A lobster phospholipase C-beta that associates with G-proteins in response to odorants.

A cDNA clone encoding a protein of 1116 amino acids with significant homology to beta-isoforms of phospholipase C was isolated from lobster olfactory organ cDNA libraries and named lobPLCbeta. This cDNA hybridized predominantly to a 9 kb transcript in RNA from olfactory organ, pereiopod, brain, and eye-eyestalk and to several smaller minor transcripts only in eye-eyestalk. An antiserum raised to the C terminus of lobPLCbeta detected immunoreactivity in a single 130 kDa band in olfactory aesthetasc hairs, olfactory organ, pereiopod, dactyl, and brain. In eye-eyestalk this 130 kDa band was abundant, and minor bands of 100, 79, and 57 kDa also were detected. In cross sections of the aesthetasc hairs, immunoreactivity was detected in the outer dendritic segments of the olfactory receptor neurons, the site of olfactory transduction. A complex odorant caused lobPLCbeta immunoreactivity to increase in membrane fractions and decrease in soluble fractions of homogenates of aesthetasc hairs. The odorant also increased the amount of lobPLCbeta in immunoprecipitates of Galphaq and Gbeta from homogenates of aesthetasc hairs. These results support the conclusion that lobPLCbeta mediates olfactory transduction.

Ionic currents and ion channels of lobster olfactory receptor neurons.

The role of the soma of spiny lobster olfactory receptor cells in generating odor-evoked electrical signals was investigated by studying the ion channels and macroscopic currents of the soma. Four ionic currents; a tetrodotoxin-sensitive Na+ current, a Ca++ current, a Ca(++)-activated K+ current, and a delayed rectifier K+ current, were isolated by application of specific blocking agents. The Na+ and Ca++ currents began to activate at -40 to -30 mV, while the K+ currents began to activate at -30 to -20 mV. The size of the Na+ current was related to the presence of a remnant of a neurite, presumably an axon, and not to the size of the soma. No voltage-dependent inward currents were observed at potentials below those activating the Na+ current, suggesting that receptor potentials spread passively through the soma to generate action potentials in the axon of this cell. Steady-state inactivation of the Na+ current was half-maximal at -40 mV. Recovery from inactivation was a single exponential function that was half-maximal at 1.7 ms at room temperature. The K+ currents were much larger than the inward currents and probably underlie the outward rectification observed in this cell. The delayed rectifier K+ current was reduced by GTP-gamma-S and AIF-4, agents which activate GTP-binding proteins. The channels described were a 215-pS Ca(++)-activated K+ channel, a 9.7-pS delayed rectifier K+ channel, and a 35-pS voltage-independent Cl- channel. The Cl- channel provides a constant leak conductance that may be important in stabilizing the membrane potential of the cell.

Distribution of G-protein alpha subunits and neurotransmitter activation of g(alphai) and g(alphaq) in the brain of the lobster Homarus americanus.

Immunocytochemistry using antisera specific for the G-protein alpha subunits G(alphai), G(alphaq), and G(alphas) revealed similar patterns of immunoreactivity in the lobster brain. Immunoreactivity was strongest in neuropil, especially the olfactory and accessory lobes, and was characterized by bundles of fine threads leading to dense concentrations of punctate staining in the glomeruli. This may reflect the concentration of G-protein alpha subunits at synapses. The major differences between the antisera were distinct patterns of staining intensity in subregions of glomeruli of the olfactory and accessory lobes. This result is potentially correlated with previous evidence that these subregions are neurochemically distinct. Neuronal cell bodies contained moderate levels of immunoreactivity at the plasma membrane and faint staining in the cytoplasm. The olfactory globular tract was moderately immunoreactive, but other fiber tracts were weakly immunoreactive. Immunoreactivity in the deutocerebral commissure consisted of small oval cell bodies and strands that formed a reticulated pattern, suggestive of glia. Photoaffinity labelling by using an analog of GTP demonstrated that histamine activated G(alphai) in brain homogenates. Further evidence of G-protein activation was obtained by showing that stimulation with a mixture of neuroactive substances increased the amount of phospholipase C-beta associated with membranes, G(alphaq), and G(beta). The lobster brain, especially in its neuropil regions, is richly endowed with neuromodulatory biochemical pathways involving G(alphai), G(alphaq), and G(alphas).

Lobster G-protein coupled receptor kinase that associates with membranes and G(beta) in response to odorants and neurotransmitters.

A cDNA clone (lobGRK2) encoding a protein of 690 amino acids with significant similarity to the GRK2 subfamily of G-protein coupled receptor kinases was isolated. lobGRK2 was widely expressed as a 9-kb major transcript and a protein of 80 kDa. It was most abundant in the brain and the olfactory organ but was absent in the eye/eyestalk. Immunocytochemistry revealed lobGRK2 immunoreactivity in the outer dendritic segments of the olfactory receptor neurons, the site of olfactory transduction. LobGRK2 immunoreactivity was observed in most neuronal structures in the brain, although with varying intensity. It was strongest in neuropil, especially the olfactory and accessory lobes but was also detectable in neuronal cell bodies. Stimulation of brain homogenates with a mixture of neurotransmitters increased the association of lobGRK2 with membranes and with G(beta). Similarly, stimulation of olfactory dendrite homogenates with an odorant mixture caused lobGRK2 to associate with G(beta). These results support the conclusion that lobGRK2 responds to odorants and to neurotransmitters and may act to initiate desensitization by phosphorylating G-protein-coupled receptors in the olfactory organ and the brain, respectively.

Signal transmission in lobster olfactory receptor cells: functional significance of electrotonic structure analysed by a compartmental model.

The electrotonic structure of lobster olfactory receptor cells was evaluated using general purpose simulation software in a compartmental model derived from electron-microscopic reconstruction. The model with non-uniform membrane resistance (Rm) was used to (i) simulate current spread and (ii) determine if the electronic structure of the cell improves signal recognition in the soma. The odor-evoked conductance change in dendrites was calculated according to the Michaelis-Menten equation with the assumption that the outer dendritic segments function as independent stimulus detectors. The inflection point of the concentration-response function measured in the soma was shifted to lower concentrations relative to that measured in the ciliary (outer dendritic) arbor. The shift, which was greater for inputs with lower efficacy (represented in the model by smaller Hill coefficients) and for the dynamic phase of the response than for the steady-state phase, effectively increased the selectivity of the somatic response. Randomized input distributed uniformly to progressively more restricted areas of the ciliary arbor showed that stimulation of larger areas (presumably the entire ciliary arbor) decreased the statistical variability of the somatic response.

Molecular cloning of a G-protein alpha i subunit from the lobster olfactory organ.

A G-protein alpha subunit was cloned from a lobster olfactory organ cDNA library and sequenced. The clone encodes an alpha i subunit based on the 80% identity its predicted amino acid sequence shares with mammalian alpha i subunits. On Northern blots of polyadenylated RNA, the clone hybridized to a 5 kb species from several tissues.

Functional expression of olfactory-adrenergic receptor chimeras and intracellular retention of heterologously expressed olfactory receptors.

Replacing the G-protein-coupling domains of the beta 2-adrenergic receptor with homologous domains of putative olfactory receptors produced chimeric receptors which were able to stimulate pigment dispersion in Xenopus melanophores, a G-protein-mediated pathway. A multiple replacement chimera containing the second, third and C-terminal cytoplasmic domains of receptor OR5 elevated cyclic adenosine 3':5'-monophosphate (cAMP) and suppressed production of inositol phosphates. Co-expression of G alpha olf did not alter the strength of response of this chimera. A novel rat olfactory receptor cDNA (U131) was isolated and sequenced. Expression of U131 and OR5 constructs containing an N-terminal epitope-tag or C-terminal fusion to green fluorescent protein occurred in an intracellular network but not in the plasma membrane of heterologous cells. Similarly treated beta 2-adrenergic receptors were functional and were observed in the plasma membrane and the intracellular network. These results demonstrate that the putative cytoplasmic domains of olfactory receptors are capable of functional interaction with heterologous G-proteins of the G alpha s subtype. Instead, the absence of these receptors from the plasma membrane of heterologous cells appears to explain our inability to determine if odorants can activate the olfactory receptor clones. We hypothesize that the olfactory receptors have requirements for maturation and targeting to the plasma membrane that are different from most other G-protein-coupled receptors.

Functional analysis by imaging of melanophore pigment dispersion of chimeric receptors constructed by recombinant polymerase chain reaction.

Analysis of functional aspects of the molecular structure of proteins often requires a means to selectively alter structure and subsequently analyze function. We have adapted a method of overlap extension polymerase chain reaction (PCR) to generate multiple domain replacements in G-protein coupled receptors. The examples described herein are beta 2-adrenergic receptors whose G-protein coupling domains have been replaced by homologous domains of olfactory receptors, but the procedure has also been used to produce constructs with mutations, deletions, and fusions of two complete open reading frames. The chimeric olfactory-adrenergic receptors were assayed by functional expression in clonal lines of Xenopus melanophores. The ability of G-protein coupled second messenger pathways to cause translocation of pigment organelles within melanophores allows the use of video microscopy to assay the function of the chimeric receptors. Digital automation of microscope stage, camera, and image processing allows multiple parallel experiments to be performed. Melanophores allow responses mediated by the Gs, Gq and Gi pathways to be assayed with equal efficiency and the specificity of the coupling between chimera (or receptor) and G-protein subtypes can be rapidly determined.

Consensus translational initiation sites of marine invertebrate phyla.

The efficiency of translational initiation depends upon the sequence context surrounding the AUG codon (1, 2, 3). A purine at position -3 contributes critically to context, but other neighboring nucleotides are also important. Nucleotide frequencies at these neighboring positions vary among distant taxa (4, 5). We have analyzed the translational initiation sites of cnidarian, echinoderm, molluscan, annelid, and crustacean sequences in nucleotide sequence databases. These taxa conform to the pattern of a strong preference for a purine at -3, but the frequencies of nucleotides at neighboring positions are characteristic for each taxon. The consensus translational initiation sequences of the marine invertebrate taxa are also different from those of vertebrates and single-celled eukaryotes. These consensus sequences are useful guides for predicting translational initiation sites in cDNA clones.

Histamine directly gates a chloride channel in lobster olfactory receptor neurons.

Biogenic amines mediate many types of intercellular communication in multicellular organisms. Heretofore, little direct evidence has indicated that biogenic amines produce intracellular responses other than by triggering the enzymatic production of second messengers. Our electrophysiological studies of lobster olfactory receptor neurons now reveal that one biogenic amine, histamine, can directly gate an ion channel. The channel responds to histamine concentrations of 1 microM or more, is permeable primarily to Cl-, is more active at depolarized potentials, and has a conductance of 44 pS in the American lobster and 66 pS in the Caribbean spiny lobster. The expression of this ligand-gated channel in olfactory receptor neurons implies that these neurons are targets of a regulatory or feedback process.

Lobster GABA receptor subunit expressed in neural tissues.

A cDNA encoding an ionotropic gamma-aminobutyric acid (GABA) receptor subunit was isolated from a lobster (Homarus americanus) cDNA library. A longer version of this cDNA, containing a 108-bp insert, was also detected. The two cDNAs are predicted to encode alternatively spliced proteins of 485 and 521 amino acids, respectively. The sequences were most similar to the Drosophila RDL (resistance to dieldrin) GABA subunit with 54% identity, and 30-35% identity with vertebrate ionotropic GABA receptor subunits. Only the shorter clone formed functional ion channels when transfected into human embryonic kidney (HEK) 293 cells. GABA caused a Cl(-)-selective current in the presence of GABA that was blocked by picrotoxin. The GABA-induced current was weakly sensitive to the GABA(A) antagonist, bicuculline, but was enhanced by pentobarbital. Expression of the GABA receptor mRNA was highest in brain and the olfactory organ, but was not detected in leg muscle. These data suggest that the isolated cDNAs are likely to encode proteins that comprise subunits of native GABA receptors expressed in olfactory receptor neurons and projection neurons of the olfactory deutocerebrum.

Nonselective cation channel activated by patch excision from lobster olfactory receptor neurons.

A nonselective cation channel activated by patch excision was characterized in inside-out patches from spiny lobster olfactory receptor neurons. The channel, which was permeable to Na+, K+ and Cs+, had a conductance of 320 pS and was weakly voltage dependent in the presence of micromolar divalent cations. Millimolar internal divalent cations caused a voltage- and concentration-dependent block of Na+ permeation. Analysis of the voltage dependence indicated that the proportion of the membrane's electric field sensed by Mg2+ was greater than 1, suggesting that the channel contains a multi-ion pore. Internal divalent cations also reduced the frequency of channel opening in a concentration-dependent, but not voltage-dependent, manner, indicating that different cation binding sites affect gating and conductance. While block of gating prevented determining if internal divalent cations permeate the channel, a channel highly permeable to external divalent cations was observed upon patch excision to the inside-out configuration. The monovalent and divalent cation conductances shared activation by patch excision, weak voltage dependence, and steady-state activity, suggesting that they are the same channel. These data extend our understanding of this type of channel by demonstrating permeation by monovalent cations, detailing Mg2+ block of Na+ permeation, and demonstrating the channel's presence in arthropods.

Olfactory receptor trafficking involves conserved regulatory steps.

Olfactory receptors are difficult to functionally express in heterologous cells. They are typically retained in the endoplasmic reticulum of cells commonly used for functional expression studies and are only released to the plasma membrane in mature cells of the olfactory receptor neuron lineage. A recently developed olfactory cell line, odora, traffics olfactory receptors to the plasma membrane when differentiated. We found that undifferentiated odora cells do not traffic olfactory receptors to their surface, even though they release the receptors to the Golgi apparatus and endosomes. This behavior differs from other cell lines tested thus far. Differentiated odora cells also properly traffic vomeronasal receptors of the VN1 type, which lack sequence similarity to olfactory receptors. ODR-4, a protein that is necessary for plasma membrane trafficking of a chemosensory receptor in nematodes, facilitates trafficking of rat olfactory receptor U131 in odora and Chinese hamster ovary cells. Olfactory receptor trafficking from the endoplasmic reticulum to the plasma membrane involves at least two steps whose regulation depends on the maturation state of cells in the olfactory receptor neuron lineage. These results also indicate that some components of the regulatory mechanism are conserved.

Inhibition of lobster olfactory receptor cells by an odor-activated potassium conductance.

1. Whole cell current-clamp recordings show that odors not only depolarize but may also hyperpolarize lobster olfactory receptor cells. Odor-evoked hyperpolarizations occurred in 36% of 178 receptor cells examined. Cell-attached recordings of action potentials followed by current-clamp recordings in the same cell indicate that depolarizing and hyperpolarizing responses were associated with increases (excitation) and decreases (inhibition) in action potential frequency, respectively. Since odorants that hyperpolarized one receptor cell depolarized other cells and since individual cells may be both excited and inhibited, the inhibitory and excitatory nature of the response must be conferred by the odorant-receptor and transduction processes expressed by the receptor cell. 2. The input resistance dropped from 1.73 G omega at rest to 1.45 G omega during odor-evoked hyperpolarization, and the membrane time constant correspondingly decreased from 114 to 61 ms. The increased conductance persisted throughout the stimulation period (5 s). 3. Shifting the K+ reversal to a more negative potential by lowering the [K+]o from 14 to 2.8 mM increased the magnitude of hyperpolarization. The hyperpolarization could be reversibly blocked by dendritic treatment with 5-10 mM 4-aminopyridine (4-AP) or 10 mM cesium ion, but not by 10 mM tetraethylammonium (TEA). 4. Substituting 80% of the [Cl-]o with NO3- increased the amplitude of the hyperpolarization. Based on a calculated equilibrium potential of -32 mV for chloride, an increase in chloride conductance in a low [Cl-]o environment should have decreased the magnitude of the response. Presumably the change in [Cl-]o acts through the dendritic steady-state chloride conductance to shift the membrane potential further from the reversal potential for K+.(ABSTRACT TRUNCATED AT 250 WORDS)

Melanophore pigment dispersion responses to agonists show two patterns of sensitivity to inhibitors of cAMP-dependent protein kinase and protein kinase C.

Melanophore pigment dispersion is a sensitive bioassay for activation of the adenylyl cyclase and phospholipase C second-messenger pathways. The necessity of protein kinase activation in causing pigment dispersion was confirmed for eight agonists of endogenous melanophore receptors and for two transfected receptors. All agonists and receptors previously shown to elevate intracellular cAMP in melanophores--melanocyte stimulating hormone, light, (-) norepinephrine, 5-hydroxytrptamine, and the beta2-adrenergic receptor--were able to stimulate pigment dispersion in the presence of Ro31-8220, a potent inhibitor of protein kinase C, but were blocked in the presence of H89, an inhibitor of cAMP-dependent protein kinase. The bombesin receptor, which elevates intracellular IP3 in melanophores, was unable to stimulate pigment dispersion in the presence of Ro31-8220 or H89. Agonists whose mechanism of activation of pigment dispersion are unknown were also tested. Endothelin 3 responses were blocked by both H89 and Ro31-8220, predicting coupling to phospholipase C. Vasoactive intestinal polypeptide, oxytocin, and calcitonin gene-related peptide beta responses were blocked only by H89, predicting coupling to adenylyl cyclase.

Molecular cloning of a lobster G alpha(q) protein expressed in neurons of olfactory organ and brain.

We have isolated from an American lobster (Homarus americanus) olfactory organ cDNA library a clone, hG alpha(q), with >80% identity to mammalian and arthropod G alpha(q) sequences. In brain and olfactory organ, hG alpha(q) mRNA was expressed predominantly in neurons, including virtually all the neuronal cell body clusters of the brain. G alpha(q) protein was also expressed broadly, appearing on western blots as a single band of 46 kDa in brain, eyestalk, pereiopod, dactyl, tail muscle, olfactory organ, and aesthetasc hairs. These results suggest that hG alpha(q) plays a role in a wide variety of signal transduction events. Its presence in the olfactory aesthetasc hairs, which are almost pure preparations of the outer dendrites of the olfactory receptor neurons, the expression of a single hG alpha(q) mRNA species (6 kb) in the olfactory organ, and the localization of hG alpha(q) mRNA predominantly in the olfactory receptor neurons of the olfactory organ strongly suggest that one function of hG alpha(q) is to mediate olfactory transduction.

Molecular cloning and characterization of a lobster G alphaS protein expressed in neurons of olfactory organ and brain.

We have isolated from an American lobster (Homarus americanus) olfactory organ cDNA library a clone, lobG alphaS, with >70% identity to mammalian and arthropod G alphaS sequences. In genomic Southern blots, a fragment of lobG alphaS detected only one band, suggesting the lobsters have a single G alphaS gene. In brain and olfactory organ, lobG alphaS mRNA was expressed predominantly in neurons, including many of the neuronal cell body clusters of the brain. G alphaS protein was also expressed broadly, appearing on western blots as a band of 51.8 kDa in brain, eyestalk, pereiopod, dactyl, tail muscle, olfactory organ, and aesthetasc hairs. These results suggest that lobG alphaS plays a role in a wide variety of signal transduction events. Its presence in the olfactory aesthetasc hairs, which are almost pure preparations of the outer dendrites of the olfactory receptor neurons, and the expression of lobG alphaS mRNA in the olfactory receptor neurons of the olfactory organ indicate that lobG alphaS may mediate olfactory transduction. That virtually all ORNs express lobG alphaS mRNA equally predicts that hyperpolarizing odor responses mediated by cyclic AMP are a property of all lobster olfactory receptor neurons.

Truncation releases olfactory receptors from the endoplasmic reticulum of heterologous cells.

Olfactory receptors are difficult to express functionally in heterologous cells. We found that olfactory receptors traffic poorly to the plasma membrane even in cells with neuronal phenotypes, including cell lines derived from the olfactory epithelium. Other than mature olfactory receptor neurons, few cells appear able to traffic olfactory receptors to the plasma membrane. In human embryonic kidney 293 cells and Xenopus fibroblasts, olfactory receptor immunoreactivity overlapped with a marker for the endoplasmic reticulum (ER) but not with markers for the Golgi apparatus or endosomes. Except for the ER, olfactory receptors were therefore absent from organelles normally involved in the plasma membrane trafficking of receptors. Olfactory receptors truncated prior to transmembrane domain VI were expressed in the plasma membrane, however. Co-expression of the missing C-terminal fragment with these truncated receptors prevented their expression in the plasma membrane. Intramolecular interactions between N- and C-terminal domains joined by the third cytoplasmic loop appear to be responsible for retention of olfactory receptors in the ER of heterologous cells. Our results are consistent with misfolding of the receptors but could also be explained by altered trafficking of the receptors.

Molecular cloning of a lobster Gbeta subunit and Gbeta expression in olfactory receptor neuron dendrites and brain neuropil.

We have isolated from the olfactory organ of the American lobster (Homarus americanus) two cDNA clones with homology to beta subunits of G proteins. LobGbeta1 contained a complete open reading frame that predicted an amino acid sequence with >80% identity to Gbeta sequences from other species. LobGbeta2 was a fragment of an open reading frame whose predicted amino acid sequence had 65-69% identity to other Gbeta sequences. LobGbeta2 mRNA was not detectable in the brain, eye plus eyestalk, leg, dactyl, olfactory organ, or tail muscle. In contrast, lobGbeta1 was expressed in all these tissues as a single mRNA species of 6.4 kb and a protein of 37 kD. In the brain and olfactory organ, Gbeta immunoreactivity was almost exclusively confined to neurites: the neuropil regions of the brain and the outer dendrites of the olfactory receptor neurons. Coimmunoprecipitation revealed that lobster Gbeta interacted with both Galpha s and Galpha q. LobGbeta1 is likely to be involved in a wide range of signaling events including olfactory transduction and synaptic transmission in the brain.