Botulinum C2 toxin ADP-ribosylates actin

ADP-ribosylation of regulatory proteins is an important pathological mechanism by which various bacterial toxins affect eukaryotic cell functions. While diphtheria toxin catalyses the ADP-ribosylation of elongation factor 2, which results in inhibition of protein synthesis, cholera toxin and pertussis toxin ADP-ribosylate Ns and Ni, respectively, the GTP-binding regulatory components of the adenylate cyclase system, thereby modulating the bidirectional hormonal regulation of the adenylate cyclase. Botulinum C2 toxin is another toxin which has been reported to possess ADP-ribosyltransferase activity. This extremely toxic agent is produced by certain strains of Clostridium botulinum and induces hypotension, an increase in intestinal secretion, vascular permeability and haemorrhaging in the lungs. In contrast to botulinum neurotoxins, the botulinum C2 toxin apparently lacks any neurotoxic effects. Here we report that botulinum C2 toxin ADP-ribosylates a protein of relative molecular mass 43,000 (43K) in intact cells and in cell-free preparations. We present evidence that the 43K protein substrate is actin, which is apparently mono-ADP-ribosylated by the toxin. Botulinum C2 toxin also ADP-ribosylated purified liver G-actin, whereas liver F-actin was only poorly ADP-ribosylated and skeletal muscle actin was not ADP-ribosylated in either its G form or its F form. ADP-ribosylation of liver G-actin by botulinum C2 toxin resulted in a drastic reduction in viscosity of actin polymerized in vitro.     

Altered Gs and adenylate cyclase activity in human GH-secreting pituitary adenomas

Gs and Gi are guanine nucleotide-binding, heterotrimer proteins that regulate the activity of adenylate cyclase, and are responsible for transferring stimulatory and inhibitory hormonal signals, respectively, from cell surface receptors to the enzyme catalytic unit. These proteins can be directly activated by agents such as GTP and analogues, fluoride and magnesium. Decreased amounts of Gs and Gi, and even the absence of Gs, have been described, whereas an altered Gs has been reported in a cultured cell line (UNC variant of S49 lymphoma cells), but has never been observed in human disease states. We have found a profoundly altered Gs protein in a group of human growth hormone-secreting pituitary adenomas, characterized by high secretory activity and intracellular cyclic AMP levels. In the membranes from these tumours no stimulation of adenylate cyclase activity by growth hormone-releasing hormone, by GTP or by fluoride was observed. Indeed, the last two agents caused an inhibition, probably mediated by Gi. In contrast, adenylate cyclase stimulation by Mg2+ was enormously increased. This altered pattern of adenylate cyclase regulation was reproduced when a cholate extract of the tumour membranes (which contains G proteins) was reconstituted with Gs-free, cyc- S49 cell membranes. Inasmuch as secretion from somatotrophic cells is known to be a cAMP-dependent function, the alteration of Gs could be the direct cause of the high secretory activity of the tumours in which it occurs.     

Antisense oligodeoxynucleotides to GS protein alpha-subunit sequence accelerate differentiation of fibroblasts to adipocytes.

Fully-differentiated mouse 3T3-L1 fibroblasts accumulate large amounts of lipid at 7-10 days after induction by insulin or by dexamethasone and a methyl xanthine. G proteins mediate transmembrane signalling from a diverse group of cell-surface receptors to effector units that include phospholipase C, adenylyl cyclase and ion channels. They are also targets of regulation themselves. 3T3-L1 fibroblasts display marked changes in levels of G protein when induced to differentiate to adipocytes. Here we show that cholera toxin, which ADP-ribosylates and activates the G protein subunit Gs alpha, blocks the induction of differentiation, whereas increasing intracellular cyclic AMP directly with the dibutyryl analogue or indirectly with pertussis toxin or forskolin does not affect differentiation. Oligodeoxynucleotides antisense to the sequence encoding Gs alpha accelerate differentiation markedly. The time course of adipogenesis declined from 7-10 days in controls to roughly 3 days in cultures treated with antisense-Gs alpha oligodeoxynucleotides, whereas oligodeoxynucleotides, antisense to Gi alpha 1, Gi alpha 3, and sense and missense to Gs alpha, had no such effect. Antisense-Gs alpha alone induced differentiation by day 7, indicating that Gs alpha activity modulates differentiation in 3T3-L1 cells, acting in a new role which is independent of increased intracellular cAMP.     

Hormonal stimulation of adenylyl cyclase through Gi-protein beta gamma subunits.

Agonist-bound receptors activate heterotrimeric (alpha beta gamma) G proteins by catalysing replacement by GTP of GDP bound to the alpha subunit, resulting in dissociation of alpha-GTP from the beta gamma subunits. In most cases, alpha-GTP carries the signal to effectors, as in hormonal stimulation and inhibition of adenylyl cyclase by alpha s and alpha i respectively. By contrast, genetic evidence in yeast and studies in mammalian cells suggest that beta gamma subunits of G proteins may also regulate effector pathways. Indeed, of the four recombinant mammalian adenylyl cyclases available for study, two, adenylyl cyclases II and IV, are stimulated by beta gamma. This effect of beta gamma requires costimulation by alpha s-GTP. This conditional pattern of effector responsiveness led to the prediction that receptors coupled to many G proteins will mediate elevation of cellular cyclic AMP, provided that Gs is also active. We now confirm this prediction. Coexpression of mutationally active alpha s with adenylyl cyclase II converted agonists that act through 'inhibitory' receptors (coupled to Gi) into stimulators of cAMP synthesis. Experiments using pertussis toxin and a putative scavenger of beta gamma, the alpha subunit of transducin, suggest that beta gamma subunits of the Gi proteins mediated this stimulation. These findings assign a new signalling function to beta gamma subunits of Gi proteins, the conditional stimulation of cAMP synthesis by adenylyl cyclase II.     

Mutant alpha subunits of Gi2 inhibit cyclic AMP accumulation

One or more of three Gi proteins, Gi1-3, mediates hormonal inhibition of adenylyl cyclase. Whether this inhibition is mediated by the alpha or by the beta gamma subunits of Gi proteins is unclear. Mutations inhibiting the intrinsic GTPase activity of another G protein, the stimulatory regulator of adenylyl cyclase (Gs), constitutively activate it by replacing either of two conserved amino acids in its alpha subunit (alpha s). These mutations create the gsp oncogene which is found in human pituitary and thyroid tumours. In a second group of human endocrine tumours, somatic mutations in the alpha subunit of Gi2 replace a residue cognate to one of those affected by gsp mutations. This implies that the mutations convert the alpha i2 gene into a dominantly acting oncogene, called gip2, and that the mutant alpha i2 subunits are constitutively active. We have therefore assessed cyclic AMP accumulation in cultured cells which stably or transiently express exogenous wild-type alpha i2 complementary DNA or either of two mutant alpha i2 cDNAs. The results show that putatively oncogenic mutations in alpha i2 constitutively activate the protein's ability to inhibit cAMP accumulation.     

Serotonin stimulates DNA synthesis in fibroblasts acting through 5-HT1B receptors coupled to a Gi-protein

Growth factors can be divided into two classes which act through distinct signal transduction pathways. One class including epidermal growth factor, platelet derived growth factor and fibroblast growth factor activates receptor tyrosine kinases, and the second class, including thrombin, bombesin, bradykinin and vasopressin activates a phosphoinositide-specific phospholipase C through GTP-binding proteins which can be inactivated by pertussis toxin. In Chinese hamster lung fibroblasts, thrombin-induced mitogenicity seems to correlate well with phospholipase C activation and both events are sensitive to pertussis toxin. Thrombin, like the other mitogens in this class, simultaneously inhibits adenylate cyclase. This involves an inhibitory G protein (Gi), a well established pertussis toxin substrate. The relative contributions of the two signalling pathways to mitogenicity has not been evaluated so far. We report here that the neurotransmitter serotonin (5-hydroxytryptamine), a contracting agent and mitogen for smooth muscle cells, activates phospholipase C, inhibits adenylate cyclase and stimulates DNA synthesis in fibroblasts. These events are sensitive to pertussis toxin. We show that the mitogenicity of 5-hydroxytryptamine can be uncoupled from phospholipase C activation that is mediated by 5-HT2 receptors, but correlates perfectly with inhibition of adenylate cyclase through 5-HT1B receptor. We propose that inhibition of adenylate cyclase or activation of an undefined effector system by Gi is important in 5-hydroxytryptamine induced DNA synthesis and contributes to the strong mitogenicity of the other members of this family of growth factors.     

Abolition of the expression of inhibitory guanine nucleotide regulatory protein Gi activity in diabetes

Many cell-surface receptors for hormones appear to exert their effects on target cells by interacting with specific guanine nucleotide binding regulatory proteins (G-proteins) which couple receptors to their second-messenger signal generation systems. A common intracellular second messenger, which is used by many hormones, is cyclic AMP. This is produced by adenylate cyclase, whose activity is controlled by two G-proteins, Gs which mediates stimulatory effects and Gi inhibitory effects on adenylate cyclase activity. In liver, the hormone glucagon increases intracellular cAMP concentrations by activating adenylate cyclase by a Gs-mediated process. This effect of glucagon is antagonised by the hormone insulin, although the molecular mechanism by which insulin elicits its actions is obscure. However, insulin receptors exhibit a tyrosyl kinase activity and appear to interact with G-proteins, perhaps by causing phosphorylation of them. In type I diabetes, circulating insulin levels are abnormally low, giving rise to gross perturbations of metabolism as well as to a variety of complications such as ionic disturbances, neuropathies of the nervous system, respiratory and cardiovascular aberrations and predisposition to infection. We show here that experimentally-induced type I diabetes leads to the loss of expression of Gi in rat liver. As it has been suggested that Gi may couple receptors to K+-channels as well as mediating the inhibition of adenylate cyclase, aberrations in the control of expression of this key regulatory protein in type I diabetes may be expected to lead to pleiotropic effects.     

Chronic ethanol causes heterologous desensitization of receptors by reducing alpha s messenger RNA

One of the biochemical results of ethanol exposure is a change in the amount of the intracellular second messenger cyclic AMP (cAMP) produced in response to receptor stimulation. In general, acute ethanol exposure increases the amount of cAMP produced on stimulation of receptors coupled to the enzyme adenylyl cyclase via the GTP-binding protein Gs, whereas chronic ethanol exposure has the opposite effect (results for receptors coupled via Gi have been more variable). We previously reported that adaptation to continuous ethanol exposure reduces receptor-stimulated cAMP production by 25-35% in a neuroblastoma cell line (NG108-15), and an even greater reduction of 75% was observed in lymphocytes taken from actively-drinking alcoholics. This reduction in receptor-stimulated cAMP levels was recently confirmed in platelets from alcoholics. None of these studies, however, determined whether more than one receptor coupled to adenylyl cyclase activity was affected in the same cell. Here we report that chronic ethanol exposure causes desensitization of heterologous receptors coupled to Gs as cAMP production mediated by prostaglandin E1 as well as by adenosine is reduced by approximately 30% in NG108-15 cells. We show that, after chronic ethanol exposure, the activity of the alpha subunit of Gs is decreased by 29%, the amount of alpha s protein is decreased by 38.5%, and alpha s messenger RNA is decreased by 30%. Thus, cellular adaptation to ethanol involves a reduction in alpha s mRNA and, as a consequence, reduced cAMP production by heterologous receptors coupled to Gs. Such changes in cAMP production may account for the tolerance and physical dependence on ethanol in alcoholism.     

Pituitary hyperplasia and gigantism in mice caused by a cholera toxin transgene.

Cyclic AMP is thought to act as an intracellular second messenger, mediating the physiological response of many cell types to extracellular signals. In the pituitary, growth hormone (GH)-producing cells (somatotrophs) proliferate and produce GH in response to hypothalamic GH-releasing factor, which binds a receptor that stimulates Gs protein activation of adenylyl cyclase. We have now determined whether somatotroph proliferation and GH production are stimulated by cAMP alone, or require concurrent, non-Gs-mediated induction of other regulatory molecules by designing a transgene to induce chronic supraphysiological concentrations of cAMP in somatotrophs. The rat GH promoter was used to express an intracellular form of cholera toxin, a non-cytotoxic and irreversible activator of Gs. Introduction of this transgene into mice caused gigantism, elevated serum GH levels, somatotroph proliferation and pituitary hyperplasia. These results support the direct triggering of these events by cAMP, and illustrate the utility of cholera toxin transgenes as a tool for physiological engineering.     

A nucleotide regulatory site for somatostatin inhibition of adenylate cyclase in S49 lymphoma cells

The cyc- variants of S49 lymphoma cells have served as powerful tools for studying the components and mechanisms of hormone-induced adenylate cyclase stimulation, as these cells are deficient in the guanine nucleotide regulatory site (Ns) mediating hormone, guanine nucleotide, cholera toxin and fluoride-induced stimulations of the enzyme. Because of this deficiency, membranes of these cells have been used for reconstitution of the system by inserting the coupling component derived from other cell types. The hormone-sensitive adenylate cyclase is not only stimulated by hormones but can also be inhibited by a wide variety of hormones and neurotransmitters, and there is some evidence that hormonal inhibition may be mediated by a distinct guanine nucleotide regulatory site. Studies in cyc- cells lacking a functional Ns may therefore answer this unresolved, important question. We have recently observed that stable GTP analogues can inhibit cyc- adenylate cyclase stimulated by purified, preactivated Ns or forskolin, which can activate adenylate cyclase even in the absence of a functional Ns (ref. 10). The data indicated that these Ns-deficient cells contain an inhibitory guanine nucleotide site, Ni. To strengthen this concept, we investigated whether the cyc- adenylate cyclase can be inhibited by a hormone. We report here that somatostatin decreases cyclic AMP levels in cyc- cells, inhibits the forskolin-stimulated adenylate cyclase and causes a concomitant increase in a high affinity GTPase activity in cyc- membranes. The data strongly suggest that both the hormone- and guanine nucleotide-induced adenylate cyclase inhibitions in cyc- cells are mediated by Ni and that the mechanisms of activation and inactivation of Ni are similar to those established for Ns.     

Stimulation and inhibition of adenylyl cyclases mediated by distinct regulatory proteins

Adenylyl cyclases are under positive and negative control by guanine nucleotides and hormones. Stimulatory responses are mediated by a guanine nucleotide- and Mg-binding regulatory component (Ns), a protein that has been purified to homogeneity. Inhibitory responses have been hypothesized to be mediated by an analogous regulatory component (Ni) distinct from Ns, but definitive proof for this is lacking and these effects may result from modulation of Ns activity. Recently, Bordetella pertussis toxin has been shown to ADP-ribosylate a peptide that is not part of Ns, and this coincides with attenuation of hormonal inhibition of adenylyl cyclase. We show here that cyc- S49 cells contain a substrate for ADP-ribosylation by pertussis toxin and that the toxin alters GTP dependent inhibition of cyc- adenyl cyclase activity. As cyc- S49 cells do not contain Ns by several criteria, we conclude that Ni is a distinct and separate regulatory component of adenylyl cyclase.     

Lactose binding to heat-labile enterotoxin revealed by X-ray crystallography.

Recognition of the oligosaccharide portion of ganglioside GM1 in membranes of target cells by the heat-labile enterotoxin from Escherichia coli is the crucial first step in its pathogenesis, as it is for the closely related cholera toxin. These toxins have five B subunits, which are essential for GM1 binding, and a single A subunit, which needs to be nicked by proteolysis and reduced, yielding an A1-'enzyme' and an A2-'linker' peptide. A1 is translocated across the membrane of intestinal epithelial cells, possibly after endocytosis, upon which it ADP-ribosylates the G protein Gs alpha. The mechanism of binding and translocation of these toxins has been extensively investigated, but how the protein is orientated on binding is still not clear. Knowing the precise arrangement of the ganglioside binding sites of the toxins will be useful for designing drugs against the diarrhoeal diseases caused by organisms secreting these toxins and in the development of oral vaccines against them. We present here the three-dimensional structure of the E. coli heat-labile enterotoxin complexed with lactose. This reveals the location of the binding site of the terminal galactose of GM1, which is consistent with toxin binding to the target cell with its A1 fragment pointing away from the membrane. A small helix is identified at the carboxy terminus of A2 which emerges through the central pore of the B subunits and probably comes into contact with the membrane upon binding, whereas the A1 subunit is flexible with respect to the B pentamer.     

Activation of Escherichia coli prohaemolysin to the mature toxin by acyl carrier protein-dependent fatty acylation

Haemolysin secreted by pathogenic Escherichia coli binds to mammalian cell membranes, disrupting cellular activities and lysing cells by pore-formation. It is synthesized as nontoxic prohaemolysin (proHlyA), which is activated intracellularly by a mechanism dependent on the cosynthesized HlyC. Haemolysin is one of a family of membrane-targeted toxins, including the leukotoxins of Pasteurella and Actinobacillus and the bifunctional adenylate cyclase haemolysin of Bordetella pertussis, which require this protoxin activation 1-5. HlyC alone cannot activate proHlyA, but requires a cytosolic activating factor6. Here we report the cytosolic activating factor is identical to the acyl carrier protein and that activation to mature toxin is achieved by the transfer of a fatty acyl group from acyl carrier protein to proHlyA. Only acyl carrier protein, not acyl-CoA, can promote HlyC-directed proHlyA acylation, but a range of acyl groups are effective.     

AB5 subtilase cytotoxin inactivates the endoplasmic reticulum chaperone BiP

AB5 toxins are produced by pathogenic bacteria and consist of enzymatic A subunits that corrupt essential eukaryotic cell functions, and pentameric B subunits that mediate uptake into the target cell. AB5 toxins include the Shiga, cholera and pertussis toxins and a recently discovered fourth family, subtilase cytotoxin, which is produced by certain Shiga toxigenic strains of Escherichia coli. Here we show that the extreme cytotoxicity of this toxin for eukaryotic cells is due to a specific single-site cleavage of the essential endoplasmic reticulum chaperone BiP/GRP78. The A subunit is a subtilase-like serine protease; structural studies revealed an unusually deep active-site cleft, which accounts for its exquisite substrate specificity. A single amino-acid substitution in the BiP target site prevented cleavage, and co-expression of this resistant protein protected transfected cells against the toxin. BiP is a master regulator of endoplasmic reticulum function, and its cleavage by subtilase cytotoxin represents a previously unknown trigger for cell death.     

Involvement of heterotrimeric G protein in signal transduction of extracellular calmodulin in regulatingrbcS expression

The role of heterotrimeric G protein in signal transduction pathway of extracellular calmodulin in regulating rbcS expression was examined in suspension-cultured cells of transgenic tobacco. Pharmalogical experiments indicated that G protein agonist cholera toxin enhanced rbcS expression and heterotrimeric G protein antagonist pertussis toxin inhibited rbcS expression in transgenic tobacco cells. Pertussis toxin also inhibited the enhancement effect caused by exogenous purified calmodulin on rbcS expression, whereas cholera toxin completely reversed the inhibitory effects caused by anti-calmodulin serum on rbcS expression. The right side-out vesicles from tobacco cell membrane were purified, which contained all of substrates for fluometric assay of GTPase activity. Exogenous purified calmodulin, when adding directly to the medium of plasma membrane vesicles, significantly activated GTPase activity in the right side-out plasma membrane vesicles, and this increase in GTPase activity was completely inhibited both by heterotrimeric G proteins antagonist pertussis toxin and nonhy-drolyzable GTP analogs GMP-PCP. These results provided the evidence that heterotrimeric G proteins may be involved in signal transduction pathways of extracellular calmodulin to regulate rbcS gene expression.     

Pertussis toxin reverses adenosine inhibition of neuronal glutamate release

Adenosine and its analogues are potent inhibitors of synaptic activity in the central and peripheral nervous system. In the central nervous system (CNS), this appears to arise primarily by inhibition of presynaptic release of transmitters, including glutamate, which is possibly the major excitatory transmitter in the brain. In addition, postsynaptic effects of adenosine have been reported which would also serve to reduce neurotransmission. The mechanism by which adenosine inhibits CNS neurotransmission is unknown, although it appears to exert its effect via an A1 receptor which in some systems is negatively coupled to adenylate cyclase. In an attempt to elucidate the mechanism of inhibition, we have examined the effect of pertussis toxin (PTX) on the ability of the stable adenosine analogue (-)phenylisopropyladenosine (PIA) to inhibit glutamate release from cerebellar neurones maintained in primary culture. PTX, by ADP-ribosylating the nucleotide-binding protein Ni, prevents coupling of inhibitory receptors such as the A1 receptor to adenylate cyclase. As reported here, we found that PTX, as well as preventing inhibition of adenylate cyclase by PIA, also converts the PIA-induced inhibition of glutamate release to a stimulation. Our results suggest strongly that purinergic inhibitory modulation of transmitter release occurs by inhibition of adenylate cyclase.     

Stimulation of specific GTP binding and hydrolysis activities in lymphocyte membrane by interleukin-2

Interleukin-2 (IL-2) is a polypeptide growth factor which stimulates the proliferation and differentiation of T lymphocytes. The receptor for IL-2 is expressed on activated T lymphocytes, cloned IL-2 dependent cells and several other cell types. Analysis of the primary structure and of immune-precipitated receptor suggests that this molecule has no intrinsic signal transduction function, unlike other growth factors. IL-2 interaction with a high affinity receptor has been shown, however, to activate the calcium/phospholipid-dependent protein kinase C (PK-C) presumably via phosphoinositide hydrolysis. Members of a family of closely related guanine nucleotide binding proteins (G proteins) regulate a diverse group of metabolic events. Two of them, Gs and Gi, stimulate and inhibit adenylate cyclase activity respectively, and other G proteins are involved in diverse signal transduction system. Another member, Go, has no known function and activation of phospholipase C has been attributed to the action of an unidentified G protein, Gp. Since it has been observed that IL-2 inhibits the catalytic activity of adenylate cyclase and that agents such as PGE2 which stimulate adenylate cyclase activity inhibit the lymphoproliferative response to IL-2, association of GTP binding proteins with IL-2 signal transduction was investigated. In this report we describe for the first time the participation of a GTP binding protein in the action of a polypeptide growth factor, interleukin-2.     

Normal p21N-ras couples bombesin and other growth factor receptors to inositol phosphate production

Many receptors, in response to ligand activation, trigger inositol phospholipid breakdown, which leads to rapid intracellular responses. The sustained activation of this pathway is believed to be at least one of the factors involved in the stimulation of cell growth and there has been much speculation that certain oncogenes use this pathway to effect uncontrolled cellular proliferation. It has been suggested, by analogy with the receptor-mediated control of adenylate cyclase, that the receptor stimulation of inositol phospholipid metabolism is mediated through a guanine nucleotide regulatory protein (G-protein) called Gp (or Np). Although such a species has not been identified, there is now strong experimental evidence that this process is mediated by a G-protein distinct from the stimulatory and inhibitory G-proteins (Gs and Gi, respectively). The ras genes code for a plasma membrane protein, p21, whose only known biochemical property is a high-affinity GTPase activity. We show here that the expression of normal p21N-ras in NIH 3T3 fibroblasts leads to the coupling of certain growth factor receptors to stimulated inositol phosphate production. We propose that the N-ras proto-oncogene encodes a protein which couples the receptors for certain growth factors to the stimulation of phospholipase C. Thus, N-ras p21 may be the putative Gp or a functionally related protein.     

Olfactory dysfunction in humans with deficient guanine nucleotide-binding protein

The guanine nucleotide-binding stimulatory protein (Gs) couples hormone-receptor interaction to the activation of adenylate cyclase and the generation of cyclic AMP. Studies using frog neuroepithelium indicate that the sense of smell is mediated by a Gs-adenylate cyclase system, and this prompted us to test olfaction in the only known model of Gs deficiency in the animal kingdom, Gs-deficient (type 1a) pseudohypoparathyroidism (PHP), which occurs in humans. Such patients are resistant to the cAMP-mediated actions of several hormones. (Although Henkin has reported disturbances in the sense of smell in six patients with PHP, currently available biochemical measurements such as the cAMP response to parathyroid hormone (PTH) and determination of Gs activity were not reported and olfactory testing was limited.) In the present study, we found that all Gs-deficient patients had impaired olfaction when compared with PHP patients who had normal Gs activity (type 1b PHP, in which patients are resistant only to the action of PTH in the kidney). This is the first evidence of human olfactory impairment which can be related to Gs deficiency and suggests that Gs-deficient PHP patients may be resistant to cAMP-mediated actions in other non-endocrine systems.     

Activation of two signal-transduction systems in hepatocytes by glucagon

The ability of glucagon to stimulate glycogen breakdown in liver played a key part in the classic identification of cyclic AMP and hormonally stimulated adenylate cyclase. But several observations indicate that glucagon can exert effects independent of elevating intracellular cAMP concentrations. These effects are probably mediated by an elevation of the intracellular concentration of free Ca2+ although the mechanism by which this occurs is unknown. We show here that glucagon, at the low concentrations found physiologically, causes both a breakdown of inositol phospholipids and the production of inositol phosphates. Indeed, we show that the glucagon analogue, (1-N-alpha-trinitrophenylhistidine,12-homoarginine)glucagon (TH-glucagon), which does not activate adenylate cyclase or cause any increase in cAMP in hepatocytes yet can fully stimulate glycogenolysis, gluconeogenesis and urea synthesis, stimulates the production of inositol phosphates. This stimulation of inositol phospholipid metabolism by low concentrations of glucagon provides a mechanism whereby glucagon can exert cAMP-independent actions on target cells. We suggest that hepatocytes possess two distinct receptors for glucagon, a GR-1 receptor coupled to stimulate inositol phospholipid breakdown and a GR-2 receptor coupled to stimulate adenylate cyclase activity.