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Dive into the research topics where Karin A. Eidne is active.

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Featured researches published by Karin A. Eidne.


Nature Methods | 2006

Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET)

Kevin D. G. Pfleger; Karin A. Eidne

Bioluminescence resonance energy transfer (BRET) is a straightforward biophysical technique for studying protein-protein interactions. It requires: (1) that proteins of interest and suitable controls be labeled with either a donor or acceptor molecule, (2) placement of these labeled proteins in the desired environment for assessing their potential interaction, and (3) use of suitable detection instrumentation to monitor resultant energy transfer. There are now several possible applications, combinations of donor and acceptor molecules, potential assay environments and detection system perturbations. Therefore, this review aims to demystify and clarify the important aspects of the BRET methodology that should be considered when using this technique.


Nature Structural & Molecular Biology | 2005

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

Richard J. Brown; Julian J. Adams; Rebecca Pelekanos; Yu Wan; William J. McKinstry; Kathryn Palethorpe; Ruth M. Seeber; Thea Monks; Karin A. Eidne; Michael W. Parker; Michael J. Waters

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.


Biochemical Journal | 2005

Monitoring the formation of dynamic G-protein-coupled receptor–protein complexes in living cells

Kevin D. G. Pfleger; Karin A. Eidne

GPCRs (G-protein-coupled receptors) play an extremely important role in transducing extracellular signals across the cell membrane with high specificity and sensitivity. They are central to many of the bodys endocrine and neurotransmitter pathways, and are consequently a major drug target. It is now clear that GPCRs interact with a range of proteins, including other GPCRs. Identifying and elucidating the function of such interactions will significantly enhance our understanding of cellular function, with the promise of new and improved pharmaceuticals. Biophysical techniques involving resonance energy transfer, namely FRET (fluorescence resonance energy transfer) and BRET (bioluminescence resonance energy transfer), now enable us to monitor the formation of dynamic GPCR-protein complexes in living cells, in real time. Their use has firmly established the concept of GPCR oligomerization, as well as demonstrating GPCR interactions with GPCR kinases, beta-arrestins, adenylate cyclase and a subunit of an inwardly rectifying K+ channel. The present review examines recent technological advances and experimental applications of FRET and BRET, discussing particularly how they have been adapted to extract an ever-increasing amount of information about the nature, specificity, stoichiometry, kinetics and agonist-dependency of GPCR-protein interactions.


Nature Protocols | 2006

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

Kevin D. G. Pfleger; Ruth M. Seeber; Karin A. Eidne

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.


Molecular and Cellular Endocrinology | 1993

Cloning and sequencing of the sheep pituitary gonadotropin-releasing hormone receptor and changes in expression of its mRNA during the estrous cycle

J. Brooks; Philip L. Taylor; Philippa T. K. Saunders; Karin A. Eidne; W.J. Struthers; Alan S. McNeilly

We have isolated a full length cDNA clone coding for the sheep GnRH receptor (GnRH-R). The amino acid sequence shows greater homology to the human GnRH-R sequence than the two rodent receptors published so far. We have also carried out physiological studies investigating the pattern of expression of the GnRH-R mRNA throughout the estrous cycle. GnRH receptor mRNA and GnRH binding levels were both significantly (P < 0.05) increased over luteal levels up until the time of the preovulatory LH surge, whilst post-surge, a significant (P < 0.05) decline was seen. These changes were related to increased follicle estradiol production in the follicular phase. In contrast, no changes in the abundance of LH beta mRNA were seen throughout the estrous cycle, but the pituitary content of LH significantly (P < 0.05) decreased after the preovulatory LH surge. These results suggest that there is a close relationship between the abundance of GnRH-R mRNA and translation of the GnRH-R in sheep.


Trends in Endocrinology and Metabolism | 2002

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

Karin A. Eidne; Karen M. Kroeger; Aylin C. Hanyaloglu

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.


Journal of Biological Chemistry | 2004

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

Amelia K. Scaffidi; Nenad Petrovic; Yuben Moodley; Mirjana Fogel-Petrovic; Karen M. Kroeger; Ruth M. Seeber; Karin A. Eidne; Philip J. Thompson; Darryl A. Knight

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.


Pharmacology & Therapeutics | 2008

G protein-coupled receptor dimers: Functional consequences, disease states and drug targets

Matthew B. Dalrymple; Kevin D. G. Pfleger; Karin A. Eidne

With an ever-expanding need for reliable therapeutic agents that are highly effective and exhibit minimal deleterious side effects, a greater understanding of the mechanisms underlying G protein-coupled receptor (GPCR) regulation is fundamental. GPCRs comprise more than 30% of all therapeutic drug targets and it is likely that this will only increase as more orphan GPCRs are identified. The past decade has seen a dramatic shift in the prevailing concept of how GPCRs function, in particular the growing acceptance that GPCRs are capable of interacting with one another at a molecular level to form complexes, with significantly different pharmacological properties to their monomeric selves. While the ability of like-receptors to associate and form homodimers raises some interesting mechanistic issues, the possibility that unlike-receptors could heterodimerise in certain tissue types, producing a functionally unique signalling complex that binds specific ligands, provides an invaluable opportunity to refine and redefine pharmacological interventions with greater specificity and efficacy.


Frontiers in Neuroendocrinology | 2003

G-protein coupled receptor oligomerization in neuroendocrine pathways

Karen M. Kroeger; Kevin D. G. Pfleger; Karin A. Eidne

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.


Molecular Pharmacology | 2008

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

Aron Chakera; Ruth M. Seeber; Alison E. John; Karin A. Eidne; David R. Greaves

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.

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Kevin D. G. Pfleger

University of Western Australia

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Ruth M. Seeber

University of Western Australia

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Karen M. Kroeger

University of Western Australia

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Matthew B. Dalrymple

University of Western Australia

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Esther Lim

University of Western Australia

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Heng B. See

University of Western Australia

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