Inhibition of basic secretagogue-induced signaling in mast cells by cell permeable G alpha i-derived peptides.

BACKGROUND: Basic secretagogues of connective tissue mast cells act as receptor mimetic agents that trigger mast cells by activating G proteins. This leads to simultaneous propagation of two signaling pathways: one that culminates in exocytosis, while the other involves protein tyrosine phosphorylation and leads to release of arachidonic acid metabolites. We have previously shown that introduction of a peptide that comprises the C-terminal end of G alpha i3 into permeabilized mast cells inhibits basic secretagogue-induced exocytosis [Aridor et al., Science 1993;262:1569-1572]. We investigated whether cell-permeable peptides, composed of the C-terminus of G alpha i3 fused with importation sequences, affect mast cell function. METHODS: Following preincubation with the fused peptides, rat peritoneal mast cells were activated by compound 48/80 and analyzed for histamine and prostaglandin D2 release and protein tyrosine phosphorylations. RESULTS: We demonstrate that out of three importation sequences tested only G alpha i3 peptide fused with the Kaposi fibroblast growth factor importation sequence (ALL1) inhibited release of histamine. ALL1 as well as a cell-permeable peptide that corresponds to G alpha i2 also blocked compound 48/80-stimulated protein tyrosine phosphorylation, though the latter did not block histamine release. ALL1 effect was G protein-specific, as it was incapable of blocking protein tyrosine phosphorylation stimulated by pervanadate. CONCLUSION: ALL1, a transducible G alpha i3-corresponding peptide, blocks the two signaling pathways in mast cells: histamine release and protein tyrosine phosphorylation. Cell permeable peptides that block these two signaling cascades may constitute a novel approach for preventing the onset of the allergic reaction.

Activation of muscarinic M4 receptor augments NGF-induced pro-survival Akt signaling in PC12 cells.

Survival or death of neurons during development is mediated by the integration of a diverse array of signal transduction cascades that are controlled by the availability and acquisition of neurotrophic factors and agonists acting at G protein-coupled receptors (GPCRs). Recent studies have demonstrated that GPCRs can modulate signals elicited by receptor tyrosine kinases (RTK) and vice versa. Here, we examined the activity of pro-survival Akt kinase, in response to stimulation by muscarinic acetylcholine receptors (mAChRs) and co-activation with the nerve growth factor (NGF) receptor in PC12 cells endogenously expressing Gi-coupled M4 mAChR and Gq-coupled M1 and M5 mAChRs. Western blotting analysis using a phosphospecific anti-Akt antibody revealed a dose- and time-dependent increase in Akt phosphorylation in cells stimulated with mAChR specific agonist carbachol (CCh). Co-stimulation with CCh and NGF resulted in augmentation of Akt activity in a pertussis toxin (PTX)-sensitive manner, suggesting that M4 mAChR, but not M1 and M5 mAChRs, was associated with this synergistic Akt activation. The use of transducin as a Gbetagamma scavenger indicated that Gbetagamma subunits rather than Galphai/o acted as the signal transducer. Additional experiments showed that CCh treatment augmented NGF-induced phosphorylation and degradation of the Akt-regulated translation regulator tuberin. This augmentation was also inhibited by PTX pre-treatment or overexpression of transducin. Finally, co-stimulation of PC12 cells with CCh and NGF resulted in enhancement of cell survival. This is the first study that demonstrates the augmentation effect between M4 mAChR and NGF receptor, and the regulatory role of mAChR on tuberin.

A conformational switch in the inhibitory gamma-subunit of PDE6 upon enzyme activation by transducin.

In response to light, a photoreceptor G protein, transducin, activates cGMP-phosphodiesterase (PDE6) by displacing the inhibitory gamma-subunits (Pgamma) from the enzyme's catalytic sites. Evidence suggests that the activation of PDE6 involves a conformational change of the key inhibitory C-terminal domain of Pgamma. In this study, the C-terminal region of Pgamma, Pgamma-73-85, has been targeted for Ala-scanning mutagenesis to identify the point-to-point interactions between Pgamma and the PDE6 catalytic subunits and to probe the nature of the conformational change. Pgamma mutants were tested for their ability to inhibit PDE6 and a chimeric PDE5-conePDE6 enzyme containing the Pgamma C-terminus-binding site of cone PDE. This analysis has revealed that in addition to previously characterized Ile86 and Ile87, important inhibitory contact residues of Pgamma include Asn74, His75, and Leu78. The patterns of mutant PDE5-conePDE6 enzyme inhibition suggest the interaction between the PgammaAsn74/His75 sequence and Met758 of the cone PDE6alpha' catalytic subunit. This interaction, and the interaction between the PgammaIle86/Ile87 and PDE6alpha'Phe777/Phe781 residues, is most consistent with an alpha-helical structure of the Pgamma C-terminus. The analysis of activation of PDE6 enzymes containing Pgamma mutants with Ala-substituted transducin-contact residues demonstrated the critical role of PgammaLeu76. Accordingly, we hypothesize that the initial step in PDE6 activation involves an interaction of transducin-alpha with PgammaLeu76. This interaction introduces a bend into the alpha-helical structure of the Pgamma C-terminus, allowing transducin-alpha to further twist the C-terminus thereby uncovering the catalytic pocket of PDE6.

Beta gamma-mediated enhancement of corticotropin-releasing hormone-stimulated adenylyl cyclase activity by activation of gamma-aminobutyric acid(B) receptors in membranes of rat frontal cortex.

A number of studies have shown that activation of gamma-aminobutyric acid(B) (GABA(B)) receptors potentiates neurotransmitter-induced accumulation of cyclic AMP in brain slices, but the mechanisms involved in the facilitatory effect have not been fully elucidated. In the present study, we showed that in membranes of rat frontal cortex the GABA(B) receptor agonist (-)baclofen increased basal adenylyl cyclase activity and potentiated the maximal enzyme stimulation elicited by corticotropin-releasing hormone (CRH). The less active enantiomer (+)baclofen had no effect on cyclic AMP formation, whereas the natural agonist GABA mimicked the stimulatory action of (-)baclofen. In radioligand-binding experiments, the affinity and maximal binding capacity of (125)I-Tyr-CRH was not affected by (-)baclofen. The GABA(B) receptor antagonist CGP 55845A competitively counteracted the (-)baclofen potentiation of CRH-stimulated adenylyl cyclase activity with a pA(2) value of 6.70. Moreover, both (-)baclofen and GABA, but not (+)baclofen, caused a concentration-dependent stimulation of [(35)S]GTP gamma S binding to membrane G-proteins. The intracerebral injection of pertussis toxin significantly reduced the facilitatory effects of (-)baclofen on both basal and CRH-stimulated adenylyl cyclase activities. Moreover, membrane incubation with the GDP-bound form of the alpha subunit of transducin, a scavenger of G protein beta gamma subunits, blocked the stimulatory effects of (-)baclofen. The data indicate that in rat frontal cortex activation of GABA(B) receptors potentiates the CRH stimulation of adenylyl cyclase activity through a mechanism involving the beta gamma subunits of the pertussis toxin-sensitive G protein G(i)/G(o).

The gain of rod phototransduction: reconciliation of biochemical and electrophysiological measurements.

We have resolved a central and long-standing paradox in understanding the amplification of rod phototransduction by making direct measurements of the gains of the underlying enzymatic amplifiers. We find that under optimized conditions a single photoisomerized rhodopsin activates transducin molecules and phosphodiesterase (PDE) catalytic subunits at rates of 120-150/s, much lower than indirect estimates from light-scattering experiments. Further, we measure the Michaelis constant, Km, of the rod PDE activated by transducin to be 10 microM, at least 10-fold lower than published estimates. Thus, the gain of cGMP hydrolysis (determined by kcat/Km) is at least 10-fold higher than reported in the literature. Accordingly, our results now provide a quantitative account of the overall gain of the rod cascade in terms of directly measured factors.

An In vitro assay useful to determine the potency of several bitter compounds.

Gustducin and transducin are guanine nucleotide binding regulatory proteins (G proteins) expressed in taste receptor cells and implicated in transducing taste cell responses to certain compounds that humans consider bitter or sweet. These G proteins can be activated in vitro by taste receptor-containing membranes plus any of several bitter compounds. This activation can be monitored using limited trypsin digestion, sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Scanning of the autoradiograms enables one to quantitate the level of activation (defined as an activation index), obtain dose-response profiles and estimate the potency of the tastant. This assay may provide a useful substitute for, or adjunct to, the time-consuming human psychophysical analysis and costly animal studies typically used in taste sensory analysis. It may be used to identify and determine the concentration-response function of many bitter components of oral pharmaceuticals and food ingredients. A potential limitation of the assay is that only about half of all bitter compounds tested demonstrated in vitro activity, perhaps due to the presence of multiple transduction pathways. Nevertheless, the rapid throughput and microsample handling capability of this assay make it an ideal method to screen for high-potency bitterness inhibitors.

Involvement of betagamma subunits of G(q/11) in muscarinic M(1) receptor potentiation of corticotropin-releasing hormone-stimulated adenylyl cyclase activity in rat frontal cortex.

In the present study, we investigated the involvement of betagamma subunits of G(q/11) in the muscarinic M(1) receptor-induced potentiation of corticotropin-releasing hormone (CRH)-stimulated adenylyl cyclase activity in membranes of rat frontal cortex. Tissue exposure to either one of two betagamma scavengers, the QEHA fragment type II adenylyl cyclase and the GDP-bound form of the alpha subunit of transducin, inhibited the muscarinic M(1) facilitatory effect. Moreover, like acetylcholine (ACh), exogenously added betagamma subunits of transducin potentiated the CRH-stimulated adenylyl cyclase activity, and this effect was not additive with that elicited by ACh. Western blot analysis indicated the expression in frontal cortex of both type II and type IV adenylyl cyclases, two isoforms stimulated by betagamma subunits in synergism with activated G(s). The M(1) receptor-induced enhancement of the adenylyl cyclase response to CRH was counteracted by the G(q/11) antagonist GpAnt-2A but not by GpAnt-2, a preferential G(i/o) antagonist. In addition, the muscarinic facilitatory effect was inhibited by membrane preincubation with antiserum directed against the C terminus of the alpha subunit of G(q/11), whereas the same treatment with antiserum against either G(i1/2) or G(o) was without effect. These data indicate that in membranes of rat frontal cortex, activation of muscarinic M(1) receptors potentiates CRH-stimulated adenylyl cyclase activity through betagamma subunits of G(q/11).

[The calcium-mobilizing effect of inositol-1,4,5-triphosphate in rod outer segments and disks]. / O Ca2+ mobilizuiushchem deistvii inozit-1,4,5-trifosfata v naruzhnykh segmentakh palochki i diskakh.

The effect of inositol-1,4,5-trisphosphate (IP3) on the release of calcium ions from retinal rod discs was studied. It was shown that the release of Ca2+ from discs is an electroneutral process. The intradiscal calcium concentration during the release of the ion from the organelle decreases by 1 mM. It was found that the IP3-dependent release of Ca2+ ions from discs is activated by guanosine triphosphate and beta gamma-transducin. The increase in calcium concentration in the medium also activates the IP3-dependent release of Ca2+ ions from discs, which probably is due to the stimulation of phospholipase C. It is suggested that the functional role of the release of ions in related not to phototransduction but to slow regulatory and adaptation processes in the photoreceptor cell.

GABA(B) receptor-mediated stimulation of adenylyl cyclase activity in membranes of rat olfactory bulb.

Previous studies have shown that GABA(B) receptors facilitate cyclic AMP formation in brain slices likely through an indirect mechanism involving intracellular second messengers. In the present study, we have investigated whether a positive coupling of GABA(B) receptors to adenylyl cyclase could be detected in a cell-free preparation of rat olfactory bulb, a brain region where other Gi/Go-coupled neurotransmitter receptors have been found to stimulate the cyclase activity. The GABA(B) receptor agonist (-)-baclofen significantly increased basal adenylyl cyclase activity in membranes of the granule cell and external plexiform layers, but not in the olfactory nerve-glomerular layer. The adenylyl cyclase stimulation was therefore examined in granule cell layer membranes. The (-)-baclofen stimulation (pD2=4.53) was mimicked by 3-aminopropylphosphinic acid (pD2=4.60) and GABA (pD2=3.56), but not by (+)-baclofen, 3-aminopropylphosphonic acid, muscimol and isoguvacine. The stimulatory effect was counteracted by the GABA(B) receptor antagonists CGP 35348 (pA2=4.31), CGP 55845 A (pA2=7.0) and 2-hydroxysaclofen (pKi=4.22). Phaclofen (1 mM) was inactive. The (-)-baclofen stimulation was not affected by quinacrine, indomethacin, nordihydroguaiaretic acid and staurosporine, but was completely prevented by pertussis toxin and significantly reduced by the alpha subunit of transducin, a betagamma scavenger. The betagamma subunits of transducin stimulated the cyclase activity and this effect was not additive with that produced by (-)-baclofen. In the external plexiform and granule cell layers, but not in the olfactory nerve-glomerular layer, (-)-baclofen enhanced the adenylyl cyclase stimulation elicited by the neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) 38. Conversely, the adenylyl cyclase activity stimulated by either forskolin or Ca2+/calmodulin-(Ca2+/CaM) was inhibited by (-)-baclofen in all the olfactory bulb layers examined. These data demonstrate that in specific layers of rat olfactory bulb activation of GABA(B) receptors enhances basal and neurotransmitter-stimulated adenylyl cyclase activities by a mechanism involving betagamma subunits of Gi/Go. This positive coupling is associated with a widespread inhibitory effect on forskolin- and Ca2+/CaM-stimulated cyclic AMP formation.

Mediation by G protein betagamma subunits of the opioid stimulation of adenylyl cyclase activity in rat olfactory bulb.

In the rat olfactory bulb, activation of opioid receptors enhances basal adenylyl cyclase (EC 4.6.1.1) activity and potentiates enzyme stimulation by Gs-coupled neurotransmitter receptors in a pertussis toxin-sensitive manner. In the present study, we investigated the involvement of G protein betagamma subunits by examining the effects of betagamma scavengers and exogenously added betagamma subunits of transducin (betagamma(t)). The QEHA fragment of type II adenylyl cyclase (50 microM), a peptide that binds to and inactivates betagamma, inhibited the maximal stimulation of adenylyl cyclase activity elicited by Leu-enkephalin (Leu-enk) by about 50%. Similarly, the GDP-bound form of the alpha subunit of transducin (5 nM-1.5 microM), another betagamma scavenger, reduced both the opioid stimulation of basal adenylyl cyclase activity and the potentiation of vasoactive intestinal peptide-stimulated enzyme activity. Under the same experimental conditions, these agents failed to affect the stimulation of the enzyme activity elicited by activation of beta-adrenergic receptors with 1-isoproterenol. Moreover, the addition of betagamma(t)(400 nM) stimulated basal adenylyl cyclase by 80%, and this effect was not additive with that produced by Leu-enk. The data indicate that opioids enhance adenylyl cyclase activity in rat olfactory bulb by promoting the release of betagamma subunits from pertussis toxin-sensitive G proteins Gi/Go.

Role of G protein betagamma subunits in muscarinic receptor-induced stimulation and inhibition of adenylyl cyclase activity in rat olfactory bulb.

In the olfactory bulb, muscarinic receptors exert a bimodal control on cyclic AMP, enhancing basal and Gs-stimulated adenylyl cyclase activities and inhibiting the Ca2+/calmodulin- and forskolin-stimulated enzyme activities. In the present study, we investigated the involvement of G protein betagamma subunits by examining whether the muscarinic responses were reproduced by the addition of betagamma subunits of transducin (betagamma(t)) and blocked by putative betagamma scavengers. Membrane incubation with betagamma(t) caused a stimulation of basal adenylyl cyclase activity that was not additive with that produced by carbachol. Like carbachol, betagamma(t) potentiated the enzyme stimulations elicited by vasoactive intestinal peptide and corticotropin-releasing hormone. RT-PCR analysis revealed the expression of mRNAs encoding both type II and type IV adenylyl cyclase, two isoforms stimulated by betagamma synergistically with activated Gs. In addition, betagamma(t) inhibited the Ca2+/calmodulin- and forskolin-stimulated enzyme activities, and this effect was not additive with that elicited by carbachol. Membrane incubation with either one of two betagamma scavengers, the GDP-bound form of the alpha subunit of transducin and the QEHA fragment of type II adenylyl cyclase, reduced both the stimulatory and inhibitory effects of carbachol. These data provide evidence that in rat olfactory bulb the dual regulation of cyclic AMP by muscarinic receptors is mediated by betagamma subunits likely acting on distinct isoforms of adenylyl cyclase.

Ablation of Go alpha-subunit results in a transformed phenotype and constitutively active phosphatidylcholine-specific phospholipase C.

Modulation of the components involved in mitogenic signaling cascades is critical to the regulation of cell growth. GTP-binding proteins and the stimulation of phosphatidylcholine (PC) hydrolysis have been shown to play major roles in these cascades. One of the enzymes involved in PC hydrolysis, a PC-specific phospholipase C (PC-PLC) has received relatively little attention. In this paper we examined the role of a particular heterotrimeric GTP-binding protein, Go, in the regulation of cell growth and PC-PLC-mediated hydrolysis of PC in IIC9 fibroblasts. The Go alpha-subunit was ablated in IIC9 cells by stable expression of antisense RNA. These stably transfected cells acquired a transformed phenotype as indicated by: (a) the formation of multiple foci in monolayer cultures, (b) the acquisition of anchorage-independent growth in soft agar; and (c) an increased level of thymidine incorporation in the absence of added mitogens. These data implicate Goalpha as a novel tumor suppressor. Interestingly, PC-PLC activity was constitutively active in the Goalpha-ablated cells as evidenced by the chronically elevated levels of diacylglycerol and phosphorylcholine in the absence of growth factors. In contrast, basal activities of PC-phospholipase D, phospholipase A2, or phosphoinositol-PLC were not affected. These data demonstrate, for the first time, a role for Go in regulating cell growth and provide definitive evidence for the existence of a PC-PLC in eukaryotic cells. The data further indicate that a subunit of Go, is involved in regulating this enzyme.

Structure and function in rhodopsin: peptide sequences in the cytoplasmic loops of rhodopsin are intimately involved in interaction with rhodopsin kinase.

Phosphorylation of light-activated rhodopsin by the retina-specific enzyme, rhodopsin kinase (RK), is the primary event in the initiation of desensitization in the visual system. RK binds to the cytoplasmic face of rhodopsin, and the binding results in activation of the enzyme which then phosphorylates rhodopsin at several serine and threonine residues near the carboxyl terminus. To map the RK binding sites, we prepared two sets of rhodopsin mutants in the cytoplasmic CD and EF loops. In the first set, peptide sequences in both loops were either deleted or replaced by indifferent sequences. In the second set of mutants, the charged amino acids (E134, R135, R147, E239, K245, E247, K248, and E249) were replaced by neutral amino acids in groups of 1-3 per mutant. The deletion and replacement mutants in the CD loop showed essentially no phosphorylation, and they appeared to be defective in binding of RK. Of the mutants in the EF loop, that with a deletion of 13 amino acids, was also defective in binding to RK while the second mutant containing a replacement sequence bound RK but showed a reduction of about 70% in Vmax for phosphorylation. The mutants containing charged to neutral amino acid replacements in the CD and EF loops were all phosphorylated but to different levels. The charge reversal mutant E134R/R135E showed a 50% reduction in Vmax relative to wild-type rhodopsin. Replacements of charged residues in the EF loop decreased the Km by 5-fold for E239Q and E247Q/K248L/E239Q. In summary, both the CD and EF cytoplasmic loops are intimately involved in binding and interaction of RK with light-activated rhodopsin.

Molecular diversity of adenylyl cyclases in human and rat myometrium. Correlation with global adenylyl cyclase activity during mid- and term pregnancy.

Expression and regulation of myometrial adenylyl cyclases (AC) were studied during pregnancy. Hybridization of poly(A)+ RNA with specific cDNA probes for enzyme types I-IX indicated 1) the presence of transcripts encoding types II-VI and type IX in rat and human, and type VII in rat and 2) the absence of detectable mRNA for types I and VIII in both species. No substantial change was observed in the amount of specific mRNA and basal AC activity from mid-pregnancy to term. However, activation of the alpha2-adrenergic receptor/Gi protein pathway resulted in potentiation of Gs-stimulated AC activity at mid-pregnancy but not at term (Mhaouty, S., Cohen-Tannoudji, J., Bouet-Alard, R., Limon-Boulez, I., Maltier, J. P., and Legrand, C. (1995) J. Biol. Chem. 270, 11012-11016). We demonstrate in the present work that betagamma scavengers transducin-alpha and QEHA peptide abolished this positive input. On the other hand, increasing submicromolar concentrations of free Ca2+, a situation that mimics late term, reduced the forskolin-stimulated AC activity with an IC50 of 3.9 microM. Thus, the presence in myometrium of AC II family (types II, IV, VII) confers ability to G inhibitory proteins to stimulate enzyme activity via betagamma complexes at mid-pregnancy, whereas expression of AC III, V, and VI isoforms confers to the myometrial AC system a high sensitivity to inhibition by Ca2+-dependent processes at term. These data suggest that in the pregnant myometrium, the expression of different species of AC with distinct regulatory properties provides a mechanism for integrating positively or negatively the responses to various hormonal inputs existing either during pregnancy or in late term.

Receptor-independent G protein activation may account for the stimulatory effects of first-generation H1-receptor antagonists in HL-60 cells, basophils, and mast cells.

The first-generation histamine H1-receptor antagonists, chlorpheniramine (CPHE) and diphenhydramine (DPH), may activate histamine release from basophils and mast cells. Because CPHE and DPH are cationic-amphiphilic and because several substances with such physicochemical properties activate heterotrimeric regulatory guanine nucleotide-binding proteins (G-proteins) in a receptor-independent manner, we asked the question of whether or not H1-receptor antagonists could be G-protein activators as well. In dibutyryl cAMP-differentiated HL-60 cells, CPHE and DPH increased cytosolic Ca2+ concentration and azurophilic granule release in pertussis toxin (PTX)-sensitive manners. In HL-60 membranes, PTX-sensitive stimulations of GTPase [E.C. 3.6.1.] and binding of guanosine 5'-[gamma-thio]triphosphate by H1 receptor antagonists were observed. CPHE and DPH also increased GTP hydrolysis by the purified PTX-sensitive G-protein, transducin. In all-trans-retinoic acid-differentiated HL-60 cells and rat basophilic leukemia cells (RBL 2H3 cells), H1-receptor antagonists induced, unlike in dibutyryl cAMP-differentiated HL-60 cells, Ca2+ influx without Ca2+ mobilization from intracellular stores. CPHE and DPH also induced serotonin release from RBL 2H3 cells. Our data indicate that first-generation H1-receptor antagonists are receptor-independent G-protein activators and that such a mechanism of action accounts for their stimulatory effects in HL-60 cells, basophils, and mast cells.

betagamma-Transducin stimulates hydrolysis and synthesis of phosphatidylinositol 4,5-bisphosphate in bovine rod outer segment membranes.

T betagamma was shown to stimulate the hydrolysis and synthesis of PtdInsP2 in dark-adapted bovine retinal rod outer segments. In contrast, T alphaGDP blocked the effect of betagamma-transducin. It was also demonstrated that T betagamma was a stimulator of 32P incorporation into PtdInsP2 in ROS. These findings explain the modulating actions of GTP and light on PtdInsP2 hydrolysis and synthesis in ROS. The possible existence of cross-talk between the cGMP and phosphoinositide cascades in retinal rods was discussed.

Characterization of mammalian Gs-alpha proteins expressed in yeast.

The guanine nucleotide regulatory protein, GS, mediates transmembrane signaling by coupling membrane receptors to the stimulation of adenylyl cyclase activity. The full length coding sequences for the M(r) = 42-45,000, short form (S), and M(r) = 46-52,000, long form (L), of the alpha-subunits of rat GS were placed in yeast expression vectors under the regulatory control of the copper-inducible CUP1 promoter and transformed into Saccharomyces cerevisiae. In the presence of 100 microM CuSO4, the transformed yeast expressed GS-alpha mRNAs and proteins. In reconstitution experiments, rat GS-alpha(S and L), solubilized from yeast membranes with 1% cholate, conferred NaF-, (-)isoproterenol-, and guanine nucleotide-dependent sensitivity to adenylyl cyclase catalytic units in S49 lymphoma cyc- cell membranes, which are devoid of endogenous GS-alpha. GS-alpha (S) demonstrated twice the activity of GS-alpha(L) in reconstitution assays of fluoride-stimulated adenylyl cyclase activity. Comparison of GS-alpha (S) expressed in yeast with GS purified from rabbit liver or human erythrocytes showed that the crude recombinant protein was fully competent in reconstituting NaF-stimulated adenylyl cyclase activity, but was only 2-5% as potent as purified GS. Addition of bovine brain beta gamma subunits during reconstitution enhanced all parameters of adenylyl cyclase activity for GS-alpha(S and L) obtained from yeast. In contrast, transducin beta gamma only enhanced agonist-stimulated adenylyl cyclase activity for GS-alpha (S and L) following reconstitution. These results demonstrate that the expression of functional mammalian GS-alpha subunits in yeast may be useful for their biochemical characterization.

[Oligonucleotide-directed mutagenesis of inhibitory gamma-subunits of cGMP phosphodiesterase from bovine outer rod segments. A new hypothesis on mechanisms for inhibiting catalytic subunits by gamma-subunits and activation of a holoenzyme by transducin]. / Oligonukleotidnapravlennyi mutagenez ingibitornoi gamma-sub''edinitsy fosfodiésterazy cGMP iz naruzhnykh segmentov palochek setchatki byka. Novaia gipoteza o mekhanizmakh ingibirovaniia kataliticheskikh sub''edinitz gamma-sub''edinitsei ii aktivatsii kholofermenta transdutsinom.

Two mutants of the phosphodiesterase (PDE) gamma subunit (PDE gamma) from bovine retinal rods were synthesized by sequential transcription and translation in vitro. PDE gamma mutants R24E and H79L exhibited inhibitory properties similar to those of the wild-type PDE gamma (wtPDE gamma). At the same time, affinity to the rod outer segment (ROS) membranes is lower for R24E and higher for H79L in comparison with wtPDE gamma. The transducin alpha subunit (in a complex with the GTP non-hydrolyzable analogue, GTP gamma S) activates the trypsin-treated PDE (tPDE) inhibited by wtPDE gamma weaker than tPDE inhibited by R24E and stronger than tPDE inhibited by H79L. To explain the properties of these and earlier studied PDE gamma mutants, a new hypothesis on the mechanisms of inhibition of the PDE catalytic subunit dimer (PDE alpha beta) by PDE gamma and mechanism of the PDE holoenzyme (PDE alpha beta gamma 2) activation by the transducin alpha subunit in a complex with GTP (T alpha.GTP) is proposed: 1) two sites on PDE alpha beta for the PDE gamma binding (A- and the B-site) are different in structure. Sites on PDE gamma interacting with A- and the B-sites on PDE alpha beta are also different in structure. The site on PDE gamma interacting with the B-site partially overlaps with the T alpha.GTP binding site; 2) PDE gamma bound to the B-site provides the main contribution to inhibition of the enzyme catalytic activity; 3) T alpha.GTP first interacts with the PDE gamma bound to the A-site in the PDE holoenzyme and removes this PDE gamma in a PDE gamma.(T alpha.GTP) complex. This results in a slight increase of the catalytic activity of the PDE alpha beta gamma complex remaining bound to the ROS membranes; 4) after removal of PDE gamma from the A-site, another T alpha.GTP molecule is enabled to interact with both PDE alpha beta and PDE gamma bound to the B-site on PDE alpha beta. This interaction results in the formation of a ROS membrane-bound fully catalytically active triple complex PDE alpha beta.PDE gamma.(T alpha.GTP).

The molecular biology of taste transduction.

Taste cells respond to a wide variety of chemical stimuli: certain ions are perceived as salty (Na+) or sour (H+); other small molecules are perceived as sweet (sugars) and bitter (alkaloids). Taste has evolutionary value allowing animals to respond positively (to sweet carbohydrates and salty NaCl) or aversively (to bitter poisons and corrosive acids). Recently, some of the proteins involved in taste transduction have been cloned. Several different G proteins have been identified and cloned from taste tissue: gustducin is a taste cell specific G protein closely related to the transducins. Work is under way to clone additional components of the taste transduction pathways. The combination of electrophysiology, biochemistry and molecular biology is being used to characterize taste receptor cells and their sensory transduction mechanisms.