Farnesylated gamma-subunit of photoreceptor G protein indispensable for GTP-binding

Transducin, composed of subunits T alpha, T beta and T gamma, is a member of a heterotrimeric G-protein family, and transduces the light signal in visual cells. We have recently found that bovine T beta gamma can be separated into two components. T beta gamma-1 and T beta gamma-2, each of which has its own gamma-subunit, T gamma-1 and T gamma-2, respectively. T beta gamma-2 enhances the binding of GTP to T alpha in the presence of metarhodopsin II by about 30-fold compared with T beta gamma-1. Here we show that a farnesyl moiety is attached to a sulphur atom of the C-terminal cysteine of T gamma-2 (active form), a part of which is additionally methyl-esterified at the alpha-carboxyl group. In T gamma-1 (inactive form), however, such modifications are missing. Thus, the farnesyl moiety attached to the gamma-subunit is indispensable for the GTP-binding activity of transducin. This suggests that a similar modification may occur in the gamma-subunits of other heterotrimeric G proteins involved in biological signal transduction processes.     

Sequence homology of the LFA-1 and Mac-1 leukocyte adhesion glycoproteins and unexpected relation to leukocyte interferon

Cell-surface adherence reactions are fundamental to the biology of lymphocytes, monocytes and granulocytes. The lymphocyte function-associated 1 (LFA-1) and macrophage 1 (Mac-1) glycoproteins mediate differing types of adhesion reactions on these cells. LFA-1 participates in T-lymphocyte and natural killer-cell adhesion to target cells, whereas the Mac-1 antigen is identical to the complement receptor type 3, which mediates adhesion of monocytes and granulocytes to C3bi-sensitized particles. Deficiency of these proteins, in a heritable disease, results in multiple adhesion-related leukocyte defects. LFA-1 and Mac-1 resemble one another in overall structure, having alpha-subunits of relative molecular mass (Mr) 180,000 and 170,000, respectively, which are non-covalently associated with beta-subunits of Mr 95,000 in alpha 1 beta 1 complexes. Peptide mapping and immunological cross-reactivity have shown that the beta-subunits are highly related if not identical, but have revealed no similarities between the alpha-subunits. Nonetheless, the shared beta-subunit suggested that LFA-1 and Mac-1 might be members of a protein family containing diversified but evolutionarily related alpha-subunits. Therefore, we examine here the structure of the alpha-subunits by N-terminal amino-acid sequencing. Sequence homology shows that the alpha-subunits are members of a novel leukocyte adhesion protein family, and suggests that their evolution occurred by gene duplication. A search for similarities to previously sequenced proteins reveals a further unexpected homology between LFA-1 and leukocyte (alpha) interferons.     

G-protein beta gamma-subunits activate the cardiac muscarinic K+-channel via phospholipase A2

Muscarinic receptors of cardiac pacemaker and atrial cells are linked to a potassium channel (IK.ACh) by a pertussis toxin-sensitive GTP-binding protein. The dissociation of G-proteins leads to the generation of two potential transducing elements, alpha-GTP and beta gamma. IK.ACh is activated by G-protein alpha- and beta gamma-subunits applied to the intracellular surface of inside-out patches of membrane. beta gamma has been shown to activate the membrane-bound enzyme phospholipase A2 in retinal rods. Arachidonic acid, which is produced from the action of phospholipase A2 on phospholipids, is metabolized to compounds which may act as second messengers regulating ion channels in Aplysia. Muscarinic receptor activation leads to the generation of arachidonic acid in some cell lines. We therefore tested the hypothesis that beta gamma activates IK.ACh by stimulation of phospholipase A2. When patches were first incubated with antibody that blocks phospholipase A2 activity, or with the lipoxygenase inhibitor, nordihydroguaiaretic acid, beta gamma failed to activate IK.ACh. Arachidonic acid and several of its metabolites derived from the 5-lipoxygenase pathway, activated the channel. Blockade of the cyclooxygenase pathway did not inhibit arachidonic acid-induced channel activation. We conclude that the beta gamma-subunit of G-proteins activates IK.ACh by stimulating the production of lipoxygenase-derived second messengers.     

Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid

A variety of hypophysiotropic peptides or proteins have been reported to be present in mammalian gonads. Inhibin, a hormone that under most circumstances selectively suppresses the secretion of follicle-stimulating hormone (FSH) but not luteinizing hormone (LH), has been isolated from the gonadal fluids of several species and characterized as a heterodimeric protein consisting of alpha- and beta-polypeptides associated by disulphide bonds. The complete amino-acid sequences of the precursors of porcine and human inhibin alpha-subunits and two distinct porcine inhibin beta-subunits (beta A and beta B) have been deduced from complementary DNA sequences. Gonadotropin releasing peptides have also been found in the gonad and have generally been shown to be active in radioreceptor assays for gonadotropin releasing hormone (GnRH) but to exhibit different chromatographic and immunological characteristics from those of GnRH. During our purification of inhibin from porcine follicular fluid, we noted fractions that could stimulate the secretion of FSH by cultured anterior pituitary cells. We report here the purification of an FSH releasing protein (FRP) and its characterization by SDS-polyacrylamide gel electrophoresis under non-reducing and reducing conditions and by partial sequence analysis. Our results indicate that porcine gonadal FRP is a homodimer consisting of two inhibin beta A-chains linked by disulphide bonds. FRP is highly potent (50% effective concentration (EC50) approximately 25 pM) in stimulating the secretion and biosynthesis of FSH but not of LH or any other pituitary hormone. In contrast to the effects of GnRH and other reported gonadal gonadotropin releasing fractions, the action of FRP is not mediated by GnRH receptors.     

Tyrosine-containing motif that transduces cell activation signals also determines internalization and antigen presentation via type III receptors for IgG.

Type III receptors for IgG (Fc gamma RII; ref. 1), high-affinity IgE receptors (Fc epsilon RI; ref. 2), as well as the T- and B-cell antigen receptors, consist of multiple components with specialized ligand-binding and signal transduction functions. Fc gamma RII alpha (ligand-binding) and gamma (signal-transducing) subunits are expressed in macrophages, a cell type involved in the uptake of antigen, its processing and the presentation of the resulting peptides to major histocompatibility complex class II-restricted T lymphocytes. Here we show that murine Fc gamma RIII, transfected into Fc gamma R-negative antigen-presenting B-lymphoma cells, mediate rapid ligand internalization and strongly increase the efficiency of antigen presentation when antigen is complexed to IgG. Efficient internalization and antigen presentation via Fc gamma RIII did not require the cytoplasmic domain of the ligand-binding alpha-chain, but did require the gamma-subunit. Using chimaeric molecules, we show that gamma-chain contains a signal for receptor internalization and that the mutation of either of the two tyrosine residues present in its cytoplasmic domain prevents efficient internalization and antigen presentation of immune complexes. Thus, associated chains and their tyrosine-containing motif are not exclusively involved in cell activation, but also determine multimeric receptor internalization.     

Subunits beta gamma of heterotrimeric G protein activate beta 2 isoform of phospholipase C.

The activation of heterotrimeric G proteins results in the exchange of GDP bound to the alpha-subunit for GTP and the subsequent dissociation of a complex of the beta- and gamma-subunits (G beta gamma). The alpha-subunits of different G proteins interact with a variety of effectors, but less is known about the function of the free G beta gamma complex. G beta gamma has been implicated in the activation of a cardiac potassium channel, a retinal phospholipase A2 (ref. 9) and a specific receptor kinase, and in vitro reconstitution experiments indicate that the G beta gamma complex can act with G alpha subunit to modulate the activity of different isoforms of adenylyl cyclase. Of two phospholipase activities that can be separated in extracts of HL-60 cells, purified G beta gamma is found to activate one of them. Here we report that in co-transfection assays G beta gamma subunits specifically activate the beta 2 and not the beta 1 isoform of phospholipase, which acts on phosphatidylinositol. We use transfection assays to show also that receptor-mediated release of G beta gamma from G proteins that are sensitive to pertussis toxin can result in activation of the phospholipase. This effect may be the basis of the pertussis-toxin-sensitive phospholipase C activation seen in some cell systems (reviewed in refs 13 and 14).     

Activation of the beta 1 isozyme of phospholipase C by alpha subunits of the Gq class of G proteins.

Many hormones, neurotransmitters and growth factors, on binding to G protein-coupled receptors or receptors possessing tyrosine kinase activity, increase intracellular levels of the second messengers inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. This is due to activation of phosphoinositide-specific phospholipase(s) C (PLC), the isozymes of which are classified into groups, alpha, beta, gamma and delta. The beta, gamma and delta groups themselves contain PLC isozymes which have both common and unique structural domains. Only the gamma 1 isozyme has been implicated in a signal transduction mechanism. This involves association with, and tyrosine phosphorylation by, the ligand-bound epidermal growth factor and platelet-derived growth factor receptors, probably by means of the PLC-gamma 1-specific src homology (SH2) domain. Because EGF receptor-mediated tyrosine phosphorylation of PLC-gamma 1 stimulates catalytic activity in vitro and G proteins have been implicated in the activation of PLC, we investigated which PLC isozymes are subject to G protein regulation. We have purified an activated G protein alpha subunit that stimulates partially purified phospholipase C and now report that this G protein specifically activates the beta 1 isozyme, but not the gamma 1 and delta 1 isozymes of phospholipase C. We also show that this protein is related to the Gq class of G protein alpha subunits.     

Isozyme-selective stimulation of phospholipase C-beta 2 by G protein beta gamma-subunits.

Hydrolysis by phospholipase C (PLC) of phosphatidylinositol 4,5-bisphosphate is a key mechanism by which many extracellular signalling molecules regulate functions of their target cells. At least eight distinct isozymes of PLC are recognized in mammalian cells. Receptor-controlled PLC is often regulated by G proteins, which can be modified by pertussis toxin in some cells but not in others. In the latter cells, PLC-beta 1, but not PLC-gamma 1 or PLC-delta 1, may be activated by members of the alpha q-subfamily of the G protein alpha-subunits. An unidentified PLC in soluble fractions of cultured human HL-60 granulocytes is specifically stimulated by G protein beta gamma subunits purified from retina and brain. Identification of a second PLC-beta complementary DNA (PLC-beta 2) in an HL-60 cell cDNA library prompted us to investigate the effect of purified G protein beta gamma subunits on the activities of PLC-beta 1 and PLC-beta 2 transiently expressed in cultured mammalian cells. We report here that PLC-beta 1 and PLC-beta 2 were stimulated by free beta gamma subunits and that PLC-beta 2 was the most sensitive to beta gamma stimulation. Thus stimulation of PLC by beta gamma subunits is isozyme-selective and PLC-beta 2 is a prime target of beta gamma stimulation. Activation of PLC-beta 2 by beta gamma subunits may be an important mechanism by which pertussis toxin-sensitive G proteins stimulate PLC.     

Cloning, sequencing and expression of cDNA for a novel subunit of acetylcholine receptor from calf muscle

The nicotinic acetylcholine receptor (AChR) from fish electric organ has a subunit structure of alpha 2 beta gamma delta, and this is thought to be also the case for the mammalian skeletal muscle AChR. By cloning and sequencing the complementary or genomic DNAs, we have previously elucidated the primary structures of all four subunits of the Torpedo californica electroplax and calf muscle AChR and of the alpha- and gamma-subunits of the human muscle AChR; the primary structures of the gamma-subunit of the T. californica AChR and the alpha-subunit of the Torpedo marmorata AChR have also been deduced elsewhere. We have now cloned DNA complementary to the calf muscle messenger RNA encoding a novel polypeptide (the epsilon-subunit) whose deduced amino-acid sequence has features characteristic of the AChR subunits and which shows higher sequence homology with the gamma-subunit than with the other subunits. cDNA expression studies indicate that the calf epsilon-subunit, as well as the calf gamma-subunit, can replace the Torpedo gamma-subunit to form the functional receptor in combination with the Torpedo alpha-, beta- and delta-subunits.     

T-type calcium channel regulation by specific G-protein betagamma subunits

Low-voltage-activated (LVA) T-type calcium channels have a wide tissue distribution and have well-documented roles in the control of action potential burst generation and hormone secretion. In neurons of the central nervous system and secretory cells of the adrenal and pituitary, LVA channels are inhibited by activation of G-protein-coupled receptors that generate membrane-delimited signals, yet these signals have not been identified. Here we show that the inhibition of alpha1H (Ca(v)3.2), but not alpha(1G) (Ca(v)3.1) LVA Ca2+ channels is mediated selectively by beta2gamma2 subunits that bind to the intracellular loop connecting channel transmembrane domains II and III. This region of the alpha1H channel is crucial for inhibition, because its replacement abrogates inhibition and its transfer to non-modulated alpha1G channels confers beta2gamma2-dependent inhibition. betagamma reduces channel activity independent of voltage, a mechanism distinct from the established betagamma-dependent inhibition of non-L-type high-voltage-activated channels of the Ca(v)2 family. These studies identify the alpha1H channel as a new effector for G-protein betagamma subunits, and highlight the selective signalling roles available for particular betagamma combinations.     

Mechanically driven ATP synthesis by F1-ATPase

ATP, the main biological energy currency, is synthesized from ADP and inorganic phosphate by ATP synthase in an energy-requiring reaction. The F1 portion of ATP synthase, also known as F1-ATPase, functions as a rotary molecular motor: in vitro its gamma-subunit rotates against the surrounding alpha3beta3 subunits, hydrolysing ATP in three separate catalytic sites on the beta-subunits. It is widely believed that reverse rotation of the gamma-subunit, driven by proton flow through the associated F(o) portion of ATP synthase, leads to ATP synthesis in biological systems. Here we present direct evidence for the chemical synthesis of ATP driven by mechanical energy. We attached a magnetic bead to the gamma-subunit of isolated F1 on a glass surface, and rotated the bead using electrical magnets. Rotation in the appropriate direction resulted in the appearance of ATP in the medium as detected by the luciferase-luciferin reaction. This shows that a vectorial force (torque) working at one particular point on a protein machine can influence a chemical reaction occurring in physically remote catalytic sites, driving the reaction far from equilibrium.     

Primary structure of the alpha-subunit of transducin and its relationship to ras proteins

A group of membrane-associated guanine nucleotide binding proteins (G-proteins) are essential for transducing signals generated at cell-surface receptors into changes in cellular function and metabolism. These proteins are a complex of three subunits designated alpha, beta and gamma. The alpha-subunit is responsible for binding guanine nucleotides and seems to be characteristic of each protein. Transducin, a member of this protein family, mediates visual transduction by coupling the signal of photolysed rhodopsin with activation of a cyclic GMP phosphodiesterase. We have now cloned and sequenced the complementary DNA encoding the alpha-subunit of bovine retinal transducin and from this we have deduced the complete amino-acid sequence. The transducin alpha-subunit shares several homologous amino-acid sequences with ras gene products. The homologous segments correspond mostly to the regions thought to be involved in the guanine nucleotide binding and GTPase activity of ras proteins and to the ADP-ribosylation sites of the transducin alpha-subunit.     

Lipid modification at the N terminus of photoreceptor G-protein alpha-subunit.

Myristate is a fatty acid (fourteen-carbon chain with no double bonds, C14:0) linked to the amino-terminal glycine of several proteins, including alpha-subunits of heterotrimeric (alpha/beta gamma) G proteins. We report here a novel modification at the N terminus of the alpha-subunit of the photoreceptor G protein transducin, T alpha, with heterogeneous fatty acids composed of laurate (C12:0), unsaturated C14:2 and C14:1 fatty acids, and a small amount (approximately 5%) of myristate. Both the GTPase activity of T alpha/T beta gamma and the T beta gamma-dependent ADP-ribosylation of T alpha catalysed by pertussis toxin were inhibited by the lauroylated and myristoylated N-terminal peptide of T alpha. The myristoylated peptide gave 50% inhibition at a 3.5 to approximately 4.5-fold lower concentration than the lauroylated peptide in each assay, indicating that the strength of the interaction between T alpha and T beta gamma is altered by heterogeneous fatty acids linked to T alpha. This suggests that a looser subunit interaction in transducin which is due to an abundance of N-linked fatty acids other than myristate would favour the rapid turnover and catalysis essential for the visual excitation in photoreceptor cells.     

Cell adhesion receptors and cancer

Cell-to-cell and cell-to-extracellular matrix (ECM) interactions in the functions of cell adhesion and signal transduction are important in global control of cell phenotypes and cell behavior and are crucial for maintenance of homeostasis and structural/functional stabilization of tissues and organs. Cell adhesion receptors are recognized as the molecular basis of cell adhesion. Cadherin and Integrin are widely expressed adhesion receptors in most tissues. They are transmembrane glycoproteins which, through their cytoplasmic domain, bind to many proteins at the inner surface of cell membrane to form molecule-linkage complexes and then connect with the cytoskeleton. Through cell adhesion receptors a network functioning as cell adhesion and signal transduction is organized between tissue cells and cell-ECM. In this regard cell adhesion receptors play an important role in regulation of morphogenesis, cell-cell recognition, cell migration, cell sorting and the determination of cell's fate in development. They mediate cell functions and their fault expression is intimately correlated with development of disorders like cancer. Several isoforms of Integrin were found to have tumor suppressor effect. Some components in the molecule-linkage of focal contact are actin-binding proteins as well as substrates of kinase in the Integrin initiated signal pathway to play a role as signal transducer. Some of these molecules exhibited tumor suppressor effect too. Decreased expression of E-Cadherin has been demonstrated in many epithelium originated carcinomas. Cadherin associated membrane adhesion plaque molecule β-Catenin is also involved in the oncogene Wnt signal pathway. Both E-Cadherin and β-Catenin were proved respectively with tumor suppressor effect against invasiveness and metastasis. That Cadherin is important for the posttranslationally functional expression of Connexin has been supported by evidence from developmental biology and cancer cell differentiation studies to suggest that some sort of interrelation feedback control exists between the two signal pathways.     

Coexpression of two distinct muscle acetylcholine receptor alpha-subunits during development

The nicotinic acetylcholine receptor is a ligand-gated channel that mediates signalling at the vertebrate neuromuscular junction. It is a pentameric complex of four different subunits, assembled with a stoichiometry of alpha 2 beta gamma delta. Muscle-like alpha-subunits have been cloned from Torpedo, mouse, calf, rat, chicken, human and Xenopus, and only a single alpha-subunit complementary DNA from each species has been detected. We report here the cloning and characterization of a second muscle alpha-subunit cDNA from Xenopus, and show that this and a previously reported Xenopus alpha-subunit cDNA are encoded by distinct genes. The novel alpha-subunit reported here is expressed uniquely in oocytes; but both types of alpha-subunit are coexpressed throughout muscle development. This latter observation indicates that the expression of these two alpha-subunits is different from a previously reported developmental 'subunit-switch' mechanism used to generate channel diversity.     

Importance of a novel GABAA receptor subunit for benzodiazepine pharmacology

Neurotransmission effected by GABA (gamma-aminobutyric acid) is predominantly mediated by a gated chloride channel intrinsic to the GABAA receptor. This heterooligomeric receptor exists in most inhibitory synapses in the vertebrate central nervous system (CNS) and can be regulated by clinically important compounds such as benzodiazepines and barbiturates. The primary structures of GABAA receptor alpha- and beta-subunits have been deduced from cloned complementary DNAs. Co-expression of these subunits in heterologous systems generates receptors which display much of the pharmacology of their neural counterparts, including potentiation by barbiturates. Conspicuously, however, they lack binding sites for, and consistent electrophysiological responses to, benzodiazepines. We now report the isolation of a cloned cDNA encoding a new GABAA receptor subunit, termed gamma 2, which shares approximately 40% sequence identity with alpha- and beta-subunits and whose messenger RNA is prominently localized in neuronal subpopulations throughout the CNS. Importantly, coexpression of the gamma 2 subunit with alpha 1 and beta 1 subunits produces GABAA receptors displaying high-affinity binding for central benzodiazepine receptor ligands.     

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.     

Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase

The enzyme F1-ATPase has been shown to be a rotary motor in which the central gamma-subunit rotates inside the cylinder made of alpha3beta3 subunits. At low ATP concentrations, the motor rotates in discrete 120 degrees steps, consistent with sequential ATP hydrolysis on the three beta-subunits. The mechanism of stepping is unknown. Here we show by high-speed imaging that the 120 degrees step consists of roughly 90 degrees and 30 degrees substeps, each taking only a fraction of a millisecond. ATP binding drives the 90 degrees substep, and the 30 degrees substep is probably driven by release of a hydrolysis product. The two substeps are separated by two reactions of about 1 ms, which together occupy most of the ATP hydrolysis cycle. This scheme probably applies to rotation at full speed ( approximately 130 revolutions per second at saturating ATP) down to occasional stepping at nanomolar ATP concentrations, and supports the binding-change model for ATP synthesis by reverse rotation of F1-ATPase.