Splice variants of the alpha subunit of the G protein Gs activate both adenylyl cyclase and calcium channels

Signal transducing guanine nucleotide binding (G) proteins are heterotrimers with different alpha subunits that confer specificity for interactions with receptors and effectors. Eight to ten such G proteins couple a large number of receptors for hormones and neurotransmitters to at least eight different effectors. Although one G protein can interact with several receptors, a given G protein was thought to interact with but one effector. The recent finding that voltage-gated calcium channels are stimulated by purified Gs, which stimulates adenylyl cyclase, challenged this concept. However, purified Gs may have four distinct alpha-subunit polypeptides, produced by alternative splicing of messenger RNA. By using recombinant DNA techniques, three of the splice variants were synthesized in Escherichia coli and each variant was shown to stimulate both adenylyl cyclase and calcium channels. Thus, a single G protein alpha subunit may regulate more than one effector function.     

小鼠可溶性腺苷酸环化酶基因的表达特性

为研究可溶性腺苷酸环化酶（sAC）基因的组织特异性表达及其在不同周龄小鼠睾丸中的表达情况，探讨其在精子发生及获能中的作用，运用RT-PCR法分析小鼠sAC基因的组织表达情况及其发育的时序性变化。结果显示，sAC基因优先表达于睾丸和附睾，表达量随发育周龄的增长而呈增加趋势：于2周龄开始检测到它的表达，随后表达量显著增加至6周龄，再逐渐增长至一个平台期，呈现出发育阶段性，说明sAC在精子形成及获能过程中具有特殊作用。     

Facilitation of signal onset and termination by adenylyl cyclase

The alpha subunit (Gsalpha) of the stimulatory heterotrimeric guanosine triphosphate binding protein (G protein) Gs activates all isoforms of mammalian adenylyl cyclase. Adenylyl cyclase (Type V) and its subdomains, which interact with Gsalpha, promoted inactivation of the G protein by increasing its guanosine triphosphatase (GTPase) activity. Adenylyl cyclase and its subdomains also augmented the receptor-mediated activation of heterotrimeric Gs and thereby facilitated the rapid onset of signaling. These findings demonstrate that adenylyl cyclase functions as a GTPase activating protein (GAP) for the monomeric Gsalpha and enhances the GTP/GDP exchange factor (GEF) activity of receptors.     

The structure of a pH-sensing mycobacterial adenylyl cyclase holoenzyme

Class III adenylyl cyclases contain catalytic and regulatory domains, yet structural insight into their interactions is missing. We show that the mycobacterial adenylyl cyclase Rv1264 is rendered a pH sensor by its N-terminal domain. In the structure of the inhibited state, catalytic and regulatory domains share a large interface involving catalytic residues. In the structure of the active state, the two catalytic domains rotate by 55 degrees to form two catalytic sites at their interface. Two alpha helices serve as molecular switches. Mutagenesis is consistent with a regulatory role of the structural transition, and we suggest that the transition is regulated by pH.     

An M2 muscarinic receptor subtype coupled to both adenylyl cyclase and phosphoinositide turnover

To investigate whether a particular receptor subtype can be coupled to multiple effector systems, recombinant M2 muscarinic receptors were expressed in cells lacking endogenous receptor. The muscarinic agonist carbachol both inhibited adenylyl cyclase and stimulated phosphoinositide hydrolysis. The stimulation of phosphoinositide hydrolysis was significantly less efficient and more dependent on receptor levels than the inhibition of adenylyl cyclase. Both responses were mediated by guanine nucleotide binding proteins, as evidenced by their inhibition by pertussis toxin; the more efficiently coupled adenylyl cyclase response was significantly more sensitive. Thus, individual subtypes of a given receptor are capable of regulating multiple effector pathways.     

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.     

Type-specific regulation of adenylyl cyclase by G protein beta gamma subunits

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) dissociate into guanosine triphosphate (GTP)-bound alpha subunits and a complex of beta and gamma subunits after interaction with receptors. The GTP-alpha subunit complex activates appropriate effectors, such as adenylyl cyclase, retinal phosphodiesterase, phospholipase C, and ion channels. G protein beta gamma subunits have been found to have regulatory effects on certain types of adenylyl cyclase. In the presence of Gs alpha, the alpha subunit of the G protein that activates adenylyl cyclase, one form of adenylyl cyclase was inhibited by beta gamma, some forms were activated by beta gamma, and some forms were not affected by beta gamma. These interactions suggest mechanisms for communication between distinct signal-transducing pathways.     

Mutations of the adenylyl cyclase gene that block RAS function in Saccharomyces cerevisiae

The interaction between RAS proteins and adenylyl cyclase was studied by using dominant interfering mutations of adenylyl cyclase from the yeast Saccharomyces cerevisiae. RAS proteins activate adenylyl cyclase in this organism. A plasmid expressing a catalytically inactive adenylyl cyclase was found to interfere dominantly with this activation. The interfering region mapped to the leucine-rich repeat region of adenylyl cyclase, which is homologous to domains present in several other proteins and is thought to participate in protein-protein interactions.     

The coupling of neurotransmitter receptors to ion channels in the brain

Recent studies on the action of neurotransmitters on hippocampal pyramidal cells indicate that different neurotransmitter receptors that use either the same or different coupling mechanisms converge onto the same ion channel. Conversely, virtually all of the neurotransmitters act on at least two distinct receptor subtypes coupled to different ion channels on the same cell. The existence of both convergence and divergence in the action of neurotransmitters results in a remarkable diversity in neuronal signaling.     

Functional coupling of gamma-aminobutyric acid receptors to chloride channels in brain membranes

gamma-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in mammalian brain, is believed to act by increasing membrane conductance of chloride ions. In this study it was found that GABA agonists increased the uptake of chloride-36 by cell-free membrane preparations from mouse brain. This influx was rapid (less than 5 seconds), and 13 micromolar GABA produced a half-maximal effect. The GABA antagonists (bicuculline and picrotoxin) blocked the effect of GABA, whereas pentobarbital enhanced the action. This may be the first demonstration of functional coupling among GABA and barbiturate receptors and chloride channels in isolated membranes. The technique should facilitate biochemical and pharmacological studies of GABA receptor-effector coupling.     

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.     

Chimeric alpha 2-,beta 2-adrenergic receptors: delineation of domains involved in effector coupling and ligand binding specificity

The alpha 2 and beta 2 adrenergic receptors, both of which are activated by epinephrine, but which can be differentiated by selective drugs, have opposite effects (inhibitory and stimulatory) on the adenylyl cyclase system. The two receptors are homologous with each other, rhodopsin, and other receptors coupled to guanine nucleotide regulatory proteins and they contain seven hydrophobic domains, which may represent transmembrane spanning segments. The function of specific structural domains of these receptors was determined after construction and expression of a series of chimeric alpha 2-,beta 2-adrenergic receptor genes. The specificity for coupling to the stimulatory guanine nucleotide regulatory protein lies within a region extending from the amino terminus of the fifth hydrophobic domain to the carboxyl terminus of the sixth. Major determinants of alpha 2- and beta 2-adrenergic receptor agonist and antagonist ligand binding specificity are contained within the seventh membrane spanning domain. Chimeric receptors should prove useful for elucidating the structural basis of receptor function.     

Autocrine stimulation of intracellular PDGF receptors in v-sis-transformed cells

Autocrine activation of platelet-derived growth factor (PDGF) receptors is the mechanism of transformation by the v-sis oncogene. Since the addition of PDGF does not transform normal cells, autocrine mechanisms may involve unique pathways of receptor activation. In this study autocrine stimulation of the PDGF receptor was observed in v-sis-transformed normal rat kidney (NRK) cells. In contrast to receptor activation in normal cells, autocrine activation of PDGF receptors in v-sis-transformed cells occurred in intracellular compartments, disrupting receptor processing and diverting receptors and their precursors to a chloroquine-sensitive degradation pathway. These findings show that intracellular activation of receptors by autocrine mechanisms may play a role in cell transformation.     

鸡大肠杆菌FimH基因C端结构域克隆及特性分析

[目的]通过进一步研究禽大肠杆菌I型菌毛主要结构基因FimH的基因结构及其抗原特性,为深入探索鸡大肠杆菌致病机理及研制基因工程疫苗奠定必要的理论基础。[方法]本文以已发表的Fim H基因C端核酸序列为参考序列,设计并合成引物,扩增鸡大肠杆菌JS1、JS2、JS6三个菌株的Fim H相应基因,使用Lasergene软件对其进行序列比对、蛋白质结构预测以及进化树的绘制等。[结果]JS1、JS2、JS6三个菌株的核酸同源性分别为97.3%、97.7%和97.3%,而氨基酸序列的相似性分别为95.3%、97.6%和95.3%。此外,其抗原决定簇基本相似,但在第78和79位抗原决定簇不同,这说明本研究中的3个鸡大肠杆菌菌株的Fim H基因序列及氨基酸序列的变异可能对Fim H蛋白抗原结构产生了轻微影响。进化树分析发现,本实验室分离的3株鸡大肠杆菌菌株FimH基因与选择的国内鸡大肠杆菌菌株的Fim H基因同源性比较高,同处于一个进化分支中。[结论]本研究结果表明鸡大肠杆菌I型菌毛Fim H基因未发生明显变异,这为后续研究Fim H基因的功能及其基因工程亚单位疫苗研发提供了重要的实验基础。     

Modulation of the affinity of integrin alpha IIb beta 3 (GPIIb-IIIa) by the cytoplasmic domain of alpha IIb

Intracellular signaling alters integrin adhesive functions in inflammation, immune responses, hemostasis, thrombosis, and retinal development. By truncating the cytoplasmic domain of alpha IIb, the affinity of integrin alpha IIb beta 3 for ligand was increased. Reconstitution with the cytoplasmic domain from integrin alpha 5 did not reverse the increased affinity. Thus, the cytoplasmic domain of the alpha subunit of GPIIb-IIIa controls ligand binding affinity, which suggests mechanisms for inside-out transmembrane signaling through integrins. These findings imply the existence of hitherto unappreciated hereditary and acquired thrombotic disorders in humans.     

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.     

beta-Adrenergic regulation of alpha 2-adrenergic receptors in the central nervous system

Incubation of rat cerebral cortical slices with the beta-adrenergic agonist isoproterenol causes an increase in alpha 2-adrenergic receptor binding in addition to a decrease in beta-adrenergic receptor binding. The effects are rapid and reversible, show a parallel time course, and are blocked by sotalol, a beta-adrenergic receptor antagonist. The beta-mediated regulation of alpha 2-receptor sensitivity at brain norepinephrine synapses may be a mechanism for the homeostatic control of central noradrenergic activity.     

新城疫病毒V蛋白C端酵母双杂交诱饵质粒的构建与鉴定

为构建新城疫病毒(NDV)V蛋白C端酵母双杂交诱饵质粒,用RNA试剂盒提取NDV F48E9株的RNA,通过反转录试剂盒将抽提的RNA反转录,以反转录合成的eDNA为模板,通过PCR扩增,获得V蛋白C端基因(VCTD).通过酶切连接的方法将V蛋白C端基因克隆至pGBKT7载体上,命名为pGBKT7-VCTD.将pGBKT7-VCTD转化酵母Y2H Gold酵母感受态细胞,转化产物涂布营养缺陷型平板,观察平板上菌落生长情况,判定诱饵质粒有无自激活能力:同时挑取平板上的生长的菌落接种液体培养基培养,绘制生长曲线,判定pGBKT7-VCTD诱饵质粒有无毒性.酶切和测序结果表明,成功构建pGBKT7-VCTD诱饵质粒,且该诱饵质粒在酵母细胞中无毒性和自激活能力.本研究成功构建了pGBKT7-VCTD诱饵质粒,为筛选NDVV蛋白C端纳米抗体奠定基础.