Secretory granule and synaptic vesicle formation.

Sorting of secreted proteins into dense-core secretory granules may involve selective aggregation of regulated secretory proteins, rather than a conventional sortase. Synaptic vesicles, which mediate paracrine communication between adjacent cells, appear to arise by a modification of the early endosome pathway. Targeting to the cell surface involves the actin-based cytoskeleton and small GTP-binding proteins.     

GUST27 and Closely Related G-protein-coupled Receptors are Localized in Taste Buds Together with Gi-protein {alpha}-Subunit

Gustatory, like olfactory signalling is probably mediated byseven-transmembrane receptors and coupling GTP-binding proteins(G proteins). We investigated the expression of a subset ofthese receptors and the Gi protein     

G-protein Subunits Expressed in Catfish Olfactory Receptor Neurons

Olfactory signal transduction in a number of species has beenshown to be mediated by heterotrimeric GTP-binding proteins(G-proteins). The expression of different G-proteins in channelcatfish (Ictalurus punctatus) olfactory epithelium was investigatedusing antibodies to both the     

Stimulation of Escherichia coli adenylate cyclase activity by elongation factor Tu, a GTP-binding protein essential for protein synthesis

A unique feature of eucaryotic adenylate cyclases is their interaction with GTP-binding proteins that mediate hormonal responses. Until now, there has been no evidence for regulation of Escherichia coli adenylate cyclase by a GTP-binding protein. We describe here that the most abundant protein in E. coli, the GTP-binding protein EF-Tu, which is important as an elongation factor in protein synthesis, also serves as a stimulator of adenylate cyclase activity. Homogeneous EF-Tu specifically increased the activity of purified adenylate cyclase as much as 70%; other E. coli GTP-binding proteins had no effect on enzyme activity. A study of the guanine nucleotide specificity for EF-Tu-mediated stimulation of adenylate cyclase activity suggested that the preferred activator is EF-Tu X GDP. To account for the GTP-specific stimulation of adenylate cyclase activity observed in intact cells, we propose that the nucleotide specificity for EF-Tu-dependent activation of adenylate cyclase is governed by other factors in the cell.     

The sensory basis of olfactory search behavior in banded kokopu (<Emphasis Type="Italic">Galaxias fasciatus</Emphasis>)

The sensory basis of olfactory search behavior was investigated in the banded kokopu, Galaxias fasciatus, using a flow tank. In the presence of a 2 cm s(-1) current flow, banded kokopu use both water current and chemical information to locate a food odor source. The superficial neuromasts of the lateral line system mediate the rheotactic component of the odor search. A physical block of one olfactory nostril did not affect the olfactory search strategy employed by banded kokopu in still water or in the presence of a current flow. Thus, there is no evidence that banded kokopu perform a bilateral comparison of the olfactory stimulus during their odor search. Previously, olfaction and gustation have been the only sensory systems shown to directly mediate orientation and movement towards odor sources in fish. The use of hydrodynamic cues by fish in location of an olfactory source has been previously proposed, but without direct experimental identification of the sensory systems employed. This study identifies the contributing roles of both olfactory and hydrodynamic sensory systems to the olfactory search repertoire of fish.     

Identification of stimuli and input pathways mediating food-attraction conditioning in the snail, Helix

Snails become conditioned by a single feeding episode to locate foods which they were unable to locate prior to feeding. To identify which of the different stimulus parameters of the food mediate learning, snails were presented with isolated stimulus components during feeding and re-tested the next day for their ability to locate the food. None of the individual components was sufficient to promote conditioning. Odor combined with a bulk stimulus conditioned the animals, as indicated by their subsequently locating the food. Elimination of the olfactory sensory inputs from the anterior and/or posterior tentacles prior to conditioning revealed that the acquisition of the olfactory memory requires olfactory stimulation of the sensory epithelia on the anterior tentacles. Recall of memory during olfactory orientation requires functional epithelia on the posterior tentacles, which suggests that the same odor is processed by different input pathways under different situations. Animals with the olfactory epithelia functional on the same side during conditioning and food searching were able to locate the conditioned food. Animals with different epithelia functional during conditioning and food searching failed, which suggests that olfactory memory is stored within one side of the nervous system and cannot be accessed from the contralateral side. Accepted: 28 November 1997     

A possible role for caveolin as a signaling organizer in olfactory sensory membranes

Fast kinetics and sensitivity of olfactory signaling raise the question of whether the participating proteins may be associated in supramolecular transduction complexes. We found evidence that caveolin proteins could play an important role in organizing signaling elements in olfactory sensory neurons. Western blot analysis indicated that caveolins are highly enriched in olfactory sensory membranes, where they co-localize in detergent-insoluble complexes with key components of the signaling pathways. Furthermore, the results of immunoprecipitation experiments suggest that G proteins and effector enzyme form preassembled subcellular complexes with caveolins. Since anti-caveolin antibodies and synthetic peptides derived from the scaffolding domains of caveolin-1 and caveolin-2 effectively attenuated second messenger responses in sensory cilia preparations in a characteristic manner, the data led to the suggestion that caveolins could mediate the assembly of signaling complexes within specialized membrane microdomains of olfactory sensory neurons.     

Thirty years of stimulus-secretion coupling: from Ca(2+) toGTP in the regulation of exocytosis

Calcium, initially considered as the universal link between receptor stimulation and the onset of exocytosis in secretory cells, is now recognised as only one of a number of intracellular activators. In cells of haematopoietic origin (including mast cells), the key activator is one or more GTPases. Cells of this class, stimulated with GTPgammaS can undergo exocytosis in the effective absence of Ca(2+). A number of GTP-binding proteins that mediate exocytosis (G(E)) have been proposed but the best evidence supports roles for members of the Rho family of monomeric GTPases and for betagamma-subunits derived from G(i3). While preactivated Rac and Cdc42 can induce secretion from permeabilised mast cells in the absence of a guanine nucleotide betagamma-subunits only act to enhance the secretion induced by other GTP-binding proteins (likely to be members of the Rho family of monomeric GTPases). Further work is required to identify downstream effectors activated by these GTP-binding proteins and to show how they interact with the SNAP and SNARE isoforms known to be present in these cells.     

The regulation of mitochondrial physiology by organelle-associated GTP-binding proteins

Recent studies have shown that GTP-binding proteins can modulate mitochondrial membrane fusion and fission. Furthermore, GTP-binding proteins can regulate the binding of ribosomes to the mitochondrial membrane and may facilitate the import of proteins through contact points between inner and outer mitochondrial membranes. Mitochondrial GTP-binding proteins therefore appear to have the potential to modulate physiological function of the organelle and may also be involved in cellular processes such as cellular transformation. A beginning has been made on the characterization of mitochondrial GTP-binding proteins and the DNA sequence of one protein has become newly available. Future studies are needed to determine whether GTP-binding proteins are interacting with cell signalling molecules such as protein kinases in the mitochondria.     

Activation of moesin and adducin by Rho-kinase downstream of Rho

The Rho GTPase (Rho) is a member of the Rho family, which belongs to the Ras superfamily of GTP-binding proteins. Like other GTP-binding proteins, Rho exists in two conformational states, an inactive GDP-bound form and an active GTP-bound form. Active Rho interacts with specific effectors to regulate the actin cytoskeleton and to mediate a variety of biological functions in cells. Rho-associated kinase (Rho-kinase) is the most studied Rho-effector, and studies of its biochemical and cell biological functions have provided us with useful information for understanding the molecular mechanisms of the actions of Rho. This review aims to summarize the roles of Rho and Rho-kinase in the regulation of the cytoskeletons.     

A monoclonal antibody against the rod outer segment guanyl nucleotide-binding protein, transducin, blocks the stimulatory and inhibitory G proteins of adenylate cyclase

GTP-binding proteins have been implicated as transducers of a variety of biological signaling processes. These proteins share considerable structural as well as functional homology. Due to these similarities, it was thought that a monoclonal antibody that inhibits the light activation of the rod outer segment GTP-binding protein, tranducin (Gt), might exert some functional effect upon the G proteins that regulate the adenylate cyclase system. Antibody 4A, raised against the alpha subunit of Gt, cross-reacted (by hybridization on nitrocellulose) with purified alpha subunits of other G proteins (Gi and Gs, regulatory guanyl nucleotide-binding proteins that mediate inhibition and stimulation of adenylate cyclase, respectively) as long as they were not denatured. This antibody, which interferes with rod outer segment cGMP phosphodiesterase activation by blocking interaction between rhodopsin and Gt, also interfered with actions of both the stimulatory and inhibitory G proteins of adenylate cyclase from rat cerebral cortex membranes. Effects of monoclonal antibody (mAb) 4A were dose-dependent and not reversed by washing. mAb 4A also blocked the Gi-mediated inhibition of adenylate cyclase in the cyc- variant of S49 lymphoma and in doing so raised the level of adenylate cyclase activity in both the cyc- variant and the S49 wild type. There was no effect of mAb 4A on adenylate cyclase activity of the resolved catalytic subunit. These results suggest that the well known sequence homologies among the G proteins involved in cellular signal transduction may extend to the sites that interact with other members of signal-transducing cascades (receptors and effector molecules). Therefore, antibody 4A may serve as a useful tool to probe the similarities and differences among the various systems.     

Synergistic Interaction of Muscarinic and Opioid Receptors with Gs-Linked Neurotransmitter Receptors to Stimulate Adenylyl Cyclase Activity of Rat Olfactory Bulb

We reported previously that in homogenates of rat olfactory bulb muscarinic and opioid receptor agonists stimulate adenylyl cyclase activity. In the present study we show that carbachol (CCh) and Leu-Enkephalin act synergistically with vasoactive intestinal peptide (VIP) and corticotropin-releasing hormone (CRH), but not with /-isoproterenol, in increasing cyclic AMP formation. The synergistic interaction consists of an increase in the maximal a0denylyl cyclase activation without a significant change in the potency of each agonist. CCh also fails to affect ¹²⁵ICRH binding to olfactory bulb membranes. The synergism requires micromolar concentrations of GTP. Substitution of the stable GTP analog guanosine 5′-O-(3′-thiotriphosphate) for GTP allows the CRH stimulation, but abolishes the CCh enhancement of both basal and CRH-stimulated enzyme activities. Moreover, in vivo treatment of olfactory bulbs with pertussis toxin completely prevents the muscarinic and opioid effects. Thus, the synergistic interaction appears to result from opioid- and muscarinic-induced activation of a pertussis toxin-sensitive GTP-binding protein which may potentiate the adenylyl cyclase stimulation by the stimulatory GTP-binding protein activated by either VIP or CRH receptors.     

Two distinct members of the ADP-ribosylation factor family of GTP-binding proteins regulate cell-free intra-Golgi transport.

We have used an intra-Golgi transport assay to identify GTP-binding proteins involved in regulation of protein traffic. Two soluble proteins of 20 kd were purified by their ability to mediate GTP gamma S-dependent inhibition of transport. These GTP-dependent Golgi binding factors, or GGBFs, exhibit a 3-fold difference in activity and are differentiated by their hydrophobicity, isoelectric points, and apparent size. Removal of 80% of GGBFs from cytosol abolishes GTP gamma S sensitivity but does not affect inhibition by aluminum fluoride. We demonstrate that GGBFs are members of the ADP-ribosylation factor (ARF) family. Recombinant ARF1 exhibits GGBF activity and myristoylation is required. The distinct biochemical properties of GGBFs indicate that members of the ARF family may have related but distinct functions in intracellular transport.     

An SH3 domain-containing GTPase-activating protein for Rho and Cdc42 associates with focal adhesion kinase.

The integrin family of cell surface receptors mediates cell adhesion to components of the extracellular matrix (ECM). Integrin engagement with the ECM initiates signaling cascades that regulate the organization of the actin-cytoskeleton and changes in gene expression. The Rho subfamily of Ras-related low-molecular-weight GTP-binding proteins and several protein tyrosine kinases have been implicated in mediating various aspects of integrin-dependent alterations in cell homeostasis. Focal adhesion kinase (FAK or pp125FAK) is one of the tyrosine kinases predicted to be a critical component of integrin signaling. To elucidate the mechanisms by which FAK participates in integrin-mediated signaling, we have used expression cloning to identify cDNAs that encode potential FAK-binding proteins. We report here the identification of a cDNA that encodes a new member of the GTPase-activating protein (GAP) family of GTPase regulators. This GAP, termed Graf (for GTPase regulator associated with FAK), binds to the C-terminal domain of FAK in an SH3 domain-dependent manner and preferentially stimulates the GTPase activity of the GTP-binding proteins RhoA and Cdc42. Subcellular localization studies using Graf-transfected chicken embryo cells indicates that Graf colocalizes with actin stress fibers, cortical actin structures, and focal adhesions. Graf mRNA is expressed in a variety of avian tissues and is particularly abundant in embryonic brain and liver. Graf represents the first example of a regulator of the Rho family of small GTP-binding proteins that exhibits binding to a protein tyrosine kinase. We suggest that Graf may function to mediate cross talk between the tyrosine kinases such as FAK and the Rho family GTPase that control steps in integrin-initiated signaling events.     

Cyclic nucleotide phosphodiesterase of the olfactory membrane in rats: its distribution and possible connection with the estradiol receptor

In the rat olfactory tissue the existence of soluble and membrane-bound forms of cyclic nucleotide phosphodiesterase (PDE) with quite similar kinetic parameters was demonstrated (KM = 220 and 200 microM, VMaX = 10 and 7 nmoles cAMP/mg protein per minute, respectively). 17 beta-estradiol (10(-7)-10(-5) M) decreased the soluble PDE activity by 25%, whereas non-hydrolysable GTP analogue (Gpp (NHp)) abolished their effect. On the other hand, this analogue in a dose-dependent manner inhibited the specific binding of 3H-estradiol to cytosolic receptors. The data indicate possible functional interrelations between the cytosolic estradiol receptors, GTP-binding proteins and PDE in the olfactory tissue which is a target organ for steroid hormones.     

An expression system for Gustatory receptors—and why it failed

A recent paper by the Dahankuar laboratory suggested that single Drosophila sugar receptors proteins accurately mediate sugar detection when ectopically expressed in olfactory neurons of the antenna. These findings contra-dict numerous previously published electrophysiological and behavioral investigations, which all point towards heteromultimeric sugar taste receptors. Here, I provide some explanation why this “pseudo-heterologous” expression system may have led to this misleading conclusion.     

rab15, a novel low molecular weight GTP-binding protein specifically expressed in rat brain.

rab3A is a low molecular weight (LMW) GTP-binding protein specifically expressed in brain and localized to synaptic vesicles. rab3A has been proposed to play a role in neurotransmitter release by regulating membrane flow in the nerve terminal. In an attempt to define other LMW GTP-binding proteins that may regulate neurotransmitter release, seven cDNA clones encoding new members of the rab family of LMW GTP-binding proteins were isolated from a rat brain cDNA library. The rab proteins contain the four conserved structural domains essential for GTP binding in addition to domains required for membrane localization and effector protein interactions. One protein, rab16, is closely related to members of the rab3 subfamily, whereas two others are assigned as the rat homologs of canine rab8 and rab10. Four additional clones, rab12, rab13, rab14, and rab15, revealed unique sequences and are new members of the rab family of LMW GTP-binding proteins. The patterns of expression of rab15 and rab3A closely overlap but differ from that observed for all other known LMW GTP-binding proteins. This data suggests that rab15 may act in concert with rab3A in regulating aspects of synaptic vesicle membrane flow within the nerve terminal.     

Interaction of GTP-binding regulatory proteins with chemosensory receptors

GTP-binding regulatory proteins (G-proteins) were identified in chemosensory membranes from the channel catfish, Ictalurus punctatus. The common G-protein beta-subunit was identified by immunoblotting in both isolated olfactory cilia and purified taste plasma membranes. A cholera toxin substrate (Mr 45,000), corresponding to the G-protein that stimulates adenylate cyclase, was identified in both membranes. Both membranes also contained a single pertussis toxin substrate. In taste membranes, this component co-migrated with the alpha-subunit of the G-protein that inhibits adenylate cyclase. In olfactory cilia, the Mr 40,000 pertussis toxin substrate cross-reacted with antiserum to the common amino acid sequence of G-protein alpha-subunits, but did not cross-react with antiserum to the alpha-subunit of the G-protein from brain of unknown function. The interaction of G-proteins with chemosensory receptors was determined by monitoring receptor binding affinity in the presence of exogenous guanine nucleotides. L-Alanine and L-arginine bind with similar affinity to separate receptors in both olfactory and gustatory membranes from the catfish. GTP and a nonhydrolyzable analogue decreased the affinity of olfactory L-alanine and L-arginine receptors by about 1 order of magnitude. In contrast, the binding affinities of the corresponding taste receptors were unaffected. These results suggest that olfactory receptors are functionally coupled to G-proteins in a manner similar to some hormone and neurotransmitter receptors.     

Expression patterns of two putative odorant-binding proteins in the olfactory organs of Drosophila melanogaster have different implications for their functions

The aqueous medium bathing the dendrites of olfactory neurons contains high concentrations of odorant-binding proteins (OBPs) whose role is still unclear. OBPs may facilitate interactions between odorants and their membrane-bound receptors, perhaps by increasing the water solubility of hydrophobic molecules. Alternatively, OBPs may be involved in the inactivation of odorants and other volatile molecules, preventing desensitization and/or protecting olfactory neurons from toxic chemicals. We report here novel features of the localization of two putative OBPs, PBPRP2 and PBPRP5, that have important and different implications for their role in olfaction. Unlike several other putative OBPs of Drosophila melanogaster that are only found in adult olfactory organs, PBPRP5 is also expressed in the larval olfactory organs, suggesting that it plays a common role in olfaction at both stages. In the adult, PBPRP5 expression is restricted to the sensillum lymph that bathes the olfactory dendrites of a subset of olfactory hairs, the basiconic sensilla. Since individual basiconic sensilla differ in olfactory specificity, PBPRP5 may be able to bind to and mediate olfactory responses to a wide range of odorants. In contrast, PBPRP2 is present in the space immediately below the antennal cuticle and in the outer cavity of approximately 30% of the double-walled coeloconic sensilla on the antennal surface. In neither case is PBPRP2 in contact with the dendritic membranes of olfactory neurons, making a carrier function unlikely for this protein. Instead, PBPRP2 may act as a sink, binding to odorants and other volatile chemicals and limiting their interactions with olfactory neurons.