Functional heterogeneity at the frog sensory-motor synapse

This article compares four models of amplitude fluctuations in postsynaptic potentials. The convolution of two binomial distributions and the beta model proved the best fit with experimentally obtained data (as compared with the binomial model). The beta model is based on the assumption that the probability of quantal transmitter release is a random variable with a beta distribution. Numbers of quantal generators as estimated by the beta model were found to resemble numbers of morphological identifiable synaptic boutons. Findings from research using this model showed that the binomial parameter n may be interpreted as the number of transmitter release sites functioning with a probability in excess of 0.2. The findings obtained confirm the postulated functional diversity of release sites at interneuronal synapses.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 21, No. 6, pp. 780–788, November–December, 1989.     

Beta gamma-subunit of bovine transducin composed of two components with distinctive gamma-subunits

During the process of transduction of a photon signal in vertebrate rod outer segments, transducin, a guanine nucleotide binding protein, mediates between a photobleaching intermediate of rhodopsin and a cGMP-phosphodiesterase. We report here that the beta gamma-subunit of bovine transducin (T beta gamma) characterized so far consists of two components (T beta gamma-1 and T beta gamma-2), which can be separated by anion exchange chromatography under nondenaturing conditions. Both components consisted of two polypeptides of Mr 36,000 (T beta) and about 8,000 (T gamma) in sodium dodecyl sulfate polyacrylamide (13%) gel electrophoresis. On a further analysis by 8 M urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, T gamma subunits of T beta gamma-1 and T beta gamma-2 showed Mr values of 8,000 (T gamma-1) and 6,000 (T gamma-2), respectively. Amino acid compositions of both T gamma-1 and T gamma-2 roughly corresponded with that of T gamma previously reported and were quite different from that of gamma-subunit of cGMP-phosphodiesterase. Western blot analysis of freshly isolated rod outer segments by an antiserum raised against a mixture of T beta gamma-1 and T beta gamma-2 revealed the presence of both components in the membranes of a starting material. This observation excludes the possibility that one of the components might be produced artificially in the course of the purification. In the presence of a photobleaching intermediate of either unphosphorylated or phosphorylated rhodopsin, the binding of guanosine 5'-(beta, gamma-imido)triphosphate (GppNHp) to the alpha-subunit of transducin (T alpha) was remarkably enhanced with increasing concentrations of purified T beta gamma-2. On the contrary, T beta gamma-1 retained little ability, if any, to enhance the GppNHp binding to T alpha; the ability of T beta gamma-1 was at least 30 times lower than that of T beta gamma-2. Such a low activity of T beta gamma-1 was attributed to inability for coupling of T alpha with a photobleaching intermediate of rhodopsin. These results indicate that T gamma-2 is essential for the GTP binding of transducin. The role of T gamma-1 in vertebrate photoreceptor cells was discussed.     

Structure and function of gamma-subunit of photoreceptor G-protein (transducin).

1. The gamma-subunit of the vertebrate photoreceptor GTP-binding protein (transducin) is S-farnesylated at the C-terminal cysteine residue, with a part of the residue being methyl-esterified at the alpha-carboxyl group. 2. Functionally, the modified cysteine residue is implicated in efficient coupling of the alpha- and beta gamma-subunits, and indispensible for expressing GTP-binding activity. 3. Similar modifications, isoprenylation and methyl-esterification of the C-terminal cysteine residue have been found in a variety of proteins involved in signal transduction and growth regulation processes. However, it seems likely that the physiological roles of the modifications are different for the various proteins.     

Modulation of retinal transducin and phosphodiesterase activities by synthetic peptides of the phosphodiesterase gamma-subunit

Synthetic peptides corresponding to various regions of the light-activated guanosine 3',5'-cyclic monophosphate phosphodiesterase (PDE) gamma-subunit (PDE gamma) from bovine retinal rod outer segments were synthesized and tested for their ability to inhibit PDE activity, and GTPase activity of transducin. One of these peptides, corresponding to PDE gamma residues 31-45, inhibited PDE activity and GTPase activity in a dose-dependent manner. The GTPase activity was inhibited by PDE gamma-3 non-competitively. This region of the PDE gamma subunit may be involved in the direct interaction of transducin and PDE alpha beta with PDE gamma.     

Carboxyl methylation and farnesylation of transducin gamma-subunit synergistically enhance its coupling with metarhodopsin II.

A heterotrimeric G-protein in vertebrate photoreceptor cells is called transducin (T alpha beta gamma), whose gamma-subunit is a mixture of two components, T gamma-1 and T gamma-2. T gamma-2 is S-farnesylated and partly carboxyl methylated at the C-terminal cysteine residue, whereas T gamma-1 lacks the modified cysteine residue. To elucidate the physiological significance of the double modifications in T gamma, we established a simple chromatographic procedure to isolate T gamma-1, methylated T gamma-2 and non-methylated T gamma-2 on a reversed phase column. Taking advantage of the high and reproducible yield of T gamma from the column, we analyzed the composition of T gamma subspecies in the T alpha-T beta gamma complex which did not bind with transducin-depleted rod outer segment membranes containing metarhodopsin II. The binding of T alpha-T beta gamma with the membranes was shown to require the S-farnesylated cysteine residue of T gamma, whose methylation further enhanced the binding. This synergistic effect was not evident when T alpha was either absent or converted to the GTP-bound form which is known to dissociate from T beta gamma. Thus we concluded that a formation of the ternary complex, T alpha-T beta gamma-metarhodopsin II, is enhanced by the farnesylation and methylation of T gamma. This suggests that the double modifications provide most efficient signal transduction in photoreceptor cells.     

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)     

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.     

Binding of the gamma-subunit of retinal rod-outer-segment phosphodiesterase with both transducin and the catalytic subunits of phosphodiesterase.

The gamma-subunit of retinal rod-outer-segment phosphodiesterase (PDE-gamma) is a multifunctional protein which interacts directly with both of the catalytic subunits of PDE (PDE alpha/beta) and the alpha-subunit of the retinal G (guanine-nucleotide-binding)-protein transducin alpha (T alpha). We have previously reported that the PDE gamma binds to T alpha at residue nos. 24-45 [Morrison. Rider & Takemoto (1987) FEBS Lett. 222, 266-270]. In vitro this results in inhibition of T alpha GTP/GDP exchange [Morrison, Cunnick, Oppert & Takemoto (1989) J. Biol. Chem. 264, 11671-11681]. We now report that the inhibitory region of PDE gamma for PDE alpha/beta occurs at PDE gamma residues 54-87. This binding results in inhibition of either trypsin-solubilized or membrane-bound PDE alpha/beta. PDE gamma which has been treated with carboxypeptidase Y, removing the C-terminus, does not inhibit PDE alpha/beta, but does inhibit T alpha GTP/GDP exchange. Inhibition by PDE gamma can be removed by T alpha-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) addition to membranes. This results in a displacement of PDE gamma, but not in removal of this subunit from the membrane [Whalen, Bitensky & Takemoto (1990) Biochem. J. 265, 655-658]. These results suggest that low levels of T alpha-GTP[S] can result in displacement of PDE gamma from the membrane in vitro as a GTP[S]-T alpha-PDE gamma complex. Further activation by high levels of T alpha-GTP[S] occurs by displacement of PDE gamma from its inhibitory site on PDE alpha/beta, but not in removal from the membrane.     

One-step purification of bacterially expressed recombinant transducin alpha-subunit and isotopically labeled PDE6 gamma-subunit for NMR analysis

Interactions between the transducin alpha-subunit (Galpha(t)) and the cGMP phosphodiesterase gamma-subunit (PDEgamma) are critical not only for turn-on but also turn-off of vertebrate visual signal transduction. Elucidation of the signaling mechanisms dominated by these interactions has been restrained by the lack of atomic structures for full-length Galpha(t)/PDEgamma complexes, in particular, the signaling-state complex represented by Galpha(t).GTPgammaS/PDEgamma. As a preliminary step in our effort for NMR structural analysis of Galpha(t)/PDEgamma interactions, we have developed efficient protocols for the large-scale production of recombinant Galpha(t) (rGalpha(t)) and homogeneous and functional isotopically labeled PDEgamma from Escherichia coli cells. One-step purification of rGalpha(t) was achieved through cobalt affinity chromatography in the presence of glycerol, which effectively removed the molecular chaperone DnaK that otherwise persistently co-purified with rGalpha(t). The purified rGalpha(t) was found to be functional in GTPgammaS/GDP exchange upon activation of rhodopsin and was used to form a signaling-state complex with labeled PDEgamma, rGalpha(t). GTPgammaS/[U-13C,15N]PDEgamma. The labeled PDEgamma sample yielded a well-resolved 1H-15N HSQC spectrum. The methods described here for large-scale production of homogeneous and functional rGalpha(t) and isotope-labeled PDEgamma should support further NMR structural analysis of the rGalpha(t)/PDEgamma complexes. In addition, our protocol for removing the co-purifying DnaK contaminant may be of general utility in purifying E. coli-expressed recombinant proteins.     

The N terminus of GTP gamma S-activated transducin alpha-subunit interacts with the C terminus of the cGMP phosphodiesterase gamma-subunit

Dynamic regulation of G-protein signaling in the phototransduction cascade ensures the high temporal resolution of vision. In a key step, the activated alpha-subunit of transducin (Galphat-GTP) activates the cGMP phosphodiesterase (PDE) by binding the inhibitory gamma-subunit (PDEgamma). Significant progress in understanding the interaction between Galphat and PDEgamma was achieved by solving the crystal structure of the PDEgamma C-terminal peptide bound to Galphat in the transition state for GTP hydrolysis (Slep, K. C., Kercher, M. A., He, W., Cowan, C. W., Wensel, T. G., and Sigler, P. B. (2001) Nature 409, 1071-1077). However, some of the structural elements of each molecule were absent in the crystal structure. We have probed the binding surface between the PDEgamma C terminus and activated Galphat bound to guanosine 5'-O-(3-thio)-triphosphate (GTPgammaS) using a series of full-length PDEgamma photoprobes generated by intein-mediated expressed protein ligation. For each of seven PDEgamma photoprobe species, expressed protein ligation allowed one benzoyl-L-phenylalaine substitution at selected hydrophobic C-terminal positions, and the addition of a biotin affinity tag at the extreme C terminus. We have detected photocross-linking from several PDEgamma C-terminal positions to the Galphat-GTPgammaS N terminus, particularly from PDEgamma residue 73. The overall percentage of cross-linking to the Galphat-GTPgammaSN terminus was analyzed using a far Western method for examining Galphat-GTPgammaS proteolytic digestion patterns. Furthermore, mass spectrometric analysis of cross-links to Galphat from a benzoyl-phenylalanine replacement at PDEgamma position 86 localized the region of photoinsertion to Galphat N-terminal residues Galphat-(22-26). This novel Galphat/PDEgamma interaction suggests that the transducin N terminus plays an active role in signal transduction.     

Functional interaction between bovine rhodopsin and G protein transducin

To elucidate the mechanisms of specific coupling of bovine rhodopsin with the G protein transducin (G(t)), we have constructed the bovine rhodopsin mutants whose second or third cytoplasmic loop (loop 2 or 3) was replaced with the corresponding loop of the G(o)-coupled scallop rhodopsin and investigated the difference in the activation abilities for G(t), G(o), and G(i) among these mutants and wild type. We have also prepared the Galpha(i) mutants whose C-terminal 11 or 5 amino acids were replaced with those of Galpha(t), Galpha(o), and Galpha(q) to evaluate the role of the C-terminal tail of the alpha-subunit on the specific coupling of bovine rhodopsin with G(t). Replacement of loop 2 of bovine rhodopsin with that of the scallop rhodopsin caused about a 40% loss of G(t) and G(o) activation, whereas that of loop 3 enhanced the G(o) activation four times with a 60% decrease in the G(t) activation. These results indicated that loop 3 of bovine rhodopsin is one of the regions responsible for the specific coupling with G(t). Loop 3 of bovine rhodopsin discriminates the difference of the 6-amino acid sequence (region A) at a position adjacent to the C-terminal 5 amino acids of the G protein, resulting in the different activation efficiency between G(t) and G(o). In addition, the binding of region A to loop 3 of bovine rhodopsin is essential for activation of G(t) but not G(i), even though the sequence of the region A is almost identical between Galpha(t) and Galpha(i). These results suggest that the binding of loop 3 of bovine rhodopsin to region A in Galpha(t) is one of the mechanisms of specific G(t) activation by bovine rhodopsin.     

Adenylate kinase and GDP-kinase activity of rod outer segments in the frog retina. Possible functional role of the T beta subunit of transducin

Pure frog retina rod outer segments (ROS) preparations (A280/A500 = 2,1-2,3) catalyze the synthesis of ATP from ADP in the presence of Mg2+. Adenylate kinase (AK) (ATP:AMP phosphotransferase, EC 2.7.4.3) specific activities for ROS preparations are within the range 2-4 mumole per hour for mg protein. The enzymatic activity of investigated preparations is due to intact, but not destroyed ROS. The component which possesses AK is found in water-soluble, but not in membranous ROS fractions and seems to be a part of the predominant ROS plasma protein--GTP-binding complex of transducin. It has been shown, that this component is the T beta subunit of transducin and its enzymatic activity is controlled by other subunits of the transducin complex. The obtained results indicate that GDP kinase (ATP:GDP phosphotransferase, EC 2.7.4.6) activity of transducin depends on the work of both of T beta and T alpha subunits. It has been shown that in the ROS preparations synthesis of the ATP from ADP and GDP phosphorylation are stimulated by a lowering of Ca2+ concentration (less than 10(-5)-10(-7) M). T beta component is suggested to play the role of phosphotransferase which phosphorylates GDP associated with the T alpha subunits and it leads to formation of a complex T alpha X GTP which is an activator of vertebrate retina ROS phosphodiesterase.     

Interaction of the gamma-subunit of retinal rod outer segment phosphodiesterase with transducin. Use of synthetic peptides as functional probes

There is considerable evidence which suggests that the gamma-subunit of cGMP phosphodiesterase (PDE gamma) is a multifunctional protein which may interact directly with both the catalytic subunits of PDE (PDE alpha beta) and the alpha-subunit of transducin (T alpha) (Whalen, M., and Bitensky, M. (1989) Biochem. J. 259, 13-19; Griswold-Prenner, I., Young, J. H., Yamane, H. K., and Fung, B. K.-K. (1988) Invest. Ophthalmol. & Visual Sci. 29, (Suppl.) 218). To determine the region of interaction between the multifunctional PDE gamma and T alpha, and to determine the significance of this interaction, peptides corresponding to various regions of PDE gamma were synthesized and tested for their ability to inhibit the GTPase activity of T alpha. One of these peptides, PDE gamma-3 (bovine amino acid residues 31-45), inhibited the GTPase activity of T alpha with an I50 of 450 microM. The peptide (PDE gamma-3) was found to inhibit the GTPase activity of T alpha by inducing the binding of transducin to the rod outer segment membrane and by altering the GTP/GDP exchange. Analogs of PDE gamma-3 were synthesized to determine the required structure of the PDE gamma-3 region needed for the interaction of PDE gamma with T alpha. The results of these studies indicated that the removal of the positively charged amino acids or any of the potential hydrogen-bonding amino acids increased the I50 for the inhibition of the GTPase activity of T alpha Substitution of the hydrophobic amino acids had no effect. These results indicate the hydrophilic interactions may be essential for the binding of PDE gamma to T alpha and for the inhibition of the GTPase activity of T alpha by PDE gamma. The observed effects of PDE gamma-3 on T alpha and on PDE suggest that PDE gamma is a multifunctional protein which may play more than one role in the deactivation of the retinal transduction cascade.     

Functional modifications of transducin induced by cholera or pertussis-toxin-catalyzed ADP-ribosylation.

Transducin (T alpha beta gamma), the heterotrimeric GTP-binding protein that interacts with photoexcited rhodopsin (Rh*) and the cGMP-phosphodiesterase (PDE) in retinal rod cells, is sensitive to cholera (CTx) and pertussis toxins (PTx), which catalyze the binding of an ADP-ribose to the alpha subunit at Arg174 and Cys347, respectively. These two types of ADP-ribosylations are investigated with transducin in vitro or with reconstituted retinal rod outer-segment membranes. Several functional perturbations inflicted on T alpha by the resulting covalent modifications are studied such as: the binding of T alpha to T beta gamma to the membrane and to Rh*; the spontaneous or Rh*-catalysed exchange of GDP for GTP or guanosine 5-[gamma-thio]triphosphate (GTP[gamma S]), the conformational switch and activation undergone by transducin upon this exchange, the activation of T alpha GDP by fluoride complexes and the activation of the PDE by T alpha GTP. ADP-ribosylation of transducin by CTx requires the GTP-dependent activation of ADP-ribosylation factors (ARF), takes place only on the high-affinity, nucleotide-free complex, Rh*-T alpha empty-T beta gamma and does not activate T alpha. Subsequent to CTx-catalyzed ADP-ribosylation the following occurs: (a) addition of GDP induces the release from Rh* of inactive CTxT alpha GDP (CTxT alpha, ADP-ribosylated alpha subunit of transducin) which remains associated to T beta gamma; (b) CTxT alpha GDP-T beta gamma exhibits the usual slow kinetics of spontaneous exchange of GDP for GTP[gamma S] in the absence of Rh*, but the association and dissociation of fluoride complexes, which act as gamma-phosphate analogs, are kinetically modified, suggesting that the ADP-ribose on Arg174 specifically perturbs binding of the gamma-phosphate in the nucleotide site; (c) CTxT alpha GDP-T beta gamma can still couple to Rh* and undergo fast nucleotide exchange; (d) CTxT alpha GTP[gamma S] and CTxT alpha GDP-AlFx (AlFx, Aluminofluoride complex) activate retinal cGMP-phosphodiesterase (PDE) with the same efficiency as their unmodified counterparts, but the kinetics and affinities of fluoride activation are changed; (e) CTxT alpha GTP hydrolyses GTP more slowly than unmodified T alpha GTP, which entirely accounts for the prolonged action of CTxT alpha GTP on the PDE; (f) after GTP hydrolysis, CTxT alpha GDP reassociates to T beta gamma and becomes inactive. Thus, CTx catalyzed ADP-ribosylation only perturbs in T alpha the GTP-binding domain, but not the conformational switch nor the domains of contact with the T beta gamma subunit, with Rh* and with the PDE.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alpha 3 beta 3 complex of thermophilic ATP synthase. Catalysis without the gamma-subunit

A complex of the alpha- and beta-subunits of thermophilic ATP synthase showed about 25% of the ATPase activity of the alpha beta gamma complex. The alpha 3 beta 3 hexamer structure was analyzed by sedimentation (11.2 S) and gel filtration (310 kDa). Dilution of the alpha beta complex caused dissociation of the complex and rapid loss of ATPase activity which was restored by addition of the gamma-subunit. A previous method using urea for isolating the subunits resulted in an alpha beta complex with lower activity than that prepared by over-expression of the genes. The alpha beta-ATP complex was formed from the alpha beta complex, ADP and Pi in the presence of dimethyl sulfoxide.     

Isolation and characteristics of cyanogen bromide peptides of transducin alpha and beta subunits

The alpha- and beta-subunits of the GTP-binding protein (transducin) from cattle retina were cleaved with cyanogen bromide. 21 peptides covering 90-100% of the amino acid sequence of the alpha- and beta-subunits were isolated from the hydrolyzate. Cyanogen bromide peptides complete or partial amino acid sequence was determined, the results were compared with those by Numa and coworkers [1] and Lochrie et al. [2] at the primary structure of the transducin alpha-subunit deduced from the nucleotide sequence of the cDNA. The structure by Lochrie is shown to differ much from the true structure of the alpha-subunit; probably, the investigators isolated cDNA, corresponding to the gene for some GTP-binding protein homologous to transducin, but not to the gene for the transducin alpha-subunit. The Numa's structure also contains an error. The final primary structure of the transducin alpha-subunit is given. The protein polypeptide chain consists of 349 amino acid residues and has an acetylmethionine residue as the N-terminal residue.