Search PubMed⌕ Search

Biomedical subjects

R Iyengar

Publications and source records attributed to R Iyengar.

At least 55 records · Page 3Linked to original sources
PubMed1994

Suppression of Ras-induced transformation of NIH 3T3 cells by activated G alpha s.

Conversion of external signals into proliferative responses may be mediated by interactions between signaling pathways that control cell proliferation. Interactions between G alpha s, the alpha subunit of the heterotrimeric guanine nucleotide binding protein that stimulates adenylyl cyclase, and Ras, an important element in growth factor signaling, were studied. Expression of activated G alpha s in NIH 3T3 cells increased intracellular concentrations of adenosine 3',5'-monophosphate (cAMP) and inhibited H-Ras-stimulated DNA synthesis and mitogen-activated protein kinase activity. Activated G alpha s and 8-Br-cAMP suppressed H-Ras-induced transformation of NIH 3T3 cells. Apparently, G alpha s inhibits proliferative signals from Ras by stimulating cAMP production and activating protein kinase A.

3T3 Cells↗
PubMed1993

Receptor-evoked Cl- current in Xenopus oocytes is mediated through a beta-type phospholipase C. Cloning of a new form of the enzyme.

Xenopus oocytes exhibit a receptor-evoked Cl- current that is mediated through the activation of phospholipase C (PLC) and release of intracellular Ca2+. The identity of PLC(s) mediating this effect is unknown. We have cloned cDNAs encoding a new form of PLC-beta from a Xenopus oocyte cDNA library. The Xenopus PLC-beta has substantial (33-64%) homology with mammalian beta 1, beta 2, beta 3, and beta 4 phospholipase C and is closest to PLC-beta 3, with 64% identity and 80% similarity. Injection of antisense oligonucleotides to a specific region of Xenopus PLC-beta results in degradation of its mRNA and significantly reduces Cl- currents evoked by both endogenous angiotensin receptors and expressed mammalian alpha 1b-adrenergic receptors and M1-muscarinic receptors as compared to responses in sense oligonucleotide-injected oocytes. Inhibition of the M1-muscarinic response by antisense oligonucleotides was nonadditive with pertussis toxin inhibition. PLC antisense oligonucleotide-injected oocytes show Cl- current responses to IP3 that are indistinguishable from sense oligonucleotide-injected oocytes. Since the receptor responses are pertussis toxin-sensitive, we conclude that we have isolated a new form of PLC-beta involved in the pertussis toxin-sensitive receptor stimulation of the Ca2+ activated Cl- current in Xenopus oocytes.

Amino Acid Sequence↗
PubMed1993

Inhibition of cloned adenylyl cyclases by mutant-activated Gi-alpha and specific suppression of type 2 adenylyl cyclase inhibition by phorbol ester treatment.

Inhibition of the various cloned Gs-sensitive adenylyl cyclases was studied. The effect of mutant-activated Gi2-alpha on types 2, 3, and 6 adenylyl cyclases was analyzed after co-transfection into COS-7 cells. These adenylyl cyclases were chosen as representative members of the distinct families of mammalian adenylyl cyclases. COS-7 cells were transfected with cDNAs encoding (a) the required type of adenylyl cyclase, (b) luteinizing hormone receptor, and (c) Q205LGi2-alpha or Q205LGo1-alpha. Human chorionic gonadotropin (hCG)-stimulated cAMP accumulation was then measured. Transfection with Q205LGi2-alpha but not Q205LGo1-alpha resulted in 50-60% inhibition of hCG-stimulated cAMP accumulation of the type 2 adenylyl cyclase. hCG stimulation of types 3 and 6 adenylyl cyclases was also extensively inhibited by co-transfection with the Q205LGi2-alpha cDNA. Treatment with 4 beta-phorbol 12-myristate 13-acetate of cells transfected with the type 2 enzyme cDNA resulted in a 2-fold increase of the hCG-stimulated cAMP accumulation and a complete suppression of the Q205LGi2-alpha inhibition of the type 2 adenylyl cyclase. However, similar treatment did not affect Q205LGi2-alpha inhibition of the type 3 and type 6 adenylyl cyclases. These data indicate that many if not all types of adenylyl cyclases may be susceptible to inhibition by Gi2-alpha, but this inhibition can be regulated by protein kinase C in a type-specific manner.

Adenylyl Cyclase Inhibitors↗
PubMed1993

Stimulation of specific types of Gs-stimulated adenylyl cyclases by phorbol ester treatment.

Regulation of adenylyl cyclases by protein kinase C was studied. Types 1-6 were transiently expressed in 293 cells. The cells were treated with 4 beta-phorbol 12-myristate 13-acetate (PMA), and adenylyl cyclase activity was measured. PMA treatment of untransfected or vector-transfected cells resulted in a 35% (mean) increase in basal and forskolin-stimulated activity. PMA treatment of type 2 enzyme-transfected cells showed a 3-fold increase in basal activity. This stimulation varied with Mg2+ concentration with up to 4.5-fold increases at low Mg2+. PMA treatment resulted in about a 60% increase in fluoride or GTP gamma S (guanosine 5'-O-(thiotriphosphate))-stimulated activity in type 2 enzyme-transfected cells. Expression of the type 1 and 3 enzymes resulted in pronounced Ca2+/calmodulin stimulation, which was significantly increased upon pretreatment with PMA. No effects of PMA treatment were observable on other activities of the type 1 enzyme, whereas all activities of the type 3 enzymes were stimulated. Expressed types 4, 5, and 6 enzymes showed modest (approximately 30%) or no increases in basal and forskolin-stimulated activities upon PMA treatment. These data indicate that specific types of adenylyl cyclases can be functional targets for protein kinase C. The pronounced effects on the basal activity of the type 2 enzyme suggest this adenylyl cyclase could in the appropriate environment be an intracellular signal generator for external signals not routed through the activation of Gs.

Adenylyl Cyclases↗
PubMed1993

Signal recognition and integration by Gs-stimulated adenylyl cyclases.

Six Gs-stimulated adenylyl cyclases have been cloned. Two additional forms have been identified as partial cDNAs. These adenylyl cyclases have distinct functional properties and are differentially regulated by protein kinases. The adenylyl cyclases have distinct patterns of distribution in peripheral tissues and various brain regions. The unique functional characteristics of the members of this effector family may allow each cell type and/or brain region to customize the signal-recognition and integrative properties of its cAMP-generation system by varying the ratios of the various adenylyl cyclases.

Adenylyl Cyclases↗
PubMed1993

Molecular and functional diversity of mammalian Gs-stimulated adenylyl cyclases.

Receptor-regulated adenylyl cyclase in mammalian systems is among the best-studied of the cell surface signaling pathways that utilize G-proteins as transducers. In addition to the multiplicity of receptors and Gs proteins that function in this pathway, recent studies have shown that substantial molecular diversity exists in the effector as well. Full-length cDNAs encoding six different G-protein-regulated adenylyl cyclases have been isolated, and partial sequences identifying two more are known. These eight mammalian adenylyl cyclases can be grouped into five distinct families. The different types share some common properties such as stimulation by Gs and the diterpene forskolin. They show very distinct patterns of regulation by the beta gamma-subunits of G-proteins and protein kinases such as protein kinase C. The different types also appear to be localized in a tissue-specific manner. This diversity of regulatory features indicates that the effector can play an important role in determining the routing of signals to the cAMP pathway. The tissue and cell type-specific localization of the individual forms suggests that effectors such as adenylyl cyclase could be potential targets for a new generation of cell and tissue-specific drugs.

Adenylyl Cyclases↗
PubMed1992

The Q205LGo-alpha subunit expressed in NIH-3T3 cells induces transformation.

The growth functions of the heterotrimeric G protein G(o) was studied by expression in heterologous systems. The alpha-subunit of G(o) was mutated to convert Gln-205 to Leu (Q205L). Mutation of this conserved glutamine residue in G protein alpha-subunits is thought to persistently activate G proteins by inhibiting their GTPase activity. The wild type and mutant G(o)-alpha subunits were expressed in NIH-3T3 fibroblasts. These cells do not contain any measurable amounts of G(o)-alpha mRNA or protein. Transfection of wild type or Q205LG(o)-alpha subunit cDNA under the control of a dexamethasone-inducible promoter results in dexamethasone-dependent transcription of the mRNA and expression of the protein. The Q205LG(o)-alpha, but not wild type G(o)-alpha, stimulates mitogenesis in NIH-3T3 fibroblasts without significantly stimulating phospholipase C activity. Continuous expression of mutant G(o)-alpha induces focus formation, whereas transfections with vector alone or vector containing the native G(o)-alpha cDNA were without significant transforming effect in NIH-3T3 cells. Q205L G(o)-alpha did not induce focus formation in RAT-1 fibroblasts. Q205LG(o)-alpha-transformed NIH-3T3 cells are capable of anchorage-independent growth, as assessed by colony formation in soft agar. Q205LG(o)-alpha transformed cells induced tumors when injected into Nu/Nu mice. These results indicate that mutant G(o)-alpha subunits whose GTPase activity is presumably inhibited can induce the neoplastic transformation of NIH-3T3 cells in a phospholipase C-independent manner.

3T3 Cells↗
PubMed1992

Two members of a widely expressed subfamily of hormone-stimulated adenylyl cyclases.

cDNA encoding a hormone- and guanine nucleotide-stimulated adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] (type 6) from rat liver and kidney has been cloned and expressed. This enzyme is stimulated by forskolin, guanosine 5'-[gamma-thio]triphosphate, and isoproterenol plus GTP but is not stimulated by beta gamma subunits of guanine nucleotide-binding proteins. A second form (type 5), which is 75% similar to type 6, has also been cloned. Both types 5 and 6 cDNAs have multiple messages. PCR-based detection of the mRNA for the type 5 and 6 enzymes indicates that both are widely distributed. Homology analyses indicate at least four distinct subfamilies of guanine nucleotide stimulatory protein-regulated adenylyl cyclases. Types 5 and 6 enzymes define one distinct subfamily of mammalian adenylyl cyclases. Diversity of one guanine nucleotide-binding protein-regulated effector may allow different modes of regulation of cell-surface signal transmission.

Adenylyl Cyclases↗
PubMed1992

Enhanced phospholipase C stimulation and transformation in NIH-3T3 cells expressing Q209LGq-alpha-subunits.

NIH-3T3 cells were transfected with cDNA encoding the native alpha-subunit of the G protein Gq(alpha q) or a mutant (Q209L) form of alpha q. Cells expressing Q209L-alpha q showed greatly enhanced basal phospholipase C activity. Stimulation of phospholipase C activity by prostaglandin F2 alpha or fetal calf serum was increased up to 10-fold in Q209L-alpha q-transfected cells. Continuous expression of Q209L-alpha q or overexpression of alpha q in NIH-3T3 cells resulted in formation of foci after 3 weeks. The number of foci was proportional to the number of transfected cells and was greater in cells expressing the Q209L-alpha q than in cells that overexpressed the wild type alpha q. Q209L-alpha q-transfected NIH-3T3 cells also formed colonies in soft agar indicating their capacity to grow in an anchorage-independent manner. Expression of Q209L-alpha q in Rat-1 cells resulted in enhanced basal and fetal calf serum-stimulated phospholipase C activity, but these cells were not transformed as assessed by either the focus formation or the soft agar colony formation assays. These results indicate that expression of continuously activated Gq-alpha can result in transformation in a cell type-specific manner.

3T3 Cells↗
PubMed1992

Inhibition of enzymatic browning in foods and beverages.

Enzymatic browning is a major factor contributing to quality loss in foods and beverages. Sulfiting agents are used commonly to control browning; however, several negative attributes associated with sulfites have created the need for functional alternatives. Recent advances in the development of nonsulfite inhibitors of enzymatic browning are reviewed. The review focuses on compositions that are of practical relevance to food use.

Enzyme Inhibitors↗
PubMed1992

Lowered responsiveness of the catalyst of adenylyl cyclase to stimulation by GS in heterologous desensitization: a role for adenosine 3',5'-monophosphate-dependent phosphorylation.

Treatment of chick hepatocytes with glucagon results in homologous and heterologous desensitization of the receptor-stimulated adenylyl cyclase. The loci of postreceptor heterologous desensitization was studied. The addition of excess purified GS to glucagon-desensitized hepatocyte membranes did not fully restore fluoride stimulation of adenylyl cyclase, even though the absolute activity was increased at least 2-fold. Treatment of chick hepatocytes with 8-bromo-cAMP resulted in a similar reduction of fluoride stimulation that could not be restored by the addition of purified GS. When membranes from control and glucagon-treated hepatocytes were treated with purified catalytic subunit of protein kinase-A (PKA), fluoride stimulation was lowered in control, but not glucagon-treated, membranes. Treatment of membranes from S49 kin- lymphoma cells with PKA also resulted in decreased fluoride- and forskolin-stimulated adenylyl cyclase activity, but activity stimulated by Mn2+ was not altered. Since previous studies from our laboratory had shown that GS and G(i) are not substrates for protein kinase-A, it appears that the catalyst of adenylyl cyclase is the likely locus of modulation. To determine if both chick hepatocytes and S49 cells contain similar types of adenylyl cyclase that could account for the similar PKA regulatory properties, we used polymerase chain reaction-based techniques to identify GS-stimulated adenylyl cyclases present in these systems. The chick liver contains both type 5 and type 6 adenylyl cyclases, while S49 cells contain the type 6 enzyme. Type 5 and 6 adenylyl cyclases are members of one widely expressed subfamily of mammalian GS-responsive adenylyl cyclases and share a predicted PKA phosphorylation site in the central cytoplasmic loop. This site is not found in other known adenylyl cyclases (types 1-4), although the olfactory-specific type 3 enzyme has a predicted site nearby. These data indicate that one component of hormone-induced desensitization of the adenylyl cyclase system can be at the level of the catalyst, where PKA-mediated phosphorylation could result in lowered responsiveness. The types 5 and 6 adenylyl cyclases are likely candidates for such regulation.

8-Bromo Cyclic Adenosine Monophosphate↗