The gene for the ligand binding chain of the human interferon gamma receptor.

In order to characterize the gene encoding the ligand binding (1(st); alpha) chain of the human IFN-gamma receptor, two overlapping cosmid clones were analyzed. The gene spans over 25 kilobases (kb) of the genomic DNA and has seven exons. The extracellular domain is encoded by exons 1 to 5 and by part of exon 6. The transmembrane region is also encoded by exon 6. Exon 7 encodes the intracellular domain and the 3' untranslated portion. The gene was located on chromosome 6q23.1, as determined by in situ hybridization. The 4 kb region upstream (5') of the gene was sequenced and analyzed for promoter activity. No consensus-matching TATA or CAAT boxes in the 5' region were found. Potential binding sites for Sp1, AP-1, AP-2, and CREB nuclear factors were identified. Compatible with the presence of the Sp1/AP-2 sites and the lack of TATA box, S1-nuclease mapping experiments showed multiple transcription initiation sites. Promoter activity of the 5' flanking region was analyzed with two different reporter genes: the Escherichia coli chloramphenicol acetyltransferase and human growth hormone. The smallest 5' region of the gene that still had full promoter activity was 692 base pairs in length. In addition, we found sequences belonging to the oldest family of Alu repeats, 2 - 3 kb upstream of the gene, which could be useful for genetic studies.

Synthesis, binding affinity and SAR of new benzolactam derivatives as dopamine D3 receptor ligands

A series of new benzolactam derivatives was synthesized and the derivatives were evaluated for theiraffinities at the dopamine D1, D2, and D3 receptors. Some of these compounds showed high D2 and/orD3 affinity and selectivity over the D1 receptor. The SAR study of these compounds revealed structuralcharacteristics that decisively influenced their D2 and D3 affinities. Structural models of the complexesbetween some of the most representative compounds of this series and the D2 and D3 receptors wereobtained with the aim of rationalizing the observed experimental results. Moreover, selected compoundsshowed moderate binding affinity on 5-HT2A which could contribute to reducing the occurrence of extrapyramidalside effects as potential antipsychotics.

Characterization of the ligand-binding site of the serotonin 5-HT3 receptor: the role of glutamate residues 97, 224, AND 235.

Ligand-gated ion channels of the Cys loop family are receptors for small amine-containing neurotransmitters. Charged amino acids are strongly conserved in the ligand-binding domain of these receptor proteins. To investigate the role of particular residues in ligand binding of the serotonin 5-HT3AS receptor (5-HT3R), glutamate amino acid residues at three different positions, Glu97, Glu224, and Glu235, in the extracellular N-terminal domain were substituted with aspartate and glutamine using site-directed mutagenesis. Wild type and mutant receptor proteins were expressed in HEK293 cells and analyzed by electrophysiology, radioligand binding, fluorescence measurements, and immunochemistry. A structural model of the ligand-binding domain of the 5-HT3R based on the acetylcholine binding protein revealed the position of the mutated amino acids. Our results demonstrate that mutations of Glu97, distant from the ligand-binding site, had little effect on the receptor, whereas mutations Glu224 and Glu235, close to the predicted binding site, are indeed important for ligand binding. Mutations E224Q, E224D, and E235Q decreased EC50 and Kd values 5-20-fold, whereas E235D was functionally expressed at a low level and had a more than 100-fold increased EC50 value. Comparison of the fluorescence properties of a fluorescein-labeled antagonist upon binding to wild type 5-HT3R and E235Q, allowed us to localize Glu235 within a distance of 1 nm around the ligand-binding site, as proposed by our model.

Mutations inducing divergent shifts of constitutive activity reveal different modes of binding among catecholamine analogues to the beta(2)-adrenergic receptor.

1. We compared the changes in binding energy generated by two mutations that shift in divergent directions the constitutive activity of the human beta(2) adrenergic receptor (beta(2)AR). 2. A constitutively activating mutant (CAM) and the double alanine replacement (AA mutant) of catechol-binding serines (S204A, S207A) in helix 5 were stably expressed in CHO cell lines, and used to measure the binding affinities of more than 40 adrenergic ligands. Moreover, the efficacy of the same group of compounds was determined as intrinsic activity for maximal adenylyl cyclase stimulation in wild-type beta(2)AR. 3. Although the two mutations had opposite effects on ligand affinity, the extents of change were in both cases largely correlated with the degree of ligand efficacy. This was particularly evident if the extra loss of binding energy due to hydrogen bond deletion in the AA mutant was taken into account. Thus the data demonstrate that there is an overall linkage between the configuration of the binding pocket and the intrinsic equilibrium between active and inactive receptor forms. 4. We also found that AA mutation-induced affinity changes for catecholamine congeners gradually lacking ethanolamine substituents were linearly correlated to the loss of affinity that such modifications of the ligand cause for wild-type receptor. This indicates that the strength of bonds between catechol ring and helix 5 is critically dependent on the rest of interactions of the beta-ethanolamine tail with other residues of the beta(2)-AR binding pocket.

Different internalization properties of the alpha1a- and alpha1b-adrenergic receptor subtypes: the potential role of receptor interaction with beta-arrestins and AP50.

The internalization properties of the alpha1a- and alpha1b-adrenergic receptors (ARs) subtypes transiently expressed in human embryonic kidney (HEK) 293 cells were compared using biotinylation experiments and confocal microscopy. Whereas the alpha1b-AR displayed robust agonist-induced endocytosis, the alpha1a-AR did not. Constitutive internalization of the alpha1a-AR was negligible, whereas the alpha1b-AR displayed significant constitutive internalization and recycling. We investigated the interaction of the alpha1-AR subtypes with beta-arrestins 1 and 2 as well as with the AP50 subunit of the clathrin adaptor complex AP2. The results from both coimmunoprecipitation experiments and beta-arrestin translocation assays indicated that the agonistinduced interaction of the alpha1a-AR with beta-arrestins was much weaker than that of the alpha1b-AR. In addition, the alpha1a-AR did not bind AP50. The alpha1b-AR mutant M8, lacking the main phosphorylation sites in the receptor C tail, was unable to undergo endocytosis and was profoundly impaired in binding beta-arrestins despite its binding to AP50. In contrast, the alpha1b-AR mutant DeltaR8, lacking AP50 binding, bound beta-arrestins efficiently, and displayed delayed endocytosis. RNA interference showed that beta-arrestin 2 plays a prominent role in alpha1b-AR endocytosis. The findings of this study demonstrate differences in internalization between the alpha1a- and alpha1b-AR and provide evidence that the lack of significant endocytosis of the alpha1a-AR is linked to its poor interaction with beta-arrestins as well as with AP50. We also provide evidence that the integrity of the phosphorylation sites in the C tail of the alpha1b-AR is important for receptor/beta-arrestin interaction and that this interaction is the main event triggering receptor internalization.

Two functional forms of the Xenopus laevis estrogen receptor translated from a single mRNA species.

Steroid receptors are nuclear proteins that regulate gene transcription in a ligand-dependent manner. Over-expression of the Xenopus estrogen receptor in a vaccinia virus-derived expression system revealed that the receptor localized exclusively in the nucleus of the infected cells, irrespective of the presence or absence of the ligand. Furthermore, two forms of the receptor were produced, a full-length and a N-terminal truncated version, which are translated from a single mRNA species by the use of two AUG within the same reading frame. These 66- and 61-kDa receptors were also observed after in vitro translation of the mRNA as well as in primary Xenopus hepatocytes. Both forms are potent estrogen-dependent transcriptional activators in transient transfection experiments, as well as in in vitro transcription assays.

Monoclonal antibodies against idiotypic determinant(s) of the T cell receptor from HPB-ALL cells induce IL2 production in Jurkat cells without apparent evidence of binding.

Two monoclonal antibodies (mAb) directed against idiotypic determinants of the T cell receptor (anti-Ti) from HPB-ALL cells induce interleukin 2 (IL2) production in Jurkat T cells without evidence of binding to these cells as judged by fluorescence-activated cell sorter (FACS) analysis, indirect antibody-binding radioimmunoassay and direct binding studies with 125I-labeled mAb. The IL2 response induced by these mAb observed both in the presence and absence of phorbol myristate acetate was in the range of that obtained when Jurkat cells were stimulated with phytohemagglutinin or anti-T3 mAb (Leu 4). The idiotypic specificity of the two anti-HPB-ALL Ti mAb was demonstrated by several criteria. Both mAb bound specifically to HPB-ALL cells as determined by radioimmunoassay or FACS analysis but not with 8 other T cell lines. The anti-HPB-ALL Ti mAb precipitated a disulfide-linked heterodimer of 85 kDa only from 125I-labeled HPB-ALL cells and not from other cell lines tested. Incubation of HPB-ALL cells with anti-T3 abrogated the expression of T3 and induced co-modulation of the idiotypic structures detected by the two anti-HPB-ALL Ti mAb. Conversely, incubation of HPB-ALL cells with either one of the anti-Ti mAb abrogated the expression of T3 and of the idiotypic structures. Our results suggest that mAb with an apparent unique specificity for the receptor of the immunizing T cell line HPB-ALL can activate Jurkat cells by a very weak cross-reaction with these cells, which is not detectable by conventional binding tests.

Homeodomain binding motifs modulate the probability of odorant receptor gene choice in transgenic mice.

Odorant receptor (OR) genes constitute with 1200 members the largest gene family in the mouse genome. A mature olfactory sensory neuron (OSN) is thought to express just one OR gene, and from one allele. The cell bodies of OSNs that express a given OR gene display a mosaic pattern within a particular region of the main olfactory epithelium. The mechanisms and cis-acting DNA elements that regulate the expression of one OR gene per OSN - OR gene choice - remain poorly understood. Here, we describe a reporter assay to identify minimal promoters for OR genes in transgenic mice, which are produced by the conventional method of pronuclear injection of DNA. The promoter transgenes are devoid of an OR coding sequence, and instead drive expression of the axonal marker tau-β-galactosidase. For four mouse OR genes (M71, M72, MOR23, and P3) and one human OR gene (hM72), a mosaic, OSN-specific pattern of reporter expression can be obtained in transgenic mice with contiguous DNA segments of only ~300 bp that are centered around the transcription start site (TSS). The ~150bp region upstream of the TSS contains three conserved sequence motifs, including homeodomain (HD) binding sites. Such HD binding sites are also present in the H and P elements, DNA sequences that are known to strongly influence OR gene expression. When a 19mer encompassing a HD binding site from the P element is multimerized nine times and added upstream of a MOR23 minigene that contains the MOR23 coding region, we observe a dramatic increase in the number of transgene-expressing founders and lines and in the number of labeled OSNs. By contrast, a nine times multimerized 19mer with a mutant HD binding site does not have these effects. We hypothesize that HD binding sites in the H and P elements and in OR promoters modulate the probability of OR gene choice.

Retinoid X receptor and peroxisome proliferator-activated receptor activate an estrogen responsive gene independent of the estrogen receptor.

Estrogen receptors regulate transcription of genes essential for sexual development and reproductive function. Since the retinoid X receptor (RXR) is able to modulate estrogen responsive genes and both 9-cis RA and fatty acids influenced development of estrogen responsive tumors, we hypothesized that estrogen responsive genes might be modulated by RXR and the fatty acid receptor (peroxisome proliferator-activated receptor, PPAR). To test this hypothesis, transfection assays in CV-1 cells were performed with an estrogen response element (ERE) coupled to a luciferase reporter construct. Addition of expression vectors for RXR and PPAR resulted in an 11-fold increase in luciferase activity in the presence of 9-cis RA. Furthermore, mobility shift assays demonstrated binding of RXR and PPAR to the vitellogenin A2-ERE and an ERE in the oxytocin promoter. Methylation interference assays demonstrated that specific guanine residues required for RXR/PPAR binding to the ERE were similar to residues required for ER binding. Moreover, RXR domain-deleted constructs in transfection assays showed that activation required RXR since an RXR delta AF-2 mutant completely abrogated reporter activity. Oligoprecipitation binding studies with biotinylated ERE and (35)S-labeled in vitro translated RXR constructs confirmed binding of delta AF-2 RXR mutant to the ERE in the presence of baculovirus-expressed PPAR. Finally, in situ hybridization confirmed RXR and PPAR mRNA expression in estrogen responsive tissues. Collectively, these data suggest that RXR and PPAR are present in reproductive tissues, are capable of activating estrogen responsive genes and suggest that the mechanism of activation may involve direct binding of the receptors to estrogen response elements.

MM-GBSA binding free energy decomposition and T cell receptor engineering.

Recognition by the T-cell receptor (TCR) of immunogenic peptides (p) presented by class I major histocompatibility complexes (MHC) is the key event in the immune response against virus infected cells or tumor cells. The major determinant of T cell activation is the affinity of the TCR for the peptide-MHC complex, though kinetic parameters are also important. A study of the 2C TCR/SIYR/H-2Kb system using a binding free energy decomposition (BFED) based on the MM-GBSA approach had been performed to assess the performance of the approach on this system. The results showed that the TCR-p-MHC BFED including entropic terms provides a detailed and reliable description of the energetics of the interaction (Zoete and Michielin, 2007). Based on these results, we have developed a new approach to design sequence modifications for a TCR recognizing the human leukocyte antigen (HLA)-A2 restricted tumor epitope NY-ESO-1. NY-ESO-1 is a cancer testis antigen expressed not only in melanoma, but also on several other types of cancers. It has been observed at high frequencies in melanoma patients with unusually positive clinical outcome and, therefore, represents an interesting target for adoptive transfer with modified TCR. Sequence modifications of TCR potentially increasing the affinity for this epitope have been proposed and tested in vitro. T cells expressing some of the proposed TCR mutants showed better T cell functionality, with improved killing of peptide-loaded T2 cells and better proliferative capacity compared to the wild type TCR expressing cells. These results open the door of rational TCR design for adoptive transfer cancer therapy.

Polarity and specific sequence requirements of peroxisome proliferator-activated receptor (PPAR)/retinoid X receptor heterodimer binding to DNA. A functional analysis of the malic enzyme gene PPAR response element.

The malic enzyme (ME) gene is a target for both thyroid hormone receptors and peroxisome proliferator-activated receptors (PPAR). Within the ME promoter, two direct repeat (DR)-1-like elements, MEp and MEd, have been identified as putative PPAR response elements (PPRE). We demonstrate that only MEp and not MEd is able to bind PPAR/retinoid X receptor (RXR) heterodimers and mediate peroxisome proliferator signaling. Taking advantage of the close sequence resemblance of MEp and MEd, we have identified crucial determinants of a PPRE. Using reciprocal mutation analyses of these two elements, we show the preference for adenine as the spacing nucleotide between the two half-sites of the PPRE and demonstrate the importance of the two first bases flanking the core DR1 in 5'. This latter feature of the PPRE lead us to consider the polarity of the PPAR/RXR heterodimer bound to its cognate element. We demonstrate that, in contrast to the polarity of RXR/TR and RXR/RAR bound to DR4 and DR5 elements respectively, PPAR binds to the 5' extended half-site of the response element, while RXR occupies the 3' half-site. Consistent with this polarity is our finding that formation and binding of the PPAR/RXR heterodimer requires an intact hinge T region in RXR while its integrity is not required for binding of the RXR/TR heterodimer to a DR4.

Catechol-binding serines of beta(2)-adrenergic receptors control the equilibrium between active and inactive receptor states.

The binding free energy for the interaction between serines 204 and 207 of the fifth transmembrane helix of the beta(2)-adrenergic receptor (beta(2)-AR) and catecholic hydroxyl (OH) groups of adrenergic agonists was analyzed using double mutant cycles. Binding affinities for catecholic and noncatecholic agonists were measured in wild-type and mutant receptors, carrying alanine replacement of the two serines (S204A, S207A beta(2)-AR), a constitutive activating mutation, or both. The free energy coupling between the losses of binding energy attributable to OH deletion from the ligand and from the receptor indicates a strong interaction (nonadditivity) as expected for a direct binding between the two sets of groups. However, we also measured a significant interaction between the deletion of OH groups from the receptor and the constitutive activating mutation. This suggests that a fraction of the decrease in agonist affinity caused by serine mutagenesis may involve a shift in the conformational equilibrium of the receptor toward the inactive state. Direct measurements using a transient transfection assay confirm this prediction. The constitutive activity of the (S204A, S207A) beta(2)-AR mutant is 50 to 60% lower than that of the wild-type beta(2)-AR. We conclude that S204 and S207 do not only provide a docking site for the agonist, but also control the equilibrium of the receptor between active (R*) and inactive (R) forms.

Cannabinoid 1 receptor promotes cardiac dysfunction, oxidative stress, inflammation, and fibrosis in diabetic cardiomyopathy.

Endocannabinoids and cannabinoid 1 (CB(1)) receptors have been implicated in cardiac dysfunction, inflammation, and cell death associated with various forms of shock, heart failure, and atherosclerosis, in addition to their recognized role in the development of various cardiovascular risk factors in obesity/metabolic syndrome and diabetes. In this study, we explored the role of CB(1) receptors in myocardial dysfunction, inflammation, oxidative/nitrative stress, cell death, and interrelated signaling pathways, using a mouse model of type 1 diabetic cardiomyopathy. Diabetic cardiomyopathy was characterized by increased myocardial endocannabinoid anandamide levels, oxidative/nitrative stress, activation of p38/Jun NH(2)-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs), enhanced inflammation (tumor necrosis factor-α, interleukin-1β, cyclooxygenase 2, intracellular adhesion molecule 1, and vascular cell adhesion molecule 1), increased expression of CB(1), advanced glycation end product (AGE) and angiotensin II type 1 receptors (receptor for advanced glycation end product [RAGE], angiotensin II receptor type 1 [AT(1)R]), p47(phox) NADPH oxidase subunit, β-myosin heavy chain isozyme switch, accumulation of AGE, fibrosis, and decreased expression of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA2a). Pharmacological inhibition or genetic deletion of CB(1) receptors attenuated the diabetes-induced cardiac dysfunction and the above-mentioned pathological alterations. Activation of CB(1) receptors by endocannabinoids may play an important role in the pathogenesis of diabetic cardiomyopathy by facilitating MAPK activation, AT(1)R expression/signaling, AGE accumulation, oxidative/nitrative stress, inflammation, and fibrosis. Conversely, CB(1) receptor inhibition may be beneficial in the treatment of diabetic cardiovascular complications.

Transcriptional repression of peroxisome proliferator-activated receptor beta/delta in murine keratinocytes by CCAAT/enhancer-binding proteins.

The roles of peroxisome proliferator-activated receptors (PPARs) and CCAAT/enhancer-binding proteins (C/EBPs) in keratinocyte and sebocyte differentiation suggest that both families of transcription factors closely interact in the skin. Initial characterization of the mouse PPARbeta promoter revealed an AP-1 site that is crucial for the regulation of PPARbeta expression in response to inflammatory cytokines in the skin. We now present evidence for a novel regulatory mechanism of the expression of the PPARbeta gene by which two members of the C/EBP family of transcription factors inhibit its basal promoter activity in mouse keratinocytes. We first demonstrate that C/EBPalpha and C/EBPbeta, but not C/EBPdelta, inhibit the expression of PPARbeta through the recruitment of a transcriptional repressor complex containing HDAC-1 to a specific C/EBP binding site on the PPARbeta promoter. Consistent with this repression, the expression patterns of PPARbeta and C/EBPs are mutually exclusive in keratinocytes of the interfollicular epidermis and hair follicles in mouse developing skin. This work reveals the importance of the regulatory interplay between PPARbeta and C/EBP transcription factors in the control of proliferation and differentiation in this organ. Such insights are crucial for the understanding of the molecular control regulating the balance between proliferation and differentiation in many cell types including keratinocytes.

Initiation and limitation of Ly-49A NK cell receptor acquisition by T cell factor-1.

The establishment of clonally variable expression of MHC class I-specific receptors by NK cells is not well understood. The Ly-49A receptor is used by approximately 20% of NK cells, whereby most cells express either the maternal or paternal allele and few express simultaneously both alleles. We have previously shown that NK cells expressing Ly-49A were reduced or almost absent in mice harboring a single or no functional allele of the transcription factor T cell factor-1 (TCF-1), respectively. In this study, we show that enforced expression of TCF-1 in transgenic mice yields an expanded Ly-49A subset. Even though the frequencies of Ly-49A(+) NK cells varied as a function of the TCF-1 dosage, the relative abundance of mono- and biallelic Ly-49A cells was maintained. Mono- and biallelic Ly-49A NK cells were also observed in mice expressing exclusively a transgenic TCF-1, i.e., expressing a fixed amount of TCF-1 in all NK cells. These findings suggest that Ly-49A acquisition is a stochastic event due to limiting TCF-1 availability, rather than the consequence of clonally variable expression of the endogenous TCF-1 locus. Efficient Ly-49A acquisition depended on the expression of a TCF-1 isoform, which included a domain known to associate with the TCF-1 coactivator beta-catenin. Indeed, the proximal Ly-49A promoter was beta-catenin responsive in reporter gene assays. We thus propose that Ly-49A receptor expression is induced from a single allele in occasional NK cells due to a limitation in the amount of a transcription factor complex requiring TCF-1.