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R T Premont

Publications and source records attributed to R T Premont.

51 records · Page 3Linked to original sources
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↗
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

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↗
PubMed1989

The G alpha z gene product in human erythrocytes. Identification as a 41-kilodalton protein.

A cDNA encoding a previously unknown G protein alpha-subunit lacking the site for pertussis toxin-catalyzed ADP-ribosylation was recently cloned and its putative protein product named Gz (Fong, H. K. W., Yoshimoto, K. K., Eversole-Cire, P., and Simon, M. I. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 3066-3070) or Gx (Matsuoka, M., Itoh, H. Kozasa, T., and Kaziro, Y. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5384-5388). A synthetic peptide corresponding to the deduced carboxyl-terminal decapeptide of this putative protein (alpha z) has been synthesized and used to prepare a polyclonal rabbit antiserum directed against the protein. The specificity and cross-reactivity of this antiserum was assessed using bacterially expressed recombinant G protein alpha-subunit fusion proteins (r alpha). The crude antiserum strongly recognizes r alpha z in immunoblots. Pretreatment of antiserum with antigen peptide greatly reduces the interaction of the antiserum with r alpha z. Affinity purified antiserum strongly recognizes expressed r alpha z, does not recognize r alpha s1, r alpha s1, r alpha o, or r alpha i3, and very weakly interacts with r alpha i1 and r alpha i2. In contrast, the alpha-subunits of purified bovine brain Gi1 and human erythrocyte Gi2 and Gi3 did not react with the alpha z-antiserum. Partially purified mixtures of human erythrocyte G proteins contain a 41-kDa protein that reacts specifically in immunoblots with both crude and affinity purified alpha z-specific antiserum. Quantitative immunoblotting using r alpha z as a standard indicates that there is 60-100 ng of alpha z/micrograms of 40/41-kDa alpha-subunit protein in partially purified human erythrocyte G protein preparations. We conclude that we have identified the alpha z gene product as a 41-kDa trace protein in human erythrocytes.

Amino Acid Sequence↗
PubMed1989

Heterologous desensitization of the liver adenylyl cyclase: analysis of the role of G-proteins.

Glucagon treatment of chick hepatocytes in primary culture results in a rapid 20-30% decrease in NaF-stimulated adenylyl cyclase activity. This heterologous desensitization is rapidly reversible upon removal of hormone from the culture medium. This decrease in NaF-stimulated adenylyl cyclase activity agrees well with the decrease in Gs activity, as assessed by reconstitution of the cyc- S49 cell adenylyl cyclase. Recovery from heterologous desensitization within 30 min results in restoration of Gs activity to control levels. No changes in Gs-alpha protein levels could be determined by immunoblotting using an alpha s-specific antiserum during heterologous desensitization. Similarly, no change in the beta gamma-subunits or Gi-alpha-subunits were detected by immunoblotting analysis using subunit-specific antisera. Pretreatment of cells with pertussis toxin did not block the onset of either heterologous or homologous desensitization, indicating the lack of involvement of Gi in glucagon-induced desensitization. cAMP-dependent phosphorylation of G-proteins does not account for heterologous desensitization, since none of the G-protein subunits appears to be phosphorylated even in vitro. However, Gs as the locus of change in heterologous desensitization was confirmed by the observation that addition of purified Gs to desensitized cell membranes results in enhancement of NaF-stimulated adenylyl cyclase activity. From these data we conclude that postreceptor heterologous desensitization in chick hepatocytes results from a reversible decrease in Gs activity in the cell surface membrane.

Adenosine Diphosphate Ribose↗
PubMed1988

Glucagon-induced desensitization of adenylyl cyclase in primary cultures of chick hepatocytes. Evidence for multiple pathways.

Chick hepatocytes in primary culture have been used to study the homologous and heterologous pathways of glucagon-induced desensitization of adenylyl cyclase. Scatchard analysis and guanine nucleotide effects on dissociation kinetics indicate that the initial phase of homologous desensitization, an increase in low affinity glucagon receptors due to the rapid uncoupling of the receptor from Gs, is essentially complete within 5 min. These receptors recouple within 20 min upon removal of glucagon. Upon prolonged (2 h or more) exposure of hepatocytes to glucagon, disappearance of low affinity receptors from cell surface membranes constitutes the second phase of homologous desensitization. Recovery of these lost and presumably internalized receptors requires more than 12 h following the removal of glucagon but is not dependent on new protein synthesis. The heterologous phase of desensitization is slower, requiring 20-30 min of glucagon treatment to reach completion. Stimulation of adenylyl cyclase by hormonal and nonhormonal effectors is similarly reduced, indicating a common defect in this desensitized state. Agonist occupancy of other hormone receptors coupled to adenylyl cyclase in hepatocytes, such as beta-adrenergic, prostaglandin E1, and vasoactive intestinal peptide, results in heterologous desensitization. Heterologous desensitization is rapidly reversed (within 30 min) upon partial removal of glucagon, under conditions allowing the maintenance of the homologously desensitized state. Neither onset of nor recovery from heterologous desensitization requires protein synthesis. These data indicate that homologous and heterologous desensitization occurs by independent mechanisms. Homologous desensitization involves uncoupling of the glucagon receptor from Gs, followed by removal of these uncoupled receptors from the cell surface. Heterologous desensitization represents a second level of cellular control of hormonal responsiveness to be turned on when the cell is subjected to prolonged hormonal stimulation and withdrawn when hormone levels are lowered.

Adenylyl Cyclases↗
PubMed1988

Glucagon receptor-mediated activation of Gs is accompanied by subunit dissociation.

The effect of the glucagon receptor on the activation of the stimulatory GTP-binding protein of adenylyl cyclase (Gs) in the native rat liver membrane environment was studied. The activated state of Gs was assessed by its ability to reconstitute the cyc- S49 cell membrane adenylyl cyclase. The Gs protein was activated by saturating concentrations of guanosine 5'-thiotriphosphate (GTP gamma S) or guanyl-5'-yl imidodiphosphate in a hormone-dependent manner at 0.4 mM Mg2+ in native membranes or in membranes that had been treated with 1 mM N-ethylmaleimide to eliminate the catalytic activity of adenylyl cyclase. At 50 mM Mg2+, Gs was fully activated by GTP gamma S in the absence of hormone. The unactivated Gs protein migrates around 4 S, whereas activated Gs migrates around 2 S on sucrose density gradients. When pure Gs is analyzed on sucrose density gradients, it is found that the unactivated protein migrates at 4.1 S. Gs was activated by saturating concentrations of GTP gamma S and Mg2+, and the alpha subunit of Gs was chromatographically purified. The resolved alpha subunit of Gs that is capable of stimulating the cyc- adenylyl cyclase migrates at 2.1 S. From these data, we conclude that activation of Gs results in the dissociation of this protein in the membrane environment and that the hormone-occupied receptor promotes this dissociation process under conditions where Mg2+ ions are limiting.

Adenylyl Cyclases↗
PubMed1988

Beta gamma subunits of GTP-binding proteins inhibit muscarinic receptor stimulation of phospholipase C.

This study examines the mechanism of guanine nucleotide-binding protein (G protein) coupling of receptors to phospholipase C. The Xenopus oocyte has a muscarinic receptor-activated Cl- current that is mediated by inositol 1,4,5-trisphosphate. Modulation of the muscarinic receptor-evoked Cl- current was examined under voltage clamp in oocytes injected with resolved G-protein subunits. The presence of an alpha subunit of G proteins in oocytes was shown by pertussis toxin-labeling of a 41-kDa band in oocyte membranes. The presence of the beta subunit of G proteins was demonstrated by immunoblotting experiments with an antiserum (U-49) that is specific for the beta subunit. Pertussis toxin treatment of oocytes resulted in the uncoupling of muscarinic receptors from activation of the Cl- current. Cells microinjected with 1.5 ng of human erythrocyte beta gamma-subunit complex or 1.0 ng of bovine brain beta gamma-subunit complex showed approximately a 95% reduction in the evoked Cl- current. Cells injected with equal volumes of protein storage vehicle showed no change in response. Cells injected with boiled beta gamma subunits, bovine serum albumin, or resolved alpha subunits also showed no reduction in response. Cells injected with various concentrations of beta gamma subunits showed a concentration dependence with half-maximal inhibition of the muscarinic activated Cl- current at about 10 nM. Cells injected with 1.0 ng of bovine brain beta gamma subunits could not respond to bath-applied agonist but could generate the Cl- current on intracellular injection of inositol 1,4,5-trisphosphate. These observations suggest that there is a G protein responsible for muscarinic receptor-mediated signal transduction through phospholipase C and that it is an alpha beta gamma heterotrimer. It appears that the mode of action of the G protein in the phospholipase C system may be similar to that of the hormone-activated adenylyl cyclase.

Animals↗
PubMed1988

A whole-cell and single-channel study of the voltage-dependent outward potassium current in avian hepatocytes.

Voltage-dependent membrane currents were studied in dissociated hepatocytes from chick, using the patch-clamp technique. All cells had voltage-dependent outward K+ currents; in 10% of the cells, a fast, transient, tetrodotoxin-sensitive Na+ current was identified. None of the cells had voltage-dependent inward Ca2+ currents. The K+ current activated at a membrane potential of about -10 mV, had a sigmoidal time course, and did not inactivate in 500 ms. The maximum outward conductance was 6.6 +/- 2.4 nS in 18 cells. The reversal potential, estimated from tail current measurements, shifted by 50 mV per 10-fold increase in the external K+ concentration. The current traces were fitted by n2 kinetics with voltage-dependent time constants. Omitting Ca2+ from the external bath or buffering the internal Ca2+ with EGTA did not alter the outward current, which shows that Ca2+-activated K+ currents were not present. 1-5 mM 4-aminopyridine, 0.5-2 mM BaCl2, and 0.1-1 mM CdCl2 reversibly inhibited the current. The block caused by Ba was voltage dependent. Single-channel currents were recorded in cell-attached and outside-out patches. The mean unitary conductance was 7 pS, and the channels displayed bursting kinetics. Thus, avian hepatocytes have a single type of K+ channel belonging to the delayed rectifier class of K+ channels.

4-Aminopyridine↗
PubMed1987

The hepatic glucagon receptor: a comparative study of the regulatory and structural properties.

Guanine nucleotide and Mg2+ ion regulation of [125I-Tyr10]monoiodoglucagon ([125I]MIG) binding to liver plasma membranes from chicken, rat, and rabbit was studied. It was found that [125I]MIG binding to chicken liver membranes was increased by the addition of Mg2+ ion, while binding to rat and rabbit liver membranes was unaffected. In the chicken liver membranes, the Mg2+ ion induced high affinity binding which was sensitive to guanine nucleotides, while the low affinity binding in the absence of Mg2+ ion was not. Maximal effects of Mg2+ ion were observed at 1 mM. Glucagon binding to rat liver membrane receptors was GTP sensitive regardless of whether Mg2+ ion was added. Glucagon binding to rabbit liver membranes was insensitive to both Mg2+ ions and GTP. This lack of GTP effect was not due to degradation of GTP; no effect of the nonhydrolyzable analog guanyl-5'-yl-imidodiphosphate was observable. Glucagon stimulation of rabbit liver adenylyl cyclase, however, was dependent on GTP, as was the case with all of the other liver adenylyl cyclases studied here. The Kact of GTP for the rabbit liver system was very similar to that for rat liver membranes. The glucagon receptor was covalently labeled with [125I]MIG using p-hydroxysuccinimidyl azidobenzoate and analyzed by sodium dodecyl sulfate-gel electrophoresis. In all cases, a major labeled band at 63,000 daltons was observed. The levels of glucagon receptor and stimulatory (Ns) and inhibitory (Ni) regulatory proteins of adenylyl cyclase were measured. The highest levels of glucagon receptor were measured in rat liver membranes, while the levels in chicken and rabbit membranes were 30-40% lower. Rabbit liver membrane had the highest levels of Ns, while rat liver membranes had 2-fold lower and chick liver membrane 4-fold lower levels than rabbit liver membranes. The levels of Ni was similar in the three systems. Thus, the ratio of Ns to glucagon receptor was highest in the rabbit. In the rat, this ratio was 3-fold lower than that in the rabbit. In the chicken membranes, the ratio was about 60% of that in the rat. These data suggest that the observed differences in effects of GTP on hormone binding can be explained by alterations in the ratio of the receptor and Ns proteins among the various species.

Adenylate Cyclase Toxin↗
PubMed1986

Properties of an intermediate-sized androgen receptor: association with RNA.

This study identifies an intermediate-sized androgen receptor and characterizes its relationship with the 9.1S and 4.4S receptor forms. Under low ionic conditions, at 2-4 degrees C, there exists a 9.1S (+/- 0.17) (n = 30) oligomeric form which does not bind to DNA. Under high ionic conditions, this form dissociates to a 4.4S (+/- 0.08) (n = 18) monomeric form. When the salt concentration is lowered, the 4.4S monomer converts to a species with an intermediate sedimentation coefficient of 7.7S (+/- 0.15) (n = 17) which binds to DNA. Unlike the 9.1S oligomer the 7.7S form is not maintained by sodium molybdate under high ionic conditions but rather dissociates to the 4.4S monomer. To determine whether these forms were associated with RNA, the 7.7S form was incubated with RNase A and analyzed by density gradient centrifugation. The 7.7S form was digested fully by RNase to the 4.4S monomer. The 7.7S form demonstrated a buoyant density of 1.2459 +/- 0.014 g/cm3 (n = 6) in metrizamide gradients, suggesting a ribonucleoprotein component. The sedimentation coefficient of the 9.1S form was unaffected by RNase. These data suggest that the intermediate 7.7S receptor form is composed of 4.4S monomer associated with a ribonucleoprotein molecule(s).

Animals↗