hoxX (hypX) is a functional member of the Alcaligenes eutrophus hyp gene cluster

The role of HoxX in hydrogenase biosynthesis of Alcaligenes eutrophus H16 was re-examined. The previously characterized hoxX deletion mutant HF344 and a newly constructed second hoxX mutant carrying a smaller in-frame deletion were studied. The second mutant was impaired in the activity of both the soluble and the membrane-bound hydrogenase. The two hydrogenase activities were reduced by approximately 50% due to delayed processing of the active-site-containing large subunits, while hydrogenase gene expression was not affected. We conclude that the mutation in mutant HF344 causes polarity resulting in the observed regulatory phenotype of this mutant. The data presented in this report point to an enhancing function of HoxX in the conversion of the soluble hydrogenase and of the membrane-bound hydrogenase large-subunit precursor. Thus, hoxX encodes a member of the Hyp proteins that are required for the formation of active hydrogenase and was accordingly renamed hypX.     

Inverse localization of Ca2+ and La3+ high affinity binding sites of the red cell membrane

Glutaraldehyde fixation in the presence of both 90 mM La3+ resulted in opaque deposits were also seen in specimens fixed in a glutaraldehyde-CaCl2 medium devoid of La3+. In that case only small amounts of a moderately opaque substance had accumulated or remained at the external surface of the erythrocyte membrane. Specimens previously fixed with Ca2+, La3+ supplemented glutaraldehyde lost their internal deposits completely during postifixation with OsO4. The findings provide evidence of Ca2+ high affinity binding sites at the internal surface of the erythrocyte membrane presumably identical with the filamentous matrix. Glycocalyx constituents are considered external Ca2+ low affinity binding sites, however, capable of accumulating high amounts of La3+ during glutaraldehyde fixation.     

The affinity of cholera toxin for Ni2+ ion

Cholera toxin (CT) was shown to bind to immobilized Ni2+ ion. The affinity of CT for the complex required the presence of the Ni2+ ion, since CT was unable to bind in its absence. Binding was mediated by the B-subunit (CTB) as both CT and CTB bound to the resin, but not the A-subunit (CTA). Binding was reversible in the presence of imidazole and suggested that the affinity of CT for the Ni2+ ion was mediated by His residues. The heat-labile enterotoxin of Escherichia coli (LT), which is closely related to CT, was unable to bind to the Ni2+ ion. Comparison of amino acid sequences revealed the presence of three His residues in CT (positions 13, 57 and 94), but only one in LT (position 57). To confirm that the residues at positions 13 and 94 of CTB were responsible for the binding, they were changed to residues found in LTB. Changing His13-->Arg completely abrogated the ability of CTB to bind to Ni2+ ion. In contrast, the mutation of His 94-->Asn reduced, but did not abrogate, the ability of CTB to bind to Ni2+ ion. Based on calculated interatomic distances, it is unlikely that His13 and His94 are part of the same complex. There appear to be two separate binding sites, with the principal site involving His13 and a much weaker site involving His94. This latter site can only participate in binding if the complex involving His13 has formed.     

Structural basis for the high affinity of amino-aromatic SH2 phosphopeptide ligands

When vertebrate somatic cells are selectively irradiated in the nucleus during late prophase (<30 min before nuclear envelope breakdown) they progress normally through mitosis even if they contain broken chromosomes. However, if early prophase nuclei are similarly irradiated, chromosome condensation is reversed and the cells return to interphase. Thus, the G2 checkpoint that prevents entry into mitosis in response to nuclear damage ceases to function in late prophase. If one nucleus in a cell containing two early prophase nuclei is selectively irradiated, both return to interphase, and prophase cells that have been induced to returned to interphase retain a normal cytoplasmic microtubule complex. Thus, damage to an early prophase nucleus is converted into a signal that not only reverses the nuclear events of prophase, but this signal also enters the cytoplasm where it inhibits e.g., centrosome maturation and the formation of asters. Immunofluorescent analyses reveal that the irradiation-induced reversion of prophase is correlated with the dephosphorylation of histone H1, histone H3, and the MPM2 epitopes. Together, these data reveal that a checkpoint control exists in early but not late prophase in vertebrate cells that, when triggered, reverses the cell cycle by apparently downregulating existing cyclin-dependent kinase (CDK1) activity.     

Relationship of low affinity [3]ryanodine binding sites to high affinity sites on the skeletal muscle Ca2+ release channel

Both high and low affinity binding sites for [3H]ryanodine exist in sarcoplasmic reticulum membranes derived from rabbit skeletal muscle. Negatively cooperative binding of [3H]ryanodine at one of four initially identical sites cannot account for some of the kinetic features of the binding to high and low affinity sites. The presence of excess unlabeled ryanodine greatly slows the rate at which [3H]ryanodine bound at the high affinity site dissociates. An examination of the rate of dissociation of [3H]ryanodine bound at increasing [3H]ryanodine concentrations reveals the existence of a second site, occupied only at high ligand concentrations. The occupation of this site correlates well with the conversion of the high affinity site from a site with a dissociation rate constant of approximately 0.0025 min-1 to one with a dissociation rate constant of less than 0.00025 min-1. The low affinity site itself has a dissociation rate constant of 0.013 min-1 and dissociation from this site is unaffected by the presence of 100 microM unlabeled ryanodine. These data suggest that the two binding sites are different but are either allosterically or sterically coupled. Association experiments support this interpretation. Low affinity binding sites for [3H]ryanodine exist in transverse tubule (t-tubule) as well as sarcoplasmic reticulum membranes. High concentrations of both ryanodine and ruthenium red inhibit the binding of [3H]PN200-110 to the dihydropyridine-binding protein in t-tubule membranes. Whether the low affinity site in t-tubule membranes is related to that found in sarcoplasmic reticulum membranes is not yet known.     

Molecular basis and species specificity of high affinity binding of vasoactive intestinal polypeptide by the rat secretin receptor

The affinity and specificity of the binding interaction between ligands and their receptors are key for appropriate hormonal regulation of target tissues. However, it is now apparent that vasoactive intestinal polypeptide (VIP) binds to the rat secretin receptor with similar affinity to that for its natural ligand, secretin (Holtmann et al., 1995). In this report, we establish that this is not a characteristic of the human secretin receptor, and use rat-human secretin receptor chimeras, site mutants and truncated receptor constructs to establish the molecular basis for this unusual binding interaction. Of note, isolated N-terminal domains of the rat secretin and the VIP receptors are capable of high affinity binding of VIP. In the recently recognized secretin family of receptors, this domain has six conserved cysteine residues and disulfide bonds that are likely important to achieve the complex conformation critical for this binding. A single acidic residue (Asp98) present in the rat secretin receptor appears to be critical, because a site-mutant changing this to the polar, but uncharged residue present in that position in the human receptor (Asn) eliminates the high affinity binding of VIP. Of interest, a previously identified critical basic residue in VIP (Lys15) provides a candidate for charge-pairing with this residue, potentially aligning the peptide ligand in a nonproductive orientation within this receptor.     

Location of a high affinity Zn2+ binding site in the channel of alpha1beta1 gamma-aminobutyric acidA receptors

Zn2+ inhibits currents through gamma-aminobutyric acid (GABA)A receptors. Its affinity depends on the subunit composition; alpha1beta1 receptors are inhibited with high affinity (IC50 = 0.54 micro M). We sought to identify the residues that form this high affinity Zn2+ binding site. beta1His267 aligns with alpha1Ser272, a residue near the extracellular end of the M2 membrane-spanning segment that we previously demonstrated to be exposed in the channel. The Zn2+ affinity of alpha1beta1 H267S was reduced by 300-fold (IC50 = 161 micro M). Addition of a histidine at the aligned position in alpha1 creates a receptor, alpha1S272Hbeta1, that should have five channel-lining histidines; the Zn2+ affinity was increased 20-fold (IC50 = 0.025 micro M). Shifting the position of the histidine from the beta1 subunit to the aligned position in alpha1 with the two mutants alpha1S272Hbeta1H267S reduced the affinity (IC50 = 26 micro M) compared with wild-type. We infer that the high affinity Zn2+ binding site involves beta1His267 from at least two subunits. For two histidines to interact with a Zn2+ ion, the alpha carbons must be separated by <13 A. This limits the separation of the subunits and provides a constraint on the possible quaternary structures of the channel. The ability of a divalent cation to penetrate from the extracellular end of the channel to beta1His267 implies that the charge-selectivity filter, the structure that discriminates between anions and cations, is located at a more cytoplasmic position than beta1His267; this is consistent with our previous work that showed that positively charged sulfhydryl-specific reagents reacted with an engineered cysteine residue as cytoplasmic as alpha1T261C.     

Microenvironment of the high affinity ATP-binding site of Na+/K+-ATPase is slightly acidic

Fluorescein-5'-isothiocyanate (FITC) was used to study the high-affinity ATP-binding site of Na+/K+-ATPase. The molar ratio of specifically bound FITC per alpha-subunit of Na+/K+-ATPase was found to be 0.5 as followed from pretreatment experiments with another specific E1ATP-inhibitor Cr(H2O)4AdoPP[CH2]P. This indicated an existence of one high affinity ATP-binding site (E1ATP-binding site) in the native (alphabeta)2-diprotomer of Na+/K+-ATPase. Fluorescence dual-excitation ratio of specifically bound FITC revealed that at external pH 7.5, the pH value inside the E1ATP-binding site is 6.95 +/- 0.18. In addition, FITC fluorescence quenching by anti-fluorescein and by iodide choline indicated the limited access of water into the small pocket of the E1ATP-binding site.     

C-terminal extension of the H2-activating subunit, HoxH, directs maturation of the NAD-reducing hydrogenase in Alcaligenes eutrophus

Idiopathic myelofibrosis, or agnogenic myeloid metaplasia, is a chronic myeloproliferative disorder characterized by clonal expansion and marrow fibrosis. Although marrow fibrosis appears to be a reactive process, it substantially contributes to impaired haemopoiesis. During the last few years the implication of megakaryocyte-derived growth factors in its pathogenesis has been documented. We previously reported increased expression of TGF-beta in patients with idiopathic myelofibrosis. In the present study we show that circulating megakaryocytic cells from such patients expressed high levels of basic fibroblast growth factor (bFGF). An increased expression of bFGF was also detected in patients' platelets. Under culture conditions, bFGF present in megakaryocytic cells was not exported into the medium. consistent with the fact that bFGF is devoid of a secretion peptide signal. Interestingly, this lack of bFGF secretion was observed in all patients but one, who was in an accelerated phase of the disease and presented an important percentage of circulating megakaryoblasts.     

A conformational rearrangement upon binding of IgE to its high affinity receptor

One of the critical steps in the allergic reaction is the binding of immunoglobulin E (IgE) to its high affinity receptor (FcepsilonRI). FcepsilonRI is a tetrameric complex composed of an alpha-chain, a beta-chain, and a dimeric gamma-chain. The extracellular portion of the alpha-chain (alpha-t) is sufficient for the binding of IgE. The Fc portion of IgE contains two copies of the FcepsilonRI binding sites. In contrast, the binding stoichiometry is 1:1. Previously, it was hypothesized that the binding of FcepsilonRI to IgE results in a conformational change in IgE that precludes the binding of a second molecule (Presta, L., Shields, R., O'Connel, L., Lahr, S., Porter, J. , Gorman, C., and Jardieu, P.(1994) J. Biol. Chem. 269, 26368-26373). Here we characterize the secondary structure of IgE and alpha-t and analyze their interaction by circular dichroism spectroscopy. Binding experiments show that when IgE interacts with alpha-t there is a 15-26% decrease of the negative ellipticity at 217 nm. Together, the absence of an alpha-helix element in alpha-t and the small contribution of alpha-t to the spectra of the complex indicate that upon binding, a major conformational rearrangement must occur on IgE. In addition, we analyze the thermal unfolding of alpha-t, IgE, and their complex. Despite the several domains that constitute IgE and alpha-t, these molecules unfold cooperatively with two-state kinetics.     

The fifth transmembrane segment of the neuromedin B receptor is critical for high affinity neuromedin B binding

The two bombesin receptor subtypes, neuromedin B (NMB-R) and gastrin releasing peptide (GRP-R) receptors, bind their respective ligands with high affinity. To identify molecular components mediating high affinity NMB binding, four mutant receptors were constructed, in which different parts of the NMB-R were replaced with the corresponding regions of the GRP-R. When stably expressed in Balb 3T3 fibroblasts, all four NMB-R/GRP-R chimeras were functional and showed NMB-induced stimulation of inositol phosphate (IP) formation. Results of 125I-[D-Tyr0]NMB displacement assays using unlabeled NMB for competition indicated that high affinity NMB binding was determined by amino acid sequences in transmembrane domain V (TM-V) of the NMB-R. To identify which amino acid(s) in TM-V of NMB-R contributed to high affinity NMB binding, four additional NMB-R mutants were constructed where non-conserved amino acids in TM-V of NMB-R were replaced by the corresponding GRP-R amino acids. Three of the mutations, TyrPheLeu220-222-->PheTyrVal, Ile230-->Val, and His234-->Phe, did not affect high affinity NMB binding. The Ile216-->Ser substitution, however, abolished high affinity NMB binding and severely impaired the ability of the mutant receptor to stimulate NMB-dependent inositol phosphate formation. These results suggest that ILe216 in TM-V of NMB-R may be critical for high affinity NMB binding.     

Identification of the high affinity receptor binding region in human immunoglobulin E

We have investigated the capacity of N- and C-terminally truncated and chimeric human (h) IgE-derived peptides to inhibit the binding of 125I-labeled hIgE, and to engage cell lines expressing high and low affinity receptors (Fc-epsilon-RI/II). The peptide sequence Pro343-Ser353 of the hC-epsilon-3 domain is common to all h-epsilon-chain peptides that recognize hFc-epsilon-RI. This region in IgE is homologous to the A loop in C-gamma-2 that engages the rat neonatal IgG receptor. Optimum Fc-epsilon-RI occupancy by hIgE occurs at pH 6.4, with a second peak at 7.4. N- or C-terminal truncation has little effect on the association rate of the ligands with this receptor. Dissociation markedly increases following C-terminal deletion, and hFc-epsilon-RI occupancy at pH 6.4 is diminished. His residue(s) in the C-terminal region of the epsilon-chain may thus contribute to the high affinity of interaction. Grafting the homologus rat epsilon-chain sequence into hIgE maintains hFc-epsilon-RI interaction without conferring binding to rat Fc-epsilon-RI. hFc-epsilon-RII interaction is lost, suggesting that these residues also contribute to hFc-epsilon RII binding. h-epsilon-chain peptides comprising only this sequence do not block hIgE/hFc-epsilon-RI interaction or engage the receptor. Therefore, sequences N- or C-terminal to this core peptide provide structures necessary for receptor recognition.     

The Ca2+-dependent binding of calmodulin to an N-terminal motif of the heterotrimeric G protein beta subunit

Ca2+ ion concentration changes are critical events in signal transduction. The Ca2+-dependent interactions of calmodulin (CaM) with its target proteins play an essential role in a variety of cellular functions. In this study, we investigated the interactions of G protein betagamma subunits with CaM. We found that CaM binds to known betagamma subunits and these interactions are Ca2+-dependent. The CaM-binding domain in Gbetagamma subunits is identified as Gbeta residues 40-63. Peptides derived from the Gbeta protein not only produce a Ca2+-dependent gel mobility shifting of CaM but also inhibit the CaM-mediated activation of CaM kinase II. Specific amino acid residues critical for the binding of Gbetagamma to CaM were also identified. We then investigated the potential function of these interactions and showed that binding of CaM to Gbetagamma inhibits the pertussis toxin-catalyzed ADP-ribosylation of Galphao subunits, presumably by inhibiting heterotrimer formation. Furthermore, we demonstrated that interaction with CaM has little effect on the activation of phospholipase C-beta2 by Gbetagamma subunits, supporting the notion that different domains of Gbetagamma are responsible for the interactions of different effectors. These findings shed light on the molecular basis for the interactions of Gbetagamma with Ca2+-CaM and point to the potential physiological significance of these interactions in cellular functions.     

Calcium pools in Ehrlich carcinoma cells. A major, high affinity Ca2+ pool is sensitive to both inositol 1,4,5-trisphosphate and thapsigargin

To investigate the presence and the size of different non-mitochondrial Ca2+ pools of Ehrlich ascites tumor cells (EATCs), digitonin-permeabilized cells were allowed to accumulate Ca2+ in the presence of mitochondrial inhibitors and treated with the reticular Ca(2+)-ATPase inhibitor thapsigargin, IP3 and the Ca2+ ionophore A23187. Emptying of thapsigargin-sensitive Ca2+ stores prevented any Ca2+ release by IP3, and, after IP3 addition, little or no Ca2+ was released by thapsigargin. In both instances, a further Ca2+ release was accomplished by A23187. The IP3-thapsigargin-sensitive pool and the residual A23187-sensitive one corresponded to approximately 60 and 37% of non-mitochondrial stored Ca2+, respectively. In intact EATCs, IP3-dependent agonists and thapsigargin discharged Ca2+ pools almost completely overlapping, and A32187 released a minor residual Ca2+ pool. The IP3-insensitive pool appeared to have a relatively low affinity for Ca2+ (below 600 nM). The high affinity, IP3-sensitive Ca2+ pool was discharged in a 'quantal' manner following step additions of sub maximal [IP3], and the IP3-induced fractional Ca2+ release was more marked at higher concentrations of stored (luminal) Ca2+, The IP3-sensitive Ca2+ pool appeared to be devoid of the Ca(2+)-activated Ca2+ release channel since caffeine did not released any Ca2+ in intact and permeabilized EATCs, and Western blot analyses of EATC microsomal membranes failed to detect any known ryanodine receptor isoform.     

Stereospecific, high affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin by hepatic cytosol. Evidence that the binding species is receptor for induction of aryl hydrocarbon hydroxylase

We previously hypothesized that the genetic trait of aromatic hydrocarbon nonresponsiveness (the failure in certain inbred strains of mice of polycyclic hydrocarbons to induce aryl hydrocarbon hydroxylase activity, and the diminished sensitivity to the more potent inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is due to mutation which results in an induction receptor with a diminished affinity for the inducing compound. Following the intraperitoneal administration of [14C]TCDD (6 nmol/kg), hepatic accumulation of the radiolabel was greatest in C57BL/6J mice, intermediate in the hybrid B6D2F1/J mice, and least in DBA/2J mice, a pattern which mirrors the strain sensitivity to hydroxylase induction by TCDD (C57BL/6J greater than B6D2F1/J greater than DBA/2J). These data are compatible with receptor mutation theory and suggested that the hepatic uptake of TCDD is determined by the affinity of the receptor. In vitro experiments on the binding of [3H]TCDD to hepatic cytosol from C57BL/6J mice revealed a small pool of high affinity sites which stereospecifically and reversibly bind TCDD. The specific binding of [3H]TCDD to hepatic cytosol had an equilibrium dissociation constant KD of 0.27 nM and a maximum binding capacity of 84 fmol/mg of cytosol protein. Much less high affinity specific binding of [3H]TCDD was observed in hepatic cytosol from DBA/2J mice, but the KD was not estimated because of the limited aqueous solubility of the ligand. The binding affinity of 23 halogenated dibenzo-p-dioxins and dibenzofurans for this hepatic cytosol-binding species closely correlated with the potencies of these compounds as inducers of hepatic aryl hydrocarbon hydroxylase activity. The polycyclic hydrocarbons that induce hepatic hydroxylase activity competed with [3H]TCDD for hepatic cytosol binding, but phenobarbital, pregnenolone-16alpha-carbonitrile, and the steroid hormones had no specific binding. The data suggest that the hepatic cytosol species which binds TCDD is the receptor for the induction of hepatic aryl hydrocarbon hydroxylase activity, and that the mutation in nonresponsive mice results in an altered receptor with a diminished affinity for inducing compounds.     

Kringle 2 mediates high affinity binding of plasminogen to an internal sequence in streptococcal surface protein PAM

Many cells express receptors for plasminogen (Pg), although the responsible molecules in most cases are poorly defined. In contrast, the group A streptococcal surface protein PAM contains a domain with two 13-amino acid residue long repeated sequences (a1 and a2) responsible for Pg binding. Here we identify the region in Pg that interacts with PAM. A radiolabeled proteolytic plasminogen fragment containing the first three kringles (K1-K3) interacted with streptococci expressing PAM or a chimeric surface protein harboring the a1a2 sequence. In contrast, plasminogen fragments containing kringle 4 or kringle 5 and the activable serine proteinase domain failed to bind to PAM-expressing group A streptococci. A synthetic and a recombinant polypeptide containing the a1a2 sequence both bound to immobilized recombinant K2 (rK2) but not to rK1 or rK3. The interaction between the a repeat region and rK2 was reversible, and rK2 completely blocked the binding of Pg to the a1a2 region. The binding of the a repeat containing polypeptide to K2 occurred with an equilibrium association constant of 4.5 x 10(7) M-1, as determined by surface plasmon resonance, a value close to that (1.6 x 10(7) M-1) calculated for the a1a2-Pg interaction. Inhibition experiments suggested involvement of the lysine-binding site of K2 in the interaction. These data demonstrate that K2 contains the major Pg-binding site for PAM, providing the first well defined example of an interaction between an internal Pg-binding region in a protein and a single kringle domain.     

Identification of a motif within the 5' regulatory region of pS2 which is responsible for AP-1 binding and TCDD-mediated suppression

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related compounds modulate several endocrine systems by altering hormone synthesis, enhancing ligand metabolism, and down-regulating receptor levels/binding activity. Previous studies have demonstrated that TCDD inhibits the 17beta-estradiol (E2)-induction of pS2, a human breast cancer prognostic marker. This inhibition occurs at the gene expression level and is Ah receptor (AhR)-mediated. Analysis of the 5' regulatory region has identified three motifs which resemble dioxin response element (DRE) core sequences. pS2-regulated luciferase deletion constructs identified the DRE-like motif located at -527 to -514 as being required for TCDD-mediated suppression. A point mutation within this core motif (T-518C) abolished the inhibition by TCDD while UV-induced protein-DNA cross-linking and competitive gel retardation assays demonstrated AhR complex binding to this motif. Further study of this region also revealed an adjacent putative AP-1 site, diverging by one base pair from the consensus sequence. Gel retardation assays using TPA-treated MCF-7 cell nuclear extracts showed an induced complex binding to the AP-1-like site. Competition studies and antibody supershifts confirmed that the retarded complex consists of AP-1-like proteins. pS2-regulated luciferase constructs containing mutations specific to the AP-1-like motif greatly diminished the inducibility in response to E2. These results suggest that an interaction between AhR complexes and AP-1-like proteins may be responsible for TCDD-mediated inhibition of E2-induced pS2 expression.     

The second and third extracellular domains of FcgammaRI (CD64) confer the unique high affinity binding of IgG2a

FcgammaRI (CD64) is functionally unique as it is the only FcgammaR able to bind monomeric IgG with high affinity. FcgammaRI is also structurally distinct, containing an extracellular Ig-interactive region of three Ig-like domains in contrast to the two domains of the low affinity receptors FcgammaRII and FcgammaRIII. Previous studies have demonstrated that the third domain of FcgammaRI plays a crucial role in high affinity IgG binding of the receptor, with the first and second domains together forming a low affinity IgG binding motif. In this study the individual functional contributions of the first and second domains of FcgammaRI to IgG binding have been investigated. Chimeric FcgammaR were generated by exchanging extracellular domains between mouse FcgammaRI and the structurally related yet distinct low affinity receptor for IgG, mouse FcgammaRII. The replacement of both domains 1 and 2 of FcgammaRI with domains 1 and 2 of FcgammaRII results in a dramatic change in IgG binding characteristics, as this receptor loses the capacity to bind monomeric IgG with high affinity and also demonstrates a broader specificity (binding not only IgG2a but also IgG1 and 2b. IgG3 was not tested). However, the substitution of FcgammaRII domain 2 of this chimeric receptor with domain 2 of FcgammaRI (generating a chimeric receptor with domain 1 of FcgammaRII linked to domains 2 and 3 of FcgammaRI) was found to reconstitute the specific high affinity monomeric IgG2a binding of wild-type FcgammaRI, albeit with a slightly reduced affinity (1.8-fold lower than wild-type FcgammaRI). These findings suggest that it is the specific interaction between domains 2 and 3 of FcgammaRI, with domain 1 playing a supporting role in maintaining the conformational stability of the receptor, that is the major structural requirement to confer the unique Ig binding characteristics of FcgammaRI.