Distinct Recognition Sites Research Articles

Global GacA-steered control of cyanide and exoprotease production in Pseudomonas fluorescens involves specific ribosome binding sites.

The conserved two-component regulatory system GacS/GacA determines the expression of extracellular products and virulence factors in a variety of Gram-negative bacteria. In the biocontrol strain CHA0 of Pseudomonas fluorescens, the response regulator GacA is essential for the synthesis of extracellular protease (AprA) and secondary metabolites including hydrogen cyanide. GacA was found to exert its control on the hydrogen cyanide biosynthetic genes (hcnABC) and on the aprA gene indirectly via a posttranscriptional mechanism. Expression of a translational hcnA'-'lacZ fusion was GacA-dependent whereas a transcriptional hcnA-lacZ fusion was not. A distinct recognition site overlapping with the ribosome binding site appears to be primordial for GacA-steered regulation. GacA-dependence could be conferred to the Escherichia coli lacZ mRNA by a 3-bp substitution in the ribosome binding site. The gene coding for the global translational repressor RsmA of P. fluorescens was cloned. RsmA overexpression mimicked partial loss of GacA function and involved the same recognition site, suggesting that RsmA is a downstream regulatory element of the GacA control cascade. Mutational inactivation of the chromosomal rsmA gene partially suppressed a gacS defect. Thus, a central, GacA-dependent switch from primary to secondary metabolism may operate at the level of translation.

Particle size determines the specificity of apolipoprotein E-containing triglyceride-rich emulsions for the LDL receptor versus hepatic remnant receptor in vivo

Apolipoprotein E (apoE) is an important determinant for the uptake of triglyceride-rich emulsions and lipoproteins by the liver, and exerts affinity for both the LDL receptor (LDLr) and a distinct liver-specific recognition site. Our current aim was to assess the mechanism underlying the receptor-specificity of apoE-carrying lipoproteins. Triglyceride-rich emulsions were synthesized, with mean sizes of 50, 80, and 150 nm. These fractions efficiently acquired apoE from rat serum, and were processed by LPL in vivo with a similar efficiency. Upon injection of the [5H]cholesteryl oleate-labeled emulsions into rats, the liver association rate was positively correlated with particle size (24 +/- 2%, 54 +/- 1%, and 64 +/- 3% of the injected dose at 20 min after injection, respectively) and the liver uptake was predominantly exerted by parenchymal cells. The role of the LDLr in emulsion clearance was established in wild-type versus LDLr knockout mice. In the absence of the LDLr, an 8-fold increased serum half-life was observed for the small emulsion, concomitant with a 6- and 15-fold decreased uptake by the liver and adrenals at 60 min after injection, respectively. In contrast, the in vivo behavior of the large emulsion was independent of the LDLr. Both the ratio of apoE:C on the emulsions upon serum incubation and the alpha-helical content of apoE were inversely correlated with particle size, indicating that these factors may be involved in the emulsion size-dependent receptor specificity in vivo. It is concluded that the contribution of the LDLr to the apoE-mediated clearance of emulsions by the liver and adrenals strongly increases with decreasing particle size, while large particles initially associate with a distinct liver-specific recognition site. As these emulsions mimic chylomicrons, we anticipate that the apoE-dependent metabolic behavior of chylomicrons (remnants) is largely dependent on their size.

Evidence for a multiple binding mode of bispyridinium-type allosteric modulators of muscarinic receptors

The ligand binding properties of muscarinic receptors can be modulated by allosterically acting compounds. Here, a set of novel bispyridinium-type compounds was investigated which were designed to study structure-activity relationships and to provide more insight into the molecular events underlying the allosteric delay of the dissociation of [ 3H] N-methylscopolamine from muscarinic M 2 receptors in porcine cardiac membranes. The parent compound, a non-substituted bispyridinium oxime, displayed a weak allosteric potency and was unable to prevent radioligand dissociation at maximum concentrations. Introduction of either a phthalimidomethyl-moiety or a dichlorobenzyl-moiety at one end of the parent compound led to a considerable increase of the allosteric activity with regard to both the potency and the maximum effect. In these unilaterally ring-substituted bispyridiniums, homologous contralateral non-aromatic modifications were accompanied by divergent potency shifts depending on whether the unilateral ring was phthalimidomethyl or dichlorobenzyl. The findings point to a multiple binding mode of bispyridinium compounds at M 2 receptors in the [ 3H] N-methylscopolamine-occupied state, i.e., different orientations of the compounds at the allosteric binding area or even an interaction with distinct allosteric recognition sites.

Modulation of 45Ca2+ influx into cells stably expressing recombinant human NMDA receptors by ligands acting at distinct recognition sites.

A 45Ca2+ influx assay has been used to investigate the pharmacology of stably expressed recombinant human NR1a/NR2A and NR1a/NR2B N-methyl-D-aspartate (NMDA) receptors. Inhibition of glutamate-stimulated 45Ca2+ influx by six glycine-site antagonists and inhibition of glycine-stimulated 45Ca2+ influx by five glutamate-site antagonists revealed no significant differences between affinity values obtained for NR1a/NR2A and NR1a/NR2B receptors. The polyamine site agonist spermine showed differential modulation of glutamate- and glycine-stimulated 45Ca2+ influx for recombinant NMDA receptors, inhibiting and stimulating 45Ca2+ influx into cells expressing NR1a/NR2A receptors (IC50 = 408 microM) and NR1a/NR2B receptors (EC50 = 37.3 microM), respectively. The antagonist ifenprodil was selective for NR1a/NR2B receptors (IC50 = 0.099 microM) compared with NR1a/NR2A receptors (IC50 = 164 microM). The effects of putative polyamine site antagonists, redox agents, ethanol, and Mg2+ and Zn2+ ions were also compared between NR1a/NR2A and NR1a/NR2B receptors. This study demonstrates the use of 45Ca2+ influx as a method for investigating the pharmacology of the numerous modulatory sites that regulate the function of recombinant human NMDA receptors stably expressed in L(tk-) cells.

Different superantigens interact with distinct sites in the Vbeta domain of a single T cell receptor.

CD4 T cell receptors (TCRs) recognize antigenic peptides presented by self major histocompatibility complex (MHC) class II molecules as well as non-self MHC class II molecules. The TCRs can also recognize endogenous retroviral gene products and bacterial toxins known collectively as superantigens (SAGs) that act mainly on the Vbeta gene segment-encoded portion of the Vbeta domain; most SAGs also require MHC II class for presentation. We have studied the interaction of the TCR from a well-characterized CD4 T cell line with SAGs by mutational analysis of its Vbeta domain. This appears to separate viral (v)SAG from bacterial (b)SAG recognition. T cells having a TCR with glycine to valine mutation in amino acid residue 51 (G51V) in complementarity determining region 2 of the TCR Vbeta domain fail to respond the bSAGs staphylococcal enterotoxin B (SEB), SEC1, SEC2, and SEC3, whereas they retain the ability to respond to non-self MHC class II molecules and to foreign peptides presented by self MHC class II molecules. It is interesting to note that T cells expressing mutations of both G51V and G53D of V beta regain the response to SEB and partially that to SEC1, but do not respond to SEC2, and SEC3, suggesting that different bacterial SAGs are viewed differently by the same TCR. These results are surprising, because it has been generally believed that SAG recognition by T cells is mediated exclusively by hypervariable region 4 on the exposed, lateral face of the TCR Vbeta domain. Response to the vSAG Mtv-7 was generated by mutation in Vbeta residue 24 (N24H), confirming previously published data. These data show that the vSAG Mtv-7 and bSAGs are recognized by different regions of the TCR Vbeta domain. In addition, various bSAGs are recognized differently by the same TCR. Thus, these mutational data, combined with the crystal structure of the TCR beta chain, provide evidence for distinct recognition sites for vSAG and bSAG.

Anti-cooperative Biphasic Equilibrium Binding of Transcription Factor Upstream Stimulatory Factor to Its Cognate DNA Monitored by Protein Fluorescence Changes

Upstream stimulatory factor USF is a human transcriptional activation factor, which uses a basic/helix-loop-helix/ leucin zipper (b/HLH/Z) motif to homodimerize and recognize specific sequences in the promoter region of both nuclear and viral genes transcribed by RNA polymerase II. Steady state fluorescence spectroscopy demonstrated that the basic/helix-loop-helix/leucin zipper domain of USF binds its DNA targets with high affinity and specificity, whereas removal of the leucine zipper yielding the basic/helix-loop-helix minimal DNA binding region reduces both affinity and specificity. Stopped flow method provided kinetic evidence for a two-step binding process involving rapid formation of a protein-DNA intermediate followed by a slow isomerization step, which is consistent with the basic region undergoing a random coil to alpha-helix folding transition on specific DNA recognition. The leucine zipper is also necessary for USF to function as a bivalent homotetramer, capable of binding two distinct recognition sites simultaneously and mediating DNA looping under physiologic conditions. Titration studies revealed that the first binding event has a equilibrium constant Keq = (2.2 +/- 2.0) x 10(9) M-1 for major late promoter DNA, whereas the second binding event occurs with a remarkable reduced affinity, Keq = (1.2 +/- 0.8) x 10(8) M-1. This anticooperative feature of DNA binding by the homotetramer suggests that USF stimulates transcription by mediating DNA looping between nearby recognition sites located in class II nuclear and viral gene promoters.

The generation of nitric oxide and its roles in neurotransmission and neurotoxicity.

The N-methyl-D-aspartate (NMDA) receptor plays a key role in synaptic plasticity and is thought to underlie memory, learning and development of the nervous system. The NMDA receptor is a ligand-gated ion channel complex that contains distinct recognition sites for endogenous and exogenous ligands, including glutamate, glycine, Mg2+, Zn2+ and noncompetitive blockers such as MK-801. In the central nervous system, nitric oxide (NO) is produced in some neurons following activation of excitatory amino acids receptors, particularly those of the NMDA receptor. Nitric oxide is synthesized from a L-arginine by the cytoplasmic enzyme nitric oxide synthase (NOS) which is a calcium dependent enzyme, and this pathway is inhibited by the analogues of L-arginine such as NG-monomethyl-L-arginine (L-NMMA) and is augmented by NMDA receptor activation. Activation of the NMDA receptor results in the elevation of intracellular calcium ([Ca2+]i) which in turn activates NOS via the calcium-calmodulin complex. Nitric oxide is not a classical neurotransmitter in the central nervous system since it is not released by exocytosis and does not interact with a receptor protein but rather diffuses rapidly across the membrane and binds with the iron in heme-containing proteins. Nitric oxide can serve as both an oxidizing and reducing agent. It has strong affinity for heme proteins such as guanylyl cyclase, but there is evident that NO may have a regulatory role by oxidizing sulfhydryl groups of non-heme proteins such as those on the NMDA receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Polyamines modulate [ 3H]L-689 binding to the glycine site of the NMDA receptor from rat brain

The N- methyl- d-aspartate (NMDA) receptor complex possesses distinct recognition sites for glutamate, glycine and polyamines, which appear to be allosterically linked. We have investigated the effects of polyamines on the binding of the glycine site antagonist [ 3 H](±)-4-(trans)-2- carboxy-5,7- dichloro-4- phenylaminocarbonylamino-1,2,3,4- tetrahydroquinoline ([ 3H]L-689,560), using rat cortex/hippocampus P 2 membranes. Spermine and spermidine partially inhibited [ 3H]L-689-560 binding under non-equilibrium conditions, with IC 50 values of 25.9 and 106 μM, respectively. The putative polyamine site antagonists arcaine, 1,10-diaminodecane, diethylenetriamine and putrescine had no effect on [ 3H]L-689,560 binding per se at 1 mM. The inhibition of [ 3H]L-689,560 binding by spermine was antagonised by arcaine in a competitive manner, but not by 1,10-diaminodecane, diethylenetriamine or putrescine. Kinetic analysis revealed that spermine (100 μM) decreased the association and dissociation rates of [ 3H]L-689,560 binding. In saturation experiments 100 μM spermine increased the K D for [ 3H]L-689,560 binding from 1.99 nM to 4.03 nM, with no effect on the number of binding sites. Spermine increased the affinity of glycine site agonists in displacing [ 3H]L-689,560 binding, with no effect on inhibition by partial agonists or antagonists, suggesting that spermine promotes an ‘agonist-preferring’ state. Modulation of [ 3H]L-689,560 binding by agonists for the polyamine and glutamate sites on the NMDA receptor did not appear to be additive in nature.

A deletion mutation in the third cytoplasmic loop of the mouse m1 muscarinic acetylcholine receptor unmasks cryptic G-protein binding sites.

Mutations were introduced in the highly conserved carboxyl-terminal region of the third cytoplasmic loop of the mouse m1 muscarinic acetylcholine receptor (mAChR) gene by site-directed mutagenesis. The effects of these mutations on ligand binding and on mAChR coupling to phosphoinositide turnover have been examined following expression in mouse Y1 adrenal carcinoma cells, Chinese hamster ovary (CHO) cells, and Rat-2 fibroblasts. Point mutations in the region proximal to the sixth transmembrane domain had no effect on antagonist binding but did result in decreased agonist affinity. A deletion of four amino acids in the same region effectively uncoupled the receptor from phosphoinositol turnover in Y1 adrenal carcinoma cells but had no effect in either CHO cells or Rat-2 fibroblasts. Differential sensitivity to pertussis toxin indicates that the m1 mAChR can interact with multiple G-proteins in CHO cells and Rat-2 cells via distinct recognition sites on the receptor. These data demonstrate multiple G-proteins can interact with an individual receptor, that the same receptor may couple to different second messenger pathways, and that these responses can vary in a cell type-specific manner.

Electrophysiological evidence for the existence of NMDA and non-NMDA receptors on rat ventral tegmental dopamine neurons.

In vitro extracellular single-unit recordings from rat midbrain slices were used to assess the effects of excitatory amino acid agonists on the activity of A10 dopamine neurons. N-methyl-D-aspartic acid (NMDA), kainic acid (KA), and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) elicited dose-dependent increases in firing rates. The relative potencies for the 3 compounds was AMPA > KA > NMDA. None of the excitations was accompanied by burst firing, but frequently periods of nonrecordable activity occurred following pronounced stimulation. Concurrent application of the excitatory amino acid antagonist CGS 19755 (cis-4-phosphonomethyl-2-piperidine carboxylate) selectively blocked the excitations elicited by NMDA but not by KA or AMPA. Likewise the selective non-NMDA antagonist NBQX [2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline] blocked only the excitatory effects of AMPA and KA but not those elicited by NMDA. NBQX appeared to be less potent at antagonizing KA than AMPA. These results suggest that mesolimbic-mesocortical dopamine neurons possess both NMDA and non-NMDA receptors, and possibly distinct AMPA and KA recognition sites.

Temperature and anion dependence of allosteric interactions at the γ-aminobutyric acid-benzodiazepine receptor

The temperature dependence of [ 3H]flunitrazepam ([ 3H]FNZ) binding to rat brain membranes was examined in the presence of the anaesthetics, pentobarbitone, alphaxalone and propofol. Van't Hoff plots showed the binding of FNZ to be largely enthalpy driven. Alphaxalone and propofol increased the entropy of the binding reaction but not the enthalpy and therefore did not show temperature dependence in their efficacy. In contrast, pentobarbitone increased the enthalpy of FNZ binding and, therefore, is more efficacious at low temperatures. The EC 50 values of all three modulators increased with temperature indicating that their interactions with the receptor may be enthalpy driven. The EC 50 values of all three modulators were also anion dependent, showing a decrease in the presence of γ-aminobutyric acid (GABA A)-channel permeant anions. The efficacies of alphaxalone and pentobarbitone, but not that of propofol, also increased with increasing chloride ion concentration. The results indicate that all three modulators interact with the GABA A receptor at distinct recognition sites.

Mapping T-cell epitopes in group A streptococcal type 5 M protein

Group A streptococcal cell surface M proteins elicit highly protective, serotype-specific opsonic antibodies and many serotypes also elicit host cross-reactive antibodies, which may contribute to the pathogenesis of poststreptococcal autoimmune disease. To date, studies aimed at designing safe (non-host-cross-reactive, defined-epitope) M vaccines have focused almost exclusively on antibody epitopes. Here we identify T-cell epitopes recognized by T cells from BALB/c, C57BL/6, and CBA/Ca mice immunized with purified, recombinant serotype 5 M protein (rM5). The responses of rM5-specific, major histocompatibility complex class II-restricted, T-cell clones to synthetic peptides representing most of the M5 sequence identified at least 13 distinct T-cell recognition sites, including sites recognized by more than one major histocompatibility complex haplotype of mice. Although none of these sites appeared to be strongly immunodominant, an N-terminal peptide, sM5[1-35], was recognized by lymph node T cells of rM5-immunized mice and by a larger proportion of rM5-specific T-cell clones than any other individual peptide. The fine specificity of these clones was mapped with subpeptides to a single site at or overlapping the sequence ELENHDL at residues 21 to 27, which is in close proximity to previously mapped protective antibody epitopes. Other T-cell recognition sites are distributed throughout the M protein and include several in the highly conserved C-terminal region of the molecule. One of these C-terminal sites, located within residues 300 to 319, was recognized by a significant proportion of T-cell clones from two strains of mice. Helper T-cell epitopes located in the C-terminal region of M5 are likely to be widely conserved between different M serotypes and could be particularly useful in designing multivalent, defined-epitope M vaccines.

Opioid inhibition of nicotine-induced 45ca 2+-uptake into cultured bovine adrenal medullary cells

The ability of a number of opioid agonists and antagonists to affect nicotine-induced 45Ca 2+-uptake into cultured bovine adrenal medullary cells has been investigated. High (10 μM) concentrations of the opioid agonist bremazocine produced a significant inhibition of nicotine-induced 45Ca 2+-uptake throughout the 15 min time course examined. The opioid subtype-selectivity of this inhibition was investigated; mu and delta selective agonists produced only minor effects whereas the kappa selective agonist U50-488H and the endogenous opioid peptides dynorphin (1-13) and metorphamide almost abolished nicotine-induced 45Ca 2+-uptake. The U50-488H inhibition was significant at 10 nM concentrations with an ic 50 of approximately 1 μm. U50-488H inhibition could not be reversed or reduced by the opioid antagonists naxolone, diprenophine or Mr2266. Furthermore, Mr2266 and its optical isomer Mr2267 also produced marked inhibition of 45Ca 2+-uptake. The inhibition was specific to nicotine-induced 45Ca 2+-uptake in that a similar level of uptake evoked by potassium depolarization was unaffected by high concentrations of U50-488H. These data indicate that opioid inhibition of nicotine-induced 45Ca 2+- uptake does not involve classical, stereospecific opioid receptors and suggests the involvement of a pharmacologically distinct opioid recognition site. It is speculated that this may be associated with the nicotine receptor-ionophore complex.

Bivalent opioid peptides synthesized from μ selective monomers display preferential selectivity for δ receptors

A series of dimeric opioid peptides derived from μ selective compounds was synthesized to investigate whether μ and δ receptors coexist as distinct recognition sites on the same receptor complex. Some compounds were several times more potent than the corresponding monomers in the MVD (mouse vas deferens) smooth muscle preparation, which is rich in δ receptors, but yet retaining substantial activity in the GPI (guinea pig ileum). It is suggested that δ receptors might differ from μ receptors in that they possess an additional accessory recognition site, to which could bind a particular amino acid residue present in the second halves of these bivalent ligands. This residue could play the role of “address” at δ opioid receptors.

Differentiation of 5-hydroxytryptamine2 receptor subtypes using 125I-R- (-)2,5-dimethoxy-4-iodo-phenylisopropylamine and 3H-ketanserin

The radioligand binding characteristics of 125I-R-(-)4-iodo-2,5-dimethoxyphenylisopropylamine [125I-R-(-)DOI] and 3H-ketanserin were compared in rat and bovine cortical membranes. In rat cortex, 125I-R-(-)DOI labels a relatively low density of binding sites (Bmax = 2.5 +/- 0.2 pmol/gm tissue) with high affinity (KD = 0.63 +/- 0.09 nM). In bovine cortex, specific binding of 125I-R-(-)DOI represents less than 20% of total binding at radioligand concentrations above 0.6 nM, and, therefore, the data cannot be analyzed adequately by Scatchard transformation. By contrast, 3H-ketanserin displays saturable, specific high-affinity binding in both rat cortex (KD = 1.0 +/- 0.1 nM; Bmax = 11 +/- 0.4 pmol/gm tissue) and bovine cortex (KD = 1.2 +/- 0.2 nM; Bmax = 5.3 +/- 0.4 pmol/gm tissue). Ki values for 30 drugs were determined for 125I-R-(-)DOI-labeled sites in rat cortex and 3H-ketanserin-labeled sites in bovine cortex. 5-Hydroxytryptamine (5-HT) displays 250-fold higher selectivity for the 125I-R-(-)DOI-labeled sites (Ki = 3.0 +/- 0.7 nM) than for the 3H-ketanserin-labeled sites (Ki = 750 +/- 50 nM). Structural congeners of R-(-)DOI display 80- to 160-fold higher affinity for the 125I-R-(-)DOI binding site than for the 3H-ketanserin-labeled binding site. d-LSD and putative 5-HT2 antagonists are approximately equipotent at both sites. Significant correlations were found between drug affinities for 125I-R-(-)DOI-labeled sites in rat cortex and putative 5-HT2A sites labeled previously by 77Br-R-(-)DOB (r = 0.93, p less than 0.01), putative 5-HT2B sites labeled by 3H-ketanserin in bovine cortex (r = 0.63, p less than 0.01), and 5-HT1C binding sites that have been characterized by other investigators (r = 0.78, p less than 0.01). No significant correlations were found between drug affinities for 125I-R-(-)DOI-labeled sites in rat cortex and 5-HT1A, 5-HT1B, 5-HT1D, or 5-HT3 sites, as determined by previous investigators. We conclude that 125I-R-(-)DOI labels a novel 5-HT binding site subtype (tentatively designated the 5-HT2A binding site) that is present in rat cortex but is either absent or minimally present in bovine cortex. By contrast, 3H-ketanserin labels both the putative 5-HT2A site in rat cortex as well as a separate, distinct recognition site that is present in both rat and bovine cortex, tentatively designated the 5-HT2B site.

The γ-Aminobutyric Acid A (GABAA) Receptor Complex and Hepatic Encephalopathy

Increased neural inhibition appears to be an important component of the syndrome of hepatic encephalopathy. The pathways subserved by the gamma-aminobutyric acid (GABA)-benzodiazepine receptor complex are the principal inhibitory systems in the mammalian brain. Hyperpolarization of neural membranes is accomplished by an increase in transmembrane chloride flux through a GABA-gated chloride channel in the complex. The opening of the chloride channel is induced by the binding of GABA to its receptors, and it is potentiated by barbiturates or benzodiazepines that act at distinct recognition sites on the complex. Involvement of the GABA neurotransmitter system in hepatic encephalopathy is suggested by several findings in animal models of fulminant hepatic failure. For example, hepatic encephalopathy resembles encephalopathies induced by drugs (including benzodiazepines) that potentiate GABAergic neurotransmission. In addition, neurons from animals with hepatic encephalopathy show increased sensitivity to benzodiazepine and GABA receptor agonists. Moreover, these neurons are excited by benzodiazepine receptor antagonists at concentrations that do not affect control neurons. Also, elevated levels of a substance that inhibits radioligand binding to benzodiazepine receptors have been found in cerebrospinal fluid from animals with hepatic encephalopathy. Furthermore, manifestations of hepatic encephalopathy can be ameliorated by benzodiazepine receptor antagonists. The relevance of these findings to hepatic encephalopathy in human beings is supported by clinical observations showing that a benzodiazepine receptor antagonist can lessen the degree of hepatic encephalopathy. These findings suggest that an endogenous substance with benzodiazepine-like properties contributes to the neuropsychiatric manifestations of hepatic encephalopathy by augmenting GABAergic neurotransmission.

Alpha adrenoceptor sites in vascular smooth muscle: Differentiation by selective antagonist binding

The properties of alpha adrenoceptors in rat-tail artery membranes were studied using tritiated ligands that are selective for the α 1 and α 2 subtypes. High-affinity saturable binding was obtained for the α 1 antagonist prazosin yielding a B max of 144 ± 31.6 fmol/mg protein (mean ± SEM, N = 3) and a K d of 0.17 ± 0.04 nM, and also for the α 2 antagonist rauwolscine which yielded a B max of 141.3 ± 19.3 mol/ mg protein and a K d of 1.57 ± 0.32 nM. The [ 3H]prazosin-labelled sites displayed a pharmacological profile characteristic of an α 1 adrenoceptor, whereas the [ 3H]rauwolscine-labelled sites exhibited the expected α 2 adrenoceptor profile. Agonist affinity for [ 3H]rauwolscine sites was reduced by Gpp(NH)p and Na +, and the effects appeared synergistic for adrenaline, but non-interactive for UK-14304. Agonist interaction with [ 3H]prazosin sites in the rat-tail artery was also regulated by Gpp(NH)p and Na +, although clearly in a qualitatively and quantitatively different manner from the [ 3H]rauwolscine sites. These results suggest that distinct binding sites for [ 3H]prazosin and [ 3H]rauwolscine could be differentiated with antagonist ligands. These distinct antagonist recognition sites demonstrate the pharmacological profile expected for α 1 and α 2 adrenoceptors, and the quantitatively differing abilities of Na + and Gpp(NH)p to regulate agonist interactions with these sites are suggestive, but do not necessarily prove, that different G proteins may be involved in this regulation.

Identification of distinct C3b and C4b recognition sites in the human C3b/C4b receptor (CR1, CD35) by deletion mutagenesis.

Complementary DNA clones encoding the NH2-terminal region of human CR1 have been isolated and sequenced. The deduced complete amino acid sequence of the F allotype of human CR1 contains 2,039 residues, including a 41-residue signal peptide, an extracellular domain of 1,930 residues, a 25-amino acid transmembrane domain, and a 43-amino acid cytoplasmic region. The extracellular domain is composed exclusively of 30 short consensus repeats (SCRs), characteristic of the family of C3/C4-binding proteins. The 28 NH2-terminal SCRs are organized as four long homologous repeats (LHRs) of seven SCRs each. The newly sequenced LHR, LHR-A, is 61% identical to LHR-B in the NH2-terminal two SCRs and greater than 99% identical in the COOH-terminal five SCRs. Eight cDNA clones were spliced to form a single construct, piABCD, that contained the entire CR1 coding sequence downstream of a cytomegalovirus promoter. COS cells transfected with piABCD transiently expressed recombinant CR1 that comigrated with the F allotype of erythrocyte CR1 on SDS-PAGE and that mediated rosette formation with sheep erythrocytes bearing C4b and C3b. Recombinant CR1 also had factor I-cofactor activity for cleavage of C3(ma). Analyses of six deletion mutants expressed in COS cells indicated that the NH2-terminal two SCRs of LHR-A contained a site determining C4 specificity and the NH2-terminal two SCRs of LHR-B and -C each had a site determining C3 specificity. The presence of these three distinct sites in CR1 may enable the receptor to interact multivalently with C4b/C3b and C3b/C3b complexes generated during activation of the classical and alternative pathways.

Identification of the p53 protein domain involved in formation of the simian virus 40 large T-antigen-p53 protein complex

An expression vector utilizing the enhancer and promoter region of the simian virus 40 (SV40) DNA regulating a murine p53 cDNA clone was constructed. The vector produced murine p53 protein in monkey cells identified by five different monoclonal antibodies, three of which were specific for the murine form of p53. The murine p53 produced in monkey cells formed an oligomeric protein complex with the SV40 large tumor antigen. A large number of deletion mutations, in-frame linker insertion mutations, and linker insertion mutations resulting in a frameshift mutation were constructed in the cDNA coding portion of the p53 protein expression vector. The wild-type and mutant p53 cDNA vectors were expressed in monkey cells producing the SV40 large T antigen. The conformation and levels of p53 protein and its ability to form protein complexes with the SV40 T antigen were determined by using five different monoclonal antibodies with quite distinct epitope recognition sites. Insertion mutations between amino acid residues 123 and 215 (of a total of 390 amino acids) eliminated the ability of murine p53 to bind to the SV40 large T antigen. Deletion (at amino acids 11 through 33) and insertion mutations (amino acids 222 through 344) located on either side of this T-antigen-binding protein domain produced a murine p53 protein that bound to the SV40 large T antigen. The same five insertion mutations that failed to bind with the SV40 large T antigen also failed to react with a specific monoclonal antibody, PAb246. In contrast, six additional deletion and insertion mutations that produced p53 protein that did bind with T antigen were each recognized by PAb246. The proposed epitope for PAb246 has been mapped adjacent (amino acids 88 through 109) to the T-antigen-binding domain (amino acids 123 through 215) localized by the mutations mapped in this study. Finally, some insertion mutations that produced a protein that failed to bind to the SV40 T antigen appeared to have an enhanced ability to complex with a 68-kilodalton cellular protein in monkey cells.

Evidence that autoreactive T hybridomas recognize multiple epitopes on the I-Ak molecule.

In this study we attempt to define the number of determinants on the I-Ak molecule recognized by a panel of autoreactive I-Ak-specific T cell hybridomas. The recognition sites or histotopes of a panel of autoreactive I-Ak-restricted T cell hybridomas was determined in two ways: 1) by their ability to be activated by a panel of A betak and A alphak mutant antigen-presenting cell lines, and 2) by inhibition of activation by anti-I-Ak monoclonal antibodies. The results are most consistent with the presence of multiple distinct recognition sites on the I-Ak molecule. However, an alternative explanation, that the T cell hybridomas recognize the same self epitope on Class II molecules, but with different affinity, cannot be excluded.