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.
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.
The ability of G-protein-coupled receptors (GPCRs) to interact to form new functional structures, either forming oligomers with themselves or forming associations with other intracellular proteins, has important implications for the regulation of cellular events; however, little is known about how this occurs. Here, we have employed a newly emerging technology, bioluminescence resonance energy transfer (BRET), used to study protein-protein interactions in living cells, to demonstrate that the thyrotropin-releasing hormone receptor (TRHR) forms constitutive homo-oligomers. This formation of TRHR homo-oligomers in the absence of ligand was shown by demonstration of an energy transfer between TRHR molecules fused to either donor, Renilla luciferase (Rluc) or acceptor, enhanced yellow fluorescent protein (EYFP) molecules. This interaction was shown to be specific, since energy transfer was not detected between co-expressed tagged TRHRs and either complementary tagged gonadotropin-releasing hormone (GnRH) or  2 -adrenergic receptors. Furthermore, generation of a BRET signal between the TRHRs could only be inhibited by co-expression of the wild-type TRHR and not by other GPCRs. Agonist stimulation led to a time-and dose-dependent increase in the amount of energy transfer. Inhibition of receptor internalization by co-expression of dynamin mutant K44A did not affect the interaction between TRHRs, suggesting that clustering of receptors within clathrin-coated pits is not sufficient for energy transfer to occur. BRET also provided evidence for the agonist-induced oligomerization of another GPCR, the GnRH receptor (GnRHR), and the presence of an agonist-induced interaction of the adaptor protein, -arrestin, with TRHR and the absence of an interaction of -arrestin with GnRHR. This study supports the usefulness of BRET as a powerful tool for studying GPCR aggregations and receptor/protein interactions in general and presents evidence that the functioning unit of TRHRs exists as homomeric complexes. Thyrotropin-releasing hormone (TRH)1 is involved in controlling the production of thyroid-stimulating hormone and prolactin from the anterior pituitary gland. TRH functions via binding to its receptor subtype that belongs to the large family of G-protein-coupled receptors (GPCRs), the first of which identified (1-4) is now known as TRH receptor 1 (TRHR). As with many other GPCRs, there has been great interest in the mechanisms of regulation of TRHRs. Although the events underlying TRHR intracellular signaling and trafficking have been studied (5-11), the potential for TRHRs to undergo receptorreceptor interactions has not been previously addressed. Traditionally, GPCRs were thought to function as monomeric units, coupling to their cognate G-proteins in a 1:1 stoichiometry upon agonist activation. However, a growing body of biochemical and functional evidence supports the existence of homo-and heterodimers and oligomers and thus a critical role for GPCR-GPCR interactions in receptor function. Early functional evidence for GPCR d...
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 beta-arrestin. Using TRHR/beta-arrestin interactions, we have demonstrated improvements to all 3 generations of BRET (BRET(1), BRET(2), 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/beta-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.
Understanding the role of G protein-coupled receptor (GPCR; also known as a 7 transmembrane receptor) heteromerization in the physiology and pathophysiology of cellular function has now become a major research focus. However, there is currently a lack of cell-based assays capable of profiling the specific functional consequences of heteromerization in a ligand-dependent manner. Understanding the pharmacology specifically associated with heteromer function in contrast to monomer or homomer function enables the so-called biochemical fingerprints of the receptor heteromer to be ascertained. This is the first step in establishing the physiological relevance of heteromerization, the goal of everyone in the field, as these fingerprints can then be utilized in future endeavors to elucidate heteromer function in native tissues. The simple, robust, ligand-dependent methodology described in this study utilizes a novel configuration of components of a proximity-based reporter system. This is exemplified by the use of bioluminescence resonance energy transfer due to the advantages of real-time live cell monitoring of proximity specifically between the heteromer complex and a protein that is recruited in a ligand-dependent manner, in this case, β-arrestin 2. Further, the demonstration of Z'-factor values in excess of 0.6 shows the potential of the method for screening compounds for heteromer-selective or biased activity. Three previously characterized GPCR heteromers, the chemokine receptor heteromers CCR2-CCR5 and CCR2-CXCR4, as well as the angiotensin II receptor type 1-bradykinin receptor type 2 heteromer, have been used to illustrate the profiling capability and specificity of the GPCR heteromer identification technology.
The ␣ v  3 integrin is known to cooperate with receptor tyrosine kinases to enhance cellular responses. To determine whether ␣ v  3 regulates transforming growth factor  (TGF) 1-induced responses, we investigated the interaction between ␣ v  3 and TGF type II receptor (TGFIIR) in primary human lung fibroblasts. We report that TGF1 up-regulates cell surface and mRNA expression of ␣ v  3 in a time-and dose-dependent manner. Co-immunoprecipitation and confocal microscopy showed that TGFRII associates and clusters with ␣ v  3 , following TGF1 exposure. This association was not observed with ␣ v  5 or ␣ 5  1 . We also used a novel molecular proximity assay, bioluminescence resonance energy transfer (BRET), to quantify this dynamic interaction in living cells. TGF1 stimulation resulted in a BRET signal within 5 min, whereas tenascin, which binds ␣ v  3 , did not induce a substantial BRET signal. Co-exposure to tenascin and TGF1 produced no further increases in BRET than TGF1 alone. Cyclin D1 was rapidly induced in cells co-exposed to TGF1 and tenascin, and as a consequence proliferation induced by TGF1 was dramatically enhanced in cells co-exposed to tenascin or vitronectin. Cholesterol depletion inhibited the interaction between TGFRII and ␣ v  3 and abrogated the proliferative effect. The cyclic RGD peptide, GpenGRGD-SPCA, which blocks ␣ v  3 , also abolished the synergistic proliferative effect seen. These results indicate a new interaction partner for the ␣ v  3 integrin, the TGFIIR, in which TGF1-induced responses are potentiated in the presence ␣ v  3 ligands. Our data provide a novel mechanism by which TGF1 may contribute to abnormal wound healing and tissue fibrosis.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.