SIGNAL TRANSDUCTION: EVOLUTION OF AN IDEA Nobel Lecture, December 8, 1994 by MARTIN RODBELL National Institute of Environmental Health Scienes, P.O. Box 12933, Research Triangle Park, North Carolina, USA In general there is no set of observations conceivable which can give enough information about the past of a system to give com- plete 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. Retrospectively, 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 of which 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 concept is still at an evolutionary phase, I will con- 188 clude 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 coHagenase 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 Sutherland’s theory that receptors are structurally and functionally linked to the regulation of cyclic AMP produc- tion. Overwhelmingly persuasive was the notion that adenyl (now adenyla- 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. 189 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 ume allowed rapid, multiple assays of adenylate cyclase. When Lutz Birnbaumer 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 Mg-dependent 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- 190 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. 191 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 J 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 !?51 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 adenylate 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 !*5I-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 GIP 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 *2P-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- 192 ted the affinity of the receptor (20). Subsequently, Michael Lin and Yoram Salomon (21) demonstrated that hormone and GTP concertedly and rapid- 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 lL). Although the components of the informational processing system remained unknown, there was littke 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(NH)p Hormone Ea (1 E ~@# Ef ~<— E* GTP Hormone ES PE FE