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R R Reed

Publications and source records attributed to R R Reed.

At least 19 recordsLinked to original sources
PubMed1992

Structurally distinct and stage-specific adenylyl cyclase genes play different roles in Dictyostelium development.

We have isolated two adenylyl cyclase genes, designated ACA and ACG, from Dictyostelium. The proposed structure for ACA resembles that proposed for mammalian adenylyl cyclases: two large hydrophilic domains and two sets of six transmembrane spans. ACG has a novel structure, reminiscent of the membrane-bound guanylyl cyclases. An aca- mutant, created by gene disruption, has little detectable adenylyl cyclase activity and fails to aggregate, demonstrating that cAMP is required for cell-cell communication. cAMP is not required for motility, chemotaxis, growth, and cell division, which are unaffected. Constitutive expression in aca- cells of either ACA or ACG, which is normally expressed only during germination, restores aggregation and the ability to complete the developmental program. ACA expression restores receptor and guanine nucleotide-regulated adenylyl cyclase activity, while activity in cells expressing ACG is insensitive to these regulators. Although they lack ACA, which has a transporter-like structure, the cells expressing ACG secrete cAMP constitutively.

Adenylyl Cyclases↗
PubMed1992

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.

Adenylate Cyclase Toxin↗
PubMed1992

The Drosophila learning and memory gene rutabaga encodes a Ca2+/Calmodulin-responsive adenylyl cyclase.

Four putative adenylyl cyclase genes from Drosophila melanogaster were identified by virtue of their extensive sequence homology with mammalian cyclases. One corresponds to the learning and memory gene rutabaga and is most similar to the mammalian brain Ca2+/calmodulin (CaM)-responsive cyclase. In a mammalian expression system, rutabaga cyclase activity was stimulated approximately 5-fold by the presence of Ca2+/CaM. A point mutation, identified at this locus in rut1 mutant flies, resulted in loss of detectable adenylyl cyclase activity. New P element insertion-induced rutabaga mutations mapped to within 200 nucleotides of the 5' end of the rutabaga cDNA. These data confirm the identity of the rutabaga locus as the structural gene for the Ca2+/CaM-responsive adenylyl cyclase and show that the inactivation of this cyclase leads to a learning and memory defect.

Adenylyl Cyclases↗
PubMed1992

Preferential expression of the Drosophila rutabaga gene in mushroom bodies, neural centers for learning in insects.

Seven lines were isolated with P element insertions in the cytogenetic vicinity of the learning and memory gene, rutabaga, from an enhancer detector screen designed to mark genes preferentially expressed in mushroom bodies. Six of these lines performed poorly in learning and memory tests, and several failed to complement an existing rutabaga allele. Molecular cloning revealed that the P elements were inserted in the putative promoter of the rutabaga gene. RNA in situ hybridization and immunohistochemistry demonstrated that the expression of the rutabaga gene, which encodes a Ca2+/calmodulin-responsive adenylyl cyclase, is markedly elevated in the mushroom bodies of normal flies and that the insertion elements compromised its expression in the new rutabaga mutants. The reisolation of a known learning and memory gene, but with a heretofore unknown expression pattern, strongly supports the postulate that mushroom bodies are principal sites mediating olfactory learning and memory.

Alleles↗
PubMed1992

Signaling pathways in odorant detection.

The application of molecular genetic techniques has led to the identification of olfactory-specific proteins that represent each component in a second messenger cascade. Our current understanding of signaling in the olfactory system suggests that receptor proteins of a large family, responsible in part for the specificity of the system, converge on a relatively small number of second messenger systems. The ability to express these elements in heterologous systems should allow for the reconstitution of the signaling cascade and provide insight into the specificity of ligand binding, pathway activation, and signal termination.

Animals↗
PubMed1992

Evolution of the mammalian G protein alpha subunit multigene family.

Heterotrimeric guanine nucleotide binding proteins (G proteins) transduce extracellular signals received by transmembrane receptors to effector proteins. The multigene family of G protein alpha subunits, which interact with receptors and effectors, exhibit a high level of sequence diversity. In mammals, 15 G alpha subunit genes can be grouped by sequence and functional similarities into four classes. We have determined the murine chromosomal locations of all 15 G alpha subunit genes using an interspecific backcross derived from crosses of C57BL/6J and Mus spretus mice. These data, in combination with mapping studies in humans, have provided insight into the events responsible for generating the genetic diversity found in the mammalian alpha subunit genes and a framework for elucidating the role of the G alpha subunits in disease.

Animals↗
PubMed1992

Mechanisms of sensitivity and specificity in olfaction.

The sensitivity and specificity of the mammalian olfactory system is the result of the contributions at the anatomical, cellular, and biochemical levels. The recent identification of a large gene family encoding putative olfactory receptors suggests that genetic diversity may play an important role in this system. Future challenges lie in understanding the mechanism that regulates expression of each of the receptors and establishes the complex neural processing network that interprets the primary neuronal responses.

Animals↗
PubMed1992

The molecular basis of signal transduction in olfactory sensory neurons.

Contributions from a wide spectrum of experimental systems have resulted in a dramatic increase in our understanding of this old and most enigmatic of the sensory systems. Many of the components of the odorant-induced transduction cascade have now been cloned, and the biochemistry, pharmacology, and regulatory mechanisms are being addressed in a logical fashion. One of the first priorities is to establish that the large family of putative receptor proteins described by Buck and Axel (1991) do, in fact, bind odorants. The ability to express members of this receptor family at high levels in the mammalian expression system is a first step in this direction. Determining specific ligand-receptor relationships is an extremely challenging task given the diversity of odorants able to be perceived and the potentially large size of the family of receptors. The role of other proteins in odorant presentation and processing, such as odorant binding protein produced in the lateral nasal gland (Pevsner et al., 1988), can be explored. A fascinating issue to be resolved is that of the distribution of receptor molecules within the population of olfactory sensory neurons. Does one cell express only one receptor, a small repertoire of receptors, or indeed the entire family? These questions can now be answered using a combined approach with in situ hybridization, immunocytochemistry, and single cell PCR techniques. One model of receptor distribution would provide for discrimination of a particular odorant by higher order analysis of the pattern of receptor neuron firing within the neuroepithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Human rod photoreceptor cGMP-gated channel: amino acid sequence, gene structure, and functional expression.

Phototransduction in retinal rods involves a G-protein-mediated signaling cascade that leads to cGMP hydrolysis and the closure of a cGMP-gated channel. This channel has recently been purified from bovine retina and molecularly cloned (Kaupp et al., 1989). We report here the cloning of cDNA and genomic DNA encoding the human rod cGMP-gated channel, based upon its homology to the bovine counterpart. The human mRNA structure differs from the bovine in containing an Alu repetitive element spliced into the 5' untranslated region. The human cGMP-gated channel gene (CNCG) is located on chromosome 4 and contains at least 10 exons. One large exon encodes the carboxy-terminal two-thirds of the protein, whereas seven small exons encode the amino-terminal one-third of the protein. Alternative splicing removes one of the small exons in a subset of transcripts in the human retina, producing an internal in-frame deletion of 36 codons. When expressed in a human embryonic kidney cell line (293S), the full-length cDNA clone, but not the differentially spliced variant, produced functional ion channels broadly similar to the native channels in vertebrate rods.

Amino Acid Sequence↗
PubMed1991

Chronic lithium regulates the expression of adenylate cyclase and Gi-protein alpha subunit in rat cerebral cortex.

A possible role for adenylate cyclase and guanine nucleotide-binding proteins (G proteins) in contributing to the chronic actions of lithium on brain function was investigated in rat cerebral cortex. It was found that chronic treatment of rats with lithium (with therapeutically relevant serum levels of approximately 1 mM) increased levels of mRNA and protein for the calmodulin-sensitive (type 1) and calmodulin-insensitive (type 2) forms of adenylate cyclase and decreased levels of mRNA and protein for the inhibitory G-protein subunits Gi alpha 1 and Gi alpha 2. Chronic lithium did not alter levels of other G-protein subunits, including Go alpha, Gs alpha, and G beta. Lithium regulation of adenylate cyclase and Gi alpha was not seen in response to short-term lithium treatment (with final serum levels of approximately 1 mM) or in response to chronic treatment at a lower dose of lithium (with serum levels of approximately 0.5 mM). The results suggest that up-regulation of adenylate cyclase and down-regulation of Gi alpha could represent part of the molecular mechanism by which lithium alters brain function and exerts its clinical actions in the treatment of affective disorders.

Adenylyl Cyclases↗
PubMed1991

Molecular cloning and characterization of a Ca2+/calmodulin-insensitive adenylyl cyclase from rat brain.

Biochemical, immunological, and molecular cloning studies have suggested the existence of multiple forms of adenylyl cyclase (EC 4.6.1.1). An adenylyl cyclase cDNA clone (type II) was isolated from a rat brain library and found to encode a protein of 1090 amino acids that was homologous to but distinct from the previously described Ca2+/calmodulin-stimulated adenylyl cyclase from bovine brain. Expression of the type II cDNA in an insect cell line resulted in an increased level of adenylyl cyclase activity that was insensitive to Ca2+/calmodulin. Addition of activated Gs alpha protein to type II-containing membranes increased enzyme activity. The mRNA encoding the type II protein was expressed at high levels in brain tissue and at low levels in olfactory epithelium and lung. The existence of multiple adenylyl cyclase enzymes may provide for complex and distinct modes of biochemical regulation of cAMP levels in the brain.

Adenylyl Cyclases↗
PubMed1991

Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase.

Nitric oxide is a messenger molecule, mediating the effect of endothelium-derived relaxing factor in blood vessels and the cytotoxic actions of macrophages, and playing a part in neuronal communication in the brain. Cloning of a complementary DNA for brain nitric oxide synthase reveals recognition sites for NADPH, FAD, flavin mononucleotide and calmodulin as well as phosphorylation sites, indicating that the synthase is regulated by many different factors. The only known mammalian enzyme with close homology is cytochrome P-450 reductase.

Amino Acid Oxidoreductases↗
PubMed1991

The second messenger cascade in olfactory receptor neurons.

The molecular cloning of components involved in the cAMP second messenger cascade has allowed their biochemical characterization and revealed properties that are important for their role in sensory transduction. Recent evidence suggests inositol 1,4,5-trisphosphate functions as an additional second messenger in olfactory signalling. The interaction of these two pathways may contribute to the sensitivity of the olfactory system.

Animals↗
PubMed1991

Signal transduction in olfactory neurons.

Recent efforts in our laboratory have focused on cloning the molecular components involved in the cAMP-mediated pathway of olfactory signal transduction. These efforts have resulted in the isolation of olfactory-specific forms of a G protein, an adenylyl cyclase, and a cyclic nucleotide-gated cation channel. Functional expression of each of these proteins in vitro confirms their ability to carry out the function ascribed to them as part of a second-messenger cascade. Putative odorant-receptor molecules which constitute the first step in odorant signal transduction have now been cloned. We have generated oligonucleotide probes which recognize a population of olfactory receptors apparently more heterogeneous than those previously reported. These probes should enable us to answer questions regarding the number of different receptors expressed per cell as well as the nature of receptor-ligand specificity.

Adenylyl Cyclases↗
PubMed1990

Identification of a specialized adenylyl cyclase that may mediate odorant detection.

The mammalian olfactory system may transduce odorant information via a G protein-mediated adenosine 3',5'-monophosphate (cAMP) cascade. A newly discovered adenylyl cyclase, termed type III, has been cloned, and its expression was localized to olfactory neurons. The type III protein resides in the sensory neuronal cilia, which project into the nasal lumen and are accessible to airborne odorants. The enzymatic activity of the type III adenylyl cyclase appears to differ from nonsensory cyclases. The large difference seen between basal and stimulated activity for the type III enzyme could allow considerable modulation of the intracellular cAMP concentration. This property may represent one mechanism of achieving sensitivity in odorant perception.

Adenylyl Cyclases↗
PubMed1990

Primary structure and functional expression of a cyclic nucleotide-activated channel from olfactory neurons.

Odorant signal transduction occurs in the specialized cilia of the olfactory sensory neurons. Considerable biochemical evidence now indicates that this process could be mediated by a G protein-coupled cascade using cyclic AMP as an intracellular second messenger. A stimulatory G protein alpha subunit is expressed at high levels in olfactory neurons and is specifically enriched in the cilia, as is a novel form of adenylyl cyclase. This implies that the olfactory transduction cascade might involve unique molecular components. Electrophysiological studies have identified a cyclic nucleotide-activated ion channel in olfactory cilia. These observations provide evidence for a model in which odorants increase intracellular cAMP concentration, which in turn activates this channel and depolarizes the sensory neuron. An analogous cascade regulating a cGMP-gated channel mediates visual transduction in photoreceptor cells. The formal similarities between olfactory and visual transduction suggest that the two systems might use homologous channels. Here we report the molecular cloning, functional expression and characterization of a channel that is likely to mediate olfactory transduction.

Amino Acid Sequence↗
PubMed1990

Biochemical characterization of three stimulatory GTP-binding proteins. The large and small forms of Gs and the olfactory-specific G-protein, Golf.

The biochemical properties of three stimulatory guanine nucleotide-binding protein (G-protein) alpha subunits, the large and small forms of Gs, Gs-l (52 kDa) and Gs-s (45 kDa), and the olfactory specific G-protein, Golf, have been compared. Complementary DNAs (cDNAs) encoding each alpha subunit were independently expressed in a mammalian cell line deficient in endogenous stimulatory G-proteins (S49 cyc-kin-). Gs-l and Gs-s respond similarly to activation by the beta-adrenergic agonist isoproterenol (EC50 = 80 and 60 nM, respectively) and the receptor-independent G-protein activators guanosine 5-O-3-(thio)triphosphate) (GTP gamma S) and AlF-4. The ability of Golf to interact with the beta-adrenergic receptor was also examined. Surprisingly, Golf interacts with beta-adrenergic receptors and is activated by isoproterenol (EC50 = 120 nM). All three G-proteins respond similarly to treatment with different alpha, beta, and gamma thiophosphoryl analogs of GTP. Specifically, (R)-GTP alpha S and GTP gamma S activate each G-protein, whereas (S)-GTP alpha S and (R)- or (S)-GTP beta S are inactive. In addition, similar to Gs alpha, Golf alpha is covalently modified and constitutively activated by cholera toxin. These studies demonstrate that all three stimulatory G-proteins are functionally and structurally similar, however, subtle differences between Golf and the two forms of Gs appear to modulate their interactions with receptors.

Adenylyl Cyclases↗