scholarly journals Methods used to study the oligomeric structure of G-protein-coupled receptors

2017 ◽  
Vol 37 (2) ◽  
Author(s):  
Hui Guo ◽  
Su An ◽  
Richard Ward ◽  
Yang Yang ◽  
Ying Liu ◽  
...  
Keyword(s):  
Energy Transfer ◽  
G Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
G Protein Coupled Receptors ◽  
Experimental Techniques ◽  
Distribution Analysis ◽  
Wide Range ◽  
And Function ◽  
G Protein Coupled

G-protein-coupled receptors (GPCRs), which constitute the largest family of cell surface receptors, were originally thought to function as monomers, but are now recognized as being able to act in a wide range of oligomeric states and indeed, it is known that the oligomerization state of a GPCR can modulate its pharmacology and function. A number of experimental techniques have been devised to study GPCR oligomerization including those based upon traditional biochemistry such as blue-native PAGE (BN-PAGE), co-immunoprecipitation (Co-IP) and protein-fragment complementation assays (PCAs), those based upon resonance energy transfer, FRET, time-resolved FRET (TR-FRET), FRET spectrometry and bioluminescence resonance energy transfer (BRET). Those based upon microscopy such as FRAP, total internal reflection fluorescence microscopy (TIRFM), spatial intensity distribution analysis (SpIDA) and various single molecule imaging techniques. Finally with the solution of a growing number of crystal structures, X-ray crystallography must be acknowledged as an important source of discovery in this field. A different, but in many ways complementary approach to the use of more traditional experimental techniques, are those involving computational methods that possess obvious merit in the study of the dynamics of oligomer formation and function. Here, we summarize the latest developments that have been made in the methods used to study GPCR oligomerization and give an overview of their application.

scholarly journals Multiple GPCR Functional Assays Based on Resonance Energy Transfer Sensors

2021 ◽  
Vol 9 ◽  
Author(s):  
Yiwei Zhou ◽  
Jiyong Meng ◽  
Chanjuan Xu ◽  
Jianfeng Liu
Keyword(s):  
Energy Transfer ◽  
G Protein ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
G Protein Coupled Receptors ◽  
Functional Assays ◽  
High Throughput Drug Screening ◽  
Gpcr Activation ◽  
G Protein Coupled

G protein-coupled receptors (GPCRs) represent one of the largest membrane protein families that participate in various physiological and pathological activities. Accumulating structural evidences have revealed how GPCR activation induces conformational changes to accommodate the downstream G protein or β-arrestin. Multiple GPCR functional assays have been developed based on Förster resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) sensors to monitor the conformational changes in GPCRs, GPCR/G proteins, or GPCR/β-arrestin, especially over the past two decades. Here, we will summarize how these sensors have been optimized to increase the sensitivity and compatibility for application in different GPCR classes using various labeling strategies, meanwhile provide multiple solutions in functional assays for high-throughput drug screening.

scholarly journals Identification of dimeric and oligomeric complexes of the human oxytocin receptor by co-immunoprecipitation and bioluminescence resonance energy transfer

2003 ◽  
Vol 31 (3) ◽  
pp. 461-471 ◽  
Author(s):  
D Devost ◽  
HH Zingg
Keyword(s):  
Energy Transfer ◽  
Cell Surface ◽  
G Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Oxytocin Receptor ◽  
G Protein Coupled Receptors ◽  
Bioluminescence Resonance Energy Transfer ◽  
G Protein Coupled

The nonapeptide hormone oxytocin exerts many important biological functions, including uterine contractions during parturition and milk ejection during lactation. The manifold effects of oxytocin are mediated by a single oxytocin receptor (OTR) type, a member of the super-family of G-protein-coupled receptors. There is accumulating recent evidence that certain G-protein-coupled receptors exist in the form of oligomeric complexes. Here we demonstrate, using two different co-immunoprecipitation strategies as well as bioluminescence resonance energy transfer techniques, that the OTR is capable of forming oligomeric complexes in vivo and that these complexes exist at the cell surface membrane. The human OTR was N-terminally tagged with either a Myc or Flag epitope and transiently expressed in COS-7 cells. Cell lysates were immunoprecipitated using an anti-Flag antibody and analyzed by SDS-PAGE and Western blotting using an anti-Myc antibody, or vice versa. Either strategy provided evidence for the co-precipitation of Myc- or Flag-tagged OTR respectively.Biochemical characterization of OTR dimers showed that homodimer formation is not dependent on the establishment of disulfide bonds. The existence of OTR dimers and oligomers at the level of the cell surface was demonstrated by exposing intact living cells to an anti-Flag antibody and analyzing the immunoprecipitate by Western blotting with an anti-Myc antibody. This approach demonstrated furthermore that the presence of receptor oligomers at the cell surface is modulated by ligand in a time-dependent fashion. Finally, we obtained evidence that the OTR is forming oligomeric structures in intact living cells by observing the occurrence of bioluminescence resonance energy transfer in cells co-transfected with OTR constructs bearing at their C-terminus either a Renilla luciferase or the yellow fluorescent protein. Taken together, these data show that the OTR can form homodimers and oligomers in the cell model used and that these oligomers are present at the cell surface.

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

2008 ◽  
Vol 13 (9) ◽  
pp. 888-898 ◽  
Author(s):  
Martina Kocan ◽  
Heng B. See ◽  
Ruth M. Seeber ◽  
Karin A. Eidne ◽  
Kevin D.G. Pfleger
Keyword(s):  
Energy Transfer ◽  
G Protein ◽  
Protein Interactions ◽  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
G Protein Coupled Receptors ◽  
Live Cells ◽  
Bioluminescence Resonance Energy Transfer ◽  
G Protein Coupled

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)

Sign in / Sign up

Export Citation Format

Share Document