Control of yeast mating signal transduction by a mammalian beta 2-adrenergic receptor and Gs alpha subunit

To facilitate functional and mechanistic studies of receptor-G protein interactions, [corrected] the human beta 2-adrenergic receptor (h beta-AR) has been expressed in Saccharomyces cerevisiae. This was achieved by placing a modified h beta-AR gene under control of the galactose-inducible GAL1 promoter. After induction by galactose, functional h beta-AR was expressed at a concentration several hundred times as great as that found in any human tissue. As determined from competitive ligand binding experiments, h beta-AR expressed in yeast displayed characteristic affinities, specificity, and stereoselectivity. Partial activation of the yeast pheromone response pathway by beta-adrenergic receptor agonists was achieved in cells coexpressing h beta-AR and a mammalian G protein (Gs) alpha subunit-demonstrating that these components can couple to each other and to downstream effectors when expressed in yeast. This in vivo reconstitution system provides a new approach for examining ligand binding and G protein coupling to cell surface receptors.     

Night-Shocker: Predatory Behavior of the Pacific Electric Ray (Torpedo californica)

Diving observations off Santa Barbara, California, indicate that Pacific electric rays (Torpedo californica) enter inshore reefs at night and actively prey on fish with the aid of powerful electric discharges.     

小菜蛾烟碱型乙酰胆碱受体α亚基cDNA片段的克隆和序列分析

利用简并引物，采用PCR等分子生物学技术，对小菜蛾烟碱型乙酰胆碱受体（nAChR)α亚基的cDNA片段进行克隆，并对序列进行分析。测序结果显示，扩增的目的基因片段包含了nAChRα亚基的3种亚型（分别命名为Pxαl，Pxα2，Pxα3）。都具有典型的α亚基的结构；2个相邻的半胱氨酸，由2个半胱氨酸之间二硫键形成包含15个氨基酸的胞外环，与烟碱和α银环蛇毒素结合有关的氨基酸残基，克隆的基因片段在氨基酸序列上和其他昆虫nAChRα亚基基因之间有高达65%-99%的同源性，表明克隆的cDNA片段是小菜蛾nAChRα亚基基因的部分序列。     

Chromosomal localization of muscle nicotinic acetylcholine receptor genes in the mouse

The chromosomal localization of the genes encoding the four subunits of muscle nicotinic receptor was determined by analyzing restriction fragment length polymorphisms between two mouse species Mus musculus domesticus (DBA/2) and Mus spretus (SPE). Analysis of the progeny of the interspecies mouse backcross (DBA/2 X SPE) X DBA/2 showed that the alpha-subunit gene cosegregates with the alpha-cardiac actin gene on chromosome 17, that the beta-subunit gene is located on chromosome 11, and that the gamma- and delta-subunit genes cosegregate and are located on chromosome 1.     

Genetic reconstitution of functional acetylcholine receptor channels in mouse fibroblasts

Foreign genes can be stably integrated into the genome of a cell by means of DNA-mediated gene transfer techniques, and large quantities of homogenous cells that continuously express these gene products can then be isolated. Such an expression system can be used to study the functional consequences of introducing specific mutations into genes and to study the expressed protein in the absence of cellular components with which it is normally in contact. All four Torpedo acetylcholine receptor (AChR) subunit complementary DNA's were introduced into the genome of a mouse fibroblast cell by DNA-mediated gene transfer. A clonal cell line that stably produced high concentrations of correctly assembled cell surface AChR's and formed proper ligand-gated ion channels was isolated. With this new expression system, recombinant DNA, biochemical, pharmacological, and electrophysiological techniques were combined to study Torpedo AChR's in a single intact system. The physiological and pharmacological profiles of Torpedo AChR's expressed in mouse fibroblast cells differ in some details from those described earlier, and may provide a more accurate reflection of the properties of this receptor in its natural environment.     

Identification of an alpha subunit of dihydropyridine-sensitive brain calcium channels

Voltage-sensitive calcium channels in different tissues have diverse functional properties. Polyclonal antibodies (PAC-2) against the alpha subunits of purified rabbit skeletal muscle calcium channels immunoprecipitated calcium channels labeled with the dihydropyridine PN200-110 from both skeletal muscle and brain. The immunoreactivity of PAC-2 with the skeletal muscle channel was greater than that with the brain calcium channel and was absorbed only partially by prior treatment with the brain channel. PAC-2 specifically recognized a large peptide in synaptic plasma membranes of rabbit brain with an apparent molecular size of 169,000 daltons. This protein resembles an alpha subunit of the skeletal muscle calcium channel in apparent molecular weight, antigenic properties, and electrophoretic behavior after reduction of disulfide bonds. Thus, the dihydropyridine-sensitive calcium channel of rabbit brain has an alpha subunit that is homologous, but not identical, to those of the skeletal muscle calcium channel. The different functional properties of these two calcium channels may result from minor variations in structurally similar components.     

Identification of a family of muscarinic acetylcholine receptor genes

Complementary DNAs for three different muscarinic acetylcholine receptors were isolated from a rat cerebral cortex library, and the cloned receptors were expressed in mammalian cells. Analysis of human and rat genomic clones indicates that there are at least four functional muscarinic receptor genes and that these genes lack introns in the coding sequence. This gene family provides a new basis for evaluating the diversity of muscarinic mechanisms in the nervous system.     

A role for glycosylation of the alpha subunit in transduction of biological signal in glycoprotein hormones

The biological properties of recombinants of glycoprotein hormones in which the alpha and beta subunits were differentially deglycosylated have been investigated. Specific deglycosylation of the alpha subunit generated a recombinant that had more receptor-binding activity but did not produce hormone response in the target cells. The deglycosylated alpha + beta recombinant was also an antagonist of the action of the native hormone. Thus, the carbohydrates in the alpha subunit play a dominant role in the transduction of the hormone signal into the cell.     

The alpha subunit of the GTP binding protein Gk opens atrial potassium channels

Guanine nucleotide binding (G) proteins (subunit composition alpha beta gamma) dissociate on activation with guanosine triphosphate (GTP) analogs and magnesium to give alpha-guanine nucleotide complexes and free beta gamma subunits. Whether the opening of potassium channels by the recently described Gk in isolated membrane patches from mammalian atrial myocytes was mediated by the alpha k subunit or beta gamma dimer was tested. The alpha k subunit was found to be active, while the beta gamma dimer was inactive in stimulating potassium channel activity. Thus, Gk resembles Gs, the stimulatory regulatory component of adenylyl cyclase, and transducin, the regulatory component of the visual system, in that it regulates its effector function--the activity of the ligand-gated potassium channel--through its guanine nucleotide binding subunit.     

Mapping the main immunogenic region and toxin-binding site of the nicotinic acetylcholine receptor

The alpha-chain of the nicotinic acetylcholine receptor carries the binding sites both for cholinergic ligands and for most experimentally induced or naturally occurring antibodies to the native receptor. By means of expression cloning in Escherichia coli, fusion proteins were derived from specific fragments of a complementary DNA encoding the mouse alpha-chain, allowing the mapping of the toxin-binding site to residues 160-216 and the main immunogenic region to residues 6-85. This approach permits the independent study of different functional domains of a complex receptor molecule and should be generally applicable to other proteins for which complementary DNA clones are available.     

The alpha subunit of the GTP binding protein activates muscarinic potassium channels of the atrium

It has been debated whether the potassium channel of the atrium is activated by the alpha subunit or by the beta gamma subunits of guanine nucleotide binding (G) proteins, which dissociate on activation with guanosine triphosphate (GTP). Therefore, the channel-activating effectiveness of these subunits on isolated guinea pig atrial cells was tested. The activated alpha K subunit from human erythrocytes activated the channel in subpicomolar concentrations. The beta gamma dimer from bovine brain activated the channel in nanomolar concentrations. These results support the view that, physiologically, the alpha subunit activates the channel.     

Amantadine: neuromuscular blockade by suppression of ionic conductance of the acetylcholine receptor

Amantadine hydrochloride decreases the sensitivity of denervated mammalian muscle to iontophoretically applied acetylcholine. The drug depresses the amplitude of the end-plate current and reverses the slope of the relation between half-decay time and membrane potential suggesting that it alters the ionic conductance that is mediated by the acetylcholine receptor. Binding studies confirm that amantadine acts on the ion conductance modulator rather than the acetylcholine receptor.     

人雌激素受体cDNA在酵母细胞中的表达

根据人全长雌激素受体cDNA序列,用RT—PCR方法使乳腺癌细胞MCF-7株扩增人雌激素受体基因(hER cDNA),通过pGEM—T／hER载体克隆到酵母表达载体中,并转化到酿酒酵母细胞中,用Western blot法和免疫组化法检测hER cDNA的表达。结果表明：hER cDNA在酵母细胞中成功表达,可用于建立环境雌激素的酵母细胞检测体系。     

Modulation of acetylcholine receptor desensitization by forskolin is independent of cAMP

Biochemical and electrophysiological studies suggest that adenosine 3',5'-monophosphate (cAMP)-dependent phosphorylation of the nicotinic acetylcholine receptor channel is functionally significant because it modifies the receptor's rate of desensitization to acetylcholine. In studies that support this conclusion researchers have used forskolin to stimulate cAMP-dependent phosphorylation in intact muscle. It is now shown that although forskolin facilitated desensitization in voltage-clamped rat muscle, this effect was not correlated with the abilities of forskolin and forskolin analogs to activate adenylate cyclase or phosphorylate the receptor. Furthermore, elevation of intracellular cAMP or addition of the catalytic subunit of A-kinase failed to alter desensitization. Therefore, in intact skeletal muscle, cAMP-dependent phosphorylation does not modulate desensitization.     

Molecular cloning of complementary DNA for the alpha subunit of the G protein that stimulates adenylate cyclase

A complementary DNA clone encoding the alpha subunit of the adenylate cyclase stimulatory G protein (Gs) was isolated and identified. A bovine brain complementary DNA library was screened with an oligonucleotide probe derived from amino acid sequence common to known G proteins. The only clone that was obtained with this probe has a complementary DNA insert of approximately 1670 base pairs. An antibody to a peptide synthesized according to deduced amino acid sequence reacts specifically with the alpha subunit of Gs. In addition, RNA that hybridizes with probes made from the clone is detected in wild-type S49 cells; however, cyc- S49 cells, which are deficient in Gs alpha activity, are devoid of this messenger RNA.     

Molecular cloning and functional expression of glutamate receptor subunit genes

Three closely related genes, GluR1, GluR2, and GluR3, encode receptor subunits for the excitatory neurotransmitter glutamate. The proteins encoded by the individual genes form homomeric ion channels in Xenopus oocytes that are sensitive to glutamatergic agonists such as kainate and quisqualate but not to N-methyl-D-aspartate, indicating that binding sites for kainate and quisqualate exist on single receptor polypeptides. In addition, kainate-evoked conductances are potentiated in oocytes expressing two or more of the cloned receptor subunits. Electrophysiological responses obtained with certain subunit combinations show agonist profiles and current-voltage relations that are similar to those obtained in vivo. Finally, in situ hybridization histochemistry reveals that these genes are transcribed in shared neuroanatomical loci. Thus, as with gamma-aminobutyric acid, glycine, and nicotinic acetylcholine receptors, native kainate-quisqualate-sensitive glutamate receptors form a family of heteromeric proteins.     

Functional expression of a new pharmacological subtype of brain nicotinic acetylcholine receptor

A new type of agonist-binding subunit of rat neuronal nicotinic acetylcholine receptors (nAChRs) was identified. Rat genomic DNA and complementary DNA encoding this subunit (alpha 2) were cloned and analyzed. Complementary DNA expression studies in Xenopus oocytes revealed that the injection of messenger RNAs (mRNAs) for alpha 2 and beta 2 (a neuronal nAChR subunit) led to the generation of a functional nAChR. In contrast to the other known neuronal nAChRs, the receptor produced by the injection of alpha 2 and beta 2 mRNAs was resistant to the alpha-neurotoxin Bgt3.1. In situ hybridization histochemistry showed that alpha 2 mRNA was expressed in a small number of regions, in contrast to the wide distribution of the other known agonist-binding subunits (alpha 3 and alpha 4) mRNAs. These results demonstrate that the alpha 2 subunit differs from other known agonist-binding alpha-subunits of nAChRs in its distribution in the brain and in its pharmacology.