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.     

Ligand binding to the beta-adrenergic receptor involves its rhodopsin-like core

Recently the genes for several hormone receptors that interact with guanine nucleotide binding proteins (G proteins) have been cloned, including the hamster beta 2-adrenergic receptor (beta 2AR), a human beta AR, the turkey erythrocyte beta AR and the porcine muscarinic acetylcholine receptor (MAR). All these receptors share some amino-acid homology with rhodopsin, particularly in 7 hydrophobic stretches of residues that are believed to represent transmembrane helices. To determine whether differences in ligand specificity result from the divergence in the sequences of the hydrophilic regions of these receptors, we have expressed in mammalian cells genes for the wild-type hamster and human beta AR proteins, and a series of deletion mutant genes of the hamster beta 2AR. The pharmacology of the expressed receptors indicates that most of the hydrophilic residues are not directly involved in the binding of agonists or antagonists to the receptor. In addition, we have identified a mutant receptor that has high agonist affinity but does not couple to adenylate cyclase.     

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.     

Odorant-sensitive adenylate cyclase may mediate olfactory reception

The mechanism of the sense of smell has long been a subject for theory and speculation. More recently, the notion of odorant recognition by stereospecific protein receptors has gained wide acceptance, but the receptor molecules remained elusive. The recognition molecules are believed to be quite diverse, which would partly explain the unusual difficulties encountered in their isolation by conventional ligand-binding techniques. An alternative approach would be to probe the receptors through transductory components that may be common to all receptor types. Here we report the identification of one such transductory molecular component. This is an odorant-sensitive adenylate cyclase, present in very large concentrations in isolated dendritic membranes of olfactory sensory neurones. Odorant activation of the enzyme is ligand and tissue specific, and occurs only in the presence of GTP, suggesting the involvement of receptor(s) coupled to a guanine nucleotide binding protein (G-protein). The olfactory G-protein is independently identified by labelling with bacterial toxins, and found to be similar to stimulatory G-proteins in other systems. Our results suggest a role for cyclic nucleotides in olfactory transduction, and point to a molecular analogy between olfaction and visual, hormone and neurotransmitter reception. Most importantly, the present findings reveal new ways to identify and isolate olfactory receptor proteins.     

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.     

Cloning of the gene and cDNA for mammalian beta-adrenergic receptor and homology with rhodopsin

The adenylate cyclase system, which consists of a catalytic moiety and regulatory guanine nucleotide-binding proteins, provides the effector mechanism for the intracellular actions of many hormones and drugs. The tissue specificity of the system is determined by the particular receptors that a cell expresses. Of the many receptors known to modulate adenylate cyclase activity, the best characterized and one of the most pharmacologically important is the beta-adrenergic receptor (beta AR). The pharmacologically distinguishable subtypes of the beta-adrenergic receptor, beta 1 and beta 2 receptors, stimulate adenylate cyclase on binding specific catecholamines. Recently, the avian erythrocyte beta 1, the amphibian erythrocyte beta 2 and the mammalian lung beta 2 receptors have been purified to homogeneity and demonstrated to retain binding activity in detergent-solubilized form. Moreover, the beta-adrenergic receptor has been reconstituted with the other components of the adenylate cyclase system in vitro, thus making this hormone receptor particularly attractive for studies of the mechanism of receptor action. This situation is in contrast to that for the receptors for growth factors and insulin, where the primary biochemical effectors of receptor action are unknown. Here, we report the cloning of the gene and cDNA for the mammalian beta 2AR. Analysis of the amino-acid sequence predicted for the beta AR indicates significant amino-acid homology with bovine rhodopsin and suggests that, like rhodopsin, beta AR possesses multiple membrane-spanning regions.     

An intronless gene encoding a potential member of the family of receptors coupled to guanine nucleotide regulatory proteins

Plasma membrane receptors for hormones, drugs, neurotransmitters and sensory stimuli are coupled to guanine nucleotide regulatory proteins. Recent cloning of the genes and/or cDNAs for several of these receptors including the visual pigment rhodopsin, the adenylate-cyclase stimulatory beta-adrenergic receptor and two subtypes of muscarinic cholinergic receptors has suggested that these are homologous proteins with several conserved structural and functional features. Whereas the rhodopsin gene consists of five exons interrupted by four introns, surprisingly the human and hamster beta-adrenergic receptor genes contain no introns in either their coding or untranslated sequences. We have cloned and sequenced a DNA fragment in the human genome which cross-hybridizes with a full-length beta 2-adrenergic receptor probe at reduced stringency. Like the beta 2-adrenergic receptor this gene appears to be intronless, containing an uninterrupted long open reading frame which encodes a putative protein with all the expected structural features of a G-protein-coupled receptor.     

Functional reconstitution of purified muscarinic receptors and inhibitory guanine nucleotide regulatory protein

Muscarinic receptors trigger several different responses including an increase in concentration of cyclic GMP, a decrease in cyclic AMP concentration, breakdown of polyphosphoinositides and changes in ion permeability. It is not yet clear whether these reactions occur sequentially or independently and which directly coupled to the muscarinic receptor. Several lines of evidence indicate that muscarinic receptors in many, if not all, cell types are coupled to the inhibitory guanine nucleotide regulatory protein (Ni or Gi) of adenylate cyclase. To provide direct evidence for this coupling, we have reconstituted muscarinic receptors purified from porcine brain with Ni purified from rat brain in a phospholipid vesicle. Here, we report that the GTPase activity of Ni is stimulated by carbachol. This action is blocked by the simultaneous addition of atropine and is not observed when the Ni protein is ADP-ribosylated. We conclude that one function of the muscarinic receptor is the activation of Ni.     

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.     

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.     

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.     

The genomic clone G-21 which resembles a beta-adrenergic receptor sequence encodes the 5-HT1A receptor

The recent cloning of the complementary DNAs and/or genes for several receptors linked to guanine nucleotide regulatory proteins including the adrenergic receptors (alpha 1, alpha 2A, alpha 2B, beta 1, beta 2), several subtypes of the muscarinic cholinergic receptors, and the visual 'receptor' rhodopsin has revealed considerable similarity in the primary structure of these proteins. In addition, all of these proteins contain seven putative transmembrane alpha-helices. We have previously described a genomic clone, G-21, isolated by cross-hybridization at reduced stringency with a full length beta 2-adrenergic receptor probe. This clone contains an intronless gene which, because of its striking sequence resemblance to the adrenergic receptors, is presumed to encode a G-protein-coupled receptor. Previous attempts to identify this putative receptor by expression studies have failed. We now report that the protein product of the genomic clone, G21, transiently expressed in monkey kidney cells has all the typical ligand-binding characteristics of the 5-hydroxytryptamine (5-HT1A) receptor.     

Identification of receptor contact site involved in receptor-G protein coupling

The mammalian G proteins transduce information from extracellular signals, including neurotransmitters, hormones and sensory stimuli, into regulation of effector enzymes or ion channels within cells. Triggered by appropriate extracellular signals, receptor proteins specifically activate members of the G protein family by catalysing replacement of GDP by GTP at the guanine nucleotide binding site. Like the receptor proteins, the heterotrimeric G proteins exhibit impressive structural similarities, suggesting that all receptor-G protein interactions use homologous structural elements and a single molecular mechanism. Topologically equivalent portions of each G protein may therefore interact with the appropriate receptor. We recently predicted the secondary structure of a composite G protein alpha-chain and proposed that a predicted amphipathic alpha-helix at the extreme carboxy-terminus of the polypeptide directly contacts receptors. This proposal has now been confirmed by sequencing complementary DNAs of the gene that encodes the alpha-chain (alpha s) of the stimulatory regulator (Gs) of adenylyl cyclase in wild-type cells and in a mutant mouse S49 lymphoma cell line, unc, in which Gs cannot be activated by hormone receptors. The sequences reveal a point mutation in the unc gene that substitutes a proline residue for an arginine near the carboxy-terminus of the alpha s-polypeptide. Expression of recombinant alpha s-unc in genetically alpha s-deficient S49 cells reproduces the unc phenotype.     

Pure beta-adrenergic receptor: the single polypeptide confers catecholamine responsiveness to adenylate cyclase

The beta-adrenergic receptor binding subunits from frog erythrocytes, hamster lung and guinea pig lung have been purified to apparent homogeneity and in all cases reside on a single polypeptide. Insertion of the pure receptors into phospholipid vesicles and subsequent fusion of these vesicles with a receptor-deficient cell conveys beta-adrenergic responsiveness to the adenylate cyclase system of the acceptor cell. Such responsiveness is linearly dependent on the amount of receptor used in the fusion experiments and is independent of the receptor source. Moreover, this responsiveness displays appropriate beta-adrenergic specificity. These results indicate that the beta-adrenergic receptor polypeptide contains both the ligand binding site and the site responsible for mediating stimulation of adenylate cyclase activity, presumably via interaction with the guanine nucleotide regulatory protein.     

alpha 2-Adrenergic receptors appear in rat salivary glands after reserpine treatment

The regulation of central and peripheral adrenergic receptors by various chemical, physiological, pharmacological and pathological stimuli has been the subject of intense study. For example, drug treatments can produce relatively small changes in the density of existing receptor binding sites in a variety of tissues. The alpha-adrenergic receptors in rat salivary gland tissue have been studied using radioligand receptor binding techniques. We have recently identified and characterised alpha 1-adrenergic receptors in the rat submandibular gland, but surprisingly, alpha 2-adrenergic receptor binding was not detectable. We now report that a single treatment of reserpine results in the appearance of alpha 2-adrenergic binding sites within 12 h. Continued treatment with the drug produces further increases in the number of alpha 2-adrenergic receptors, such that after 7 days the levels of alpha 1- and alpha 2-adrenergic receptors are similar. This is the first example of a drug treatment resulting in the appearance of a receptor type which was not previously detectable.     

RGS2 regulates signal transduction in olfactory neurons by attenuating activation of adenylyl cyclase III

The heterotrimeric G-protein Gs couples cell-surface receptors to the activation of adenylyl cyclases and cyclic AMP production (reviewed in refs 1, 2). RGS proteins, which act as GTPase-activating proteins (GAPs) for the G-protein alpha-subunits alpha(i) and alpha(q), lack such activity for alpha(s) (refs 3-6). But several RGS proteins inhibit cAMP production by Gs-linked receptors. Here we report that RGS2 reduces cAMP production by odorant-stimulated olfactory epithelium membranes, in which the alpha(s) family member alpha(olf) links odorant receptors to adenylyl cyclase activation. Unexpectedly, RGS2 reduces odorant-elicited cAMP production, not by acting on alpha(olf) but by inhibiting the activity of adenylyl cyclase type III, the predominant adenylyl cyclase isoform in olfactory neurons. Furthermore, whole-cell voltage clamp recordings of odorant-stimulated olfactory neurons indicate that endogenous RGS2 negatively regulates odorant-evoked intracellular signalling. These results reveal a mechanism for controlling the activities of adenylyl cyclases, which probably contributes to the ability of olfactory neurons to discriminate odours.     

Cloning of the gene for a human dopamine D5 receptor with higher affinity for dopamine than D1.

Dopamine receptors belong to a superfamily of receptors that exert their biological effects through guanine nucleotide-binding (G) proteins. Two main dopamine receptor subtypes have been identified, D1 and D2, which differ in their pharmacological and biochemical characteristics. D1 stimulates adenylyl cyclase activity, whereas D2 inhibits it. Both receptors are primary targets for drugs used to treat many psychomotor diseases, including Parkinson's disease and schizophrenia. Whereas the dopamine D1 receptor has been cloned, biochemical and behavioural data indicate that dopamine D1-like receptors exist which either are not linked to adenylyl cyclase or display different pharmacological activities. We report here the cloning of a gene encoding a 477-amino-acid protein with strong homology to the cloned D1 receptor. The receptor, called D5, binds drugs with a pharmacological profile similar to that of the cloned D1 receptor, but displays a 10-fold higher affinity for the endogenous agonist, dopamine. As with D1, the dopamine D5 receptor stimulates adenylyl cyclase activity. Northern blot and in situ hybridization analyses reveal that the receptor is neuron-specific, localized primarily within limbic regions of the brain; no messenger RNA was detected in kidney, liver, heart or parathyroid gland. The existence of a dopamine D1-like receptor with these characteristics had not been predicted and may represent an alternative pathway for dopamine-mediated events and regulation of D2 receptor activity.     

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.     

Increase of corticotropin-releasing factor staining in rat paraventricular nucleus neurones by depletion of hypothalamic adrenaline

In response to stress, adrenocorticotropic hormone (ACTH) is released by corticotrophs in the anterior pituitary under the control of several central and peripheral factors including corticotropin-releasing factor (CRF), which was recently isolated from the brain and sequenced. Immunocytochemical studies have shown that most of the CRF-containing cell bodies that project to the median eminence are present in the hypothalamic paraventricular nucleus (PVN). A dense PNMT(phenylethanolamine-N-methyltransferase)-containing fibre network was also observed in the same region--PNMT is the final enzyme in the biosynthesis of adrenaline and has been demonstrated in the brain. In the present study we found an association of adrenergic nerve fibres and CRF neurones by immunohistochemistry using antisera to PNMT and CRF. To examine the functional significance of the adrenergic projection to the PVN, we blocked the synthesis of adrenaline using a specific inhibitor of PNMT. The depletion of adrenaline resulted in an increase in CRF immunoreactivity. The present results suggest that, as well as catecholamines which regulate ACTH release at the anterior pituitary level via a beta 2-adrenergic receptor mechanism, central catecholamines (mainly adrenaline) also affect ACTH release through their action on CRF cells. Peripheral catecholamines seem to have a direct stimulatory effect on the pituitary corticotroph cells, whereas the present findings suggest that central adrenaline-containing neurones have an inhibitory role in the physiological response to stress.     

Multiple D2 dopamine receptors produced by alternative RNA splicing

Dopamine receptor belong to a large class of neurotransmitter and hormone receptors that are linked to their signal transduction pathways through guanine nucleotide binding regulatory proteins (G proteins). Pharmacological, biochemical and physiological criteria have been used to define two subcategories of dopamine receptors referred to as D1 and D2. D1 receptors activate adenylyl cyclase and are coupled with the Gs regulatory protein. By contrast, activation of D2 receptors results in various responses including inhibition of adenylyl cyclase, inhibition of phosphatidylinositol turnover, increase in K+ channel activity and inhibition of Ca2+ mobilization. The G protein(s) linking the D2 receptors to these responses have not been identified, although D2 receptors have been shown to both copurify and functionally reconstitute with both Gi and Go related proteins. The diversity of responses elicited by D2-receptor activation could reflect the existence of multiple D2 receptor subtypes, the identification of which is facilitated by the recent cloning of a complementary DNA encoding a rat D2 receptor. This receptor exhibits considerable amino-acid homology with other members of the G protein-coupled receptor superfamily. Here we report the identification and cloning of a cDNA encoding an RNA splice variant of the rat D2 receptor cDNA. This cDNA codes for a receptor isoform which is predominantly expressed in the brain and contains an additional 29 amino acids in the third cytoplasmic loop, a region believed to be involved in G protein coupling.