Synthesis in Escherichia coli of GTPase-deficient mutants of Gs alpha

We have reduced the GTPase activity of the alpha subunit of Gs, the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase, by introduction of point mutations analogous to those described in p21ras. Mutants G49V and Q227L differ from the wild type protein in the substitution of Val for Gly49 and Leu for Gln227, respectively (analogous to positions 12 and 61 in p21ras). Wild type and mutant proteins were synthesized in Escherichia coli, purified, and characterized. The rate constants for dissociation of GDP from G49V recombinant Gs alpha (rGs alpha) (0.47/min) and Q227L rGs alpha (0.23/min) differ by no more than 2-fold from that observed for the wild type protein (0.5/min). In marked contrast, the rate constants for hydrolysis of GTP by G49V rGs alpha (0.78/min) and Q227L rGs alpha (0.03-0.06/min) are 4-fold and roughly 100-fold slower than that for wild type rGs alpha (3.5/min). These reductions in the rate of hydrolysis of GTP result in significant fractional occupancy of these proteins by GTP in the presence of the nucleotide, 0.37 for G49V rGs alpha and 0.78 for Q227L rGs alpha, compared to 0.05 for wild type rGs alpha. When reconstituted with cyc- (Gs alpha-deficient) S49 cell membranes or purified adenylyl cyclase, both mutant proteins stimulate adenylyl cyclase activity in the presence of GTP to a much greater extent than does wild type Gs alpha; their maximal ability to activate the enzyme is largely unaltered. The fractional ability of a given Gs alpha polypeptide to active adenylyl cyclase in the presence of GTP correlates well with the fractinal occupancy of the protein by the nucleotide. The mutant subunits appear to interact normally with G protein beta gamma subunits, and their ability to activate adenylyl cyclase is enhanced by interaction with beta-adrenergic receptors. These results indicate that the structural analogy that has been inferred between the guanine nucleotide-binding domains of G proteins and the p21ras family is at least generally correct. They also provide confirmation of the kinetic model of G protein function and document mutations that permit the expression in vivo of constitutively activated G protein alpha subunits.     

Gs alpha stimulates transcytosis and apical secretion in MDCK cells through cAMP and protein kinase A

Recent evidence suggests a role for heterotrimeric G proteins in vesicular transport. Cholera toxin, which activates Gs alpha by ADP- ribosylation, has been reported to stimulate both apical secretion (Pimplikar, S.W., and K. Simons. 1993. Nature (Lond.). 352:456-458) and apically directed transcytosis (Bomsel, M., and K.E. Mostov. 1993. J. Biol. Chem. 268:25824-25835) in MDCK cells, via a cAMP-independent mechanism. Here, we demonstrate that apical secretion and apically directed transcytosis are significantly stimulated by agents that elevate cellular cAMP. Forskolin, which activates adenylyl cyclase directly, and 8BrcAMP augment both transport processes in MDCK cells. The increase is not limited to receptor-mediated transport (polymeric Ig receptor), since transcytosis of ricin, a galactose-binding lectin, is similarly stimulated. The effects of elevated cellular cAMP on apical secretion and transcytosis are apparently mediated via protein kinase A (PKA), as they are inhibited by H-89, a selective PKA inhibitor. Experiments employing a 17 degrees C temperature block indicate that cAMP/PKA acts at a late, possibly rate-limiting stage in the transcytotic pathway, after translocation of internalized markers into the apical cytoplasm. However, no significant stimulus of apical recycling was observed in the presence of FSK, suggesting that cAMP/PKA either affects transcytosis at a level proximal to apical early endosomes and/or specifically increases the efficiency by which transcytosing molecules are delivered to the apical plasma membrane. Finally, we overexpressed wild-type Gs alpha and a mutant, Q227L, which constitutively activates adenylyl cyclase, in MDCK cells. Although Q227L increased transcytosis more than wild-type Gs alpha, neither construct was as effective as FSK in stimulating transcytosis, arguing against a significant role of Gs alpha in transcytosis independent of cAMP and PKA.     

The trimeric GTP-binding protein (G(q)/G(11)) alpha subunit is required for insulin-stimulated GLUT4 translocation in 3T3L1 adipocytes

To investigate the potential role of trimeric GTP-binding proteins regulating GLUT4 translocation in adipocytes, wild type and constitutively active G(q) (G(q)/Q209L), G(i) (G(i)/Q205L), and G(s) (G(s)/Q227L) alpha subunit mutants were expressed in 3T3L1 adipocytes. Although expression of neither the wild type nor G(i)/Q205L and G(s)/Q227L alpha subunit mutants had any effect on the basal or insulin-stimulated translocation of a co-expressed GLUT4-enhanced green fluorescent protein (EGFP) fusion protein, expression of G(q)/Q209L resulted in GLUT4-EGFP translocation in the absence of insulin. In contrast, microinjection of an inhibitory G(q)/G(11) alpha subunit-specific antibody but not a G(i) or G(s) alpha subunit antibody prevented insulin-stimulated endogenous GLUT4 translocation. Consistent with a required role for GTP-bound G(q)/G(11), expression of the regulators of G protein signaling (RGS4 and RGS16) also attenuated insulin-stimulated GLUT4-EGFP translocation. To assess the relationship between G(q)/G(11) function with the phosphatidylinositol 3-kinase dependent pathway, expression of a dominant-interfering p85 regulatory subunit, as well as wortmannin treatment inhibited insulin-stimulated but not G(q)/Q209L-stimulated GLUT4-EGFP translocation. Furthermore, G(q)/Q209L did not induce the in vivo accumulation of phosphatidylinositol-3,4,5-trisphosphate (PIP(3)), whereas expression of the RGS proteins did not prevent the insulin-stimulated accumulation of PIP(3). Together, these data demonstrate that insulin stimulation of GLUT4 translocation requires at least two independent signal transduction pathways, one mediated through the phosphatidylinositol 3-kinase and another through the trimeric GTP-binding proteins G(q) and/or G(11).     

Dexras1 inhibits adenylyl cyclase

Dexras1 is a steroid hormone-induced Ras family G protein that acts as a receptor-independent activator of signaling by Gi/o family heterotrimeric G proteins. We examined the effects of Dexras1 on the activity of adenylyl cylase, a target of inhibitory regulation by Gialpha x GTP. Constitutively active Gsalpha (Q227L) increased cAMP levels 43-fold above baseline, and Dexras1 expression inhibited cAMP levels by 61% (P < 0.01). Dexras1 mediated inhibition of adenylyl cyclase was blocked by treatment pertussis toxin or by co-expression of RGS4, but was not inhibited by with dominant-interfering (G203T or G204A) mutants of Gi alpha2. Dexras1 decreased forskolin-stimulated CREB activation (P < 0.01) and this activity was also inhibited by co-expression of RGS4. These findings indicate that Dexras1 expression leads to ligand-independent activation of both Gialpha- and G(beta)gamma-dependent arms of the Gi signaling cascade, and suggest that Dexras1 may exert physiologically relevant inhibitory effects on the cAMP-PKA-CREB.     

Molecular determinants of PKA-dependent inhibition of TRPC5 channel

Canonical transient receptor potential (TRPC) channels are Ca(2+)-permeable, nonselective cation channels that are widely expressed in numerous cell types. Here, we demonstrate a new mechanism of TPRC isofom 5 (TRPC5) regulation, via cAMP signaling via Gα(s). Monovalent cation currents in human embryonic kidney-293 cells transfected with TRPC5 were induced by G protein activation with intracellular perfusion of GTPγS or by muscarinic stimulation. This current could be inhibited by a membrane-permeable analog of cAMP, 8-bromo-cAMP, by isoproterenol, by a constitutively active form of Gα(s) [Gα(s) (Q227L)], and by forskolin. These inhibitory effects were blocked by the protein kinase A (PKA) inhibitors, KT-5720 and H-89, as well as by two point mutations at consensus PKA phosphorylation sites on TRPC5 (S794A and S796A). Surface expression of several mutated versions of TRPC5, quantified using surface biotinylation, were not affected by Gα(s) (Q227L), suggesting that trafficking of this channel does not underlie the regulation we report. This mechanism of inhibition was also found to be important for the closely related channel, TRPC4, in particular for TRPC4α, although TRPC4β was also affected. However, this form of regulation was not found to be involved in TRPC6 and transient receptor potential vanilloid 6 function. In murine intestinal smooth muscle cells, muscarinic stimulation-induced cation currents were mediated by TRPC4 (>80%) and TRPC6. In murine intestinal smooth muscle cells, 8-bromo-cAMP, adrenaline, and isoproterenol decreased nonselective cation currents activated by muscarinic stimulation or GTPγS. Together, these results suggest that TRPC5 is directly phosphorylated by G(s)/cAMP/PKA at positions S794 and S796. This mechanism may be physiologically important in visceral tissues, where muscarinic receptor and β(2)-adrenergic receptor are involved in the relaxation and contraction of smooth muscles.     

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.     

Role of Gln1029 in the photoactivation processes of the LOV2 domain in adiantum phytochrome3

Phototropin (phot) is a blue-light receptor in plants. The molecule has two FMN (flavin mononucleotide)-binding domains named the LOV (light-oxygen-voltage) domain, that is a subset of a PAS (per-arnt-sim) superfamily. Illumination of phot-LOV domains produces a covalent C(4a) flavin-cysteinyl adduct, which is called the S390 intermediate state. According to the crystal structures of the LOV2 domain of Adiantum phytochrome3 (phy3), a fusion protein of phot containing the phytochrome chromophoric domain, in the unphotolyzed and S390 states, and the side chain of Gln1029 switches hydrogen bonds with the FMN chromophore. Gln1029 is the hydrogen-bonding donor of the C(4)=O group of FMN in the unphotolyzed state, whereas Gln1029 is the hydrogen-bonding acceptor of the N(5)-H group of FMN in S390. In this paper, we measured the light-induced structural changes in the Q1029L mutant protein of phy3-LOV2 by means of low-temperature FTIR spectroscopy, and the obtained spectra are compared with those of the wild type. Low-temperature UV-visible spectroscopy of Q1029L detected only one intermediate state, S390, at 77-295 K, as well as the wild type. The C(4)=O stretch of FMN at 1710 cm(-1) is shifted to 1723 cm(-1) in Q1029L, presumably because of the lack of hydrogen bonds between Gln1029 and FMN. Upon formation of S390, the C(4)=O group hydrogen bond is weakened in both wild type and Q1029L. These observations are fully consistent with the X-ray crystal structures of the unphotolyzed and S390 states. On the other hand, the C(4)=O stretch of FMN and amide-I vibrations are temperature-independent in Q1029L, in contrast to wild type, in which highly temperature-dependent FTIR spectra are detected. Amide-I vibrations of Q1029L at room temperature are similar to those of the wild type at 77-150 K but not at room temperature. These facts imply that the Q1029L mutant protein lacks progressive protein structural changes following flavin-cysteinyl adduct formation in the wild type, which eventually alter structures of beta sheet and alpha helix in the protein moiety. Hydrogen-bonding interaction of Gln1029 with the FMN chromophore likely plays an important role in the protein structural changes of phy3-LOV2.     

Targeted expression of activated Q227L G(alpha)(s) in vivo

We reportthe creation of transgenic mice with an inducible, tissue-targetedexpression of a constitutively active mutant form (Q227L) ofG_s. Mice expressing activated G_s in fattissue, liver, and skeletal muscle displayed normal body mass andblunted glucose metabolism. cAMP accumulation in adipose tissue wasincreased in the basal state, but far less than would be expected.Marked adaptation to elevated cAMP levels occurred, leading to anincrease in total cAMP-specific phosphodiesterase activity, a 50%decline in cAMP-dependent protein kinase (protein kinase A) activity, and an increased expression of G_i2. The reduction inkinase activity coincided with >50% increase in the expression ofRI and RII regulatory subunits of protein kinase A, with nochange in the amount of catalytic subunit. These data demonstrate theexistence of adaptive responses of protein kinase A, phosphodiesterase, and G_i2 in tissues expressing constitutively activeG_s that may act to rectify the impact of increased cAMP accumulation.

     

An equilibrium study of the cooperative binding of adenosine cyclic 3',5'-monophosphate and guanosine cyclic 3',5'-monophosphate to the adenosine cyclic 3',5'-monophosphate receptor protein from Escherichia coli

The binding of adenosine cyclic 3',5'-monophosphate (cAMP) and guanosine cyclic 3',5'-monophosphate (cGMP) to the adenosine cyclic 3',5'-monophosphate receptor protein (CRP) from Escherichia coli was investigated by equilibrium dialysis at pH 8.0 and 20 degrees C at different ionic strengths (0.05--0.60 M). Both cAMP and cGMP bind to CRP with a negative cooperativity that is progressively changed to positive as the ionic strength is increased. The binding data were analyzed with an interactive model for two identical sites and site/site interactions with the interaction free energy--RT ln alpha, and the intrinsic binding constant K and cooperativity parameter alpha were computed. Double-label experiments showed that cGMP is strictly competitive with cAMP, and its binding parameters K and alpha are not very different from that for cAMP. Since two binding sites exist for each of the cyclic nucleotides in dimeric CRP and no change in the quaternary structure of the protein is observed on binding the ligands, it is proposed that the cooperativity originates in ligand/ligand interactions. When bound to double-stranded deoxyribonucleic acid (dsDNA), CRP binds cAMP more efficiently, and the cooperativity is positive even in conditions of low ionic strength where it is negative for the free protein. By contrast, cGMP binding properties remained unperturbed in dsDNA-bound CRP. Neither the intrinsic binding constant K nor the cooperativity parameter alpha was found to be very sensitive to changes of pH between 6.0 and 8.0 at 0.2 M ionic strength and 20 degrees C. For these conditions, the intrinsic free energy and entropy of binding of cAMP are delta H degree = -1.7 kcal . mol-1 and delta S degree = 15.6 eu, respectively.     

Synergistic effect of prostaglandin F2alpha and cyclic AMP on glucose transport in 3T3-L1 adipocytes

The combined effect of prostaglandin F2alpha (PGF2alpha) and cAMP on glucose transport in 3T3-L1 adipocytes was examined. In cells pretreated with PGF2alpha and 8-bromo cAMP for 8 h, a synergy between these two agents on glucose uptake was found. Insulin-stimulated glucose transport, on the other hand, was only slightly affected. The synergistic effect of these two agents was suppressed in the presence of cycloheximide and actinomycin D. In concord, immunoblot and Northern blot analyses revealed that GLUT1 protein and mRNA levels were both increased in cells pretreated with both PGF2alpha and 8-bromo cAMP, greater than the additive effect of each agent alone. The synergistic action of PGF2alpha with 8-bromo cAMP to enhance glucose transport was inhibited by GF109203X, a selective protein kinase C (PKC) inhibitor. In addition, in cells depleted of diacylglycerol-sensitive PKC by prolonged treatment with 4beta-phorbol 12beta-myristate 13alpha-acetate, a PKC activator, the synergistic effects of PGF2alpha and 8-bromo cAMP on glucose transport and GLUT1 mRNA accumulation were both abolished. Taken together, these results indicate that PGF2alpha may act with cAMP in a synergistic way to increase glucose transport, probably through enhanced GLUT1 expression by a PKC-dependent mechanism.     

Lack of effect of incretin hormones on insulin release from pancreatic islets in the bile duct-ligated rats

Hyperglycemia associated with obstructive jaundice seriously affects the prognosis of patients with hepatobiliary diseases. We investigated secretory properties of isolated islets from bile duct-ligated (BDL) rats. Pancreatic islets from BDL rats lost their secretory responses to glucagon-like peptide-1 (GLP-1), although their responses to glucose were normal. Loss of potentiation of insulin release was also observed in glucagon and glucose-dependent insulinotropic peptide (GIP), whereas modulation of the release by forskolin, dibutyryl cAMP, or epinephrine remained unaffected. cAMP production by BDL islets was not increased by these insulinotropic hormones. Serum levels of glucagon, but not GIP, were increased in BDL rats. GLP-1 levels were also elevated, although they did not reach statistical significance. Immunoblotting of trimeric G protein subunits demonstrated that G(s)alpha L and G(s)alpha S, but not G(i)alpha 1/2 and G(i)alpha 3/o alpha, were less expressed in BDL islets. Therefore, unresponsiveness of the beta-cell to cAMP-raising hormones is involved in glucose intolerance under cholestasis. It results from diminished expression of alpha-subunits of the relevant G protein, G(s), and desensitization of receptors of these hormones.     

8-Chloro-cAMP inhibits transforming growth factor alpha transformation of mammary epithelial cells by restoration of the normal mRNA patterns for cAMP-dependent protein kinase regulatory subunit isoforms which show disruption upon transformation.

Differential regulation of the regulatory subunits of cAMP-dependent protein kinase isozymes correlates with the growth inhibitory effect of site-selective 8-Cl-cAMP demonstrated in cancer cell lines (Ally, S., Tortora, G., Clair, T., Grieco, D., Merlo, G., Katsaros, D., Ogreid, D., D?skeland, S.O., Jahnsen, T., and Cho-Chung, Y.S. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6319-6322). Such selective modulation of protein kinase isozyme regulatory subunits was also found in the 8-Cl-cAMP-induced inhibition of both transformation and transforming growth factor alpha (TGF alpha) production in Ki-ras-transformed rat kidney fibroblasts (Tortora, G., Ciardiello, F., Ally, S., Clair, T., Salomon, D. S., and Cho-Chung, Y. S. (1989) FEBS Lett. 242, 363-367). In this work, we have demonstrated that 8-Cl-cAMP antagonizes the TGF alpha effect in TGF alpha-transformed mouse mammary epithelial cells (NOG-8TFC17) at the level of gene expression for cAMP receptor protein isoforms, RI and RII (the regulatory subunits of protein kinase isozymes). Northern blot analysis demonstrated that in the transformed NOG-8TFC17 cells, compared with the nontransformed counterpart NOG-8 cells, the mRNA levels for the RI alpha cAMP receptor protein markedly increased, whereas the mRNA levels for the RII alpha and RII beta cAMP receptor proteins decreased. 8-Cl-cAMP, which induced growth inhibition and phenotypic reversion in NOG-8TFC17 cells, caused an inverse change in the mRNA patterns of the cAMP receptor proteins; RI alpha cAMP receptor mRNA sharply decreased to levels comparable with that of the nontransformed NOG-8 cells, whereas RII beta mRNA increased to a level even greater than that in the NOG-8 cells. In addition, one mRNA species of RII alpha increased, whereas the other RII alpha mRNA species decreased during the treatment. The mRNA level for the catalytic subunit of protein kinase, however, did not change during 8-Cl-cAMP treatment. In addition, 8-Cl-cAMP brought about a reduction in both TGF alpha mRNA and protein levels. These coordinated changes in the expression of the cAMP receptor proteins and TGF alpha were not observed during cis-hydroxyprolineor TGF beta-induced growth inhibition of the NOG-8TFC17 cells. Thus, the antagonistic effect of 8-Cl-cAMP toward TGF alpha-induced transformation involves modulation of the expression of a specific set of cellular genes.     

Activated G alpha q inhibits p110 alpha phosphatidylinositol 3-kinase and Akt

Some Gq-coupled receptors have been shown to antagonize growth factor activation of phosphatidylinositol 3-kinase (PI3K) and its downstream effector, Akt. We used a constitutively active Galphaq(Q209L) mutant to explore the effects of Galphaq activation on signaling through the PI3K/Akt pathway. Transient expression of Galphaq(Q209L) in Rat-1 fibroblasts inhibited Akt activation induced by platelet-derived growth factor or insulin treatment. Expression of Galphaq(Q209L) also attenuated Akt activation promoted by coexpression of constitutively active PI3K in human embryonic kidney 293 cells. Galphaq(Q209L) had no effect on the activity of an Akt mutant in which the two regulatory phosphorylation sites were changed to acidic amino acids. Inducible expression of Galphaq(Q209L) in a stably transfected 293 cell line caused a decrease in PI3K activity in p110alpha (but not p110beta) immunoprecipitates. Receptor activation of Galphaq also selectively inhibited PI3K activity in p110alpha immunoprecipitates. Active Galphaq still inhibited PI3K/Akt in cells pretreated with the phospholipase C inhibitor U73122. Finally, Galphaq(Q209L) co-immunoprecipitated with the p110alpha-p85alpha PI3K heterodimer from lysates of COS-7 cells expressing these proteins, and incubation of immunoprecipitated Galphaq(Q209L) with purified recombinant p110alpha-p85alpha in vitro led to a decrease in PI3K activity. These results suggest that agonist binding to Gq-coupled receptors blocks Akt activation via the release of active Galphaq subunits that inhibit PI3K. The inhibitory mechanism seems to be independent of phospholipase C activation and might involve an inhibitory interaction between Galphaq and p110alpha PI3K.     

The effect of PGF2 alpha on parathyroid hormone-stimulated cyclic AMP production in mouse osteoblastic cell, MC3T3E1.

The effect of prostaglandin F2 alpha (PGF2 alpha) was investigated in MC3T3E1 cells on the succeeding cAMP response to parathyroid hormone (PTH). PGF2 alpha increased the membrane-associated protein kinase C (PKC) activity, indicating the activation of this enzyme. The effect of PTH to increase cAMP production was enhanced by pretreatment with PGF2 alpha. Phorbol 12-myristate 13-acetate also enhanced cAMP production stimulated by PTH, and PKC inhibitor H7 attenuated the enhancement of PGF2 alpha. A23187 did not reproduce the PGF2 alpha effect, and this effect was not antagonized by the calmodulin antagonist W7. PGF2 alpha did not change the ED50 nor the maximally responsive dose of PTH in stimulating cAMP production. The effect of PGF2 alpha was not affected by pertussis toxin, and PGF2 alpha also enhanced cholera toxin- or forskolin-stimulated cAMP production. In accordance with the response of cAMP to PTH, the resorption of mouse limb bones stimulated submaximally by PTH was enhanced by the concomitant presence of PGF2 alpha. These results indicate that PGF2 alpha modulates cAMP response through the activation of PKC, the target of which might be the catalytic unit of adenylate cyclase. Such interaction between signal transduction systems may have significance in modulating the effect of PTH on bone, i.e., bone resorption.     

Studies on reduction of S-nitrosoglutathione by human carbonyl reductases 1 and 3

Human carbonyl reductases 1 and 3 (CBR1 and CBR3) are monomeric NADPH-dependent enzymes of the short-chain dehydrogenase/reductase superfamily. Despite 72% identity in primary structure they exhibit substantial differences in substrate specificity. Recently, the endogenous low molecular weight S-nitrosothiol S-nitrosoglutathione (GSNO) has been added to the broad substrate spectrum of CBR1. The current study initially addressed whether CBR3 could equally reduce GSNO which was not the case. Neither the introduction of residues which contribute to glutathione binding in CBR1, i.e. K106Q and S97V/D98A, nor the exchange C143S, which prevents a theoretical disulfide bond with C227 in CBR3, could engender activity towards GSNO. However, exchanging amino acids 236-244 in CBR3 to correspond to CBR1 was sufficient to engender catalytic activity towards GSNO. Catalytic efficiency was further improved by the exchanges Q142M, C143S, P230W and H270S. Hence, the same residues previously reported as important for reduction of carbonyl compounds appear to be key to CBR1-mediated reduction of GSNO. Furthermore, for CBR1-mediated reduction of GSNO, considerable substrate inhibition at concentrations >5 K(m) was observed. Treatment of CBR1 with GSNO followed by removal of low molecular weight compounds decreased the GSNO reducing activity, suggesting a covalent modification. Treatment with dithiothreitol, but not with ascorbic acid, could rescue the activity, indicating S-glutathionylation rather than S-nitrosation as the underlying mechanism. As C227 has previously been identified as the reactive cysteine in CBR1, the variant CBR1 C227S was generated, which, in comparison to the wild-type protein, displayed a similar k(cat), but a 30-fold higher K(m), and did not show substrate inhibition. Collectively, the results clearly argue for a physiological role of CBR1, but not for CBR3, in GSNO reduction and thus ultimately in regulation of NO signaling. Furthermore, at higher concentrations, GSNO appears to work as a suicide inhibitor for CBR1, probably through glutathionylation of C227.     

G alpha 3 regulates the cAMP signaling system in Dictyostelium.

The Dictyostelium discoideum developmental program is initiated by starvation and its progress depends on G-protein-regulated transmembrane signaling. Disruption of the Dictyostelium G-protein alpha-subunit G alpha 3 (g alpha 3-) blocks development unless the mutant is starved in the presence of artificial cAMP pulses. The function of G alpha 3 was investigated by examining the expression of several components of the cAMP transmembrane signaling system in the g alpha 3- mutant. cAMP receptor 1 protein, cyclic nucleotide phosphodiesterase, phosphodiesterase inhibitor, and aggregation-stage adenylyl cyclase mRNA expression were absent or greatly reduced when cells were starved without exogenously applied pulses of cAMP. However, cAMP receptor 1 protein and aggregation-stage adenylyl cyclase mRNA expression were restored by starving the g alpha 3- cells in the presence of exogenous cAMP pulses. Adenylyl cyclase activity was also reduced in g alpha 3- cells starved without exogenous cAMP pulses compared with similarly treated wild-type cells but was elevated to a level twofold greater than wild-type cells in g alpha 3- cells starved in the presence of exogenous cAMP pulses. These results suggest that G alpha 3 is essential in early development because it controls the expression of components of the transmembrane signaling system.     

Regulated interactions of the alpha 2A adrenergic receptor with spinophilin, 14-3-3zeta,and arrestin 3

The present studies demonstrate that no single stretch of sequence in the third intracellular (3i) loop of the alpha(2A) adrenergic receptor (alpha(2A)-AR) can fully account for its previously described interactions with spinophilin (Richman, J. G., Brady, A. E., Wang, Q., Hensel, J. L., Colbran, R. J., and Limbird, L. E. (2001) J. Biol. Chem. 276, 15003-15008), 14-3-3zeta (Prezeau, L., Richman, J. G., Edwards, S. W., and Limbird, L. E. (1999) J. Biol. Chem. 274, 13462-13469), and arrestin 3 (Wu, G., Krupnick, J. G., Benovic, J. L., and Lanier, S. M. (1997) J. Biol. Chem. 272, 17836-17842), suggesting that a three-dimensional surface, rather than a linear sequence, provides the basis for these interactions as proposed for 3i loop tethering of the alpha(2A)-AR to the basolateral surface of Madin-Darby canine kidney cells (Edwards, S. W., and Limbird, L. E. (1999) J. Biol. Chem. 274, 16331-16336). Sequences at the extreme N-terminal and C-terminal ends of the 3i loop are critical for interaction with spinophilin but not for interaction with 14-3-3zeta or arrestin 3, for which the C-terminal half of the loop is more important. Competition binding for (35)S-labeled alpha(2A)-AR 3i loop binding to glutathione S-transferase (GST)-spinophilin amino acids 151-444 revealed a relative affinity of spinophilin congruent with arrestin > 14-3-3zeta for the unphosphorylated alpha(2A)-AR 3i loop. Agonist occupancy of the alpha(2A)-AR increases receptor association with spinophilin, and arrestin 3 appears to compete for this enrichment. However, when the G protein-coupled receptor kinase 2 substrate sequence was deleted from the 3i loop, arrestin 3 could not compete for the agonist-enriched binding of spinophilin to the mutant alpha(2A)-AR. These data are consistent with a model where sequential or competitive interactions among spinophilin, arrestin, and/or 14-3-3zeta play a role in alpha(2A)-AR functions.