Diversity among the beta subunits of heterotrimeric GTP-binding proteins: characterization of a novel beta-subunit cDNA.

Heterotrimeric guanine nucleotide binding proteins transduce signals from cell surface receptors to intracellular effectors. The alpha subunit is believed to confer receptor and effector specificity on the G protein. This role is reflected in the diversity of genes that encode these subunits. The beta and gamma subunits are thought to have a more passive role in G protein function; biochemical data suggests that beta-gamma dimers are shared among the alpha subunits. However, there is growing evidence for active participation of beta-gamma dimers in some G protein mediated signaling systems. To further investigate this role, we examined the diversity of the beta subunit family in mouse. Using the polymerase chain reaction, we uncovered a new member of this family, G beta 4, which is expressed at widely varying levels in a variety of tissues. The predicted amino acid sequence of G beta 4 is 79% to 89% identical to the three previously known beta subunits. The diversity of beta gene products may be an important corollary to the functional diversity of G proteins.     

The dipole moment of membrane proteins: potassium channel protein and beta-subunit.

The mechanism of ion channel opening is one of the most fascinating problems in membrane biology. Based on phenomenological studies, early researchers suggested that the elementary process of ion channel opening may be the intramembrane charge movement or the orientation of dipolar proteins in the channel. In spite of the far reaching significance of these hypotheses, it has not been possible to formulate a comprehensive molecular theory for the mechanism of channel opening. This is because of the lack of the detailed knowledge on the structure of channel proteins. In recent years, however, the research on the structure of channel proteins made marked advances and, at present, we are beginning to have sufficient information on the structure of some of the channel proteins, e.g. potassium-channel protein and beta-subunits. With these new information, we are now ready to have another look at the old hypothesis, in particular, the dipole moment of channel proteins being the voltage sensor for the opening and closing of ion channels. In this paper, the dipole moments of potassium channel protein and beta-subunit, are calculated using X-ray diffraction data. A large dipole moment was found for beta-subunits while the dipole moment of K-channel protein was found to be considerably smaller than that of beta-subunits. These calculations were conducted as a preliminary study of the comprehensive research on the dipolar structure of channel proteins in excitable membranes, above all, sodium channel proteins.     

Subunit interactions of GTP-binding proteins.

Fluorescence energy transfer [cf. F?rster, T. (1948) Ann. Phys. 6, 55-75] was tested for its suitability to study quantitative interactions of subunits of G0 with each other and these subunits or trimeric G0 with the beta 1-adrenoceptor in detergent micelles or after reconstitution into lipid vesicles [according to Feder, D., Im, M.-J., Klein, H. W., Hekman, M., Holzh?fer, A, Dees, C., Levitzki, A., Helmreich, E. J. M. & Pfeuffer, T. (1986) EMBO J. 5, 1509-1514]. For this purpose, alpha 0- and beta gamma-subunits and trimeric G0 purified from bovine brain, the beta gamma-subunits from bovine rod outer segment membranes and the beta 1-adrenoceptor from the turkey erythrocyte were all labelled with either tetramethylrhodamine maleimide or fluorescein isothiocyanate under conditions which leave the labelled proteins functionally intact. In the case of alpha 0- and beta gamma-interactions, specific high-affinity binding interactions (Kd approximately 10 nM) and nonspecific low-affinity binding interactions (Kd approximately 1 microM) could be readily distinguished by comparing fluorescence energy transfer before and after dissociation with 10 microM guanosine 5'-O-[gamma-thio]triphosphate and 10 mM MgCl2 where only low-affinity binding interactions remained. Interactions between alpha 0- and beta gamma-subunits from bovine brain or from bovine retinal transducin did not differ much. The beta gamma-subunits from bovine brain were found to bind with high transfer efficiency and comparable affinities to the hormone-activated and the nonactivated beta 1-receptor reconstituted in lipid vesicles: Kd = 100 +/- 20 and 120 +/- 20 nM, respectively; however, beta gamma-subunits from transducin appeared to bind more weakly to the beta 1-adrenoceptor than beta gamma-subunits from bovine brain. Separated purified homologous alpha 0- and beta gamma-subunits from bovine brain interfered mutually with each other in binding to the beta 1-adrenoceptor presumably because they had a greater affinity for each other than for the receptor. These findings attest to the suitability of fluorescence energy transfer for studying protein-protein interactions of G-proteins and G-protein-linked receptors. Moreover, they supported the previous finding [Kurstjens, N. P., Fr?hlich, M., Dees, C., Cantrill, R. C., Hekman, M. & Helmreich, E. J. M. (1991) Eur. J. Biochem. 197, 167-176] that beta gamma-subunits can bind to the nonactivated beta 1-adrenoceptor.     

Light-dependent GTP-binding proteins in squid photoreceptors.

Previous biochemical and electrophysiological evidence suggests that in invertebrate photoreceptors, a GTP-binding protein (G-protein) mediates the actions of photoactivated rhodopsin in the initial stages of transduction. We find that squid photoreceptors contain more than one protein (molecular masses 38, 42 and 46 kDa) whose ADP-ribosylation by bacterial exotoxins is light-sensitive. Several lines of evidence suggest that these proteins represent distinct alpha subunits of G-proteins. (1) Pertussis toxin and cholera toxin react with distinct subsets of these polypeptides. (2) Only the 42 kDa protein immunoreacts with the monoclonal antibody 4A, raised against the alpha subunit of the G-protein of vertebrate rods [Hamm & Bownds (1984) J. Gen. Physiol. 84. 265-280]. (3) In terms of ADP-ribosylation, the 42 kDa protein is the least labile to freezing. (4) Of the 38 kDa and 42 kDa proteins, the former is preferentially extracted with hypo-osmotic solutions, as demonstrated by the solubility of its ADP-ribosylated state and by the solubility of the light-dependent binding of guanosine 5'-[gamma-thio]triphosphate. The specific target enzymes for the observed G-proteins have not been established.     

Multiple opioid receptors and GTP-binding proteins.

We believed that GTP-binding protein (G-protein)-coupling receptor always transduces stimulatory signals to G-proteins. From our recent experiments using reconstitution techniques, however, it was revealed that some receptors transduce an inhibitory or no signal to G-proteins in specific tissues, despite some interaction between them. Here we discuss the molecular basis of mechanisms of such diverse modes of functional coupling between different subtypes of opioid receptors and G-proteins.     

Ultrastructural changes in the mouse fetal choroid plexuses following chronic maternal alcoholization

Female mice (RAP strain) were alcoholized for 30-50 days before mating and during pregnancy until killing, with a 20% solution of ethanol administered instead of drinking water. From foetuses of 16, 18 and 20 days and from newborn puppies (day 1) choroid plexuses were excised and electronmicroscopically examined. Chronic maternal alcoholization induced the lowering of glycogen content in the choroid cells of 16 day old foetuses, the swelling and vacuolization of mitochondria with the disappearance of cristae and enlargement of the Golgi complex--in the choroid cells at all the developmental stages controlled, the enlargement of intercellular spaces within the choroid epithelium and between the capillaries and the epithelial layer. The changes detected are presumedly due to disturbances of intracerebral fluid homeostasis and may be responsible for at least some of the CNS pathology observed in alcohol embryo- and fetopathy.     

GTP-binding proteins in bovine brain nuclear membranes.

Nuclear membranes and other subcellular fractions derived from bovine brain cortex were investigated for the existence of GTP-binding proteins. By using photolytic labeling with [alpha-32P]GTP a 29 kDa GTP-binding protein was shown to be present in nuclear membranes which was not present in the plasma membranes nor in microsomal or cytosolic fractions. Two-dimensional gel electrophoresis revealed that this protein is rather acidic with a pI lower than 4.5. Members of the heterotrimeric Gi/o family are not present in the nuclear envelope: a 39 kDa protein, ADP ribosylated by pertussis toxin, was shown to originate from plasma membrane contamination.     

Small molecular weight GTP-binding proteins in human platelet membranes. Purification and characterization of a novel GTP-binding protein with a molecular weight of 22,000

When guanosine 5'-(3-O-[35S]thio)triphosphate (GTP gamma S)-binding activity was assayed in the particulate and cytosol fractions of human platelets, most activity was found in the particulate fraction. GTP-binding proteins (G proteins) were extracted from the particulate fraction by sodium cholate and purified by several column chromatographies. At least three G proteins with Mr values of about 21,000, 22,000, and 24,000 (21K G, 22K G, and 24K G, respectively) were separated in addition to the stimulatory (Gs) and inhibitory (Gi) regulatory GTP-binding proteins of adenylate cyclase. Among them, the amount of 22K G was more than 10-fold of those of other G proteins. 22K G was purified to near homogeneity and characterized. 22K G specifically bound GTP gamma S, GTP, and GDP, with a Kd value for GTP gamma S of about 50 nM. [35S]GTP gamma S binding to 22K G was inhibited by pretreatment with N-ethylmaleimide. 22K G hydrolyzed GTP to liberate Pi, with a turnover number of 0.01 min-1. 22K G was not copurified with the beta gamma subunits of Gs and Gi and was not recognized by the antibodies against the ADP-ribosylation factor for Gs and the ras protein. The peptide map of 22K G was different from those of the smg-25A and rho proteins, which we have purified from bovine brain membranes. 21K G was identified to be the c-ras protein, but 24K G was unidentified. These results indicate that there are multiple G proteins in platelet membranes and that a novel G protein (22K G) is a major G protein in platelets.     

The beta-subunit of the signal recognition particle receptor is a novel GTP-binding protein without intrinsic GTPase activity

The beta-subunit of the signal recognition particle receptor (SRbeta), a member of the Ras family of small molecular weight GTPases, is involved in the targeting of nascent polypeptide chains to the protein translocation machinery in the endoplasmic reticulum membrane. We purified SRbeta from an expressing strain of Escherichia coli and investigated the properties of the isolated GTPase. We find that, unlike other Ras family GTPases, most SRbeta purifies bound to GTP, and SRbeta-bound GTP is not easily exchanged with solution GTP. SRbeta possesses no detectable GTPase activity. Although a stable interaction between SRbeta and ribosomes is observed, SRbeta is not stimulated to hydrolyze GTP when incubated with ribosomes or ribosome-nascent chains. A GTPase mutant harboring a mutation in a region predicted to be functionally important, based on observations made in related GTPases, binds GTP with faster kinetics and appears to be a less stable protein but otherwise displays similar properties to the wild-type SRbeta GTPase. Our results demonstrate that as an isolated GTPase, SRbeta functions differently from the Arf- and Ras-type GTPases that it is most closely related to by sequence.     

GTP-binding proteins in intracellular transport

One of the most exciting recent discoveries in the area of intracellular protein transport is the finding that many organelles involved in exocytic and endocytic membrane traffic have one or more Ras-like GTP-binding proteins on their cytoplasmic face that are specific for each membranous compartment. These proteins are attractive candidates for regulators of transport vesicle formation and the accurate delivery of transport vesicles to their correct targets.     

Affinity labeling of GTP-binding proteins in cellular extracts.

GTP-binding proteins in cellular extracts from Escherichia coli, Thermus thermophilus, yeast, wheat germ or calf thymus were identified using in situ periodate-oxidized [alpha-32P]GTP as affinity label. Site-specific reaction of individual GTP-binding proteins was achieved by cross-linking the protein-bound 2',3'-dialdehyde derivative of GTP with the single lysine residue of the conserved NKXD sequence through Schiff's base formation and subsequent cyanoborohydride reduction. Labeled GTP-binding proteins from prokaryotic or eukaryotic cell homogenates were separated by polyacrylamide gel electrophoresis and visualized by autoradiography. In addition cross-linking of [alpha-32P]GTP with GTP-binding proteins was demonstrated in model systems using different purified GTPases, human c-H-ras p21, transducin from bovine retina, polypeptide elongation factor Tu (EF-Tu) from T. thermophilus and initiation factor 2 (IF2) from T. thermophilus. The described affinity labeling technique can serve as an analytical method for the identification of GTPases belonging to the classes of ras-proteins, elongation and initiation factors, and heterotrimeric signal transducing G-proteins.     

GTP-binding proteins as possible targets for protein kinase C action

The alpha-subunits of two guanine nucleotide binding proteins Gi and transducin, as well as the beta-subunit of transducin, serve as substrates for phosphorylation by the Ca2+- and phospholipid-dependent protein kinase C (PKC). Phosphorylation of the alpha-subunit of transducin is strictly dependent on its conformation and it is only the inactive form that is subjected to phosphorylation by PKC. This review will focus on the proposition that G proteins may serve as cellular targets for modulatory actions of PKC.     

Pollination-induced senescence in phalaenopsis petals. Relationship of ethylene sensitivity to activity of GTP-binding proteins and protein phosphorylation

Treatments of cut phalaenopsis ( Phalaenopsis hybrid , cv. 'Herbert Hager') flowers with cholera toxin or guanosine-5-0-(3-thiotriphosphate), compounds that modulate GTP-binding protein activity, increased the sensitivity of the flowers to ethylene. Guanosine-5-0-(2-thiodiphosphate) which does not affect the activity of GTP-binding proteins, had no affect on the sensitivity to ethylene. Western blot analysis of microsomal proteins, revealed that a peptide with a molecular mass of ca 42 kDa cross-reacts with antibodies against a well-conserved amino acid sequence (G_α-commun peptide) of mammalian G-proteins. Calcium ions, known co-factors of protein kinases, also increased the sensitivity of the flowers to ethylene, while EGTA, a chelator of calcium, decreased it. Phorbol 12-myrisate 13-acetate, a phorbol ester, had no effect on the sensitivity to ethylene. Protein phosphorylation in petal microsomal membranes was doubled in the presence of calcium ions, but was unaffected by phorbol ester. Ten h after pollination, at the peak of ethylene sensitivity, a significant increase of ca 20% was measured in the binding of GTP to the membranes. Protein phosphorylation in flowers increased significantly following pollination, with a single peptide of ca 30 kDa most heavily phosphorylated. These observations may indicate a direct involvement of GTP-binding proteins, and protein phosphorylation, two major components of the cellular signal transduction pathway, in the regulation of pollination induced ethylene sensitivity in phalaenopsis petals.     

Distribution of the stimulatory GTP-binding proteins, Gs and Golf, within olfactory neuroepithelium

Several lines of evidence suggest that, for certain odorants,olfactory signal transduction may be mediated by a stimulatoryG-protein coupled adenylate cyclase cascade. Two stimulatoryG-proteins, G_olf and G_s, are expressed in olfactory tissue.To evaluate their relative contributions to the process of odorantsignal transduction, specific antisera were used to determinethe distribution and relative abundance of G_olf and G_s in ratolfactory neuroepithelium and olfactory sensory cilia. Theseanalyses demonstrate that (1) Golf is far more abundant thanG_s in olfactory neuroepithelium and (2) G_olf is essentiallythe only stimulatory G-protein present in olfactory sensorycilia. ¹Present address: Gene Expression Laboratory, The Salk Institute,PO Box 85800, San Diego, CA 92138, USA