Ruth M. Seeber

Model for growth hormone receptor activation based on subunit rotation within a receptor dimer

Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.

Bioluminescence resonance energy transfer (BRET) for the real-time detection of protein-protein interactions

A substantial range of protein-protein interactions can be readily monitored in real time using bioluminescence resonance energy transfer (BRET). The procedure involves heterologous coexpression of fusion proteins, which link proteins of interest to a bioluminescent donor enzyme or acceptor fluorophore. Energy transfer between these proteins is then detected. This protocol encompasses BRET1, BRET2 and the recently described eBRET, including selection of the donor, acceptor and substrate combination, fusion construct generation and validation, cell culture, fluorescence and luminescence detection, BRET detection and data analysis. The protocol is particularly suited to studying protein-protein interactions in live cells (adherent or in suspension), but cell extracts and purified proteins can also be used. Furthermore, although the procedure is illustrated with references to mammalian cell culture conditions, this protocol can be readily used for bacterial or plant studies. Once fusion proteins are generated and validated, the procedure typically takes 48–72 h depending on cell culture requirements.

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.

Plasma Leptin-Binding Activity and Hypothalamic Leptin Receptor Expression During Pregnancy and Lactation in the Rat

Abstract Leptin, the 16-kDa peptide hormone product of the ob gene, regulates body weight via the hypothalamus but also influences several aspects of reproductive function. Results of previous studies have suggested that pregnancy is a state of leptin resistance, because food consumption remains stable or increases despite a progressive rise in plasma leptin across most of gestation. In the present study, we assessed whether this apparent leptin resistance during rat pregnancy was due to either increased plasma leptin-binding activity and/or reduced expression of hypothalamic leptin receptor. Plasma leptin increased from 2.2 ± 0.4 ng/ml before pregnancy to a maximum at midgestation (4.2 ± 0.8 ng/ml on Day 12) and then fell by Day 22 and remained low throughout lactation. Despite the higher plasma leptin levels in pregnancy, food consumption increased from a minimum of 13.6 ± 0.5 g/day before pregnancy to a peak of 21.9 ± 0.6 g/day on Day 19, then fell before parturition (11.9 ± 0.4 g/day on Day 22). At least part of the increase in plasma leptin during pregnancy was attributable to a marked increase (P < 0.001) in plasma leptin-binding activity between diestrus and late pregnancy, which then fell after birth but remained at midpregnancy levels to at least Day 12 of lactation. Hypothalamic expression of mRNA encoding the long form of the leptin receptor (Ob-Rb) was elevated in early pregnancy (Day 7) but returned to prepregnancy levels by midgestation and remained stable thereafter. The results of this study confirm that pregnancy in the rat is a state of relative leptin resistance, which is due primarily to increased plasma leptin-binding activity rather than to changes in hypothalamic Ob-Rb expression.

The Duffy Antigen/Receptor for Chemokines Exists in an Oligomeric Form in Living Cells and Functionally Antagonizes CCR5 Signaling through Hetero-Oligomerization

The Duffy antigen/receptor for chemokines (DARC) is an unusual chemokine receptor that binds a large number of inflammatory chemokines of both the CC and CXC families with nanomolar affinity, yet it lacks the ability to signal upon ligand binding. Using bioluminescent resonant energy transfer, we have demonstrated for the first time that DARC exists as a constitutive homo-oligomer in living cells and furthermore that DARC hetero-oligomerizes with the CC chemokine receptor CCR5. DARC-CCR5 interaction impairs chemotaxis and calcium flux through CCR5, whereas internalization of CCR5 in response to ligand binding remains unchanged. These results suggest a novel mechanism by which DARC could modulate inflammatory responses to chemokines in vivo.

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.

Demonstration of Improvements to the Bioluminescence Resonance Energy Transfer (BRET) Technology for the Monitoring of G Protein–Coupled Receptors in Live Cells:

The bioluminescence resonance energy transfer (BRET) technique has become extremely popular for studying protein-protein interactions in living cells and real time. Of particular interest is the ability to monitor interactions between G protein–coupled receptors, such as the thyrotropin-releasing hormone receptor (TRHR), and proteins critical for regulating their function, such as β-arrestin. Using TRHR/β-arrestin interactions, we have demonstrated improvements to all 3 generations of BRET (BRET1, BRET2, and eBRET) by using the novel forms of luciferase, Rluc2 and Rluc8, developed by the Gambhir laboratory. Furthermore, for the 1st time it was possible to use the BRET2 system to detect ligand-induced G protein–coupled receptor/β-arrestin interactions over prolonged periods (on the scale of hours rather than seconds) with a very stable signal. As demonstrated by our Z′-factor data, these luciferases increase the sensitivity of BRET to such an extent that they substantially increase the potential applicability of this technology for effective drug discovery high-throughput screening. (Journal of Biomolecular Screening 2008:888-898)

Cytoplasmic terminus of vacuolar type proton pump accessory subunit Ac45 is required for proper interaction with V(0) domain subunits and efficient osteoclastic bone resorption.

Solubilization of mineralized bone by osteoclasts is largely dependent on the acidification of the extracellular resorption lacuna driven by the vacuolar (H+)-ATPases (V-ATPases) polarized within the ruffled border membranes. V-ATPases consist of two functionally and structurally distinct domains, V1 and V0. The peripheral cytoplasmically oriented V1 domain drives ATP hydrolysis, which necessitates the translocation of protons across the integral membrane bound V0 domain. Here, we demonstrate that an accessory subunit, Ac45, interacts with the V0 domain and contributes to the vacuolar type proton pump-mediated function in osteoclasts. Consistent with its role in intracellular acidification, Ac45 was found to be localized to the ruffled border region of polarized resorbing osteoclasts and enriched in pH-dependent endosomal compartments that polarized to the ruffled border region of actively resorbing osteoclasts. Interestingly, truncation of the 26-amino acid residue cytoplasmic tail of Ac45, which encodes an autonomous internalization signal, was found to impair bone resorption in vitro. Furthermore, biochemical analysis revealed that although both wild type Ac45 and mutant were capable of associating with subunits a3, c, c″, and d, deletion of the cytoplasmic tail altered its binding proximity with a3, c″, and d. In all, our data suggest that the cytoplasmic terminus of Ac45 contains elements necessary for its proper interaction with V0 domain and efficient osteoclastic bone resorption.

Identification and profiling of CXCR3–CXCR4 chemokine receptor heteromer complexes

The C‐X‐C chemokine receptors 3 (CXCR3) and C‐X‐C chemokine receptors 4 (CXCR4) are involved in various autoimmune diseases and cancers. Small antagonists have previously been shown to cross‐inhibit chemokine binding to CXCR4, CC chemokine receptors 2 (CCR2) and 5 (CCR5) heteromers. We investigated whether CXCR3 and CXCR4 can form heteromeric complexes and the binding characteristics of chemokines and small ligand compounds to these chemokine receptor heteromers.

Identification and Profiling of Novel α1A-Adrenoceptor-CXC Chemokine Receptor 2 Heteromer

Background: Receptor heteromers are macromolecular complexes containing at least two different receptor subunits, resulting in distinct pharmacology. Results: The observed α1AAR-CXCR2 heteromer recruits β-arrestin strongly upon activation with norepinephrine, in contrast to α1AAR alone. Conclusion: Heteromerization with CXCR2 dramatically changes α1AAR pharmacology, revealing the potential for heteromer-specific biased agonism. Significance: Such heteromer-specific novel pharmacology has important implications for drug discovery. We have provided the first evidence for specific heteromerization between the α1A-adrenoceptor (α1AAR) and CXC chemokine receptor 2 (CXCR2) in live cells. α1AAR and CXCR2 are both expressed in areas such as the stromal smooth muscle layer of the prostate. By utilizing the G protein-coupled receptor (GPCR) heteromer identification technology on the live cell-based bioluminescence resonance energy transfer (BRET) assay platform, our studies in human embryonic kidney 293 cells have identified norepinephrine-dependent β-arrestin recruitment that was in turn dependent upon co-expression of α1AAR with CXCR2. These findings have been supported by co-localization observed using confocal microscopy. This norepinephrine-dependent β-arrestin recruitment was inhibited not only by the α1AR antagonist Terazosin but also by the CXCR2-specific allosteric inverse agonist SB265610. Furthermore, Labetalol, which is marketed for hypertension as a nonselective β-adrenoceptor antagonist with α1AR antagonist properties, was identified as a heteromer-specific-biased agonist exhibiting partial agonism for inositol phosphate production but essentially full agonism for β-arrestin recruitment at the α1AAR-CXCR2 heteromer. Finally, bioluminescence resonance energy transfer studies with both receptors tagged suggest that α1AAR-CXCR2 heteromerization occurs constitutively and is not modulated by ligand. These findings support the concept of GPCR heteromer complexes exhibiting distinct pharmacology, thereby providing additional mechanisms through which GPCRs can potentially achieve their diverse biological functions. This has important implications for the use and future development of pharmaceuticals targeting these receptors.