High-affinity Receptor Binding Sites Research Articles

Efficacy of Omalizumab therapy in a case of severe atopic dermatitis

Background Atopic dermatitis (AD) is a common skin disease of childhood which may cause debilitating symptoms and greatly impair the quality of life of the patient and his relatives [1]. Treatment of chronic AD usually focuses on topical regimen of emolients and immunosuppressants, although systemic immunosuppressive therapy is sometimes required in more severe cases. Omalizumab is a humanized monoclonal anti- IgE antibody that binds at the high-affinity receptor (FceRI) binding site that has revealed some potential in the treatment of severe and recalcitrant AD [2]. Case Here, we present the case of a 11-years-old girl who has been under treatment with Omalizumab for the past five years. The patient first presented at 2 months of age with a global and severe AD involving. She was severely atopic with total IgE levels of 121,000, mild asthma, and multiple food allergies. Treatment with oral prednisone, cyclosporin, azathioprine and intravenous immunoglobulins did not improve her skin symptoms significantly. She was hospitalised multiple times for skin infections attributed to the disease and immunosuppressive medication. Treatment with Omalizumab was initiated at 6 years of age. Four months later, SCORAD index improved significantly. Since, her follow-up has been almost free of any remarkable event and treatment with Omalizumab has been well tolerated.

Recalcitrant Atopic Dermatitis Treated with Omalizumab

Atopic dermatitis (AD) is a chronic cutaneous inflammatory disease. Various categories of therapeutic medications are used for treating AD. Omalizumab is a monoclonal anti-IgE antibody that binds to IgE molecules at the high-affinity receptor (FcepsilonRI) binding site. Therefore, omalizumab can be used as a potential new systemic treatment agent for recalcitrant AD patients with elevated IgE levels. A 34-year-old man had been treated for AD with several topical and oral agents. However, he was refractory to these therapies and his serum IgE levels were very high. We treated him with omalizumab. After 8 months of the treatment, his symptoms were notably improved and the SCORAD index was decreased. Thus, we report on the first case of recalcitrant AD that was successfully treated with omalizumab in Korea.

Interaction of coagulation factor VIII with members of the low-density lipoprotein receptor family follows common mechanism and involves consensus residues within the A2 binding site 484–509

Coagulation factor VIII interacts with several members of the low-density lipoprotein receptor family including low-density lipoprotein receptor-related protein, low-density lipoprotein receptor, and very low-density lipoprotein receptor. The present study was aimed to compare the mechanisms of factor VIII interaction with low-density lipoprotein receptor-related protein, megalin, low-density lipoprotein receptor, and very low-density lipoprotein receptor in order to reveal a general mode of these interactions. Binding of plasma-derived factor VIII and its fragments to recombinant soluble ligand-binding domain of low-density lipoprotein receptor (sLDLR1-7) and purified megalin was studied in solid phase and surface plasmon resonance assays. Full-length factor VIII and its light chain bound to the receptors with similar affinities (KD = 260 +/- 9 and 156 +/- 4 nmol/l, respectively, for megalin and KD = 210 +/- 3 and 174 +/- 13 nmol/l, respectively, for sLDLR1-7). Von Willebrand factor inhibited factor VIII binding to both receptors. In contrast to the light chain, exposure of the high-affinity receptor-binding site within the heavy chain (KD = 22 +/- 4 nmol/l for megalin and 17 +/- 3 nmol/l for sLDLR1-7) required proteolytic cleavage by thrombin. This site was mapped to the A2 domain residues 484-509, based on the inhibitory effects of anti-A2 monoclonal antibody 413, and is shared by all four receptors. Using a panel of A2 mutants, we identified key amino acid residues- positively charged K466, R471, R489 and R490, and hydrophilic residues Y487 and S488- which form the frame of this 'consensus' binding site. We conclude that interaction of factor VIII with the members of the low-density lipoprotein receptor family follows the general mode, requires dissociation of factor VIII from von Willebrand factor, and is activation sensitive.

Evaluation of omalizumab from a health plan perspective.

To review the pathophysiology of allergic asthma and information on the pharmacology, clinical efficacy, safety profile, and direct drug costs for omalizumab to provide a basis for a defined role of this agent in allergic asthma therapy in managed care organizations. Omalizumab is a monoclonal antibody targeting the high-affinity receptor binding site on human immunoglobulin E (IgE). When bound by omalizumab, IgE does not bind to basophils. As a result, degranulation is attenuated and allergic asthma symptoms are reduced. In asthma trials, omalizumab reduced inhaled corticosteroid and rescue medication requirements and improved asthma control and asthma quality of life in moderate-to-severe allergic asthmatics with disease poorly controlled by inhaled corticosteroids. Omalizumab has generally been well tolerated. However, injection site reactions occur in nearly 1 of every 2 patients, a problem that generally becomes less with continued dose administration. Severe injection site reactions are reported in 12% of patients. Other adverse events commonly reported in clinical trials include viral infections (23%), upper respiratory infections (20%), sinusitis (16%), headache (15%), and pharyngitis (11%). Because the acquisition cost of omalizumab is high (generally $15,000 to $44,000 per patient per year, before contractual discounts), its use is cost-prohibitive in all but the most severe, poorly controlled allergic asthmatic patients who are utilizing large amounts of emergency health care resources to manage exacerbations. Experience with use of this drug beyond 52 weeks is lacking. Although omalizumab has demonstrated efficacy and safety in adults and adolescents with uncontrolled moderate-to-severe allergic asthma, its use should be restricted to a narrowly defined population of allergic asthmatics who utilize large amounts of health care resources. If targeted only toward this population, cost-of-care studies suggest that the high cost of this product in these patients could be offset by savings resulting from the less frequent use of high-intensity medical services for asthma exacerbations. The use of omalizumab beyond 52 weeks needs evaluation.

Alternative views of functional protein binding epitopes obtained by combinatorial shotgun scanning mutagenesis

Combinatorial shotgun scanning mutagenesis was used to analyze two large, related protein binding sites to assess the specificity and importance of individual side chain contributions to binding affinity. The strategy allowed for cost-effective generation of a plethora of functional data. The ease of the technology promoted comprehensive investigations, in which the classic alanine-scanning approach was expanded with two additional strategies, serine- and homolog-scanning. Binding of human growth hormone (hGH) to the hGH receptor served as the model system. The entire high affinity receptor-binding sites (site 1) of wild-type hGH (hGHwt) and of an affinity-improved variant (hGHv) were investigated and the results were compared. The contributions that 35 residue positions make to binding were assessed on each hormone molecule by both serine- and homolog-scanning. The hormone molecules were displayed on the surfaces of bacteriophage, and the 35 positions were randomized simultaneously to allow equal starting frequencies of the wild-type residue and either serine or a homologous mutation in separate libraries. Functional selections for binding to the hGH receptor shifted the relative wild-type/mutant frequencies at each position to an extent characteristic of the functional importance of the side chain. Functional epitope maps were created and compared to previous maps obtained by alanine-scanning. Comparisons between the different scans provide insights into the affinity maturation process that produced hGHv. The serine and homolog-scanning results expand upon and complement the alanine-scanning results and provide additional data on the robustness of the high affinity receptor-binding site of hGH.

Receptors for VIP and PACAP in Guinea Pig Cerebral Cortex: Effects on Cyclic AMP Synthesis and Characterization by <SUP>125</SUP>I-VIP Binding

Receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) in guinea pig cerebral cortex were characterized by (1) radioreceptor binding of 125I-labeled VIP (human/rat/porcine), and (2) cyclic AMP (cAMP) formation. Saturation analysis of 125I-VIP binding to membranes of guinea pig cerebral cortex resulted in a linear Scatchard plot, suggesting the presence of a single class of high-affinity receptor-binding sites, with a Kd of 0.63 nM and a B(max) of 77 fmol/mg protein. Various peptides from the PACAP/VIP/secretin family displaced the specific binding of 125I-VIP to guinea pig cerebrum with the relative rank order of potency: chicken VIP (cVIP) > or = PACAP38 approximately PACAP27 approximately guinea pig VIP (gpVIP) > or = mammalian (human/rat/porcine) VIP (mVIP) > peptide histidine-methionine (PHM) > peptide histidine-isoleucine (PHI) > secretin. Analysis of the competition curves revealed displacement of 125I-VIP from high- and lower-affinity binding sites, with IC50 values in the picomolar and the nanomolar range, respectively. About 70% of the specific 125I-VIP-binding sites in guinea pig cerebral cortex were sensitive to Gpp(NH)p, a nonhydrolyzable analog of GTP. Pituitary adenylate cyclase-activating polypeptide 38 (PACAP38), PACAP27, cVIP, gpVIP, mVIP, PHM, and PHI stimulated cAMP production in [3H]adenine-prelabeled slices of guinea pig cerebral cortex in a concentration-dependent manner. Of the tested peptides, the most effective were PACAP38 and PACAP27, which at a 1 microM concentration produced a 17- to 19-fold rise in cAMP synthesis, increasing the nucleotide production to approx 11% conversion above the control value. The three forms of VIP (cVIP, mVIP, and gpVIP) at the highest concentration used, i.e., 3 microM, produced net increases in cAMP production in the range of 8-9% conversion, whereas 5 microM PHM and PHI, by, respectively, 6.7% and 4.9% conversion. It is concluded that cerebral cortex of guinea pig contains VPAC- type receptors positively linked to cAMP formation. In addition, the observed stronger action of PACAP (both PACAP38 and PACAP27), when compared to any form of VIP, on cAMP production in this tissue, suggests its interaction with both PAC1 and VPAC receptors.

Receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide in turkey cerebral cortex: characterization by 6125I9-VIP binding and effects on cyclic AMP synthesis

Receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) in turkey cerebral cortex were characterized using two approaches: (1) in vitro radioreceptor binding of [125I]-VIP, and (2) effects of peptides from the PACAP/VIP/secretin family on cyclic AMP formation. The binding of [125I]-VIP to turkey cortical membranes was rapid, stable, and reversible. Saturation analysis resulted in a linear Scatchard plot, suggesting binding to a single class of high affinity receptor binding sites with a Kd of 0.70 nM and a Bmax of 52 fmol/mg protein. Various peptides displaced the specific binding of 0.12 nM [125I]-VIP to turkey cerebral cortical membranes in a concentration-dependent manner. The relative rank order of potency of the tested peptides to inhibit [125I]-VIP binding to turkey cerebrum was: PACAP38 ≈ PACAP27 ≈ chicken VIP ≈ mammalian VIP ⋙ PHI ≫ secretin, chicken VIP16-28 (inactive). About 65% of specific [125I]-VIP binding sites in turkey cerebral cortex was sensitive to Gpp(NH)p, a nonhydrolysable analogue of GTP. PACAP38, PACAP27, chicken VIP and, to a lesser extent, mammalian VIP potently stimulated cyclic AMP formation in turkey cerebral cortical slices in a concentration-dependent manner, displaying EC50 values of 8.7 nM (PACAP38), 21.3 nM (PACAP27), 67.4 nM (chicken VIP), and 202 nM (mammalian VIP). On the other hand, PHI and secretin very weakly affected the nucleotide production. The obtained results indicate that cerebral cortex of turkey contains VPAC type receptors that are positively linked to cyclic AMP-generating system and are labeled with [125I]-VIP.

Receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide in turkey cerebral cortex: characterization by [ 125I]-VIP binding and effects on cyclic AMP synthesis

Receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) in turkey cerebral cortex were characterized using two approaches: (1) in vitro radioreceptor binding of [ 125I]-VIP, and (2) effects of peptides from the PACAP/VIP/secretin family on cyclic AMP formation. The binding of [ 125I]-VIP to turkey cortical membranes was rapid, stable, and reversible. Saturation analysis resulted in a linear Scatchard plot, suggesting binding to a single class of high affinity receptor binding sites with a K d of 0.70 nM and a B max of 52 fmol/mg protein. Various peptides displaced the specific binding of 0.12 nM [ 125I]-VIP to turkey cerebral cortical membranes in a concentration-dependent manner. The relative rank order of potency of the tested peptides to inhibit [ 125I]-VIP binding to turkey cerebrum was: PACAP38 ≈ PACAP27 ≈ chicken VIP ≈ mammalian VIP ⋙ PHI ≫ secretin, chicken VIP16-28 (inactive). About 65% of specific [ 125I]-VIP binding sites in turkey cerebral cortex was sensitive to Gpp(NH)p, a nonhydrolysable analogue of GTP. PACAP38, PACAP27, chicken VIP and, to a lesser extent, mammalian VIP potently stimulated cyclic AMP formation in turkey cerebral cortical slices in a concentration-dependent manner, displaying EC 50 values of 8.7 nM (PACAP38), 21.3 nM (PACAP27), 67.4 nM (chicken VIP), and 202 nM (mammalian VIP). On the other hand, PHI and secretin very weakly affected the nucleotide production. The obtained results indicate that cerebral cortex of turkey contains VPAC type receptors that are positively linked to cyclic AMP-generating system and are labeled with [ 125I]-VIP.

Cortical cholinergic dysfunction after human head injury.

Loss of cholinergic neurotransmission is implicated in memory impairment and cognitive dysfunction after head injury. The aim of the present study was to investigate presynaptic markers, particularly in relation to cholinergic neurotransmission in human postmortem brain from patients who died following a head injury and age-matched controls. Choline acetyltransferase activity and high-affinity nicotinic receptor binding sites were assayed in the inferior temporal gyrus, cingulate gyrus, and superior parietal cortex of 16 head-injured patients and 8 controls. Synaptophysin immunoreactivity was determined in the left cingulate gyrus from the same patient groups. In the head-injured group, choline acetyltransferase activity was consistently reduced in each cortical region compared to control subjects. The presence of a subdural haematoma and a prolonged survival period after head injury tended to be associated with lower choline acetyltransferase activity. In contrast to the marked reduction in choline acetyltransferase activity, nicotine receptor binding was unchanged in head-injured compared to control patients. Synaptophysin immunoreactivity in the cingulate gyrus was reduced by approximately 30% (p < 0.05) in the head-injured group compared to controls. Correlation of choline acetyltransferase activity with synaptophysin immunoreactivity indicated there is a deficit of cholinergic presynaptic terminals in postmortem human brain following head injury.

Plasminogen Activator Inhibitor 1 Contains a Cryptic High Affinity Receptor Binding Site that Is Exposed upon Complex Formation with Tissue-type Plasminogen Activator ,

The low density lipoprotein receptor-related protein (LRP), a multi-functional endocytic receptor, mediates the cellular internalization of tissue-type (t-PA) and urokinase-type (u-PA) plasminogen activator and their complexes with plasminogen activator inhibitor type 1 (PAI-1). LRP preferentially binds the complexed forms, exemplified by equilibrium dissociation constants (KD) that are at least an order of magnitude lower than those of the free components. To understand the molecular interactions, underlying the preference of the receptor for complexes rather than for the free components, we have performed a detailed analysis of the affinity and kinetics of the binding of PAI-1 and t-PA:PAI-1 complexes to the receptor, using surface plasmon resonance. To assess the involvement of the heparin-binding domain of PAI-1 for the interaction with LRP, we determined the equilibrium dissociation constants for the binding to LRP of a panel of PAI-1 mutants with single- and multiple amino-acid substitutions of the basic residues that constitute the heparin binding site of PAI-1 (K65, K69, R76, K80 and K88). The binding of these PAI-1 mutants was partially reduced with a 2 to 4 fold increase in KD values for single (K80, K88) and combined (K80, 88) substitution mutant proteins respectively. LRP binding of complexes, composed of t-PA with either wild type PAI-1 or any one of the single PAI-1 mutants indicated a major role of lysine 69 (K69) for the binding of t-PA:PAI-1 complexes to LRP (KD values of 6.1, 3.7. 75.4, 5.4, 12.5 and 8.1 nM for wild type, K65A, K69A, R76A, K80A and K88A complexes, respectively). Since the KD for the binding of free t-PA to LRP is 158 nM, we conclude that the PAI-1 moiety harbors the major determinant for t-PA:PAI-1 complex binding to LRP. The in vitro binding studies were extended by binding and clearance studies with COS-1 cells. Degradation of both 125I-t-PA:PAI-1 K69A and 125I-t-PA:PAI-1 K69A K80A K88A complexes after 2 h of incubation was reduced compared to the degradation of 125I-t-PA:PAI-1 complexes. We conclude that PAI-1 contains a cryptic binding site (lysine 69) for LRP, that is specifically expressed upon t-PA:PAI-1 complex formation.

A 10-amino acid sequence of fibroblast growth factor 2 is sufficient for its mitogenic activity on neural progenitor cells.

During development of the central nervous system, neurons and glia are generated from immature neural progenitor cells (NPCs). Basic fibroblast growth factor (FGF-2) is a mitogen for these cells both in vitro and in vivo. However, it is not known whether other members of the FGF family have similar mitogenic effects on NPCs. We have found that FGF-4, in addition to FGF-2, is a mitogen for NPCs isolated from fetal and adult central nervous systems. Other family members have no proliferative effects on these cells. FGFs transduce signals to the cytoplasm through a family of transmembrane tyrosine kinase receptors (FGFR-1-4) or their isoforms. The high-affinity receptor binding sites are found in two regions of the FGF-2 molecule. We have examined the involvement of these sites in mitogenic signaling. Synthetic peptides corresponding to sequences in FGF-2 receptor binding sites were examined in [3H]thymidine incorporation assays for their agonist or antagonist activity. A 10-aa sequence present in the first receptor binding domain has been found to act as an antagonist, blocking the mitogenic effects of FGF-2. Chemical crosslinking studies using 125I-labeled FGF-2 showed specific reduction in binding of radiolabeled FGF-2 to its receptors present on the membranes of NPCs. The identification of this sequence will assist in the study of pathways involved in signal transduction for mitogenesis in these cells and elucidate the role of FGF-2 and FGF-4 during normal development and in the pathogenesis of disease.

An Androgen Response Element in a Far Upstream Enhancer Region Is Essential for High, Androgen-Regulated Activity of the Prostate-Specific Antigen Promoter

Prostate-specific antigen (PSA) is expressed at a high level in the luminal epithelial cells of the prostate and is absent or expressed at very low levels in other tissues. PSA expression can be regulated by androgens. Previously, two functional androgen-response elements were identified in the proximal promoter of the PSA gene. To detect additional, more distal control elements, DNaseI-hypersensitive sites (DHSs) upstream of the PSA gene were mapped in chromatin from the prostate-derived cell line LNCaP grown in the presence and absence of the synthetic androgen R1881. In a region 4.8 to 3.8 kb upstream of the transcription start site of the PSA gene, a cluster of three DHSs was detected. The middle DNAseI-hypersensitive site (DHSII, at ∼−4.2 kb) showed strong androgen responsiveness in LNCaP cells and was absent in chromatin from HeLa cells. Further analysis of the region encompassing DHSII provided evidence for the presence of a complex, androgen-responsive and cell-specific enhancer. In transient transfected LNCaP cells, PSA promoter constructs containing this upstream enhancer region showed approximately 3000-fold higher activity in the presence than in the absence of R1881. The core region of the enhancer could be mapped within a 440-bp fragment. The enhancer showed synergistic cooperation with the proximal PSA promoter and was found to be composed of at least three separate regulatory regions. In the center, a functionally active, high-affinity androgen receptor binding site (GGAACATATTGTATC) could be identified. Mutation of this element almost completely abolished PSA promoter activity. Transfection experiments in prostate and nonprostate cell lines showed largely LNCaP cell specificity of the upstream enhancer region, although some activity was found in the T47D mammary tumor cell line.

A Neural Substrate of Hyperactivity in Borna Disease: Changes in Brain Dopamine Receptors

Rats experimentally infected with the neurotropic RNA virus, Borna disease virus, have a hyperactive movement disorder. Because locomotor activity is modulated by the nucleus accumbens (N. Acc.) dopamine (DA) system, high-affinity DA uptake, DA D1, D2, and D3 receptor binding sites were examined in N. Acc. subregions of normal and infected rats by quantitative receptor autoradiography. The N. Acc. of infected rats had decreased mazindol and D2 and D3 radioligand binding in the core and decreased D3 radioligand binding in rostral subregions. The abnormalities observed in the N. Acc. DA system of infected rats may offer insights into the potential viral pathogenesis of psychiatric conditions with a dopaminergic substrate such as schizophrenia and affective disorders.

A Domain of the even-skipped Protein Represses Transcription by Preventing TFIID Binding to a Promoter: Repression by Cooperative Blocking

We examined the mechanism by which the C-terminal 236 amino acids of the even-skipped protein (region CD) repress transcription. A fusion protein, CDGB, was created that contains region CD fused to the glucocorticoid receptor DNA binding domain. This protein repressed transcription in an in vitro system containing purified fractions of the RNA polymerase II general transcription factors, and repression was dependent upon the presence of high-affinity glucocorticoid receptor binding sites in the promoter. Repression by CDGB was prevented when the promoter DNA was preincubated with TFIID or TBP, whereas preincubation of the template DNA with CDGB prevented TFIID binding. Together, these results strongly imply that CDGB represses transcription by inhibiting TFIID binding, and further experiments suggested a mechanism by which this may occur. Region CD can mediate cooperative interactions between repressor molecules such that molecules bound at the glucocorticoid receptor binding sites stabilize binding of additional CDGB molecules to low-affinity binding sites throughout the basal promoter. Binding to some of these low-affinity sites was shown to contribute to repression. Further experiments suggested that the full-length eve protein also represses transcription by the same mechanism. We speculate that occupancy of secondary sites within the basal promoter by CDGB or the eve protein inhibits subsequent TFIID binding to repress transcription, a mechanism we term cooperative blocking.

DNA sequences that act as high affinity targets for the vitamin D3 receptor in the absence of the retinoid X receptor.

DNA binding site discrimination within a subgroup of nuclear receptors, including the human vitamin D3 receptor (hVDR), appears to be influenced primarily by spacing and orientation differences of response element half-sites, since many receptors recognize and bind to the same hexameric half-site sequence, AGGTCA. Small sequence differences within half-sites, however, may also play an important role in distinguishing between different receptor complexes. Several laboratories have reported that the AGGTCA element in a direct repeat (DR) configuration appears to be a high affinity recognition site for only nuclear receptor-9 retinoid X receptor (RXR) heterodimers. However, we have previously shown that a closely related, but distinct, element (AGTTCA; essentially the mouse osteopontin [Spp-1] vitamin D response element) acts as a high affinity target for purified hVDR in the absence of RXR. This suggests that some half-site sequences could be targets for hVDR alone while others serve as recognition elements for hVDR-RXR complexes. In this report, we test this hypothesis by selecting, using purified hVDR only, for high affinity receptor binding sites in a complex DNA mixture which should by chance contain such sequences. We find that the purified receptor selects a heptameric sequence resembling a half-site of the osteopontin vitamin D response element, consistent with osteopontin-like sequences acting as high affinity targets for hVDR in the absence of RXR. We directly test this by comparing the in vitro DNA binding activity of purified hVDR to DR+3 elements comprised of osteopontin-like AGTTCA or AGGTCA half-sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of melatonin receptors in the brain of the frog Rana pipiens as revealed by in vitro autoradiography

Melatonin binding sites were identified in the brain of the frog Rana pipiens using in vitro autoradiography. Coronal sections were incubated for 1 h in 100 pM 2- 125I-iodomelatonin. Specific binding was displaced with 1 μM nonradioactive melatonin. Autoradiographic labeling of 3H-Hyperfilm was observed in areas that receive primary, secondary, and tertiary visual input: the superficial layers of the optic tectum, anterior and posterior thalamic nuclei, striatum, medial pallium, and interpeduncular nucleus. Other areas that demonstrated binding included the medial and lateral septal nuclei, medial preoptic area, suprachiasmatic region, and anterodorsal tegmental nucleus. Binding was also apparent in the distribution of the lateral olfactory tract (lateral pallium), and in tracts associated with visual pathways: optic nerve, chiasm and tract, and lateral and medial forebrain bundles. A high degree of melatonin binding was observed in the left habenular nucleus, but not in the habenulum of the right side of the brain. Radioreceptor binding assays of frog whole-brain homogenate demonstrated specific saturable melatonin binding ( K d = 70 pM, B max = 0.80 fmol/mg protein). Melatonin and 6-chloromelatonin were potent displacers of 2- 125I-iodomelatonin, while 5-hydroxytryptamine, 5-methoxytryptamine, and N-acetylserotonin were much less potent. Melatonin inhibited the forskolin-stimulated increase in cAMP synthesis in optic tectum expiants. These results suggest that high-affinity melatonin receptor binding sites are widely distributed in the telencephalon, diencephalon, and mesencephalon and are very prominent in areas of the frog brain that are associated with visual processing.

Opioid-inhibited adenylyl cyclase in rat brain membranes: lack of correlation with high-affinity opioid receptor binding sites.

Opioid agonists bind to GTP-binding (G-protein)-coupled receptors to inhibit adenylyl cyclase. To explore the relationship between opioid receptor binding sites and opioid-inhibited adenylyl cyclase, membranes from rat striatum were incubated with agents that block opioid receptor binding. These agents included irreversible opioid agonists (oxymorphone-p-nitrophenylhydrazone), irreversible antagonists [naloxonazine, beta-funaltrexamine, and beta-chlornaltrexamine (beta-CNA)], and phospholipase A2. After preincubation with these agents, the same membranes were assayed for high-affinity opioid receptor binding [3H-labeled D-alanine-4-N-methylphenylalanine-5-glycine-ol-enkephalin (mu), 3H-labeled 2-D-serine-5-L-leucine-6-L-threonine enkephalin (delta), and [3H]ethylketocylazocine (EKC) sites] and opioid-inhibited adenylyl cyclase. Although most agents produced persistent blockade in binding of ligands to high-affinity mu, delta, and EKC sites, no change in opioid-inhibited adenylyl cyclase was detected. In most treated membranes, both the IC50 and the maximal inhibition of adenylyl cyclase by opioid agonists were identical to values in untreated membranes. Only beta-CNA blocked opioid-inhibited adenylyl cyclase by decreasing maximal inhibition and increasing the IC50 of opioid agonists. This effect of beta-CNA was not due to nonspecific interactions with G(i), Gs, or the catalytic unit of adenylyl cyclase, as neither guanylylimidodiphosphate-inhibited, NaF-stimulated, nor forskolin-stimulated activity was altered by beta-CNA pretreatment. Phospholipase A2 decreased opioid-inhibited adenylyl cyclase only when the enzyme was incubated with brain membranes in the presence of NaCl and GTP. These results confirm that the receptors that inhibit adenylyl cyclase in brain do not correspond to the high-affinity mu, delta, or EKC sites identified in brain by traditional binding studies.

Regulation of prostaglandin E 2 binding to a murine macrophage cell line, P388D 1, by insulin

Preincubation of murine macrophage-like P388D 1 cells with physiological amounts of insulin resulted in an increase in prostaglandin E 2 binding to these cells, by approximately 2-fold, when compared to untreated cells. Scatchard analysis of the binding of PGE 2 to insulin-treated cells indicated that the enhanced binding was due to an increase in receptor number (from 0.30 ± 0.02 to 0.63 ± 0.03 fmol/10 6 cells for the high affinity receptor binding sites, and from 2.4 ± 0.31 to 5.0 ± 0.41 fmol/10 6 cells for the low affinity receptor binding sites) rather than to an increase in the affinity of the binding sites. The insulin-stimulation of PGE 2 binding appeared to be associated with a lowering of the cAMP level in these cells; treatment of cells with insulin lowered the cAMP level by increasing the cAMP phosphodiesterase activity of both the membrane and cytosolic fractions. However, enhanced PGE 2 binding to the cells resulted in an increase in cAMP level in the cells. This increase in cAMP level may help to enhance the immunosuppressive action of this prostanoid, as PGE 2 is known to suppress many steps in the immune response, including interleukin-1 expression, by raising cAMP levels via activation of receptor-linked adenylate cyclase. Our data suggest that insulin at physiological concentrations may enhance the immunosuppressive action of PGE 2.

Interleukin-1 inhibits PGE 2 binding to macrophage-like P388D 1 cells by a cyclic AMP-independent process

The interaction between interleukin IL-1α and PGE 2 on P388D 2 on cells has been investigated. Preincubation of murine macrophage-like cells, P388D 1, with IL-1α (0–73 pM) reduced the binding of PGE 2 to these cells in a concentration-dependent manner. Scatchard analysis showed that IL-1α decreased the PGE 2 binding by lowering both the high and low affinity receptor binding capacities (from 0.31 ± 0.02 to 0.12 ± 0.01 fmol/10 6 cells for the high affinity receptor binding sites and from 2.41 ± 0.12 to 1.51 ± 0.21 fmol/10 6 cells for the low affinity receptor binding sites). However, the dissociation constants of the receptor of the IL-1α-treated cells remained unchanged. Inhibition of PGE 2 binding IL-1α did not involve changes in either protein phosphorylation or intracellular cyclic AMP levels. Our data clearly show that IL-1α inhibits the binding of PGE 2 to monocytes/macrophages and may thereby counter the immunosuppressive actions of PGE 2.

Monoclonal antibodies against human IgE. identification of an epitope sharing properties with the high-affinity receptor binding site

Three monoclonal antibodies to human IgE are described. One of them recognizes an epitope located within a region of 76 amino acids that has been shown to contain the fc ϵRI binding site. That epitope is shown to be susceptible to heating and to alkylation of cysteines involved in inter heavy chain bonds, but not to their reduction alone. In addition, this monoclonal antibody, although having a high affinity for free IgE, is unable to bind Fc ϵRI-linked IgE. Based on these results, we discuss the possibility that the antibody recognizes the fc ϵRI binding site of the IgE molecule.