmitters act through receptors that regulate the pro-
duction of 3'5' adenosine monophosphate (cyclic AMP) , considered to be
the intracellular messenger that mediates the actions of hormones on all
aspects of cellular metabolism, growth, and differentiation. The perceptions
of Monod and colleagues that led to their incisive theory of allosteric regu-
lation (8) blended beautifully with Sutberlancl's theory that receptors are
structurally and functionally linked to the regulation of cyclic AMP procluc-
tion. Overwhelmingly persuasive was the notion that aclenyl (now atlenyla-
te or adenylyl) cyclase) is an allosterically-regulated enzyme system consisting
of two distinct sites, receptors and catalytic. Located at the surface or plasma
membrane of cells, the assymetric positioning of these sites- the allosteric
hormone-sensing sites on the exterior and ATP-utilizing catalytic sites at the
interior surfaces of the membrane- provided a logical framework for investi-
gating the molecular basis for hormone action. My attention shifted from
insulin to those hormones known to stimulate the production of cyclic AMP
in fat cells.

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THE MULTI-RECEPTOR ADENYLATE CYCLASE SYSTEM IN
ADIPOCYTES

At the time, the only specific assay for cyclic AMP production relied on a
complicated, time consuming bioassay. Gopal Krishna (9) and later Salomon
(10) developed relatively simple chromatographic assays which for the first
time allowed rapid, multiple assays of adenylate cyclase. When Lutz
Birnbaumcr arrived in my laboratory in 1967, that assay literally danced
under his extraordinary prowess, yielding information that laid the founda-
tion for the concept of transducers. Prior to his coming, I had developed a
rapid method for obtaining fat cell membranes (called "ghosts") responsive
not only to insulin but also to various hormones that stimulate cyclic AMP
production and resultant lipolysis in fat cells (11). These hormones included
epinephrine, ACTH, TSH, LH, secretin, and glucagon. ACTH and fluoride
ion. The latter, shown previously to stimulate adenylate cyclase in a variety of
cell membranes (6)) activated the fat cell system by a Mgdependent process
displaying a Hill coefficient of 2.0, suggesting that the system may contain at
least two sites of Mg action, one certainly a Mg-ATP complex at the catalytic
site. That a regulatory site for Mg exists was suggested by the finding that
both ACTH and fluoride markedly reduced the concentration of Mg ions
necessary for stimulation of activity (12). The kinetics of ATP action proved
too complicated for interpretation at the time. Not realizing that ATP was
contaminated with GTP, we couldn't interpret what later proved to be the sti-
mulatory and inhibitory actions of GTP on adenylate cyclase systems. The
lesson is clear to me today; never attempt to interpret a hyperbolic curve; it
describes the behavior of the entire universe!

DEMONSTRATION OF DISTINCT HORMONE RECEPTORS.

Much of our energy and time was devoted to delineating the receptors for
the hormones that stimulated the cyclase system. The pharmacology of the
peptide hormones receptor was essentially unknown and necessitated a vari-
ety of indirect tests, including the effects of proteases, inhibitory analogs,
and differential ion dependencies, which combined suggested that each of
the hormones stimulated cyclase through distinct receptor types. Since the
enzyme system and the receptors were contained in the same cell, these fin-
dings allowed us to test a fundamental question; do all of the hormones ope-
rate on the same enzyme or, as depicted in the Sutherland model, is each
hormone receptor coupled to separate cyclase models. The various hormo-
nes were tested at maximal and submaximal concentrations alone or comb-
ined with the other hormones. Synergy was seen with some combinations
but, most importantly, additivity of response was not obtained with maximal
concentrations of the hormones (13). Similar findings were reported simult-
aneously (14). Although not proof, we argued that it is likely that the fat cell
cyclase system consists of multiple receptors interacting with a common cata-

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lytic unit. Conceptually, the picture that emerged is that each receptor con-
tains specific binding regions and some common structural element that
interacts with the catalytic component to stimulate conversion of MgATP to
CAMP. At that time we considered that the catalytic component contains the
regulatory site for Mg ions and is the site of action of fluoride ion. Lipids
were somehow involved in the structural interactions between receptors and
catalytic unit because, unlike fluoride action, hormone action was exquisite-
ly sensitive to agents (phospholipases, detergents) that affect membrane
structure (15 ). It was clear that hormone action involved a more complex
structural and regulatory enzyme system than originally conceived. It was
inconceivable to me that several hormone receptors could be structurally
annealed to the same enzyme (I referred to this problem as "too many angels
on a pinhead"). A new concept of hormone action had to be considered.

INFORMATIONAL PROCESSING: THE CONCEPT OF TRANSDUCTION

At that time my thinking on the subject of how hormonal information is
transferred across the cell membrane and translated into action was greatly
influenced by the  theories of informational processing proposed by
Norbert Wiener (16), the originator of cybernetic theory . This subject was
introduced to me by Oscar Hechter who had previously proposed several
important theoretical considerations concerning hormone action. He was
the first to question the proposition that hormones directly acted on the ade-
nylate cyclase enzyme (17). Through lengthy discussions at a downtown
hotel bar in Washington,D.C. prior to a meeting that I had organized at NIH
to honor Sutherland, we arrived at the concept of transduction as a means
of coupling information between signal-activated receptor and regulation of
adenylate cyclase. Given the paucity of knowledge at that time, the concept
of informational processing was put in abstract cybernetic terms: discrimina-
tor for receptor, a transducer, and an amplifier representing adenylate cycla-
se because of the large increase in cyclic AMP generated when converted to
its activated state. The transducer is a coupling device designed to allow com-
munication between discriminator and amplifier. At the meeting I presented
this idea, illustrated (but without participation of Mg and GTP at that time)
in Fig. 1. We considered the possibility that Mg ions and lipids participated
in the transduction process, but we realized that the transducer concept
required fleshing out with more evidence on the structure/functional rela-
tionships between receptor and enzyme.

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THE ACTIONS OF GTP AND GLUCAGON ON LIVER CYCLASE

Because of the experimental complexity of studying the multi-receptor ade-
nylate cyclase system in rat adipocytes, my colleagues ( Birnbaumer, Pohl,
Krans) and I turned our attention to the glucagon-sensitive adenylate cycla-
se system in liver. To some extent this change was made because of the his-
torical significance of the hepatic system in hormone action and, coinci-
dentally, because David Neville (18) at NIH had reported purification of rat
liver plasma membranes by a relatively simple procedure. As importantly, we
radio-labeled glucagon with `251 making it possible to investigate both the
nature of the glucagon receptor and the relationship between hormone bin-
ding and hormonal activation of adenylate cyclase. .

Michiel Krans began the glucagon-binding studies with our findings that
hormonal activation of atlenylate cyclase in liver membranes rises within
seconds and falls rapidly when the hormone is displaced by an antagonist
such as des-his-glucagon, which proved later to be a weak partial agonist. Our
expectations were that binding of 1251-glucagon would proceed rapidly (wit-
hin seconds) and would be reversed easily by washing the membranes free of
medium containing the hormone. Instead, Krans observed that binding was
extremely slow requiring at least 20 minutes before reaching a plateau.
Extensive washing under a variety of conditions failed to remove the bound
material. None of the binding characteristics fit with the kinetics of hormo-
ne action. However, the medium used for binding contained nothing but salt
and buffer whereas the cyclase assay medium contained multiple compo-
nents including the substrate, MgATP. A dramatic change resulted when all
of the cyclase-ingredients were added to the hormone-binding medium. The
level of bound hormone at "steady-state" was drastically reduced; maximal
binding was attained within seconds. We subsequently found that ATP was
the principal culprit. Realizing from painful experience as a graduate stu-
dent that commercial preparations of ATP contain a variety of contaminating
nucleotides, I tested many types of purine and pyrimidine nucleotides. GTP,
GDP, and ITP were the only nucleotides that mimicked the effects of ATP.
Most importantly, the guanine nucleotides acted at concentrations much
lower (two to three orders of magnitude) than ATP. GppCp, a poorly hydro-
lyzable analog, also acted although its effects required much higher concen-
trations compared to GTP or GDP. Each of the nucleotides induced rapid
release of pre-bound glucagon from its receptor. We established that guani-
ne nucleotides act by lowering the affinity of receptor for the hormone
(19)).

At that point the central question was the possible relationship of this
effect of GTP on hormone binding to the actions of glucagon on adenylate
cyclase activity. To avoid the problem of contaminating GTP in the assay for
the enzyme, we prepared 3*P-App(NH)p as substrate using a biosynthetic
method. This analog proved stable to degradation by ATPases in the mem-
brane, Under these conditions, glucagon did not stimulate adenylate cyclase
unless GTP was present in approximately the same concentrations that affec-

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ted the affinity of the receptor (20). Subsequently, Michael Lin and Yoram
Salomon (21) demonstrated that hormone and GTP concertedly and rapicl-
ly induced the active form of the enzyme. Glucagon, moreover, reduced the
small lag in activation given by activating nucleotide alone. The die was cast;
logically GTP acts at the transduction process along with Mg ions (Fig 1).
Although the components of the informational processing system remained
unknown, there was little doubt in our minds that a transducer exists and
that this crucial component mediates the transfer of information between
receptor and enzyme.

GTP HYDROLYSIS.

Because GTP was susceptible to hydrolysis by nucleotidases in membranes,
our next objective was to substitute GTP with a non-hydrolyzable derivative.
Taking a cue from our experience with App(NH)p, Gpp(NH)p) was synthe-
sized. A few months later, we found that Gpp(NH)p caused the enzyme's
activity to "take off' to an extent not even seen with fluoride ion. To our ama-
zement, the normally unstable cyclase system remained fully active even after
three days at room temperature. We then tested Gpp(NH)p on a variety of
cyclase systems using every cell membrane preparation we could obtain. All
showed the same phenomenon (22). Gpp (NH) p, unlike hormone plus GTP,
stimulated activity following a rather lengthy lag period which was shortened
considerably when hormone was added (21). Yoram Salomon investigated
the binding of "*P-Gpp(NH)p to liver membranes and found substantial
guanine nucleotide- specific binding, far in excess of the number of gluca-
gon receptors (23). These findings were discounted by others because of the
seeming disparity in the levels of glucagon receptor and guanine nucleotide
binding sites. However, it was not understood at the time that multiple types
of receptors interact with several types of GTP-binding proteins; that story
evolved nearly 10 years later. The key elements of signal transduction gained
from these findings were that Gpp(NH)p binds to the liver membranes in
the absence of hormone whereas glucagon quickened the ability of the
nucleotide to activate adenylate cyclase, not vice versa. These findings plus
modeling of the kinetics of Gpp(NH)p/Mg (24 ) gave rise to a three state

             8
GPP(N'H)P   Hormone
E = E i-     Eq (1)
                    E*

GTP    Hormone

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model in which hormones act by promoting the conversion of the nucleoti-
de-bomnd E' state to the activated state (E*). However, with 21 parameters
using just Mg2+ and Gpp(NH)p concentrations as variables we realized that
this model yielded only an approximation of what must be a very complica-
ted system.

At about the same time Michael Schramm, in a series of beautifully exe-
cuted experiments, demonstrated that Gpp(NH)p acted in a pseudo-irrever-
sible fashion; i.e., removal of the nucleotide from the medium after incuba-
tion resulted in retention of the high level of cyclase activity (`25). Based on
this finding with Gpp(NH)p taken together with the inability of GTP alone
to stimulate activity, we proposed that the transducer must have the capacity
to hydrolyze GTP. When GTP was substituted for Gpp(NH)p in the modeling
of the liver system's kinetics (equation 2)) the data fit with the activated state
(E*) being the state in which GTP was converted to GDP+Pi. In this fashion,
it coulcl be understood why activation by GTP and hormone involved essen-
tially no lag period whereas with Gpp(NH)p + hormone, the lag was shorte-
ned but persisted. GTP-turnover, in this model, is required for the rapid,
reversible actions of the hormone. A few years later, Cassel and Selinger, in a
brilliant set of experiments, showed conclusively that hormones stimulated
the hydrolysis of GTP to GDP + Pi, From these findings, they elaborated the
theory that hydrolysis of GTP to GDP is the "turn-off' reaction and the resul-
tant bound GDP converts the transducer to its inhibitory state (26).
Hormones promote the displacement of GDP and its exchange with GTP;
this exchange reaction is the key to hormonal activation of Gproteins.
Nucleotide exchange and GTP-hydrolysis are fundamental to the regulation
of all types of Gproteins that have been examined to date. Not considered
in this theory, however, is that the overall turnover of GTP is a complex set
of reactions including hydrolysis and subsequent release of phosphate from
a bound state. In a detailed study of the light-activated rhodopsin system
(27)) it was suggested that hydrolysis of GTP is a very rapid process, whereas
the rate limiting step is the release of inorganic phosphate from its binding
siLcs on transducin, the G-protein responsible for activation of phosphodies-
lerase in rot1 outer segments. This proposal fits with the prolonged activation
by fluoride (complexed with aluminum or magnesium ions) which most lik-
ely acts by binding to the same Mg-phosphate binding sites on transducin.

DUAL STIMULATORY AND INHIBITORY ACTIONS OF GTP
AND FLUORIDE

The multi-receptor fat cell system proved invaluable not only for investiga-
ting the multiple actions of hormones. It provided the first insight that ade-
nylyl cyclase is both inhibited and stimulated by two independent processes
involving GTP and fluoride. Hans Liiw and Jim Harwood found that fluori-
de ion and both GTP and Gpp(NH)p induced stimulation and inhibition of
the enzyme as the concentrations of these agents were increased (28, 29 ).

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The mechanism was elusive until Hirohei Yamamura (30) noted marked dif-
ferences in the properties of the stimulatory and inhibitory phases.
Subsequent characterization of the dual process (31) and the discovery (32)
that the fat cell contained adenosine receptors that induce inhibition of ade-
nylate cyclase via a GTPdependent process finally placed the inhibitory role
of guanine nucleotides on the same level of importance as the stimulatory
process. From these studies arose the new concept of dual regulation of ade-
nylate cyclase by hormones, guanine nucleotides, and fluoride ion (33).
Implicit in the argument was the understanding that transduction involving
stimulation and inhibition must be exercised through distinct GTP-binding
proteins. We called them nucleotide regulatory proteins (abbreviated N)
because ITP was also active. Thus arose the nomenclature Ns and Ni, now
popularly known as Gs and Gi. One logical consequence of these findings is
that G-proteins are independent of both receptors and adenylate cyclase.
Pfeuffer's purification of a 42 kDa protein that he could labeled by incuba-
ting membranes with XP-NAD and cholera toxin (34,35) provided the first
tangible evidence for the existence of Gs, the cyclase stimulatory transducer.
It had been earlier discovered that cholera toxin greatly increased the pro-
duction of CAMP in intestinal cells, suggesting that the toxin acts on the ade-
nylate cyclase system (reviewed in (36). Later, pertussis toxin (37) provi-
ded the means for detecting and purifying Gi and Go. Meanwhile, in the
laboratory of Gordon Tompkins it was found that treatment of cultured lym-
phoma cells (rat S49) with cyclic AMP resulted in their death (38). Based on
this phenomenon they isolated surviving mutant forms, one of which was
eventually shown to lack the ability of Gpp(NH)p and fluoride ion to stimu-
late the enzyme; epinephrine action was also abolished (39). Using the
mutant called AC- (because it was mistakenly thought to lack adenylate cycla-
se), Gilman and his colleagues (40,41) subsequently demonstrated that sup-
plementation with extracts from wild type cells restored both hormonal
action in a GTP-dependent fashion and the actions of Gpp(NH)p and fluo-
ride. This assay proved useful for the first purification of what was then cal-
led G/F factor, now known as Gas, the transduction protein(s) responsible
for stimulating adenylate cyclase.

During this period, studies in the lab (42,43) showed that hormone
receptors linked with Gs displayed very different physical and kinetic pro-
perties from those observed when adenylate cyclase was linked (after acti-
vation) with Gs, suggesting either different states or different forms of the
GTP-regulatory process. Finally, and perhaps most critically was the discove-
ry by Bitensky and colleagues (44) that light- activation of a cyclic GMP phos-
phodiesterase in rod outer segments was mediated by a guanine nucleoticle-
dependent process, similar to the actions of guanine nucleotides on adeny-
late cyclase. By 1980 it was clear that the actions of guanine nucleotides were
not confined to the adenylate cyclase system. In a brief overview (33) I pro-
posed that there must be several types of GTP-binding proteins which I cal-
led Ns, Ni, Nt (now transducin), and Nx, that mediate the actions of hor-

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mones on a number of effecters systems. Nx was postulated when I learned
that GTP affected the binding of agonists to receptors known to alter calci-
urn uptake in liver cells (45). By 1990, those predictions have been proven
correct. However, the number and variety of GTP-binding proteins involved
in signal transduction are now greater than I had imagined.

GENERAL CHARACTERISTICS OF GUANINE NUCLEOTIDE ACTION

Within the decade of the 1970's, some of the fundamental characteristics of
receptor systems coupled through GTP-binding proteins had been delinea-
ted. What followed in the ensuing 20 years was the elaboration of the types
of G-proteins, now about 20. Beginning with transducin (46)) it emerged that
Gproteins are constructed of three types of subunits, an a-subunit uniquely
capable of binding and degrading GTP and a tightly knit complex of p and
y subunits. This discovery, eventually established for all Gproteins coupled
to receptors (47)) opened up a new chapter in signal transduction which, in
recent years, has helped to explain the pleiotropic actions of hormones.

Dr. Gilman will present much of the work on detailed structures of Gpro-
teins, including the recent x-ray crystallographic studies of Gcz,. I will now
turn to a subject that has dominated my efforts for the past 15 years.

TOPOLOGICAL DISPOSITION OF COMPONENTS.

One of the most difficult problems in membrane biology is to understand
how its components are organized or structured within the plane of the
membrane. The topological relationship of membrane proteins to the exte-
rior and interior components of the cell presents another major problem.
The "mobile receptor" concept introduced the notion that receptor proteins
are free to move rapidly within the membrane. In the case of receptors iin-
ked to Gproteins, this concept gave rise to the hypothesis that hormones act
by stimulating the engagement between receptors and Gproteins. The "col-
lision-coupling" model (48) attributes the rate of cyclase activation to the
fi-equency and efficiency of collisions between agonist-bound receptors and
G protein; in this manner any one receptor can activate a number of C pro-
teins due to the free mobility of each component. The rate of activation of
G proteins (and adenylate cyclase) are directly proportional to the number
of agonist-occupied receptors.

Although kinetic analysis can provide important insights into mechanism,
in reality the fundamental question is how the different components are con-
structed and distributed in the plane of the membrane so that they interact
with the observed efficiency and rapidity. The logistics of the encounters are
obviously better if the membrane is packed with receptors, as in the case of
rhodopsin in rods or cones which is in large excess of G proteins and effec-
tors. However, in most cells hormone receptors are present at relatively low
concentrations.

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For this reason, I have thought that receptors and G-proteins may be pre-
coupled and that hormones act by altering the nature of the coupling pro-
cess. This notion now seems justified based on biophysical studies which reve-
al that receptors are complexed with G-proteins and that such complexes are
confined within matrix-like, specialized domains(49). In fact, receptor-coup-
led signaling processes in general now seem more Bhudda-like in their struc-
tures, both in their stationary setting and the multi-component structures
which appear to interact in a flickering fashion, more in keeping with the
ephemeral relationship between action and inaction, between life and death.
The major concern in my laboratory starting in the late 70's was the struc-
ture of the hormone-sensitive cyclase systems as they exist in their native
membrane environment. I had learned of target or irradiation analysis from
a report that target analysis might be useful for discerning the nature of the
interactions between the components of the glucagon-sensitive system in
liver membanes (50). Their interpretations of the data were based on the
mobile receptor theory. Of major concern to us was the fact that irradiation
studies were carried out with freeze-dried material. We had learned that free-
ze-drying of liver membranes, for example, led to drastic reductions in hor-
monal regulation of adenylate cyclase. We decided to use this technique
employing a different protocol not involving freeze-drying.
Fortunately, on the floor above my lab dwelled a scientist with the neces-
sary credentials. Ellis Kempner had conducted his graduate thesis on the
usage of irradiation ,?nalysis, knew both its promises and its faults, and beca-
me interested in our problem. As importantly, a young scientist from
Switzerland, trained in biophysics, had just arrived in the lab looking for a
suitable research problem. Werner Schlegel and Kempner began a project
which became the focal point of our research for the past 15 years.

TARGET ANALYSIS

Schlegel and Kempner ultimately worked out procedures that fully preser-
ved activity and, indeed, provided the first detailed functional structure of
each component of the glucagon-sensitive system in liver membranes and
the hormone-sensitive, stimulatory and inhibitory structures in rat adipocy-
tes (51,52). I emphasize the phrase "functional structure" since the analysis
measures the exponential decay in activity in relation to the energy input of
electrons that bombard the system; this relationship provides the functional
mass. As reviewed recently by Kempner (53), irradiation of complex, multi-
component enzyme systems does not cause disruption of complexes, but
introduces breakages in the protein backbone along each chain of the com-
plex. Thus, although activity is !ost, the decay in activity accurately reflects
the loss in functional mass.

Most surprising and initially puzzling were the findings that irradiation of
both the liver and adipocyte systems prior to exposure to regulatory ligands-
hormones, fluoride ion, guanine nucleotides- displayed functional target


sizes of about 1500 kDa for the stimulatory processes involving glucagon +
GTP; an even larger functional size was exhibited by the inhibitory phase of
the adipocyte adenosine-receptor mediated process. Such large sizes did not
fit with the estimated sizes of receptors, G-proteins or adenylyl cyclase. When
the systems were exposed first to activating ligands and then analyzed for
their target sizes, dramatic reductions in functional mass were observed. For
example, in the presence of glucagon and GTP, the functional size was redu-
ced to about 350 kDa. In the presence of fluoride ion or Gpp(NH)p, the size
was reduced to about 250 kDa. The size of adenylate cyclase as measured with
MnATP as substrate was about 120 kDa, now supported by the structure of
cloned cyclases.

DISAGGREGATION THEORY OF HORMONE/GTP ACTION.

Out of these findings arose the postulate that the hormone-sensitive cyclase
system is composed of an oligomeric complex of receptors and G (or N) pro-
teins which, upon interaction with hormone and GTP, disaggregate into
monomers of the receptor-G complex (33).
Most importantly, target analysis led me to the conclusion that the primary
signal emanating from the actions of hormones must be a protein; this pro-
tein had to consist, minimally, of a GTP-binding protein. Not knowing that
G-proteins were heterotrimers, the estimated size of the monomer ranged
from about 120 kDa (fluoride- or Gpp(NH)p-activation) to about 220 kDa
after glucagon-treatment (correcting for the estimated mass of cyclase) . The
estimated values obtained after fluoride or Gpp(NH)p treatment were much
larger than that of GCY, (43-50 kDa). The larger value obtained after gluca-
gon treatment I conjectured as the combination of the receptor complexed
with a monomer of Gs. The monomer complex, considered to be the true
"messenger" of hormone action, reacts with adenylate cyclase resulting in eit-
her stimulation (by Gs) or inhibition (by Gi). This theory I termed the
"Disaggregation Theory of Hormone Action" (33). Incorporated are the fun-
damental ideas that the structure of the receptor/G-protein complex is a
multimer of these components, that adenylyl cyclase exists separately from
the complex, and that a "monomeric" structure derived from the disaggre-
gation is the messenger that communicates information from the hormone
bound receptor/Gprotein complex to the effector or enzyme.

In this model, I had assumed that receptors and G proteins existed in
about equal amounts and were coupled stoichiometrically. Much later when
accurate methods became available for measuring the concentrations of
receptors and Gproteins in cells, it became clear that in most cells, Gpro-
teins are present in excess of receptors, possibly as much as 1O:l. Given such
information, clearly the model must be altered in that the largest portion of
the mass of the glucagon-sensitive adenylyl cyclase (or the adenosine-sensiti-
ve, inhibitory system in adipocytes) must be attributed to that of G-proteins
i.e., Gproteins are likely multimeric structures.

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The disaggregation theory soon fell into disfavor because of the findings
that heterotrimeric G-proteins treated with Gpp(NH)p or the later more
popular GTPgS dissociated into free a-subunits and the pr complexes
(54,55). From this arose the "dissociation" theory (Gilman , 1988). On my
part, the disaggregation theory clearly needed biochemical evidence for the
existence of multimeric forms of Gproteins. The odyssey in this direction
began with two approaches: cross-linking experiments with synaptoneuroso-
mes from rat brain and extraction of G-proteins with various detergents fol-
lowed by sucrose-gradient analysis of the hydrodynamic properties of the
extracted material.

CROSS-LINKING STUDIES

Synaptoneurosome membranes were chosen for most of the studies because
brain tissue contains the bulk of all known types of Gproteins. We were gre-
atly aided in these studies by generous contributions from several colleagues
(principally, Dr. Alan Spiegel at NIH) in the field who had prepared poly-
clonal antibodies against peptide sequences of the a and p subunits of seve-
ral types of Gproteins (Gs, Gi, Go, and Gq), including subspecies of these
proteins.

We tested a variety of cross-linking agents for both their efficacy and selec-
tivity of action at low concentrations. Phenylenedimaleimide proved the
most satisfactory. In addition to all of the Gproteins tested, multimeric tubu-
lin and F-actin were the only two types of membrane-associated proteins that
were detectably cross-linked (56). After cross-linking in their membrane-
environment, the Gproteins were extracted with sodium dodecylsulfate and
chromatographed on sieving columns that allow separation of proteins over
a large range of sizes. In this manner it was found that both a- and P-subunits
of Gs, Gi, Go, and Gq were cross-linked to form structures comparable in size
to cross-linked tubulin or actin. We concluded from these studies that Gpro-
teins, most likely intact heterotrimers, are multimeric structures in associa-
tion with the plasma membrane. Such evidence provided substantial cre-
dence to our basic arguments for the disaggregation theory. Most impor-
tantly, it appeared that multimeric Gproteins are responsible for the large
ground state structures observed with target analysis.

DETERGENT STUDIES

The next stage necessitated some means of isolating the multimeric Gpro-
teins, a process necessitating the use of detergents. Aware of the fact that
detergents such as sodium cholate and Lubrol extracted intact heterotrime-
ric structures (57); i.e., monomers of the putative multimers, we considered
the possibility that these detergents may disrupt the multimeric structure.
Accordingly, we tested the sizes of G-protein structures extracted with a vari-
ety of detergents, using hydrodynamic properties on sucrose gradients as our

199


assay. Of the seven tested, octyl-b-glucoside (OG), tween 20, and digitonin
yielded structures behaving hydrodynamically larger than those given with
sodium cholate or Lubrol, after correcting for the possible contributions of
micellar forms of the detergents (58). OG extracted from liver membranes
structures that were heterodisperse, about 10 % sedimenting through sucro-
se gradients, the bulk remaining soluble in the detergent. When membranes
were treated with cholera toxin in the presence of SP-NAD (the means of
specifically labeling Ga,), the majority of labeled material appeared in the
insoluble fraction (59,60). When such labeled material in the membranes
was subjected to the combined actions of glucagon and low concentrations
of GTPgS, a large portion of the insoluble material became soluble and
appeared in a fraction similar to that of purified heterotrimeric Gs.

Based on the cross-linking and hydrodynamic studies we deduced that Gs
is likely multimeric in liver and synaptoneurosome membranes, that only
multimeric structures are altered by glucagon and low concentrations of
GTP@ in liver membranes, and that one of the primary results of their
action is the disaggregation of multimers to monomers, as predicted in the
disaggregation theory. In synaptoneurosomes high concentrations of GTPfi
caused dissociation into free a and py of heterotrimeric Gproteins dissolved
in Lubrol or sodium cholate but not in digitonin (58). Hence, our suspicions
were confirmed that the native structures of G-proteins are not preserved
with detergents used for purifying heterotrimeric forms of Gproteins.

AN EXTENDED DISAGGREGATION THEORY OF HORMONE ACTION

Target analysis provided the initial impetus for proposing the disaggregaton
theory. However, it has become clear that the theory as originally presented
has to be modified to account for the fact that Gproteins are the major com-
ponent representing the large functional mass;i.e. G-proteins form multime-
ric structures. We had also established that there are marked differences
between the regulation of Gproteins by the coupled receptors and the regu-
lation of adenylyl cyclase by Gproteins (42,43). When the structures and
regulatory properties of adenylyl cyclases became known (61), particularly
the fact that these are transmembrane proteins that have a two-cassette struc-
ture: i.e. two distinct domains on a 12 membrane-spanning structure, it beca-
me possible to construct a more coherent theory to explain the regulation
of the cyclase system (62). Two regulatory cycles, one (A) for regulation of
multimer to monomer Gproteins, the other (B) for regulation of cyclase by
a monomeric Gprotein (Gs) are illustrated in Fig. 2.


Receptor-activation   Gs-activation of
  of G-proteins    Adenylyl Cyclase

       -rllbdt
      fn-==plex               enylyl cyclus

The excursion of receptor along the multimeric Gprotein chain is governed
by the hormone- induced exchange of GTP and GDP; the GTP-occupied
monomer at one end is released, allowing it either to interact with adenylyl
cyclase or to return (after hydrolysis of GTP to GDP) to the other terminus
of the multimer. In (B), the GTP-occupied monomer interacts with the
enzyme without necessarily inducing significant changes in enzyme activity.
Activity is governed by Mg-dependent hydrolysis of bound GTP to GDP + Pi.
In this theory hydrolyis induces dissociation of a from pr; the resultant sepa-
rated subunits interact distinctively with the two cassettes or domains of ade-
nylyl cyclase. Depending on the type of adenylyl cyclase associated with the
associated Gprotein, activity is governed solely by 01, synergistically by the
combination of a, and py., or by inhibition of as- stimulation by by. Release
of Pi from its binding site on as results in re-association of as with &. The
GDP-bound Gs then re-associates with the multimer to become part of the
hormone-regulated cycle. It should be emphasized that both cycles occur in
association with the surface membrane. The principal element that differs
from other theories of hormone-regulated cyclase systems is that the con-
certed interactions of enzyme, Mg*+ and GTPase are responsible for separa-
tion of as from bg. The extent and duration of enzyme stimulation are con-
trolled by the independent actions of the separated subunits and the rate at
which Pi is released following hydrolysis.

Most people in the field will argue that hydrolysis is not necessary for acti-
vation because non-hydrolyzable analogs of GTP are fully capable of stimu-
lating cyclase activity. However, my view is that allosteric regulation by
Gpp(NH)p, a slow, hysteretic process, may involve sthilization of a Mg-indu-
ted disassociation of Gs that normally exists transiently and which tlocs not
require any participation by adenylyl cyclase in the dissociative process. In
this sense, the non-hydrolyzable analogs of GTP may have misguided many
in the G-protein field into thinking that energy derived from the splitting oi
GTP is not involved in signal transduction.

201


It should be noted in this extension of the disaggregation theory that both
disaggregation of multimers and dissociation of monomers are separate but
interrelated phenomena, both contributing to the overall dynamics of signal
transduction.

GPROTEINS ARE SIMILAR IN STRUCTURE AND REGULATION TO
CYTOSKELETAL PROTEINS.

During these studies, my attention was drawn to the striking similarities in
the properties of C-proteins with those of tubulin and actin, the major cytos-
keletal elements in cells (reviewed in (63)). For example, Gproteins, like
actin and tubulin, are associated with the inner aspect of the surface mem-
brane, adhering possibly both through intrinsic membrane proteins, such as
receptors, and to membrane lipids. Of particular interest is the fact that all
three types of multimeric proteins are subject to regulation by either GTP
(Gproteins and tubulin) or ATP (actin) and their hydrolytic products (dinu-
cleotides and Pi). Receptors regulate exchange of bound nucleotides (GDP
with GTP) and act catalytically in the process. Similarly, the excursion of a
single myosin molecule during muscle contraction along the chain of actin
multimers is governed by the exchange of bound ADP with ATP and the
hydrolysis of ATP to ADP and Pi; As stated previously, GTP-turnover (pro-
duction of GDPtPi) is essential for the rapid and sustained actions of hor-
mones; release of bound Pi is the crucial rate-limiting process in the overall
dynamics of signaling. The same is true for myosin/ actin interactions (64).

With these similarities in structure and regulation, G-proteins can be clas-
sified as part of the cytoskeletal matrix, with the primary functional diffe-
rence that G-proteins serve as chemical signaling devices whereas tubulin
and actin serve as mechano-signaling devices. The release of monomers from
multimers is the basis for chemical signaling by Gproteins. Dynamic changes
in the disaggregation-aggregation cycle of actin and tubulin multimers are
also regulatory devices designed for regulating the interactions or movement
between specialized components of cells. Based on evidence accumulated
over the past decade (reviewed in 63) all three types of cytoskeletal proteins
are connected in some manner to a variety of signaling systems that adhere
to the cytoskeletal matrix, including heterotrimeric G-proteins, so-called
small molecular weight Gproteins, protein kinases and phosphatases, and
other proteins or systems that communicate between the surface membrane
and the interior of cells. These components form web-like structures that
possibly interact in a flickering manner in response to activation of mem-
brane receptors, including those that are growth promoting. Given the extra-
ordinary complexity of signaling processes, as viewed at the biochemical
level, clearly needed are new investigatory tools. Already promising are the
microscopic imaging techniques with immunofluorescent molecules for spe-
cifically tagging and viewing structures in their living environment. I suspect
that the reductionists with their prowess in molecular biology and x-ray crys-

202


tallography and those of us attempting to view the living process at the cel-
lular level will merge with our assemblages of ideas and experiences. When
this larger, multiplex community of effort finally is consummated, a bright
new era in scientific discovery will certainly emerge.

ACKNOWLEDGMENTS

This article and the following "poem" are dedicated to all my colleagues,
former and present, who contributed heavily to the concept of signal trans-
duction. Without their efforts, the field of Gproteins would not have existed.

To my Friends: Thoughts from "On High"
Life on a roller coaster, oscillating from hither to yon,
no respite for the iconoclast, wandering from dusk to dawn.
Conjuring strange thoughts foreign and twice forbidden,
like Prometheus unbound, this Nobelist climbs in vain
to Andean peaks, seeking what most would proclaim insane.
Why, he ponders, are there no answers to protean questions
when others thinking cleanly and simply with Occam's sharp razor
proclaim what seems obvious given the beam of their unerring laser.
Nature, happily unfettered with philosophy, or with cunning, or with intent
moves relentlessly onward or even backward with energy unspent
while we mortals test and probe with twinkling machines blinking precisely
at each movement, striving to unravel its irresolute randomness, its fathom-
less, unlimited, meaningless rush into spiraling chaos,
oblivious of its multitudinous trials & errors which we pontifically believe
must be unerring truth & resolution.
The laugh is on those who, burdened with pretensions of truth, believe they
can fathom within 15 minutes of human existence what has transpired over
eons of space and time in this Universe .
So, I extol the intuitions encapsulated in the folds of my mind
from whence occasionally they hurtle to the forebrain and in a twinkling of
a proton's discharge bring to fruition a thought, an idea borne on the feat-
hery appendages of teeming neurons wedded in a seamless synergy. Those
fleeting moments are cherished as are those precious impulses imparted by
the innumerable individuals who nurtured and instilled unknowingly their
encrypted thoughts in mine.

So, with these fanciful thoughts in mind I give praise to you - my friends, my
colleagues, my soul-mates, my loved ones - for letting my soul and thoughts
meander hither and yonder in this attempt at philosophy and poetry. We
now belong to the Gods on high who praise us for our frailties and our
achievements.

203


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20.5