Karen M. Kroeger

The −308 tumor necrosis factor-α promoter polymorphism effects transcription

Since the tumor necrosis factor alpha (TNF-α) gene was found to be located in the central major histocompatibility complex (MHC) there has been much speculation concerning a genetic association between particular TNF alleles and disease susceptibility. A relationship between the MHC haplotype Al, B8, DR3, TNF-α expression levels and susceptibility to autoimmune disease has been suggested by several groups. The identification of the −308 polymorphism and its association with the HLA Al, B8, DR3 haplotype have led to speculation that the polymorphism may play a role in the altered expression of TNF-α. We have demonstrated that the region (−323 to −285) encompassing −308 in the TNF2 allele binds nuclear factors differently to the same region in the promoter of the more common TNF1 allele. The GA −308 polymorphism affected the affinity of factor binding and resulted in a factor binding to TNF2 but not TNF1. The observed differential binding was shown to be functional, with the 38 by region from TNF2 causing a two-fold greater activity of a heterologous promoter over that due to the same region in TNFI. To further substantiate the functional consequences of the TNF-α −308 polymorphism, we analysed both allelic forms of the TNF-α promoter region (−993 to +110) in a transient transfection assay, using luciferase as a reporter gene. The results showed that when present with the 3′UTR the −308A allelic form gave a two-fold greater level of transcription than the −308G form in PMA-stimulated Jurkat and U937 cells. This suggests that the −308 GA polymorphism may play a role in the altered TNF-α gene expression observed in individuals with the HLA Al, B8, DR3 haplotype.

Impact of the -308 TNF promoter polymorphism on the transcriptional regulation of the TNF gene: relevance to disease.

A biallelic G (TNF1 allele) to A (TNF2 allele) polymorphism 308 nucleotides upstream from the transcription initiation site in the tumor necrosis factor (TNF) promoter is associated with elevated TNF levels and disease susceptibilities observed in human subjects. The TNF2 allele is strongly associated with the high‐TNF‐producing autoimmune MHC haplotype HLA‐A1, B8, DR3, with elevated serum TNF levels and a more severe outcome in infectious diseases, such as cerebral malaria. A number of groups have set out to determine whether the ‐308 polymorphism could affect transcription factor binding and hence influence TNF transcription and expression levels. Although some studies have failed to show any functional difference between the two allelic forms, others have shown that the ‐308 polymorphism effected transcription factor binding to the region encompassing ‐308, with the region in the TNF2 allele showing altered binding characteristics. The ‐308 polymorphism also has been found by some groups to be functionally significant in reporter gene assays in Raji B cells, Jurkat T cells, and U937 pre‐monocytic cells. Up to fivefold differences can be measured between TNF1 and TNF2 allelic constructs when the TNF 3′UTR is present, indicating a role in the expression of the polymorphism. Although controversial, the majority of the data support a direct role for the TNF2 ‐308 allele in the elevated TNF levels observed in TNF2 homozygotes and HLA‐A1, B8, DR3 individuals. Elevated TNF levels due to the ‐308 polymorphism may alter the immune response such that it confers susceptibility to certain autoimmune and infectious diseases. J. Leukoc. Biol. 66: 562–566; 1999.

Applications of novel resonance energy transfer techniques to study dynamic hormone receptor interactions in living cells.

Many aspects of hormone receptor function that are crucial for controlling signal transduction of endocrine pathways can be monitored more accurately with the use of non-invasive, live cell resonance energy transfer (RET) techniques. Fluorescent RET (FRET), and its variation, bioluminescent RET (BRET), can be used to assess the real-time responses to specific hormonal stimuli, whilst preserving the cellular protein network, compartmentalization and spatial arrangement. Both FRET and BRET can be readily adapted to the study of membrane proteins. Here, we focus on their applications to the analysis of interactions involving the superfamily of hormone G-protein-coupled receptors. RET is also emerging as a significant tool for the determination of protein function in general. Such techniques will undoubtedly be of value in determining the functional identities of the vast array of proteins that are encoded by the human genome.

αvβ3 Integrin Interacts with the Transforming Growth Factor β (TGFβ) Type II Receptor to Potentiate the Proliferative Effects of TGFβ1 in Living Human Lung Fibroblasts

The αvβ3 integrin is known to cooperate with receptor tyrosine kinases to enhance cellular responses. To determine whether αvβ3 regulates transforming growth factor β (TGFβ) 1-induced responses, we investigated the interaction between αvβ3 and TGFβ type II receptor (TGFβIIR) in primary human lung fibroblasts. We report that TGFβ1 up-regulates cell surface and mRNA expression of αvβ3 in a time- and dose-dependent manner. Co-immunoprecipitation and confocal microscopy showed that TGFβRII associates and clusters with αvβ3, following TGFβ1 exposure. This association was not observed with αvβ5 or α5β1. We also used a novel molecular proximity assay, bioluminescence resonance energy transfer (BRET), to quantify this dynamic interaction in living cells. TGFβ1 stimulation resulted in a BRET signal within 5 min, whereas tenascin, which binds αvβ3, did not induce a substantial BRET signal. Co-exposure to tenascin and TGFβ1 produced no further increases in BRET than TGFβ1 alone. Cyclin D1 was rapidly induced in cells co-exposed to TGFβ1 and tenascin, and as a consequence proliferation induced by TGFβ1 was dramatically enhanced in cells co-exposed to tenascin or vitronectin. Cholesterol depletion inhibited the interaction between TGFβRII and αvβ3 and abrogated the proliferative effect. The cyclic RGD peptide, GpenGRGDSPCA, which blocks αvβ3, also abolished the synergistic proliferative effect seen. These results indicate a new interaction partner for the αvβ3 integrin, the TGFβIIR, in which TGFβ1-induced responses are potentiated in the presence αvβ3 ligands. Our data provide a novel mechanism by which TGFβ1 may contribute to abnormal wound healing and tissue fibrosis.

G-protein coupled receptor oligomerization in neuroendocrine pathways

Protein-protein interactions are fundamental processes for many biological systems including those involving the superfamily of G-protein coupled receptors (GPCRs). A growing body of biochemical and functional evidence supports the existence of GPCR-GPCR homo- and hetero-oligomers. In particular, hetero-oligomers can display pharmacological and functional properties distinct from those of the homodimer or oligomer thus adding another level of complexity to how GPCRs are activated, signal and traffick in the cell. Dimerization may also play a role in influencing the activity of agonists and antagonists. We are only beginning to unravel how and why such complexes are formed, the functional implications of which will have an enormous impact on GPCR biology. Future research that studies GPCRs as dimeric or oligomeric complexes will enhance not only our understanding of GPCRs in cellular function but will also be critical for novel drug design and improved treatment of the vast array of GPCR-related conditions.

αvβ3 integrin interacts with the TGFβ type II receptor to potentiate the proliferative effects of TGFβ1 in living human lung fibroblasts

The αvβ3 integrin is known to cooperate with receptor tyrosine kinases to enhance cellular responses. To determine whether αvβ3 regulates transforming growth factor β (TGFβ) 1-induced responses, we investigated the interaction between αvβ3 and TGFβ type II receptor (TGFβIIR) in primary human lung fibroblasts. We report that TGFβ1 up-regulates cell surface and mRNA expression of αvβ3 in a time- and dose-dependent manner. Co-immunoprecipitation and confocal microscopy showed that TGFβRII associates and clusters with αvβ3, following TGFβ1 exposure. This association was not observed with αvβ5 or α5β1. We also used a novel molecular proximity assay, bioluminescence resonance energy transfer (BRET), to quantify this dynamic interaction in living cells. TGFβ1 stimulation resulted in a BRET signal within 5 min, whereas tenascin, which binds αvβ3, did not induce a substantial BRET signal. Co-exposure to tenascin and TGFβ1 produced no further increases in BRET than TGFβ1 alone. Cyclin D1 was rapidly induced in cells co-exposed to TGFβ1 and tenascin, and as a consequence proliferation induced by TGFβ1 was dramatically enhanced in cells co-exposed to tenascin or vitronectin. Cholesterol depletion inhibited the interaction between TGFβRII and αvβ3 and abrogated the proliferative effect. The cyclic RGD peptide, GpenGRGDSPCA, which blocks αvβ3, also abolished the synergistic proliferative effect seen. These results indicate a new interaction partner for the αvβ3 integrin, the TGFβIIR, in which TGFβ1-induced responses are potentiated in the presence αvβ3 ligands. Our data provide a novel mechanism by which TGFβ1 may contribute to abnormal wound healing and tissue fibrosis.

Casein kinase II sites in the intracellular C-terminal domain of the thyrotropin-releasing hormone receptor and chimeric gonadotropin-releasing hormone receptors contribute to β-arrestin-dependent internalization

We have previously shown that the mammalian gonadotropin-releasing hormone receptor (GnRHR), a unique G-protein-coupled receptor (GPCR) lacking an intracellular carboxyl tail (C-tail), does not follow a β-arrestin-dependent internalization pathway. However, internalization of a chimeric GnRHR with the thyrotropin-releasing hormone receptor (TRHR) C-tail does utilize β-arrestin. Here, we have investigated the sites within the intracellular C-tail domain that are important for conferring β-arrestin-dependent internalization. In contrast to the chimeric GnRHR with a TRHR C-tail, a chimeric GnRHR with the catfish GnRHR C-tail is not β-arrestin-dependent. Sequence comparisons between these chimeric receptors show three consensus phosphorylation sites for casein kinase II (CKII) in the TRHR C-tail but none in the catfish GnRHR C-tail. We thus investigated a role for CKII sites in determining GPCR internalization via β-arrestin. Sequential introduction of three CKII sites into the chimera with the catfish C-tail (H354D,A366E,G371D) resulted in a change in the pattern of receptor phosphorylation and β-arrestin-dependence, which only occurred when all three sites were introduced. Conversely, mutation of the putative CKII sites (T365A,T371A,S383A) in the C-tail of a β-arrestin-sensitive GPCR, the TRHR, resulted in decreased receptor phosphorylation and a loss of β-arrestin-dependence. Mutation of all three CKII sites was necessary before a loss of β-arrestin-dependence was observed. Visualization of β-arrestin/GFP redistribution confirmed a loss or gain of β-arrestin sensitivity for receptor mutants. Internalization of receptors without C-tail CKII sites was promoted by a phosphorylation-independent β-arrestin mutant (R169E), suggesting that these receptors do not contain the necessary phosphorylation sites required for β-arrestin-dependent internalization. Apigenin, a specific CKII inhibitor, blocked the increase in receptor internalization by β-arrestin, thus providing further support for the involvement of CKII. This study presents evidence of a novel role for C-tail CKII consensus sites in targeting these GPCRs to the β-arrestin-dependent pathway.

Identification of an AP‐2 element in the ‐323 to ‐285 region of the TNF‐α gene

We have identified a region of the human TNF‐α promoter between nucleotides ‐323 and ‐285, capable of influencing transcriptional activity. This region encompasses the ‐308 polymorphism and contains a 10 bp sequence homologous to the consensus binding site of activator protein‐2 (AP‐2). Protein complexes derived from U937 and Jurkat cells were found to bind to this element. Competitive EMSA using a consensus AP‐2 oligonucleotide indicated that AP‐2 may be involved. Functional assays demonstrate that this region can repress activity of a heterologous promoter in the Jurkat T‐cell line, but act as an inducible enhancer of transcription in U937 cells.

Preassociation of nonactivated STAT3 molecules demonstrated in living cells using bioluminescence resonance energy transfer: a new model of STAT activation?

Signal transducers and activators of transcription (STATs) are crucial molecules in cytokine signaling. In th conventional model of STAT activation, STAT molecules are recruited from a latent pool of cytoplasmic monomers to the activated cytokine receptor. After binding to the receptor, they get tyrosine‐phosphorylated, dissociate from the receptor, and translocate to the nucleus as activation‐induced dimers. Recently, several publications questioned this model of STAT activation and showed the existence of preassociated STAT molecules before activation. We were able to demonstrate the existence of these preassociated STAT3 molecules in living mammalian cells using bioluminescence resonance energy transfer. Our results support the new hypothesis that STAT molecules exist in the cytoplasm as dimers or multimers and point to an activation‐induced change in STAT3 conformation. Therefore, we propose a new model of STAT activation and discuss a hypothetical structure of “cytoplasmic” STAT dimers as opposed to the known “activation‐induced” dimer.

Study of G-Protein-Coupled Receptor-Protein Interactions by Bioluminescence Resonance Energy Transfer

Complex networks of protein-protein interactions are key determinants of cellular function, including those regulated by G-protein-coupled receptors (GPCRs). Formation of either stable or transitory complexes are involved in regulating all aspects of receptor function, from ligand binding through to signal transduction, desensitization, resensitization and downregulation. Today, 50% of all recently launched drugs are targeted against GPCRs. This particular class of proteins is extremely useful as a drug target because the receptors are partly located outside the cell, simplifying bioavailability and delivery of drugs directed against them. However, being located within the cell membrane causes difficulties for the study of GPCR function and bioluminescence resonance energy transfer (BRET), a naturally occurring phenomenon, represents a newly emerging, powerful tool with which to investigate and monitor dynamic interactions involving this receptor class. BRET is a noninvasive, highly sensitive technique, performed as a simple homogeneous assay. involving the proximity-dependent transfer of energy from an energy donor to acceptor resulting in the emission of light. This technology has several advantages over alternative approaches as the detection occurs within live cells, in real time, and is not restricted to a particular cellular compartment. The use of such biophysical techniques as BRET, will not only increase our understanding of the nature of GPCR regulation and the protein complexes involved, but could also potentially lead to the development of novel therapeutics that modulate these interactions.