Affinity labeling of the guanine nucleotide binding site of transducin by pyridoxal 5'-phosphate

Transducin (T), a guanine nucleotide binding regulatory protein composed of -, -, and -subunits, serves as an intermediary between rhodopsin and cGMP phosphodiesterase during signaling in the visual process. Pyridoxal 5-phosphate (PLP), a reagent that has been used to modify enzymes that bind phosphorylated substrates, was probed here as an affinity label for T. PLP inhibited the guanine nucleotide binding activity of T in a concentration dependent manner, and was covalently incorporated into the protein in the presence of [³H]NaBH₄. Approximately 1 mol of ³H was bound per mol of T. GTP and GTP analogs appreciably hindered the incorporation of ³H to T, suggesting that PLP specifically modified the protein active site. Interestingly, PLP modified both the - and -subunits of T. Moreover, PLP in the presence of GDP behaved as a GTP analog, since this mixture was capable of dissociating T from T:photoactivated rhodopsin complexes.     

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.     

Distinct guanine nucleotide binding and release properties of the three Gi proteins

The native pertussis toxin sensitive GTP-binding proteins (Gi proteins) were individually resolved, and their guanine nucleotide binding and release properties were studied. Gi2 and Gi3, the two major GTP-binding proteins of human erythrocytes, were purified to apparent homogeneity by fast protein liquid chromatography. Gi1 was purified from bovine brain. The three proteins bound 0.6-0.85 mol of guanosine 5'-O-(thio-triphosphate (GTP gamma S)/mol of protein with similar affinities (KD(app) = 50-100 nM). The rate of [35S]GTP gamma S binding to Gi2 was 5-8-fold faster than to Gi1 or Gi3 at 2 mm Mg2+. There were no observable differences in the binding characteristics between bovine brain Gi1 and human erythrocyte Gi3. At 50 mM Mg2+, all three Gi proteins exhibited fast binding, although Gi1 and Gi3 were marginally slower than Gi2. All three Gi proteins exhibited different rates of [32P]GDP release at 2 mM Mg2+. GDP release from Gi2 was severalfold faster than that from Gi1 or Gi3. GDP release rates from Gi1 and Gi3 were similar, although Gi3 was somewhat (60-80%) faster than Gi1. These data indicate that rates of GDP release and GTP binding may be independently regulated for these three proteins and that the relative proportions of Gi2/Gi1 or Gi2/Gi3 will be a crucial factor in determining the kinetics of signal transduction through Gi-coupled effectors.     

Physiological correlation between nucleoside-diphosphate kinase and the enzyme-associated guanine nucleotide binding proteins

The physiological correlation between NDP-kinase and the enzyme-associated guanine nucleotide binding proteins (G1 and G2) has been studied in vitro. It was found that incubation of the phosphoenzyme (enzyme-bound high-energy phosphate intermediate) of NDP-kinases with one of the nucleoside 5'-diphosphates (NDPs) in the presence of divalent cations (Mg2+ and Ca2+) results in the formation of nucleoside 5'-triphosphates (NTPs) within 40 sec even at low temperatures (below 4 degrees C) without strict base-specificity; and high-energy phosphates on the phosphoenzyme can transfer preferentially to GDP on the guanine nucleotide binding proteins (G1, G2 and r-p21 protein) in the presence of 0.25 mM Ca2+ or 1 mM Mg2+ even if any other NDPs are present in the reaction mixtures. These observations suggest that NDP-kinase may be responsible for the phosphate-transfer between GDP on the guanine nucleotide binding proteins and its phosphoenzyme.     

Fluorescently labelled guanine nucleotide binding proteins to analyse elementary steps of GAP-catalysed reactions

Downregulation of small guanine nucleotide-binding proteins (GNBPs) requires the interaction with their corresponding GTPase-activating proteins (GAPs), which increase the slow intrinsic GTPase reaction by several orders of magnitude. On the basis of the structure of H-Ras in complex with the catalytic domain of p120-GAP, we have developed a set of site-specifically labelled Ras-variants, one of which turned out to be particularly sensitive for studying the interaction with Ras-specific GAPs. This specific fluorescent reporter group and the use of manganese to increase the rate of the chemical reaction step allowed us to identify differences in the rate-limiting step of either the GAP-334 or NF1-333 catalyzed reaction. The assay was also applied to study the interaction of the Ras-related protein Rap1B with Rap1GAP, for which no detailed kinetic analysis was available. Single-turnover experiments of this reaction show that the low affinity of the complex (50 microM) is due to a slow association rate as well as a fast dissociation rate. RapGAP promotes AlFx binding to Rap1B, even though it does not contain a catalytic arginine. The rate-limiting step of the RapGAP catalysed reaction is release of inorganic phosphate, which is about five times slower than the chemical cleavage step. Our data reveal marked differences in GAP/target interactions even between closely related systems and suggest that the fluorescent reporter group method might be generally applicable to many other GNBPs and their cognate GAPs.     

Kinetic analysis of the binding of guanine nucleotide to bovine brain smg p25A

Bovine brain smg p25A, a guanine nucleotide-binding protein with a Mr of about 25,000, bound specifically GTP, guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) and GDP. The initial velocities of the binding of GTP gamma S to GDP-bound smg p25A and the dissociation of GDP from this protein increased by decreasing Mg2+ concentrations or increasing NaCl concentrations. The initial velocity of the binding of GTP gamma S to GDP-free smg p25A was not affected by changing Mg2+ concentrations. These results indicate that the dissociation of GDP from smg p25A limits the binding of GTP to this protein, and suggest that there is a protein stimulating the dissociation of GDP from smg p25A and thereby stimulating the binding of GTP to this protein in mammalian tissues. In fact, the protein stimulating the dissociation of GDP, but not of GTP gamma S, from smg p25A was detected in bovine brain cytosol.     

The intrinsic fluorescence of the alpha subunit of transducin. Measurement of receptor-dependent guanine nucleotide exchange

We have made use of the enhancement of the intrinsic fluorescence of the alpha subunit of transducin (alpha T), which accompanies guanine nucleotide exchange, to follow the reconstituted interactions between pure rhodopsin and pure transducin in phospholipid vesicles. When the pure alpha T.GDP complex is added to lipid vesicles containing rhodopsin and the beta gamma T complex, a light- and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-dependent enhancement of the fluorescence emission of alpha T is observed. When GTP is substituted for GTP gamma S, a similar enhancement of the intrinsic fluorescence of alpha T occurs; however, this enhancement is transient and precedes a fluorescence decay which is complete in 2-5 min. The fact that the fluorescence decay is specifically induced by GTP and is not observed either with nonhydrolyzable GTP analogs or with NaF (plus AlCl3) indicates that the decay represents GTP hydrolysis in alpha T. The dose-response profiles for the effects of the beta gamma T complex on the rate and extent of the GTP gamma S-stimulated fluorescence enhancement of alpha T have also been examined. The addition of relatively low levels of beta gamma T to these reconstituted systems can promote the GTP gamma S-stimulated enhancement of the fluorescence of multiple alpha T subunits with half-maximal enhancement occurring at alpha T:beta gamma T ratios of 150:1. These findings are consistent with earlier suggestions (Fung, B. K.-K. (1983) J. Biol. Chem. 258, 10495-10502) that the beta gamma T subunit dissociates from alpha T as a result of the GDP-GTP exchange reaction and thus can act catalytically to promote the activation of a number of inactive alpha T species. However, the dependence of the rate of the GTP gamma S-stimulated fluorescence enhancement on beta gamma T is complex and cannot be explained adequately by simple models where alpha T-beta gamma T interactions (or rhodopsin-transducin interactions) are rate-limiting for the rhodopsin-stimulated activation of the alpha T subunits. Overall, the results reported here demonstrate that fluorescence spectroscopy can be used to monitor directly a receptor-catalyzed activation-deactivation cycle of a GTP-binding protein within a lipid milieu.     

Functional differences in the beta gamma complexes of transducin and the inhibitory guanine nucleotide regulatory protein

We have examined the mechanism of inhibition of adenylate cyclase using the purified alpha and beta gamma subunits of bovine brain inhibitory guanine nucleotide regulatory protein (Ni) (i.e., alpha i and beta gamma N) and bovine retinal transducin (alpha T and beta gamma T) in reconstituted phospholipid vesicle systems. The addition of beta gamma N or beta gamma T to lipid vesicles containing the pure stimulatory guanine nucleotide regulatory protein (Ns) from human erythrocytes as well as a resolved preparation of the catalytic moiety (C) of bovine caudate adenylate cyclase results in significant inhibition of guanine nucleotide stimulated cyclase activity (80-90%). The inhibition by these beta gamma subunit complexes appears to fully account for the inhibitory effects observed with holo-Ni or holotransducin. A variety of structure-function comparisons of the beta gamma N and beta gamma T complexes were performed in order to further probe the molecular mechanisms involved in the inhibitory pathway. Whereas the beta subunits of beta gamma N and beta gamma T appear to be very similar, if not identical, on the basis of comparisons of their gel electrophoretic mobility and immunological cross-reactivity, clear differences exist in the apparent structures of gamma N and gamma T. Interestingly, functional differences are observed in the effectiveness of these two beta gamma complexes to inhibit adenylate cyclase activity. Specifically, while both beta gamma N and beta gamma T are capable of effecting significant levels of inhibition of the guanine nucleotide stimulated activities, the beta gamma N complex is consistently more potent than beta gamma T in inhibiting these activities.(ABSTRACT TRUNCATED AT 250 WORDS)     

The transitory complex between photoexcited rhodopsin and transducin. Reciprocal interaction between the retinal site in rhodopsin and the nucleotide site in transducin

In the first step of the visual transduction cascade a photoexcited rhodopsin molecule, R*ret, binds to a GDP-carrying transducin molecule, TGDP. The R*-T interaction causes the opening of the nucleotide site in T and catalyzes the GDP/GTP exchange by allowing the release of the GDP. We have studied the influences on this R*-T transitory complex of the occupancies of the nucleotide site in T and the retinal site in rhodopsin. After elimination of the GDP released from the bound transducin, the complex, named R*ret-te (ret for retinal present, e for nucleotide site empty) remains stabilized almost indefinitely in a medium whose ionic composition is close to physiological. In this complex the bound Te retains a lasting ability to interact with GDP or GTP, and R*ret remains spectroscopically in the meta-II state, by contrast with free R*ret which decays to opsin and free retinal. Hence the R*-T interaction which opens the nucleotide site in T conversely blocks the retinal site in R*ret. Upon prolonged incubation in a low-ionic-strength medium the R*ret-Tc complex dissociates partially, but the liberated Te is then unable to rebind GDP or GTP, even in the presence of R*ret, it is probably denaturated. Upon treatment of the R*ret-Te complex by a high concentration of hydroxylamine, the retinal can be removed from the rhodopsin. The Re-Te complex remains stable and the complexed transducin keeps its capacity to bind GTP. TGTP then dissociates from Re. The liberated Re loses its capacity to interact with a new transducin. These data are integrated into a discussion of the development of the cascade. We stress that affinities, i.e. dissociation equilibrium constants, are insufficient to describe the flow of reactions triggered by one R*ret molecule. It depends on a few critical rapid binding and dissociation processes, and is practically insensitive to other slow ones, hence to the values of affinities that express only the ratio of kinetics constants. The effect of the R*-T interaction on the retinal site in rhodopsin is analogous to the effect of the binding of a G-protein on the apparent affinity of a receptor for its agonist.     

Thermodynamics of Ca2+ binding to calmodulin and its tryptic fragments.

The binding of Ca2+ to calmodulin and its two tryptic fragments has been studied using microcalorimetry. The binding process is accompanied by the uptake or release of protons, depending on the ionic strength. With no added salt, the total enthalpy change for the binding of four calcium ions to calmodulin is -41 kJ mol-1 but in the presence of 0.15 mM KCl delta Htot is +17 kJ mol-1. The mode of binding of Ca2+ is also completely different with and without added salt. It is also shown that for the C-terminal fragment of calmodulin, TR2C, the drastic reduction in delta Gtot for the binding process on increasing the ionic strength is largely an enthalpic effect. Domain interactions in calmodulin are indicated by the fact that the sum of the enthalpies of calcium binding to the two tryptic fragments is not the same as the total binding enthalpy to calmodulin itself. The binding of Ca2+ to calmodulin has also been studied calorimetrically at different temperatures in the range 21-37 degrees C. delta Cp is large and negative in this interval.     

Magnetic bead isolation of neutrophil plasma membranes and quantification of membrane-associated guanine nucleotide binding proteins

A protocol for isolation of neutrophil plasma membranes utilizing a plasma membrane marker antibody, anti-CD15, attached to superparamagnetic beads was developed. Cells were initially disrupted by nitrogen cavitation and then incubated with anti-CD15 antibody-conjugated superparamagnetic beads. The beads were then washed to remove unbound cellular debris and cytosol. Recovered plasma membranes were quantified by immunodetection of G(beta2) in Western blots. This membrane marker-based separation yielded highly pure plasma membranes. This protocol has advantages over standard density sedimentation protocols for isolating plasma membrane in that it is faster and easily accommodates cell numbers as low as 10(6). These methods were coupled with immunodetection methods and an adenosine 5(')-diphosphate-ribosylation assay to measure the amount of membrane-associated G(ialpha) proteins available for receptor coupling in neutrophils either stimulated with N-formyl peptides or treated to differing degrees with pertussis toxin. As expected, pertussis toxin treatment decreased the amount of membrane G protein available for signaling although total membrane G protein was not affected. In addition, activation of neutrophils with N-formyl peptides resulted in an approximately 50% decrease in G protein associated with the plasma membrane.     

Differential coupling of smooth muscle and liver vasopressin (V1) receptors to guanine nucleotide binding proteins

We report the reconstitution of the smooth muscle vasopressin V1 receptor functionally coupled to a pertussis toxin-insensitive guanine nucleotide-binding protein. This V1 receptor was spontaneously coupled to this guanine nucleotide-binding protein upon solubilization in the absence of agonist, in contrast to our earlier report on the liver V1 receptor, which required agonist for coupling. The smooth muscle V1 receptor was reconstituted as a high affinity receptor (Kd = 5 nM), with a slow rate of agonist dissociation. Upon the addition of guanosine 5'-thiotriphosphate, there was a decrease in receptor affinity (Kd = 30 nM) concomitant with an increase in the rate of ligand dissociation. The ability of the smooth muscle V1 receptor to spontaneously couple to a guanine nucleotide-binding protein(s) suggests that in the absence of agonist it exists as a high affinity receptor. The smooth muscle V1 receptor may, therefore, be more sensitive to plasma concentrations of vasopressin than its liver homologue.     

The beta-subunit of the protein-conducting channel of the endoplasmic reticulum functions as the guanine nucleotide exchange factor for the beta-subunit of the signal recognition particle receptor

Cotranslational protein transport to the endoplasmic reticulum is controlled by the concerted interaction of three GTPases: the SRP54 subunit of the signal recognition particle (SRP) and the alpha- and beta-subunits of the SRP receptor (SR). SRbeta is related to ADP-ribosylation factor (ARF)-type GTPases, and the recently published crystal structure of SRbeta-GTP in complex with the binding domain of SRalpha suggested that SRbeta, like all ARF-type GT-Pases, requires a guanine nucleotide exchange factor (GEF) for function. Searching the sequence data base, we identified significant sequence similarity between the Sec7 domain of ARF-GEFs and the cytosolic domains of the beta-subunits of the two homologous heterotrimeric protein-conducting channels in yeast. Using a fluorescence nucleotide exchange assay, we show that the beta-subunits of the heterotrimeric protein-conducting channels function as the GEFs for SRbeta. Both the cytosolic domain of Sec61beta as well as the holo-Sec61beta, when part of the isolated trimeric Sec61p complex, function as the GEF for SRbeta, whereas the same Sec61beta, when part of the heptameric complex that facilitates posttranslational protein transport, is inactive as the GEF for SRbeta     

The generation of inositolglycan mediators from rat liver plasma membranes: the role of guanine nucleotide binding proteins.

The guanine nucleotide dependence for the generation of inositolglycan second messengers from rat liver plasma membranes has been investigated. Plasma membranes, when treated with insulin release a soluble mediator substance which activates pyruvate dehydrogenase (PDH). Guanosine 5'-[3-thio]triphosphate (GTP gamma S) was found to be as potent as insulin in stimulating mediator release. The stimulatory effects of GTP gamma S required the presence of magnesium and following preincubation of membranes with guanosine 5'-[2-thio]diphosphate (GDP beta S) the stimulation of mediator release by either insulin or GTP gamma S was blocked. The activation of PDH by mediator fractions produced in response to either insulin or GTP gamma S was abolished following treatment of the fractions with anti-inositolglycan antibodies. The significance of these observations with respect to the possible involvement of a regulatory guanine-nucleotide binding protein (G-protein) in the generation of insulin mediators is discussed.