The influence of bound GDP on the kinetics of guanine nucleotide binding to G proteins

Purified guanine nucleotide-binding regulatory proteins, as either the oligomers or the isolated nucleotide-binding alpha subunits, display anomalous kinetics of nucleotide binding. This is due to the presence of tightly bound GDP in these preparations. The dissociation of bound GDP is the rate-limiting step for nucleotide binding. GDP can be removed by chromatography in the presence of 1 M (NH4)2SO4 and 20% glycerol, which yields preparations of G proteins that contain less than 0.1 mol of GDP/mol of guanosine 5'-(gamma-thio)triphosphate (GTP gamma S)-binding site. When the GDP is removed, the binding of GTP gamma S displays kinetics consistent with a bimolecular reaction.     

Agonist regulation of D(2) dopamine receptor/G protein interaction. Evidence for agonist selection of G protein subtype

The D(2) dopamine receptor has been expressed in Sf21 insect cells together with the G proteins G(o) and G(i2), using the baculovirus system. Expression levels of receptor and G protein (alpha, beta, and gamma subunits) in the two preparations were similar as shown by binding of [(3)H]spiperone and quantitative Western blot, respectively. For several agonists, binding data were fitted best by a two-binding site model in either preparation, showing interaction of expressed receptor and G protein. For some agonists, binding to the higher affinity site was of higher affinity in D(2)/G(o) than in the D(2)/G(i2) preparation. Some agonists exhibited binding data that were best fitted by a two-binding site model in D(2)/G(o) and a one-binding site model in D(2)/G(i2). Therefore, receptor/G protein interaction seemed to be stronger in the D(2)/G(o) preparation. Agonist stimulation of [(35)S]GTP gamma S (guanosine 5'-3-O-(thio)triphosphate) binding in the two preparations also gave evidence for higher affinity D(2)/G(o) interaction. In the D(2)/G(o) preparation, agonist stimulation of [(35)S]GTP gamma S binding occurred at higher potency for several agonists, and a higher stimulation (relative to dopamine) was achieved in D(2)/G(o) compared with D(2)/G(i2). Some agonists were able to stimulate [(35)S]GTP gamma S binding in the D(2)/G(o) preparation but not in D(2)/G(i2). The extent of D(2) receptor selectivity for G(o) over G(i2) is therefore dependent on the agonist used, and thus agonists may stabilize different conformations of the receptor with different abilities to couple to and activate G proteins.     

Guanine nucleotide binding characteristics of transducin: essential role of rhodopsin for rapid exchange of guanine nucleotides

Transducin (Gt) is a member of a family of receptor-coupled signal-transducing guanine nucleotide (GN) binding proteins (G-proteins). Light-activated rhodopsin is known to catalyze GN exchange on Gt, resulting in the formation of the active state of the Gt alpha-GTP complex. However, purified preparations of Gt have been shown to exchange GN in the absence of activated receptors [Wessling-Resnick, M., & Johnson, G. L. (1987) Biochemistry 26, 4316-4323]. To evaluate the role of rhodopsin in the activation of Gt, we studied GN-binding characteristics of different preparations of Gt. Gt preparations obtained rom the supernate of GTP-treated bovine rod outer segment (ROS) disks, followed by removal of free GTP on a Sephadex G-25 column, bound GTP gamma S at 30 degrees C in the absence of added exogenous rhodopsin with an activity of 1 mol of GTP gamma S bound/mol of Gt (Gt-I preparations). Binding of GTP gamma S to Gt-I preparations closely correlated with the activation of ROS disk cGMP phosphodiesterase. GN-binding activity of Gt-I preparations was dependent on reaction temperature, and no binding was observed at 4 degrees C. In the presence of 10 microM bleached rhodopsin, Gt-I preparations bound GTP gamma S at 4 degrees C. However, hexylagarose chromatography of Gt-I preparations led to a preparation of Gt that showed less than 0.1 mol/mol binding activity following 60-min incubation at 30 degrees C in the absence of rhodopsin (Gt-II preparations).(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of DNA cleavage by the restriction enzymes SalPI and PstI.

Methods for obtaining highly active, exonuclease-free, stable preparations of the Streptomyces albus P restriction enzyme SalPI are described. SalPI and its isoschizomer PstI (from the taxonomically distant Providencia stuartii 164) both cleave their recognition sequence (5'-CTGCAG-3') to generate fragments terminating in tetranucleotide 3' extensions whose sequence is 5'-TGCA-3'. Bacteriophage R4G2 DNA, protected against SalPI cleavage by pregrowth on S. albus P, is also protected against PstI cleavage; and total DNA of both S. albus P and P. stuartii 164 is resistant to cleavage by both enzymes.     

Lipid requirements for reconstitution of the delipidated beta-adrenergic receptor and the regulatory protein

The role of lipids in the interaction of the beta-adrenergic receptor (R) with the regulatory protein (Gs) was investigated. Solubilized preparations of R and of Gs from turkey erythrocytes were delipidated by gel filtration. They were subsequently combined and reconstituted by the addition of various lipids. When reconstitution was carried out in the presence of soybean lipids, Gs could be fully activated via R by addition of hormone plus GTP gamma S. In contrast, purified phospholipids or a phospholipid fraction from soybean failed to produce an active system. Fractionation of soybean lipids revealed that acetone-soluble neutral lipids are essential for the reconstitution of a hormone responsive system. The acetone fraction could be replaced by specific neutral lipids such as alpha-tocopherol or cholesteryl arachidonate while a mixture of phosphatidylethanolamine, -choline and -serine satisfied the phospholipid requirement of the system.     

High affinity quinuclidinyl benzilate binding to rat parotid membranes requires muscarinic receptor. G protein interactions

The binding of the non-selective muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) to rat parotid membranes was characterized. Under equilibrium conditions, [3H]QNB bound to a homogenous population of muscarinic receptors (Kd, 118 +/- 19 pM; Bmax, 572 +/- 42 fmol/mg membrane protein, n = 12). The addition of G protein activators AlF4- or guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) + Mg2+ increased the Kd by 77 +/- 7% (n = 4, P less than 0.05) and 83 +/- 27% (n = 7, P less than 0.05), respectively, without a change in the Bmax or homogeneity of the binding site. GTP gamma S added without exogenous Mg2+ did not affect [3H]QNB binding. Thus, optimal QNB binding requires a muscarinic receptor/G protein interaction.     

Regulation of geranylgeranyl pyrophosphate synthase in the proliferation of rat FRTL-5 cells: involvement of both cAMP-PKA and PI3-AKT pathways

We have reported that geranylgeranyl pyrophosphate (GGPP), one of the isoprenoids in the mevalonate pathway, plays an essential role for cell growth through the geranylgeranylation of Rho small GTPases, which control the degradation of P27Kip1 at G1/S transition in rat thyroid FRTL-5 cells. Since GGPP is synthesized from isopentenyl pyrophosphate (IPP) and farnesyl pyrophosphate (FPP) by GGPP synthase, we analyzed the regulatory roles of GGPP synthase in the proliferation of FRTL-5 cells stimulated by thyrotropin and insulin in the presence of 5% calf serum (TSH+Ins). We found that: (1) GGPP synthase was activated at G1/S transition with increasing mRNA accumulation followed by protein expression, (2) pravastatin, an inhibitor of HMG-CoA reductase, did not suppress the increasing activity of GGPP synthase with its protein expression although it inhibits proliferation in growth-stimulated FRTL-5 cells, (3) forskolin stimulated proliferation with activation of GGPP synthase in FRTL-5 cells, and (4) LY294002, an inhibitor of phosphatidylinositol 3-kinase, inhibited proliferation with the decreasing activity of GGPP synthase in growth-stimulated FRTL-5 cells. These data indicated that growth stimulation by TSH+Ins increased the activity of GGPP synthase with its increasing protein expression from G1/S transition, in which both cAMP-PKA and PI3-kinase pathways are involved in the proliferation of FRTL-5 cells.     

Activation of a Gi protein in mouse sperm membranes by solubilized proteins of the zona pellucida, the egg's extracellular matrix.

Zona pellucida (ZP)-induced acrosomal exocytosis in mammalian spermatozoa is thought to be mediated by signal transduction cascades similar to those found in hormonally responsive cells. In order to characterize this process further, we have examined the role of GTP-binding regulatory proteins (G proteins) in coupling sperm-ZP interaction to intracellular second messenger systems in mouse sperm. An in vitro signal transduction assay was developed to assess ZP-G protein dynamics in sperm membrane preparations. Guanosine 5'-3-O-(thio)triphosphate (GTP gamma S), a poorly hydrolyzable analogue of GTP, bound to these membranes in a specific and concentration-dependent fashion which reached saturation at 100 nM. Incubation of the membrane preparations with heat-solubilized ZP resulted in a significant increase in specific GTP gamma S binding in a concentration-dependent fashion with a half-maximal response at 1.25-2 ZP/microliters. Solubilized ZP also caused a significant increase in high affinity GTPase activity in the membranes over basal levels. Mastoparan increased specific GTP gamma S binding to the sperm membranes and stimulated high-affinity membrane GTPase activity to levels consistently greater than that seen with the solubilized ZP. Mastoparan, together with solubilized ZP, gave the same level of stimulation of GTP gamma S binding as mastoparan alone. Pertussis toxin completely inhibited the ZP-stimulated GTP gamma S binding, but only decreased mastoparan-stimulated GTP gamma S binding by 70-80%. Purified ZP3, the ZP component which possesses quantitatively all of the acrosomal exocytosis-inducing activity of the intact ZP, stimulated GTP gamma S binding to the same level as solubilized ZP; ZP1 and ZP2 did not stimulate GTP gamma S binding. ZP from fertilized eggs (ZPf), which does not possess acrosome reaction-inducing activity, also failed to stimulate GTP gamma S binding to sperm membranes. These data demonstrate the direct activation of a Gi protein in sperm membrane preparations in response to the ZP glycoprotein, ZP3, that induces the acrosome reaction. These data imply that Gi protein activation is an early event in the signal sequence leading to sperm acrosomal exocytosis.     

Rhodopsin and the retinal G-protein distinguish among G-protein beta gamma subunit forms

The beta gamma subunits of G-proteins are composed of closely related beta 35 and beta 36 subunits tightly associated with diverse 6-10 kDa gamma subunits. We have developed a reconstitution assay using rhodopsin-catalyzed guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding to resolved alpha subunit of the retinal G-protein transducin (Gt alpha) to quantitate the activity of beta gamma proteins. Rhodopsin facilitates the exchange of GTP gamma S for GDP bound to Gt alpha beta gamma with a 60-fold higher apparent affinity than for Gt alpha alone. At limiting rhodopsin, G-protein-derived beta gamma subunits catalytically enhance the rate of GTP gamma S binding to resolved Gt alpha. The isolated beta gamma subunit of retinal G-protein (beta 1, gamma 1 genes) facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha in a concentration-dependent manner (K0.5 = 254 +/- 21 nM). Purified human placental beta 35 gamma, composed of beta 2 gene product and gamma-placenta protein (Evans, T., Fawzi, A., Fraser, E.D., Brown, L.M., and Northup, J.K. (1987) J. Biol. Chem. 262, 176-181), substitutes for Gt beta gamma reconstitution of rhodopsin with Gt alpha. However, human placental beta 35 gamma facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha with a higher apparent affinity than Gt beta gamma (K0.5 = 76 +/- 54 nM). As an alternative assay for these interactions, we have examined pertussis toxin-catalyzed ADP-ribosylation of the Gt alpha subunit which is markedly enhanced in rate by beta gamma subunits. Quantitative analyses of rates of pertussis modification reveal no differences in apparent affinity between Gt beta gamma and human placental beta 35 gamma (K0.5 values of 49 +/- 29 and 70 +/- 24 nM, respectively). Thus, the Gt alpha subunit alone does not distinguish among the beta gamma subunit forms. These results clearly show a high degree of functional homology among the beta 35 and beta 36 subunits of G-proteins for interaction with Gt alpha and rhodopsin, and establish a simple functional assay for the beta gamma subunits of G-proteins. Our data also suggest a specificity of recognition of beta gamma subunit forms which is dependent both on Gt alpha and rhodopsin. These results may indicate that the recently uncovered diversity in the expression of beta gamma subunit forms may complement the diversity of G alpha subunits in providing for specific receptor recognition of G-proteins.     

Interaction of retinal transducin with guanosine triphosphate analogues: specificity of the gamma-phosphate binding region

The interaction of six hydrolysis-resistant analogues of GTP with transducin, the signal-coupling protein in vertebrate photoreceptors, was investigated. GppNHp and GppCH2p differ from GTP at the bridging position between the beta- and gamma-phosphate groups. The other analogues studied (GTP gamma F, GTP gamma OMe, GTP gamma OPh, and GTP gamma S) differ from GTP in containing a substituent on the gamma-phosphorus atom or at a nonbridging gamma-oxygen atom. Competition binding experiments were carried out by adding an analogue, [alpha-32P]GTP, and a catalytic amount of photoexcited rhodopsin (R) to transducin and measuring the amount of bound [gamma-32P]GTP. The order of effectiveness of these analogues in binding to transducin was GTP gamma S greater than GTP much greater than GppNHp greater than GTP gamma OPh greater than GTP gamma OMe greater than GppCH2p greater than GTP gamma F A second assay measured the effectiveness of GTP gamma S, GppNHp, and GppCH2p in eluting transducin from disc membranes containing R. The basis of this assay is that transducin is released from disc membranes when it is activated to the GTP form. The relative potency of these three analogues in converting transducin from a membrane-bound to a soluble form was 1000, 75, and 1, respectively. Stimulation of cGMP phosphodiesterase activity served as a third criterion of the interaction of these analogues with transducin. The order of effectiveness of these analogues in promoting the transducin-mediated activation of the phosphodiesterase was GTP gamma S greater than GTP much greater than GppNHp greater than GTP gamma OPh much greater than GppCH2p greater than GTP gamma OMe greater than GTP gamma F GTP gamma S was more than a 1000 times as potent as GTP gamma F in activating the phosphodiesterase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fc gamma receptor signal transduction in natural killer cells. Coupling to phospholipase C via a G protein-independent, but tyrosine kinase-dependent pathway.

Antibody-dependent cellular cytotoxicity is initiated when low affinity Fc receptors (Fc gamma R type III/CD16) on NK cells bind to sensitized (i.e., antibody coated) target cells. Fc gamma R cross-linkage induces the activation of phospholipase C (PLC), which hydrolyses membrane phosphoinositides, generating inositol-1,4,5-trisphosphate and sn-1,2-diacylglycerol as second messengers. However, the mechanism that couples Fc gamma R stimulation to PLC activation remains unknown. In this study, we investigated whether the Fc gamma R is coupled to PLC via a guanine nucleotide-binding (G) protein or an alternative pathway. Stimulation of electropermeabilized human NK cells with GTP gamma S induced inositol phosphate (IP) release, indicating the presence of a G protein-linked PLC activity in these cells. However, stimulation with both anti-Fc gamma R mAb and GTP gamma S provoked additive rather than synergistic increases in IP formation. Furthermore, exogenous GDP strongly inhibited GTP gamma S-stimulated IP release, but failed to inhibit the response to anti-Fc gamma R mAb stimulation. These results suggested GTP gamma S and anti-Fc gamma R mAb activated PLC through distinct regulatory mechanisms, and that Fc gamma R was not linked to PLC via a G protein. Hence, an alternative transduction mechanism for Fc gamma R-PLC coupling was considered. Antibody-mediated Fc gamma R cross-linkage was shown to rapidly stimulate tyrosine phosphorylation of multiple proteins in NK cells. Pretreatment with the tyrosine kinase inhibitor, herbimycin A, inhibited these phosphorylation events and disrupted the coupling between Fc gamma R ligation and PLC activation. These observations suggest that Fc gamma R in NK cell is coupled to PLC via a G protein-independent, but tyrosine kinase-dependent pathway.     

Hetero-oligomerization between GABAA and GABAB receptors regulates GABAB receptor trafficking

The neurotransmitter gamma-aminobutyric acid (GABA) mediates inhibitory signaling in the brain via stimulation of both GABA(A) receptors (GABA(A)R), which are chloride-permeant ion channels, and GABA(B) receptors (GABA(B)R), which signal through coupling to G proteins. Here we report physical interactions between these two different classes of GABA receptor. Association of the GABA(B) receptor 1 (GABA(B)R1) with the GABA(A) receptor gamma2S subunit robustly promotes cell surface expression of GABA(B)R1 in the absence of GABA(B)R2, a closely related GABA(B) receptor that is usually required for efficient trafficking of GABA(B)R1 to the cell surface. The GABA(B)R1/gamma2S complex is not detectably functional when expressed alone, as assessed in both ERK activation assays and physiological analyses in oocytes. However, the gamma2S subunit associates not only with GABA(B)R1 alone but also with the functional GABA(B)R1/GABA(B)R2 heterodimer to markedly enhance GABA(B) receptor internalization in response to agonist stimulation. These findings reveal that the GABA(B)R1/gamma2S interaction results in the regulation of multiple aspects of GABA(B) receptor trafficking, allowing for cross-talk between these two distinct classes of GABA receptor.     

Human progesterone receptor binding to mouse mammary tumor virus deoxyribonucleic acid: dependence on hormone and nonreceptor nuclear factor(s)

Using crude progesterone receptor preparations from T47D human breast cancer cells, we show by immunoprecipitation assay that receptor specifically and with high affinity recognizes the hormone response element (HRE) of the mouse mammary tumor virus (MMTV). The use of crude preparations minimizes alterations of receptors or loss of associated factors that may occur during purification. Specific binding was obtained at 1:1 molar ratios of receptor to DNA, and HRE sequences are recognized with an affinity at least 3 orders of magnitude greater than nonspecific DNA. We have compared the DNA-binding activities of different forms of progesterone receptors. The unliganded 8S cytosol receptor had low but detectable binding activity for MMTV DNA. Addition of hormone to cytosol produced a small but consistent 2.5-fold increase. In vitro methods of transforming cytosol receptors from an 8S to a 4S species failed to increase DNA-binding further. By contrast, 4S receptors bound by R5020 in whole cells and extracted from nuclei by salt, displayed a substantially higher (average, 11-fold) binding activity than an equal number of unliganded cytosol receptors. The dissociation constants for cytosol and nuclear receptor binding to MMTV DNA were similar (approximately 2 x 10(-9) M). Thus, nuclear receptors possess a higher capacity for binding to specific recognition sequences. These results suggest that hormone or a hormone-dependent mechanism increases the intrinsic DNA-binding activity of receptors independent of receptor transformation from 8S to 4S. Further experiments indicate that a nonreceptor activity in nuclear extracts can increase the sequence-specific DNA-binding activity of cytosol receptors. This activity is present in both T47D cells and receptor-negative MDA-231 cells. We conclude that the higher DNA-binding activity of the nuclear receptor-hormone complex is due in part to receptor interaction with other nuclear proteins or factors. Such interactions may function to maintain receptors in a disaggregated active complex or to stabilize their binding to specific DNA sites.     

Affinity of transducin for photoactivated rhodopsin: dependence on nucleotide binding state

The interaction of the rod GTP binding protein, Transducin (G(t)), with bleached Rhodopsin (R(*)) was investigated by measuring radiolabeled guanine nucleotide binding to and release from soluble and/or membrane-bound G(t) by reconstituting G(t) containing bound GDP (G(t-)GDP) or the hydrolysis-resistant GTP analog guanylyl imidodiphosphate (G(t-)p[NH]ppG) with R* under physiological conditions. Release of GDP and p[NH]ppG from G(t) occurred to the same extent and with the same light sensitivity both in the presence and absence of added GTP. Significant amounts of G(t) without bound nucleotide (G(t-)) were generated. When ROS containing bleached rhodopsin (R(*)) were centrifuged in low ionic strength buffer, G(t-) remained associated with the membrane fraction, whereas G(t-)GDP remained in the soluble fraction. These results suggest that G(t-)GDP and G(t-)p[NH]ppG have similar affinities for R(*). The results also suggest that G(t-), rather than G(t-)GDP, is the moiety which exhibits tight, "light-induced" binding to rhodopsin.     

Reconstitution of agonist-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis using purified m1 muscarinic receptor, Gq/11, and phospholipase C-beta 1.

We describe the reconstitution using purified proteins of the m1 muscarinic cholinergic pathway that activates phosphatidylinositol 4,5-bisphosphate-specific phospholipase C via the G protein Gq/11. Recombinant m1 muscarinic receptor was co-reconstituted in lipid vesicles with either hepatic Gq/11 or with cerebral alpha q/11 and beta gamma subunits. The rate of [35S]GTP gamma S binding to the reconstituted vesicles was stimulated 20-50-fold by agonist. Maximal receptor-catalyzed binding was 7 mol of GTP gamma S bound per mol of receptor. The m2 muscarinic receptor was a poor activator of Gq/11. The binding of [alpha-32P]GTP to [gamma-32P]GTP to m1/Gq/11 vesicles indicated that the receptor could maintain up to 40% of the total coupled Gq/11 in the GTP bound state. The rate of hydrolysis of bound GTP, 0.8 min-1, is consistent with the rate predicted from the GTP binding data but is 3-5-fold lower than rates reported for other trimeric G proteins. Agonist-stimulated photo-affinity labeling with gamma-(4-azidoanilido)-[alpha-32P]GTP indicated that the receptor catalyzed binding to both alpha q and alpha 11 with about equal efficiency. Receptor-catalyzed activation of Gq/11 by GTP gamma S, measured as the ability to activate purified phospholipase C-beta 1, paralleled receptor-catalyzed [35S]GTP gamma S binding. Co-reconstitution of receptor, Gq/11, and phospholipase C-beta 1 restored GTP gamma S-dependent carbachol-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate. The m1 receptor, Gq/11, and phospholipase C-beta 1 are thus sufficient to initiate the hormonal inositol trisphosphate/diacylglycerol signaling pathway without additional proteins.     

Interaction of fibrin(ogen) with leukocyte receptor alpha M beta 2 (Mac-1): further characterization and identification of a novel binding region within the central domain of the fibrinogen gamma-module

The fibrinogen gamma-module sequences, gamma190-202 or P1, and gamma377-395 or P2, were implicated in interaction with the alpha(M)I-domain of the leukocyte receptor alpha(M)beta(2). P1 is an integral part of the gamma-module central domain, while P2 is inserted into this domain forming an antiparallel beta-strand with P1. We hypothesized earlier that separation of P2 from P1 may regulate interaction of fibrin(ogen) with leukocytes during the inflammatory response. To test the relative contributions of these sequences to the interaction and the effect of their separation, we prepared the recombinant gamma-module (gamma148-411) and its halves, gamma148-286 and gamma287-411 fragments containing P1 and P2, respectively, and evaluated their affinities for the recombinant alpha(M)I-domain. In a solid-phase binding assay, the immobilized gamma-module exhibited high affinity for alpha(M)I (K(d) = 22 nM), while the affinities of the isolated gamma148-286 and gamma287-411 halves were much lower (K(d)'s = 521 and 194 nM, respectively), indicating that both halves contribute to the interaction in a synergistic manner. This is consistent with the above hypothesis. Further, we prepared the recombinant gamma148-191 and gamma192-286 fragments corresponding to the NH(2)-terminal and central domains, respectively, as well as gamma148-226 containing P1, and tested their interaction with alpha(M)I. The immobilized gamma192-286 fragment bound to alpha(M)I with K(d) = 559 nM, while both gamma148-191 and gamma148-226 failed to bind suggesting that P1 does not contribute substantially to the binding and that the binding occurs mainly through the gamma227-286 region. To further localize a putative binding sequence, we cleaved gamma192-286 and analyzed the resulting peptides. The only alpha(M)I-binding activity was associated with the gamma228-253 peptide, indicating that this region of the central domain contains a novel alpha(M)beta(2)-binding sequence.     

cGMP phosphodiesterase dependent light-induced scattering changes in suspensions of retinal disc membranes.

Light-induced GTP-dependent scattering changes are studied in suspensions of retinal disc membranes to which one or both of the purified proteins involved in the phototransduction mechanism (G-protein and cGMP phosphodiesterase) are reassociated; a scattering change which depends on the presence of both G-protein (G) and inhibited cGMP phosphodiesterase (PDE) and on an ATPase-dependent process, previously described in Bennett [(1986) Eur. J. Biochem. 157, 487-495] is compared to the signal observed in the absence of PDE or of ATP and to PDE activity. The same signal can also be induced either in the dark or in the light by addition of preactivated G in the presence of inhibited PDE. This PDE-dependent scattering change is composed of two components (fast and slow); the variation of the amplitude and kinetics of both components with PDE or G concentration is similar to the variation of the active PDE state with two activator GGTP molecules (G with GTP bound), calculated with dissociation constants previously reported for the interaction between GGTP and PDE [Bennett, N., & Clerc, A. (1989) Biochemistry 28, 7418-7424]. The two components are therefore proposed to be associated with processes which depend on the formation of the active PDE state with two activators.     

Engineering of Sulfolobus solfataricus HMG-CoA reductase to a form whose activity is regulated by phosphorylation and dephosphorylation

There are two classes of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase: the class I enzymes of eukaryotes and some archaea, and the class II enzymes of certain eubacteria. The activity of the class I Syrian hamster HMG-CoA reductase is regulated by phosphorylation-dephosphorylation of Ser871. Phosphorylation apparently prevents the active site histidine, His865, from protonating the inhibitory coenzyme A thioanion prior to its release from the enzyme. Structural evidence for this hypothesis is, however, lacking. The HMG-CoA reductase of the thermophilic archaeon Sulfolobus solfataricus, whose stability recommends it for physical studies, lacks both a phosphoacceptor serine and a protein kinase recognition motif. Consequently, its activity is not regulated by phosphorylation. We therefore employed site-directed mutagenesis to engineer an appropriately located phosphoacceptor serine and cAMP-dependent protein kinase recognition motif. Substitution of serine for Ala406, the apparent cognate of hamster Ser871, and replacement of Leu403 and Gly404 by arginine created S. solfataricus mutant enzyme L403R/G404R/A406S. The general properties of enzyme L403R/G404R/A406S (K(m) values, V(max), optimal pH and temperature) were essentially those of the wild-type enzyme. Exposure of enzyme L403R/G404R/A406S to [gamma-(32)P]ATP and cAMP-dependent protein kinase was accompanied by incorporation of (32)P(i) and by a parallel decrease in catalytic activity. Subsequent treatment with a protein phosphatase released enzyme-bound (32)P(i) and restored activity to pretreatment levels. The regulatory properties of enzyme L403R/G404R/A406S thus match those of the hamster enzyme. Solution of the three-dimensional structures of the phospho and dephospho forms of this mutant enzyme thus should reveal structural features critical for regulation of the activity of a class I HMG-CoA reductase.     

Regulation of G proteins by human 5-HT1a receptor TM3/i2 and TM5/i3 loop peptides

A bioactive synthetic 11 amino acid peptide probe (P11) was constructed according to the published sequence of the human 5HT1a receptor. The probe was used to enhance understanding of cytoplasmic loop 2/G protein coupling and activation. Additionally, two peptides (P8, P9) from the cytoplasmic loop 3 region were synthesized and studied. These probes were tested in a model system of human 5HT1a receptor stably expressed in Chinese Hamster Ovary cells. In agonist inhibition studies, P11 was active in all three receptor preparations tested: whole cells, membrane bound, and solubilized. In analyses of the membrane bound receptor system, P11 demonstrated uncompetitive inhibition characteristics. When forskolin-stimulated cAMP levels were measured, P11 was inactive in this negatively coupled system. Utilizing a [35S]gamma-S-GTP incorporation assay, P11 was unable to stimulate G protein incorporation of GTP. While P8 and P9 were also broadly active as non-competitive agonist inhibitors, their characteristics differed in the signal transduction system. P8 and P9 did not significantly change forskolin-stimulated cAMP levels. However, P8 increased [35S]gamma-S-GTP incorporation, while P9 decreased incorporation. Thus, P11, a synthetic peptide from the TM3/i2 region of the receptor, provides suggestive evidence that this receptor region is involved in G protein coupling but not activation. On the other hand, P8 and P9 activities suggest that the TM5/i3 region is involved in both coupling to and regulation of G protein activity. The current evidence from these cytoplasmic loop regions is discussed in the overall context of an emerging model for human 5HT1a receptor-G protein interactions.     

Sites of interaction between rod G-protein alpha-subunit and cGMP-phosphodiesterase gamma-subunit. Implications for the phosphodiesterase activation mechanism.

In photoreceptor cells of vertebrates light activates a series of protein-protein interactions resulting in activation of a cGMP-phosphodiesterase (PDE). Interaction between the GTP-bound form of rod G-protein alpha-subunit (alpha t) and PDE inhibitory gamma-subunit (P gamma) is a key event for effector enzyme activation. This interaction has been studied using P gamma labeled with the fluorescent probe, lucifer yellow vinyl sulfone, at Cys-68 (P gamma LY) and sites of interaction on alpha t and P gamma have been investigated. Addition of alpha tGTP gamma S to P gamma LY produced a 3.2-fold increase in the fluorescence of P gamma LY. The Kd for alpha tGTP gamma S.P gamma LY interaction was 36 nM. Addition of 1 microM alpha tGDP had no effect, but in the presence of A1F4-, alpha tGDP increased P gamma LY fluorescence by 85%. When P gamma LY was reconstituted with P alpha beta to form fluorescent holo-PDE, alpha tGTP gamma S increased the fluorescence of holo-PDE with a K0.5 = 0.7 microM. Also, alpha tGTP gamma S stimulated the activity of this PDE over an identical range of concentrations with a similar K0.5 (0.6 microM). alpha tGTP gamma S enhanced the fluorescence of a COOH-terminal P gamma fragment, P gamma LY-46-87, as well (Kd = 1.5 microM). We demonstrate that an alpha t peptide, alpha t-293-314, which activated PDE (Rarick, H. M., Artemyev, N. O., and Hamm, H. E. (1992) Science 256, 1031-1033), mediates PDE activation by interacting with the P gamma-46-87 region. Peptide alpha t-293-314 bound to P gamma LY (K0.5 = 1.2 microM) as well as to the carboxyl-terminal P gamma fragment, P gamma LY-46-87 (K0.5 = 1.7 microM) as measured by fluorescence increase, while other alpha t peptides had no effect. A peptide from the P gamma central region, P gamma-24-46, blocked the interaction between alpha tGTP gamma S and P gamma LY. The Kd for alpha tGTP gamma S.P gamma-24-46 interaction was 0.7 microM. On the other hand, P gamma-24-46 had no effect on alpha t-293-314 interaction with P gamma LY. Our data suggest that there are at least two distinct sites of interaction between alpha tGTP gamma S and P gamma. The interaction between alpha t-293-314 and P gamma-46-87 is important for PDE activation.(ABSTRACT TRUNCATED AT 400 WORDS)