Regulation of phosducin phosphorylation in retinal rods by Ca2+/calmodulin-dependent adenylyl cyclase

The phosphoprotein phosducin (Pd) regulates many guanine nucleotide binding protein (G protein)-linked signaling pathways. In visual signal transduction, unphosphorylated Pd blocks the interaction of light-activated rhodopsin with its G protein (Gt) by binding to the beta gamma subunits of Gt and preventing their association with the Gt alpha subunit. When Pd is phosphorylated by cAMP-dependent protein kinase, it no longer inhibits Gt subunit interactions. Thus, factors that determine the phosphorylation state of Pd in rod outer segments are important in controlling the number of Gts available for activation by rhodopsin. The cyclic nucleotide dependencies of the rate of Pd phosphorylation by endogenous cAMP-dependent protein kinase suggest that cAMP, and not cGMP, controls Pd phosphorylation. The synthesis of cAMP by adenylyl cyclase in rod outer segment preparations was found to be dependent on Ca2+ and calmodulin. The Ca2+ dependence was within the physiological range of Ca2+ concentrations in rods (K1/2 = 230 +/- 9 nM) and was highly cooperative (n app = 3.6 +/- 0.5). Through its effect on adenylyl cyclase and cAMP-dependent protein kinase, physiologically high Ca2+ (1100 nM) was found to increase the rate of Pd phosphorylation 3-fold compared to the rate of phosphorylation at physiologically low Ca2+ (8 nM). No evidence for Pd phosphorylation by other (Ca2+)-dependent kinases was found. These results suggest that Ca2+ can regulate the light response at the level of Gt activation through its effect on the phosphorylation state of Pd.     

The alpha subunit of Gq contributes to muscarinic inhibition of the M-type potassium current in sympathetic neurons

Rat superior cervical ganglion (SCG) neurons express low-threshold noninactivating M-type potassium channels (IK(M)), which can be inhibited by activation of M1 muscarinic receptors. This inhibition occurs via pertussis toxin-insensitive G-proteins belonging to the Galphaq family (Caulfield et al., 1994 ). We have used DNA plasmids encoding antisense sequences against the 3' untranslated regions of Galpha subunits (antisense plasmids) to investigate the specific G-protein subunits involved in muscarinic inhibition of IK(M). These antisense plasmids specifically reduced levels of the target G-protein 48 hr after intranuclear injection. In cells depleted of Galphaq, muscarinic inhibition of IK(M) was attenuated compared both with uninjected neurons and with neurons injected with an inappropriate GalphaoA antisense plasmid. In contrast, depletion of Galpha11 protein did not alter IK(M) inhibition. To determine whether the alpha or beta gamma subunits of the G-protein mediated this inhibition, we have overexpressed the C terminus of beta adrenergic receptor kinase 1 (betaARK1), which binds free beta gamma subunits. betaARK1 did not reduce muscarinic inhibition of IK(M) at a concentration of plasmid that can reduce beta gamma-mediated inhibition of calcium current (). Also, expression of beta1gamma2 dimers did not alter the IK(M) density in SCG neurons. In contrast, IK(M) was virtually abolished in cells expressing GTPase-deficient, constitutively active forms of Galphaq and Galpha11. These data suggest that Galphaq is the principal mediator of muscarinic IK(M) inhibition in rat SCG neurons and that this more likely results from an effect of the alpha subunit than the beta gamma subunits of the Gq heterotrimer.     

Residues at the subunit interfaces of the nicotinic acetylcholine receptor that contribute to alpha-conotoxin M1 binding

The two binding sites in the pentameric nicotinic acetylcholine receptor of subunit composition alpha2 beta gamma delta are formed by nonequivalent alpha-gamma and alpha-delta subunit interfaces, which produce site selectivity in the binding of agonists and antagonists. We show by sedimentation analysis that 125I-alpha-conotoxin M1 binds with high affinity to the alpha-delta subunit dimers, but not to alpha-gamma dimers, nor to alpha, gamma, and delta monomers, a finding consistent with alpha-conotoxin M1 selectivity for the alpha delta interface in the intact receptor measured by competition against alpha-bungarotoxin binding. We also extend previous identification of alpha-conotoxin M1 determinants in the gamma and delta subunits to the alpha subunit interface by mutagenesis of conserved residues in the alpha subunit. Most mutations of the alpha subunit affect affinity similarly at the two sites, but Tyr93Phe, Val188Lys, Tyr190Thr, Tyr198Thr, and Asp152Asn affect affinity in a site-selective manner. Mutant cycle analysis reveals only weak or no interactions between mutant alpha and non-alpha subunits, indicating that side chains of the alpha subunit do not interact with those of the gamma or delta subunits in stabilizing alpha-conotoxin M1. The overall findings suggest different binding configurations of alpha-conotoxin M1 at the alpha-delta and alpha-gamma binding interfaces.     

Differential activity of the G protein beta5 gamma2 subunit at receptors and effectors

The G protein beta5 subunit differs substantially in amino acid sequence from the other known beta subunits suggesting that beta gamma dimers containing this protein may play specialized roles in cell signaling. To examine the functional properties of the beta5 subunit, recombinant beta5 gamma2 dimers were purified from baculovirus-infected Sf9 insect cells using a strategy based on two affinity tags (hexahistidine and FLAG) engineered into the N terminus of the gamma2 subunit (gamma2HF). The function of the pure beta5 gamma2HF dimers was examined in three assays: activation of pure phospholipase C-beta in lipid vesicles; activation of recombinant, type II adenylyl cyclase expressed in Sf9 cell membranes; and coupling of alpha subunits to the endothelin B (ETB) and M1 muscarinic receptors. In each case, the efficacy of the beta5 gamma2HF dimer was compared with that of the beta1 gamma2HF dimer, which has demonstrated activity in these assays. The beta5 gamma2HF dimer activated phospholipase C-beta with a potency and efficacy similar to that of beta1 gamma2 or beta1 gamma2HF; however, it was markedly less effective than the beta1 gamma2HF or beta1 gamma2 dimer in its ability to activate type II adenylyl cyclase (EC50 of approximately 700 nM versus 25 nM). Both the beta5 gamma2HF and the beta1 gamma2HF dimers supported coupling of M1 muscarinic receptors to the Gq alpha subunit. The ETB receptor coupled effectively to both the Gi and Gq alpha subunits in the presence of the beta1 gamma2HF dimer. In contrast, the beta5 gamma2HF dimer only supported coupling of the Gq alpha subunits to the ETB receptor and did not support coupling of the Gi alpha subunit. These results suggest that the beta5 gamma2HF dimer binds selectively to Gq alpha subunits and does not activate the same set of effectors as dimers containing the beta1 subunit. Overall, the data support a specialized role for the beta5 subunit in cell signaling.     

Crystal structure at 2.4 angstroms resolution of the complex of transducin betagamma and its regulator, phosducin

The crystal structure of transducin's betagamma subunits complexed with phosducin, which regulates Gtbetagamma activity, has been solved to 2.4 angstroms resolution. Phosducin has two domains that wrap around Gtbetagamma to form an extensive interface. The N-terminal domain binds loops on the "top" Gtbeta surface, overlapping the Gtalpha binding surface, explaining how phosducin blocks Gtbetagamma's interaction with Gtalpha. The C-terminal domain shows structural homology to thioredoxin and binds the outer strands of Gtbeta's seventh and first blades in a manner likely to disrupt Gtbetagamma's normal orientation relative to the membrane and receptor. Phosducin's Ser-73, which when phosphorylated inhibits phosducin's function, points away from Gtbetagamma, toward a large flexible loop. Thus phosphorylation is not likely to affect the interface directly, but rather indirectly through an induced conformational change.     

Purification of four forms of the beta gamma subunit complex of G proteins containing different gamma subunits

To investigate the physiological significance of the diversity of gamma subunits of G proteins, we purified four forms of beta gamma of G proteins from bovine brain (beta gamma-B1, beta gamma-B2, beta gamma-B3), and spleen (beta gamma-S1) by the sequential chromatography on columns of DEAE-Sephacel, Ultrogel AcA 34, heptylamine-Sepharose, phenyl-5PW, and DEAE-5PW. Electrophoretic analyses showed that each beta gamma mainly contained the 36-kDa beta and a distinct but homogeneous gamma. These beta gamma complexes were subjected directly to proteolytic digestion and subsequent amino acid sequence analyses of their fragments. It was revealed that beta gamma-B1, -B2, and -B3 were identical to beta 1 gamma 7 (with a low level of beta 2 gamma 7), beta 1 gamma 2 and beta 1 gamma 3, respectively, while beta gamma-S1 was composed of beta 1 and an unidentified form of gamma. Then we examined the functional differences among these beta gamma complexes and the beta gamma of transducin (beta gamma-T, beta 1 gamma 1). Few differences were observed among all beta gamma complexes to enhance pertussis toxin-catalyzed ADP-ribosylation of the alpha subunits of G(o) and Gt. The four forms of beta gamma complexes purified from brain and spleen showed indistinguishable inhibitory effects on the release of GDP from G(o) alpha, but beta gamma-T was much less effective. Brain and spleen beta gamma complexes were equally effective in inhibiting calmodulin-stimulated adenylyl-cyclase activity, but beta gamma-T had a very weak inhibitory effect. Five forms of beta gamma facilitated metarhodopsin II-catalyzed binding of GTP gamma S to Gt alpha in a concentration-dependent manner with the following rank order of effectiveness: beta gamma-S1 > beta gamma-T > beta gamma-B1 > beta gamma-B2 > beta gamma-B3. Because the beta gamma complexes used in this study mostly contained the same beta subunit, the functional differences must be dependent on the gamma subunits. Thus, it seems likely that the receptor, the alpha subunits, and the effector are able to distinguish between the various gamma subunits.     

Glucose activates the carboxyl methylation of gamma subunits of trimeric GTP-binding proteins in pancreatic beta cells. Modulation in vivo by calcium, GTP, and pertussis toxin

The gamma subunits of trimeric G-proteins (gamma1, gamma2, gamma5, and gamma7 isoforms) were found to be methylated at their carboxyl termini in normal rat islets, human islets and pure beta [HIT-T15] cells. Of these, GTPgammaS significantly stimulated the carboxyl methylation selectively of gamma2 and gamma5 isoforms. Exposure of intact HIT cells to either of two receptor-independent agonists--a stimulatory concentration of glucose or a depolarizing concentration of K+--resulted in a rapid (within 30 s) and sustained (at least up to 60 min) stimulation of gamma subunit carboxyl methylation. Mastoparan, which directly activates G-proteins (and insulin secretion from beta cells), also stimulated the carboxyl methylation of gamma subunits in intact HIT cells. Stimulatory effects of glucose or K+ were not demonstrable after removal of extracellular Ca2+ or depletion of intracellular GTP, implying regulatory roles for calcium fluxes and GTP; however, the methyl transferase itself was not directly activated by either. The stimulatory effects of mastoparan were resistant to removal of extracellular Ca2+, implying a mechanism of action that is different from glucose or K+ but also suggesting that dissociation of the alphabetagamma trimer is conducive to gamma subunit carboxyl methylation. Indeed, pertussis toxin also markedly attenuated the stimulatory effects of glucose, K+ or mastoparan without altering the rise in intracellular calcium induced by glucose or K+. Glucose-induced carboxyl methylation of gamma2 and gamma5 isoforms was vitiated by coprovision of any of three structurally different cyclooxygenase inhibitors. Conversely, exogenous PGE2, which activates Gi and Go in HIT cells and which thereby would dissociate alpha from beta(gamma), stimulated the carboxyl methylation of gamma2 and gamma5 isoforms and reversed the inhibition of glucose-stimulated carboxyl methylation of gamma subunits elicited by cyclooxygenase inhibitors. These data indicate that gamma subunits of trimeric G-proteins undergo a glucose- and calcium-regulated methylation-demethylation cycle in insulin-secreting cells, findings that may imply an important role in beta cell function. Furthermore, this is the first example of the regulation of the posttranslational modification of G-protein gamma subunits via nonreceptor-mediated activation mechanisms, which are apparently dependent on calcium influx and the consequent activation of phospholipases releasing arachidonic acid.     

Phosphorylation of the gamma subunit of the retinal photoreceptor cGMP phosphodiesterase by the cAMP-dependent protein kinase and its effect on the gamma subunit interaction with other proteins

Cyclic GMP phosphodiesterase, a key enzyme in phototransduction, is composed of P alpha beta and two P gamma subunits. Interaction of P gamma with P alpha beta or with the alpha subunit (T alpha) of transducin is crucial for the regulation of cGMP phosphodiesterase in retinal photoreceptors. Here we have investigated phosphorylation of P gamma by cAMP-dependent protein kinase and its functional effect on the P gamma interaction with P alpha beta or T alpha in vitro. P gamma, but not P gamma complexed with T alpha (both GTP and GDP forms), is phosphorylated. Measurement of 32P radioactivity in phosphorylated P gamma, analysis of phosphorylated P gamma by laser mass spectrometry, identification of phosphoamino acid, and phosphorylation of mutant forms of P gamma indicate that only threonine 35 in P gamma is phosphorylated. Phosphorylation of P gamma mutants also reveals that the C and N terminals of P gamma which are required for the regulation of P alpha beta functions are not involved in the P gamma phosphorylation but that arginine 33, which is ADP-ribosylated by an endogenous ADP-ribosyltransferase, is required for the phosphorylation. Phosphorylated P gamma has a higher inhibitory activity for trypsin-activated cGMP phosphodiesterase than nonphosphorylated P gamma, indicating that the P gamma-P alpha beta interaction is affected by P gamma phosphorylation. Nonphosphorylated P gamma inhibits both the GTPase activity of T alpha and the binding of a hydrolysis-resistant GTP analogue to T alpha, while P gamma phosphorylation reduces these inhibitory activities. These observations suggest that a P gamma domain containing threonine 35 is involved in the P gamma-T alpha interaction, and P gamma phosphorylation regulates the P gamma-T alpha interaction. Our observation suggests that P gamma phosphorylation by cAMP-dependent protein kinase may function for the regulation of phototransduction in vertebrate rod photoreceptors.     

Mechanism of allosteric regulation of the rod cGMP phosphodiesterase activity by the helical domain of transducin alpha subunit

The G protein alpha subunit (Galpha) is composed of two distinct folding domains: a GTP-binding Ras-like domain and an alpha helical domain (HD). We have recently reported that the helical domain (HDt) of the vertebrate visual transducin alpha subunit (Galphat) synergizes activation of retinal cyclic GMP phosphodiesterase (PDE) by activated Galphat (Liu, W., and Northup, J. K., (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12878-12883). Here, we examine the molecular basis for this HD-based signaling regulation, and we provide a new model for the activation of the target effector. The HD proteins derived from visual transducin or taste gustducin alpha subunits, but no other Galpha HD proteins, each attenuate the PDE catalytic core (Palphabeta) and synergize Galphat stimulation of the holoPDE (Palphabetagamma2) with similar apparent affinities. The data from studies of both HDt-mediated attenuation and stimulation indicate that the HDt and the PDE inhibitory subunit (Pgamma) interact with PDE at independent sites and that Palphabeta contains the binding sites for HD. The saturation of both processes by HDt displays positive cooperativity with Hill coefficients of 1.5 for the attenuation of Palphabeta activity and 2.1 for synergism of holoPDE activation. Our data suggest the that Galphat-HDt regulates PDE by allosterically decreasing the affinity of Palphabeta for Pgamma and thus simultaneously facilitating the interaction of the activated Galphat-Ras-like domain with Pgamma. Thus, we propose a new model for the high efficiency of PDE activation as well as deactivation, and, overall, a novel mechanism for controlling fidelity, sensitivity, and efficacy of G protein signaling.     

Formation of the nicotinic acetylcholine receptor binding sites

Nicotinic acetylcholine receptors (AChRs) are activated by ACh binding to two sites located on different alpha subunits. The two alpha subunits, alpha gamma and alpha delta, are distinguished by their interface with gamma and delta subunits. We have characterized the formation of the ACh binding sites and found, contrary to the current model, that the sites form at different times and in a set order. The first site forms on alpha gamma subunits during the process of subunit assembly. Our data are consistent with the appearance of this site on alpha beta gamma delta subunit tetramers soon after the site for the competitive antagonist alpha-bungarotoxin has formed and delta subunits have assembled with alpha beta gamma trimers. The second site is located on alpha delta subunits and forms after AChR subunits have assembled into alpha2 beta gamma delta pentamers. By determining the order in which the ACh binding sites form, we have also identified the sites in which the delta and second alpha subunits associate during subunit assembly.     

The A326S mutant of Gialpha1 as an approximation of the receptor-bound state

Agonist-bound heptahelical receptors activate heterotrimeric G proteins by catalyzing exchange of GDP for GTP on their alpha subunits. In search of an approximation of the receptor-alpha subunit complex, we have considered the properties of A326S Gialpha1, a mutation discovered originally in Gsalpha (Iiri, T., Herzmark, P., Nakamoto, J. M., Van Dop, C., and Bourne, H. R. (1994) Nature 371, 164-168) that mimics the effect of receptor on nucleotide exchange. The mutation accelerates dissociation of GDP from the alphai1beta1gamma2 heterotrimer by 250-fold. Nevertheless, affinity of mutant Gialpha1 for GTPgammaS is high in the presence of Mg2+, and the mutation has no effect on the intrinsic GTPase activity of the alpha subunit. The mutation also uncouples two activities of betagamma: stabilization of the GDP-bound alpha subunit (which is retained) and retardation of GDP dissociation from the heterotrimer (which is lost). For wild-type and mutant Gialpha1, beta gamma prevents irreversible inactivation of the alpha subunit at 30 degreesC. However, the mutation accelerates irreversible inactivation of alpha at 37 degreesC despite the presence of beta gamma. Structurally, the mutation weakens affinity for GTPgammaS by steric crowding: a 2-fold increase in the number of close contacts between the protein and the purine ring of the nucleotide. By contrast, we observe no differences in structure at the GDP binding site between wild-type heterotrimers and those containing A326S Gialpha1. However, the GDP binding site is only partially occupied in crystals of G protein heterotrimers containing A326S Gialpha1. In contrast to original speculations about the structural correlates of receptor-catalyzed nucleotide exchange, rapid dissociation of GDP can be observed in the absence of substantial structural alteration of a Galpha subunit in the GDP-bound state.     

Light-dependent activation of rod transducin by pineal opsin

The pineal gland expresses a unique member of the opsin family (P-opsin; Max, M., McKinnon, P. J., Seidenman, K. J., Barrett, R. K., Applebury, M. L., Takahashi, J. S., and Margolskee, R. F. (1995) Science 267, 1502-1506) that may play a role in circadian entrainment and photo-regulation of melatonin synthesis. To study the function of this protein, an epitope-tagged P-opsin was stably expressed in an embryonic chicken pineal cell line. When incubated with 11-cis-retinal, a light-sensitive pigment was formed with a lambdamax at 462 +/- 2 nm. P-opsin bleached slowly in the dark (t1/2 = 2 h) in the presence of 50 mM hydroxylamine. Purified P-opsin in dodecyl maltoside activated rod transducin in a light-dependent manner, catalyzing the exchange of more than 300 mol of GTPgammaS (guanosine 5'-O-(3-thiotriphosphate))/mol of P-opsin. The initial rate for activation (75 mol of GTPgammaS bound/mol of P-opsin/min at 7 microM) increased with increasing concentrations of transducin. The addition of egg phosphatidylcholine to P-opsin had little effect on the activation kinetics; however, the intrinsic rate of decay in the absence of transducin was accelerated. These results demonstrate that P-opsin is an efficient catalyst for activation of rod transducin and suggest that the pineal gland may contain a rodlike phototransduction cascade.     

Zero-length crosslinking of the beta subunit of phosphorylase kinase to the N-terminal half of its regulatory alpha subunit

Phosphorylase kinase, a regulatory enzyme of glycogenolysis in skeletal muscle, is a hexadecameric oligomer containing four copies each of four distinct subunits: alpha, beta, gamma, and delta. By intramolecular zero-length crosslinking with transglutaminase, we have previously demonstrated that the regulatory alpha and beta subunits abut one another in the holoenzyme [Nadeau, O. W., and Carlson, G. M. (1994) J. Biol. Chem. 269, 29670-29676]. Selective partial proteolysis of the 138 kDa alpha subunit in holoenzyme that had been crosslinked by transglutaminase has revealed a high molecular weight conjugate corresponding to full-length beta subunit crosslinked to a 60 kDa N-terminal fragment of alpha (determined by SDS-PAGE, Western blotting and N-terminal sequencing). This conjugate was also observed when the enzyme was first activated by partial proteolysis of alpha and then crosslinked by transglutaminase. Both forms of the kinase, generated by either sequential crosslinking and proteolysis or the reverse, coeluted with non-crosslinked hexadecameric control enzyme in size exclusion chromatography, indicating that the crosslinking was intramolecular, i.e., within hexadecamers. This is the first demonstration of any intersubunit interaction involving the N-terminal domain of the alpha subunit and the first region of any subunit shown to interact with the beta subunit. The results are consistent with the predicted path of the polypeptide backbone of the alpha subunits within the holoenzyme and with the proposed location of the beta subunits.     

In vivo phosphorylation of the epithelial sodium channel

The activity of the epithelial sodium channel (ENaC) in the distal nephron is regulated by an antidiuretic hormone, aldosterone, and insulin, but the molecular mechanisms that mediate these hormonal effects are mostly unknown. We have investigated whether aldosterone, insulin, or activation of protein kinases has an effect on the phosphorylation of the channel. Experiments were performed in an epithelial cell line generated by stable cotransfection of the three subunits (alpha, beta, and gamma) of ENaC. We found that beta and gamma, but not the alpha subunit, are phosphorylated in the basal state. Aldosterone, insulin, and protein kinases A and C increased phosphorylation of the beta and gamma subunits in their carboxyl termini, but none of these agents induced de novo phosphorylation of alpha subunits. Serines and threonines but not tyrosines were found to be phosphorylated. The results suggest that aldosterone, insulin, and protein kinases A and C modulate the activity of ENaC by phosphorylation of the carboxyl termini of the beta and gamma subunits.     

Regulation of the rate and extent of phospholipase C beta 2 effector activation by the beta gamma subunits of heterotrimeric G proteins

The activity of mammalian phosphoinositide-specific phospholipase C beta 2 (PLC-beta 2) is regulated by the alpha q family of G proteins and by beta gamma subunits. We measured the affinity between the laterally associating PLC-beta 2 and G beta gamma on membrane surfaces by fluorescence resonance energy transfer. Using a simple model, we translated this apparent affinity to a bulk or three-dimensional equilibrium constant (Kd) and obtained a value of 3.2 microM. We confirmed this Kd by separately measuring the on and off (kf and kr) rate constants. The kf was slower than a diffusion-limited value, suggesting that conformational changes occur when the two proteins interact. The off rate shows that the PLC-beta 2.G beta gamma complexes are long-lived ( approximately 123 s) and that activation of PLC-beta 2 by G beta gamma would be sustained without a deactivating factor. The addition of alpha i1(GDP) subunits failed to physically dissociate the complex as determined by fluorescence. However, enzyme activity studies performed under similar conditions show that the addition of G alpha i1(GDP) results in reversal of PLC-beta 2 activation by G beta gamma during the time of the assay (30 s). From these results, we propose that G alpha(GDP) subunits can bind to the PLC-beta 2.G beta gamma complex to allow for rapid deactivation without complex dissociation. In support of this model, we show by fluorescence that G alpha i1(GDP).G beta gamma.PLC-beta 2 can form.     

Role of the prenyl group on the G protein gamma subunit in coupling trimeric G proteins to A1 adenosine receptors

The coupling of receptors to heterotrimeric G proteins is determined by interactions between the receptor and the G protein alpha subunits and by the composition of the betagamma dimers. To determine the role of the gamma subunit prenyl modification in this interaction, the CaaX motifs in the gamma1 and gamma2 subunits were altered to direct modification with different prenyl groups, recombinant betagamma dimers expressed in the baculovirus/Sf9 insect cell system, and the dimers purified. The activity of the betagamma dimers was compared in two assays: formation of the high affinity agonist binding conformation of the A1 adenosine receptor and receptor-catalyzed exchange of GDP for GTP on the alpha subunit. The beta1gamma1 dimer (modified with farnesyl) was significantly less effective than beta1gamma2 (modified with geranylgeranyl) in either assay. The beta1gamma1-S74L dimer (modified with geranylgeranyl) was nearly as effective as beta1gamma2 in either assay. The beta1gamma2-L71S dimer (modified with farnesyl) was significantly less active than beta1gamma2. Using 125I-labeled betagamma subunits, it was determined that native and altered betagamma dimers reconstituted equally well into Sf9 membranes containing A1 adenosine receptors. These data suggest that the prenyl group on the gamma subunit is an important determinant of the interaction between receptors and G protein gamma subunits.     

The influence of beta subunit structure on the stability of Na+/K(+)-ATPase complexes and interaction with K+

Heterologous expression of the beta subunit of H+/K(+)-ATPase (HK beta) with alpha subunits of Na+/K(+)-ATPase (NK alpha) in yeast leads to the formation of ouabain binding complexes, indicating assembly of the two subunits into active ion pumps (Eakle, K. A., Kim, K. S., Kabalin, M. A., and Farley, R. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2834-2838). Complexes of NK alpha and HK beta are less sensitive to inhibition of ouabain binding by K+, suggesting that HK beta lowers the affinity of K+ binding sites. This effect is particularly pronounced when HK beta is combined with the alpha 3 isoform of NK alpha. In this case, titration with K+ yields a biphasic curve, suggesting that there are two nonequivalent sites for K+ binding. Attempts at purifying complexes formed with either alpha 1 + HK beta or alpha 3 + HK beta using SDS extraction of microsomal membranes resulted in the loss of ouabain binding. Controls show that alpha 1 + beta 1 and alpha 3 + beta 1 complexes still retain ouabain binding after SDS extraction under the same conditions. This suggests that the HK beta subunit forms a less stable complex with NK alpha subunits. We have created chimeric beta subunits comprised of the amino-terminal cytoplasmic and transmembrane regions of HK beta combined with the carboxyl-terminal extracellular region of Na+/K(+)-ATPase beta 1 (HN beta 1) and the complementary chimera with amino-terminal cytoplasmic and transmembrane regions of beta 1 combined with the carboxyl-terminal extracellular region of HK beta (NH beta 1). When NH beta 1 is combined with either alpha 1 or alpha 3, the complexes show profiles of K+ inhibition of ouabain binding that are very similar to HK beta combined with either alpha 1 or alpha 3. The data suggest that the extracellular region of HK beta is primarily responsible for the effect on apparent K+ affinity. When the HN beta 1 subunit is expressed with the alpha 3 subunit, less than 5% of the amount of ouabain binding complexes are formed compared with HN beta 1 + alpha 1. This observation suggests that the HN beta 1 subunit either assembles poorly or forms an unstable complex with alpha 3. After SDS extraction, complexes of alpha 1 + NH beta 1 and alpha 3 + NH beta 1 retain ouabain binding, while alpha 1 + HN beta 1 complexes are sensitive to SDS extraction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Asymmetry of the alpha subunit of the chloroplast ATP synthase as probed by the binding of Lucifer Yellow vinyl sulfone

The catalytic portion of the chloroplast ATP synthase (CF1) is structurally asymmetric. Asymmetry of the otherwise symmetrical alpha3beta3 heterohexamer is induced by the presence of tightly bound nucleotides and interactions with the single-copy, smaller subunits. Lucifer Yellow vinyl sulfone (4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide-3,6-disulfonic acid) rapidly and covalently binds to lysine 378 on one alpha subunit [Nalin, C. M., Snyder, B., and McCarty, R. E., (1985) Biochemistry 24, 2318-2324] [Shapiro, A. B. (1991) Ph.D. Thesis, Cornell University, Ithaca, NY). The asymmetrical binding of Lucifer Yellow to CF1 provides a method to investigate the cause of asymmetry in the alpha subunits. The reaction of CF1 with Lucifer Yellow was monitored by total fluorescence of bound Lucifer Yellow as well as by quantitative determination of Lucifer Yellow bound to the tryptic peptide that contains lysine 378 of the alpha subunit. The total binding of Lucifer Yellow to CF1 was not affected by the presence of tightly bound nucleotides or nucleotide in the medium. Neither the total binding of Lucifer Yellow to CF1 nor the reaction of alpha-lysine 378 with Lucifer Yellow was changed by the removal of the epsilon subunit, the delta subunit, or both subunits. The extent of incorporation of Lucifer Yellow into lysine 378 of the alpha subunit in (alphabeta)n was about three times that of Lucifer Yellow incorporation into CF1. Reconstitution of (alphabeta)n with gamma restored the binding of one Lucifer Yellow per alpha3beta3gamma. Therefore, the interactions between gamma and the alphabeta heterohexamer are important in conferring asymmetry to the alpha subunits of CF1.