Asymmetric contribution of alpha and beta subunits to the activation of alphabeta heteromeric glycine receptors

This study investigated the role of beta subunits in the activation of alphabeta heteromeric glycine receptor (GlyR) chloride channels recombinantly expressed in HEK293 cells. The approach involved incorporating mutations into corresponding positions in alpha and beta subunits and comparing their effects on receptor function. Although cysteine-substitution mutations to residues in the N-terminal half of the alpha subunit M2-M3 loop dramatically impaired the gating efficacy, the same mutations exerted little effect when incorporated into corresponding positions of the beta subunit. Furthermore, although the alpha subunit M2-M3 loop cysteines were modified by a cysteine-specific reagent, the corresponding beta subunit cysteines showed no evidence of reactivity. These observations suggest structural or functional differences between alpha and beta subunit M2-M3 loops. In addition, a threonine-->leucine mutation at the 9' position in the beta subunit M2 pore-lining domain dramatically increased the glycine sensitivity. By analogy with the effects of the same mutation in other ligand-gated ion channels, it was concluded that the mutation affected the GlyR activation mechanism. This supports the idea that the GlyR beta subunit is involved in receptor gating. In conclusion, this study demonstrates that beta subunits contribute to the activation of the GlyR, but that their involvement in this process is significantly different to that of the alpha subunit.     

Molecular determinants of proton modulation of glycine receptors

Extracellular pH regulates glycine receptors through an unknown mechanism. Here we demonstrate that acidic pH remarkably inhibited glycine-activated whole-cell currents in recombinant glycine alpha1 and alpha1beta receptors transiently expressed in human embryonic kidney 293 cells. The proton effect was voltage-independent and pharmacologically competed with glycine receptor agonist glycine and antagonist strychnine. Using site-directed mutagenesis, we have identified an N-terminal domain that is essential for proton-induced inhibition of glycine current. In alpha1 homomers, removal of the hydroxyl group by mutation of residue Thr-112 to Ala or Phe abolished inhibition of glycine currents by acidification. In contrast, mutation of Thr-112 to another hydroxylated residue (Tyr) produced receptors that retained partial proton sensitivity. In alpha1beta heteromers, a single mutation of the beta subunit T135A, which is homologous to alpha1 Thr-112, reduced proton sensitivity, whereas the double mutation alpha1(T112A)beta(T135A) almost completely eliminated the proton sensitivity. In addition, the mutation alpha1 H109A greatly reduced sensitivity to protons in homomeric alpha1 receptors. The results demonstrate that extracellular pH can regulate the function of glycine alpha1 and alpha1beta receptors. An extracellular domain consisting of Thr-112 and His-109 at the alpha1 subunit and Thr-135 at the beta subunit plays a critical role in determining proton modulation of glycine receptor function.     

Molecular determinants of ginkgolide binding in the glycine receptor pore

Ginkgolides are potent blockers of the glycine receptor Cl- channel (GlyR) pore. We sought to identify their binding sites by comparing the effects of ginkgolides A, B and C and bilobalide on alpha1, alpha2, alpha1beta and alpha2beta GlyRs. Bilobalide sensitivity was drastically reduced by incorporation of the beta subunit. In contrast, the sensitivities to ginkgolides B and C were enhanced by beta subunit expression. However, ginkgolide A sensitivity was increased in the alpha2beta GlyR relative to the alpha2 GlyR but not in the alpha1beta GlyR relative to the alpha1 GlyR. We hypothesised that the subunit-specific differences were mediated by residue differences at the second transmembrane domain 2' and 6' pore-lining positions. The increased ginkgolide A sensitivity of the alpha2beta GlyR was transferred to the alpha1beta GlyR by the G2'A (alpha1 to alpha2 subunit) substitution. In addition, the alpha1 subunit T6'F mutation abolished inhibition by all ginkgolides. As the ginkgolides share closely related structures, their molecular interactions with pore-lining residues were amenable to mutant cycle analysis. This identified an interaction between the variable R2 position of the ginkgolides and the 2' residues of both alpha1 and beta subunits. These findings provide strong evidence for ginkgolides binding at the 2' pore-lining position.     

Two novel residues in M2 of the gamma-aminobutyric acid type A receptor affecting gating by GABA and picrotoxin affinity

An amino acid residue was found in M2 of gamma-aminobutyric acid (GABA) type A receptors that has profound effects on the binding of picrotoxin to the receptor and therefore may form part of its binding pocket. In addition, it strongly affects channel gating. The residue is located N-terminally to residues suggested so far to be important for channel gating. Point mutated alpha1beta(3) receptors were expressed in Xenopus oocytes and analyzed using the electrophysiological techniques. Coexpression of the alpha(1) subunit with the mutated beta(3) subunit beta(3)L253F led to spontaneous picrotoxin-sensitive currents in the absence of GABA. Nanomolar concentrations of GABA further promoted channel opening. Upon washout of picrotoxin, a huge transient inward current was observed. The reversal potential of the inward current was indicative of a chloride ion selectivity. The amplitude of the inward current was strongly dependent on the picrotoxin concentration and on the duration of its application. There was more than a 100-fold decrease in picrotoxin affinity. A kinetic model is presented that mimics the gating behavior of the mutant receptor. The point mutation in the neighboring residue beta(3)A252V resulted in receptors that displayed an about 6-fold increased apparent affinity to GABA and an about 10-fold reduced sensitivity to picrotoxin.     

Resistance to fipronil in Drosophila simulans: influence of two point mutations in the RDL GABA receptor subunit

The Eyguieres 42 strain of Drosophila simulans, obtained by laboratory selection, displayed approximately 20,000-fold resistance to the insecticide fipronil. Molecular cloning of the cDNA encoding the RDL GABA receptor subunit of this strain revealed the presence of two mutations: the Rdl mutation (A301G) and an additional mutation in the third transmembrane domain (T350M). In order to assess the individual and combined roles of the two mutations in fipronil resistance, the functional properties of wild-type, A301G, T350M and A301G/T350M homomultimeric RDL receptors were compared by expression in Xenopus oocytes. In wild-type receptors, the inhibition of GABA (EC(30))-induced currents by fipronil and picrotoxin was enhanced by repeated GABA applications. The A301G mutation nearly abolished this effect, decreased the sensitivity to fipronil and picrotoxin and increased the reversibility of inhibition. The T350M mutation also reduced the sensitivity to both antagonists. Of the four receptor variants tested, the double mutant showed the highest resistance to fipronil, following repeated GABA applications. In conclusion, the present study emphasizes new aspects of the pharmacological alterations induced by the Rdl mutation and shows that resistance to GABA receptor-directed insecticides may implicate a mutation distinct from Rdl.     

Ivermectin, an unconventional agonist of the glycine receptor chloride channel

The effects of the antihelmintic, ivermectin, were investigated in recombinantly expressed human alpha(1) homomeric and alpha(1)beta heteromeric glycine receptors (GlyRs). At low (0.03 microm) concentrations ivermectin potentiated the response to sub-saturating glycine concentrations, and at higher (> or =0.03 microm) concentrations it irreversibly activated both alpha(1) homomeric and alpha(1)beta heteromeric GlyRs. Relative to glycine-gated currents, ivermectin-gated currents exhibited a dramatically reduced sensitivity to inhibition by strychnine, picrotoxin, and zinc. The insensitivity to strychnine could not be explained by ivermectin preventing the access of strychnine to its binding site. Furthermore, the elimination of a known glycine- and strychnine-binding site by site-directed mutagenesis had little effect on ivermectin sensitivity, demonstrating that the ivermectin- and glycine-binding sites were not identical. Ivermectin strongly and irreversibly activated a fast-desensitizing mutant GlyR after it had been completely desensitized by a saturating concentration of glycine. Finally, a mutation known to impair dramatically the glycine signal transduction mechanism had little effect on the apparent affinity or efficacy of ivermectin. Together, these findings indicate that ivermectin activates the GlyR by a novel mechanism.     

Mutation of a single TMVI residue, Phe(282), in the beta(2)-adrenergic receptor results in structurally distinct activated receptor conformations

We showed previously that Phe(303) in transmembrane segment (TM) VI of the alpha(1B)-adrenergic receptor (alpha(1B)-AR), a residue conserved in many G protein-coupled receptors (GPCRs), is critically involved in coupling agonist binding with TM helical movement and G protein activation. Here the equivalent residue, Phe(282), in the beta(2)-AR was evaluated by mutation to glycine, asparagine, alanine, or leucine. Except for F282N, which exhibits attenuated basal and maximal isoproterenol stimulation, the Phe(282) mutants display varying degrees of constitutive activity (F282L > F282A > F282G), and as shown by the results of substituted cysteine accessibility method (SCAM) studies, induce movement of endogenous cysteine(s) into the water-accessible ligand-binding pocket. For F282A, movement is confined to Cys(285) in TMVI, whereas F282L induces movement of both Cys(285) in TMVI and Cys(327) in TMVII. Further, engineered cysteine-sensor studies indicate that F282L causes movement of TMVI, both above and below an apparent kink-inducing TMVI proline (Pro(288)), whereas that due to F282A is confined to the domain below Pro(288). A plausible interpretation of these data is that receptor activation involves rigid body movement of TMVI which, because of its Pro(288)-induced kink, acts as a pivot to transduce and amplify the agonist-induced conformational change in the upper domain, to a change in the lower domain required for productive receptor-G protein coupling.     

Point mutation in the first transmembrane region of the beta 2 subunit of the gamma--aminobutyric acid type A receptor alters desensitization kinetics of gamma--aminobutyric acid- and anesthetic-induced channel gating

A conserved glycine residue in the first transmembrane (TM1) domain of the beta2 subunit has been identified to be involved with desensitization induced by gamma-aminobutyric acid (GABA) and anesthetics. Recombinant GABA(A) receptors expressed in Sf9 cells were recorded using semi-fast agonist application. Upon direct activation by GABA or anesthetics, the main effect of the TM1 point mutation on the beta2 subunit (G219F) was to slow the time constant (tau) of desensitization. At GABA concentrations eliciting maximum currents, the corresponding median tau values were 0.87 s (25-75% interval (0.76; 1.04 s)), 0.93 s (0.76; 1.23 s), and 1.36 s (1.17; 1.57 s) for alpha1beta2gamma2, alpha1(G223F)beta2gamma2, and alpha1beta2(G219F)gamma2, respectively. The tau value for the beta2-mutant receptor was significantly longer than alpha1beta2gamma2 (p < 0.01) and alpha1(G223F)beta2gamma2 (p < 0.05). For pentobarbital-induced currents (500 microm), the corresponding median tau values were 1.36 s (0.81; 1.41 s), 1.47 s (1.31; 2.38 s), and 2.82 s (2.21; 5.56 s) for alpha1beta2gamma2, alpha1(G223F)beta2gamma2, and alpha1beta2(G219F)gamma2, respectively. The tau value for the beta2-mutant receptor was significantly longer than that for alpha1beta2gamma2 (p < 0.01). The present findings suggest that this TM1 glycine residue is critical for the rate at which desensitization occurs and that both GABA and intravenous anesthetics implement an analogous pathway for generating desensitization.     

Stalk region of beta-chain enhances the coreceptor function of CD8

CD8 glycoproteins are expressed as either alphaalpha homodimers or alphabeta heterodimers on the surface of T cells. CD8alphabeta is a more efficient coreceptor than the CD8alphaalpha for peptide Ag recognition by TCR. Each CD8 subunit is composed of four structural domains, namely, Ig-like domain, stalk region, transmembrane region, and cytoplasmic domain. In an attempt to understand why CD8alphabeta is a better coreceptor than CD8alphaalpha, we engineered, expressed, and functionally tested a chimeric CD8alpha protein whose stalk region is replaced with that of CD8beta. We found that the beta stalk region enhances the coreceptor function of chimeric CD8alphaalpha to a level similar to that of CD8alphabeta. Surprisingly, the beta stalk region also restored functional activity to an inactive CD8alpha variant, carrying an Ala mutation at Arg(8) (R8A), to a level similar to that of wild-type CD8alphabeta. Using the R8A variant of CD8alpha, a panel of anti-CD8alpha Abs, and three MHC class I (MHCI) variants differing in key residues known to be involved in CD8alpha interaction, we show that the introduction of the CD8beta stalk leads to a different topology of the CD8alpha-MHCI complex without altering the overall structure of the Ig-like domain of CD8alpha or causing the MHCI to employ different residues to interact with the CD8alpha Ig domain. Our results show that the stalk region of CD8beta is capable of fine-tuning the coreceptor function of CD8 proteins as a coreceptor, possibly due to its distinct protein structure, smaller physical size and the unique glycan adducts associated with this region.     

Insights into the structural basis for zinc inhibition of the glycine receptor

Histidines 107 and 109 in the glycine receptor (GlyR) alpha1 subunit have previously been identified as determinants of the inhibitory zinc-binding site. Based on modeling of the GlyR alpha1 subunit extracellular domain by homology to the acetylcholine-binding protein crystal structure, we hypothesized that inhibitory zinc is bound within the vestibule lumen at subunit interfaces, where it is ligated by His107 from one subunit and His109 from an adjacent subunit. This was tested by co-expressing alpha1 subunits containing the H107A mutation with alpha1 subunits containing the H109A mutation. Although sensitivity to zinc inhibition is markedly reduced when either mutation is individually incorporated into all five subunits, the GlyRs formed by the co-expression of H107A mutant subunits with H109A mutant subunits exhibited an inhibitory zinc sensitivity similar to that of the wild type alpha1 homomeric GlyR. This constitutes strong evidence that inhibitory zinc is coordinated at the interface between adjacent alpha1 subunits. No evidence was found for beta subunit involvement in the coordination of inhibitory zinc, indicating that a maximum of two zinc-binding sites per alpha1beta receptor is sufficient for maximal zinc inhibition. Our data also show that two zinc-binding sites are sufficient for significant inhibition of alpha1 homomers. The binding of zinc at the interface between adjacent alpha1 subunits could restrict intersubunit movements, providing a feasible mechanism for the inhibition of channel activation by zinc.     

Allosteric activation mechanism of the alpha1beta2gamma2 gamma-aminobutyric acid type A receptor revealed by mutation of the conserved M2 leucine

A conserved leucine residue in the midpoint of the second transmembrane domain (M2) of the ligand-activated ion channel family has been proposed to play an important role in receptor activation. In this study, we assessed the importance of this leucine in the activation of rat alpha1beta2gamma2 GABA receptors expressed in Xenopus laevis oocytes by site-directed mutagenesis and two-electrode voltage clamp. The hydrophobic conserved M2 leucines in alpha1(L263), beta2(L259), and gamma2(L274) subunits were mutated to the hydrophilic amino acid residue serine and coexpressed in all possible combinations with their wild-type and/or mutant counterparts. The mutation in any one subunit decreased the EC(50) and created spontaneous openings that were blocked by picrotoxin and, surprisingly, by the competitive antagonist bicuculline. The magnitudes of the shifts in GABA EC(50) and picrotoxin IC(50) as well as the degree of spontaneous openings were all correlated with the number of subunits carrying the leucine mutation. Simultaneous mutation of the GABA binding site (beta2Y157S; increased the EC(50)) and the conserved M2 leucine (beta2L259S; decreased the EC(50)) produced receptors with the predicted intermediate agonist sensitivity, indicating the two mutations affect binding and gating independently. The results are discussed in light of a proposed allosteric activation mechanism.     

A picrotoxin-specific conformational change in the glycine receptor M2-M3 loop

The external loop linking the M2 and M3 transmembrane domains is crucial for coupling agonist binding to channel gating in the glycine receptor chloride channel (GlyR). A substituted cysteine accessibility scan previously showed that glycine activation increased the surface accessibility of 6 contiguous residues (Arg271-Lys276) toward the N-terminal end of the homomeric alpha1 GlyR M2-M3 loop. In the present study we used a similar approach to determine whether the allosteric antagonist, picrotoxin, could impose conformational changes to this domain that cannot be induced by varying agonist concentrations alone. Picrotoxin slowed the reaction rate of a sulfhydryl-containing compound (MTSET) with A272C, S273C, and L274C. Before interpreting this as a picrotoxin-specific conformational change, it was necessary to eliminate the possibility of steric competition between picrotoxin and MTSET. Accordingly, we showed that picrotoxin and the structurally unrelated blocker, bilobalide, were both trapped in the R271C GlyR in the closed state and that a point mutation to the pore-lining Thr6' residue abolished inhibition by both compounds. We also demonstrated that the picrotoxin dissociation rate was linearly related to the channel open probability. These observations constitute a strong case for picrotoxin binding in the pore. We thus conclude that the picrotoxin-specific effects on the M2-M3 loop are mediated allosterically. This suggests that the M2-M3 loop responds differently to the occupation of different binding sites.     

Efficient coupling of ligand binding to channel opening by the binding domain of a modulatory (beta) subunit of the olfactory cyclic nucleotide-gated channel.

CNG channels in vivo are heteromers of homologous alpha and beta subunits that each contain a six-transmembrane segment domain and a COOH-terminal cytoplasmic cyclic nucleotide binding domain (BD). In heterologous expression systems, heteromeric alphabeta channels activate with greater sensitivity to ligand than do homomeric alpha channels; however, ligand-gating of channels containing only beta subunit BDs has never been studied because beta subunits cannot form functional homomeric CNG channels. To characterize directly the contribution of the beta subunit BD to ligand-gating, we constructed a chimeric subunit, X-beta, whose BD sequence was that of the beta subunit CNG5 from rat, but whose sequence outside the BD was derived from alpha subunits. For comparison, we constructed another chimera, X-alpha, whose sequence outside the BD was identical to that of X-beta, but whose BD sequence was that of the alpha subunit CNG2 from catfish. When expressed in Xenopus oocytes, X-beta and X-alpha each formed functional homomeric channels activated by both cAMP and cGMP. This is the first demonstration that the beta subunit BD can couple ligand binding to activation in the absence of alpha subunit BD residues. Notably, both agonists activate X-beta more effectively than X-alpha (higher opening efficacy and lower K(1/2)). The BD is believed to comprise two functionally distinct subdomains: (1) the roll subdomain (beta-roll and flanking A- and B-helices) and (2) the C-helix subdomain. Opening efficacy was previously believed to be controlled primarily by the C-helix, but when we made additional chimeras by exchanging the subdomains between X-beta and X-alpha, we found that both subdomains contain significant determinants of efficacy and agonist selectivity. In particular, only channels containing the roll subdomain of the beta subunit had high efficacy. Thermodynamic linkage analysis shows that interaction between the two subdomains accounts for a significant portion of their contribution to activation energetics.     

An asymmetric contribution to gamma-aminobutyric type A receptor function of a conserved lysine within TM2-3 of alpha1, beta2, and gamma2 subunits

Mutations that impair the expression and/or function of gamma-aminobutyric acid type A (GABAA) receptors can lead to epilepsy. The familial epilepsy gamma2(K289M) mutation affects a basic residue conserved in the TM2-3 linker of most GABAA subunits. We investigated the effect on expression and function of the Lys --> Met mutation in mouse alpha1(K278M), beta2(K274M), and gamma2(K289M) subunits. Compared with cells expressing wild-type and alpha1beta2gamma2(K289M) receptors, cells expressing alpha1(K278M)beta2gamma2 and alpha1beta2(K274M)gamma2 receptors exhibited reduced agonist-evoked current density and reduced GABA potency, with no change in single channel conductance. The low current density of alpha1beta2(K274M)gamma2 receptors coincided with reduced surface expression. By contrast the surface expression of alpha1(K278M)beta2gamma2 receptors was similar to wild-type and alpha1beta2gamma2(K289M) receptors suggesting that the alpha1(K278M) impairs function. In keeping with this interpretation GABA-activated channels mediated by alpha1(K278M)beta2gamma2 receptors had brief open times. To a lesser extent gamma2(K289M) also reduced mean open time, whereas beta2(K274M) had no effect. We used propofol as an alternative GABAA receptor agonist to test whether the functional deficits of mutant subunits were specific to GABA activation. Propofol was less potent as an activator of alpha1(K278M)beta2gamma2 receptors. By contrast, neither beta2(K274M) nor gamma2(K289M) affected the potency of propofol. The beta2(K274M) construct was unique in that it reduced the efficacy of propofol activation relative to GABA. These data suggest that the alpha1 subunit Lys-278 residue plays a pivotal role in channel gating that is not dependent on occupancy of the GABA binding site. Moreover, the conserved TM2-3 loop lysine has an asymmetric function in different GABAA subunits.     

Catalytic site of F1-ATPase of Escherichia coli. Lys-155 and Lys-201 of the beta subunit are located near the gamma-phosphate group of ATP in the presence of Mg2+.

The catalytic site of Escherichia coli F1 was probed using a reactive ATP analogue, adenosine triphosphopyridoxal (AP3-PL). For complete loss of enzyme activity, about 1 mol of AP3-PL bound to 1 mol of F1 was estimated to be required in the presence or absence of Mg2+. About 70% of the label was bound to the alpha subunit and the rest to the beta subunit in the absence of Mg2+, and the alpha Lys-201 and beta Lys-155 residues, respectively, were the major target residues (Tagaya, M., Noumi, T., Nakano, K., Futai, M., and Fukui, T. (1988) FEBS Lett. 233, 347-351). Addition of Mg2+ decreased the AP3-PL concentration required for half-maximal inhibition, and predominant labeling of the beta subunit (beta Lys-155 and beta Lys-201) with the reagent. ATP and ADP were protective ligands in the presence and absence of Mg2+. The alpha subunit mutation (alpha Lys-201----Gln or alpha Lys-201 deletion) were active in oxidative phosphorylation. However, purified mutant F1s showed impaired low multi-site activity, although their uni-site catalyses were essentially normal. Thus alpha Lys-201 is not a catalytic residue, but may be important for catalytic cooperativity. Mutant F1s were inhibited less by AP3-PL in the absence of Mg2+, and consistent with this, modifications of their alpha subunits by AP3-PL were reduced. AP3-PL was more inhibitory to the mutant enzymes in the presence of Mg2+, and bound to the beta Lys-155 and beta Lys-201 residues of mutant F1 (alpha Lys-201----Gln). These results strongly suggest that alpha Lys-201, beta Lys-155, and beta Lys-201 are located close together near the gamma-phosphate group of ATP bound to the catalytic site, and that the two beta residues and the gamma-phosphate group become closer to each other in the presence of Mg2+.     

Identification of a novel residue within the second transmembrane domain that confers use-facilitated block by picrotoxin in glycine alpha 1 receptors.

The central nervous system convulsant picrotoxin (PTX) inhibits GABA(A) and glutamate-gated Cl(minus sign) channels in a use-facilitated fashion, whereas PTX inhibition of glycine and GABA(C) receptors displays little or no use-facilitated block. We have identified a residue in the extracellular aspect of the second transmembrane domain that converted picrotoxin inhibition of glycine alpha1 receptors from non-use-facilitated to use-facilitated. In wild type alpha1 receptors, PTX inhibited glycine-gated Cl(minus sign) current in a competitive manner and had equivalent effects on peak and steady-state currents, confirming a lack of use-facilitated block. Mutation of the second transmembrane domain 15'-serine to glutamine (alpha1(S15'Q) receptors) converted the mechanism of PTX blockade from competitive to non-competitive. However, more notable was the fact that in alpha1(S15'Q) receptors, PTX had insignificant effects on peak current amplitude and dramatically enhanced current decay kinetics. Similar results were found in alpha1(S15'N) receptors. The reciprocal mutation in the beta2 subunit of alpha1beta2 GABA(A) receptors (alpha1beta2(N15'S) receptors) decreased the magnitude of use-facilitated PTX inhibition. Our results implicate a specific amino acid at the extracellular aspect of the ion channel in determining use-facilitated characteristics of picrotoxin blockade. Moreover, the data are consistent with the suggestion that picrotoxin may interact with two domains in ligand-gated anion channels.     

Complementation analysis demonstrates that insulin cross-links both alpha subunits in a truncated insulin receptor dimer

The insulin receptor is a homodimer composed of two alphabeta half receptors. Scanning mutagenesis studies have identified key residues important for insulin binding in the L1 domain (amino acids 1-150) and C-terminal region (amino acids 704-719) of the alpha subunit. However, it has not been shown whether insulin interacts with these two sites within the same alpha chain or whether it cross-links a site from each alpha subunit in the dimer to achieve high affinity binding. Here we have tested the contralateral binding mechanism by analyzing truncated insulin receptor dimers (midi-hIRs) that contain complementary mutations in each alpha subunit. Midi-hIRs containing Ala(14), Ala(64), or Gly(714) mutations were fused with Myc or FLAG epitopes at the C terminus and were expressed separately by transient transfection. Immunoblots showed that R14A+FLAG, F64A+FLAG, and F714G+Myc mutant midi-hIRs were expressed in the medium but insulin binding activity was not detected. However, after co-transfection with R14A+FLAG/F714G+Myc or F64A+FLAG/F714G+Myc, hybrid dimers were obtained with a marked increase in insulin binding activity. Competitive displacement assays revealed that the hybrid mutant receptors bound insulin with the same affinity as wild type and also displayed curvilinear Scatchard plots. In addition, when hybrid mutant midi-hIR was covalently cross-linked with (125)I(A14)-insulin and reduced, radiolabeled monomer was immunoprecipitated only with anti-FLAG, demonstrating that insulin was bound asymmetrically. These results demonstrate that a single insulin molecule can contact both alpha subunits in the insulin receptor dimer during high affinity binding and this property may be an important feature for receptor signaling.     

Split leucine-specific domain of leucyl-tRNA synthetase from the hyperthermophilic bacterium Aquifex aeolicus

Leucyl-tRNA synthetase (LeuRS) from Aquifex aeolicus is the only known heterodimer synthetase. It is named LeuRS alphabeta;, and its alpha and beta subunits contain 634 and 289 residues, respectively. Like Thermus thermophilus LeuRS, LeuRS alphabeta has a large extra domain, the leucine-specific domain, inserted into the catalytic domain. The subunit split site is exactly in the middle of the leucine-specific domain and may have a unique function. Here, a series of mutants of LeuRS alphabeta consisting of either mutated alpha subunits and wild-type beta subunits or wild-type alpha subunits and mutated beta subunits were constructed and purified. ATP-PPi exchange and aminoacylation activities and the ability of the mutants to charge minihelix(Leu) were assayed. Interaction of the mutants with the tRNA was assessed by gel shift. Two peptides of eight and nine amino acid residues in the domain located in the alpha subunit were found to be essential for the enzyme's activity. We also showed that the domain in LeuRS alphabeta plays an important role in minihelix(Leu) recognition. Additionally, the domain was found to have little impact on the assembly of the heterodimer, to play a role in the thermal stability of the whole enzyme, and to interact with the cognate tRNA in the predicted manner.     

Gating of heteromeric retinal rod channels by cyclic AMP: role of the C-terminal and pore domains

Cyclic nucleotide-gated channels are tetramers composed of homologous alpha and beta subunits. C-terminal truncation mutants of the alpha and beta subunits of the retinal rod channel were expressed in Xenopus oocytes, and analyzed for cGMP- and cAMP-induced currents (single-channel records and macroscopic currents). When the alpha subunit truncated downstream of the cGMP-binding site (alpha D608stop) is co-injected with truncated beta subunits, the heteromeric channels present a drastic increase of cAMP sensitivity. A partial effect is observed with heteromeric alpha R656stop-containing channels, while alpha K665stop-containing channels behave like alpha wt/beta wt. The three truncated alpha subunits have wild-type activity when expressed alone. Heteromeric channels composed of alpha wt or truncated alpha subunits and chimeric beta subunits containing the pore domain of the alpha subunit have the same cAMP sensitivity as alpha-only channels. The results disclose the key role of two domains distinct from the nucleotide binding site in the gating of heteromeric channels by cAMP: the pore of the beta subunit, which has an activating effect, and a conserved domain situated downstream of the cGMP-binding site in the alpha subunit (I609-K665), which inhibits this effect.