TY - JOUR
T1 - Use of fluorescence resonance energy transfer to analyze oligomerization of G-protein-coupled receptors expressed in yeast
AU - Overton, Mark C.
AU - Blumer, Kendall J.
N1 - Funding Information:
This work was supported by a grant from the National Institutes of Health (K.J.B). We thank Tom Baranski for providing comments on the manuscript.
PY - 2002
Y1 - 2002
N2 - Oligomerization or dimerization of G-protein-coupled receptors (GPCRs) has emerged as an important theme in signal transduction. This concept has recently gained widespread interest due to the application of direct and noninvasive biophysical techniques such as fluorescence resonance energy transfer (FRET), which have shown unequivocally that several types of GPCR can form dimers or oligomers in living cells. Current challenges are to determine which GPCRs can self-associate and/or interact with other GPCRs, to define the molecular principles that govern these specific interactions, and to establish which aspects of GPCR function require oligomerization. Although these questions ultimately must be addressed by using GPCRs expressed endogenously in their native cell types, analysis of GPCR oligomerization in heterologous expression systems will be useful to survey which GPCRs can interact, to conduct structure-function studies, and to identify peptides or small molecules that disrupt GPCR oligomerization and function. Here, we describe methods employing scanning fluorometry to detect FRET between GPCRs tagged with enhanced cyan and yellow fluorescent proteins (CFP and YFP) in living yeast cells. This approach provides a powerful means to analyze oligomerization of a variety of GPCRs that can be expressed in yeast, such as adrenergic, adenosine, C5a, muscarinic acetylcholine, vasopressin, opioid, and somatostatin receptors.
AB - Oligomerization or dimerization of G-protein-coupled receptors (GPCRs) has emerged as an important theme in signal transduction. This concept has recently gained widespread interest due to the application of direct and noninvasive biophysical techniques such as fluorescence resonance energy transfer (FRET), which have shown unequivocally that several types of GPCR can form dimers or oligomers in living cells. Current challenges are to determine which GPCRs can self-associate and/or interact with other GPCRs, to define the molecular principles that govern these specific interactions, and to establish which aspects of GPCR function require oligomerization. Although these questions ultimately must be addressed by using GPCRs expressed endogenously in their native cell types, analysis of GPCR oligomerization in heterologous expression systems will be useful to survey which GPCRs can interact, to conduct structure-function studies, and to identify peptides or small molecules that disrupt GPCR oligomerization and function. Here, we describe methods employing scanning fluorometry to detect FRET between GPCRs tagged with enhanced cyan and yellow fluorescent proteins (CFP and YFP) in living yeast cells. This approach provides a powerful means to analyze oligomerization of a variety of GPCRs that can be expressed in yeast, such as adrenergic, adenosine, C5a, muscarinic acetylcholine, vasopressin, opioid, and somatostatin receptors.
KW - Cyan fluorescent protein
KW - Dimerization
KW - Fluorescence resonance energy transfer
KW - G-protein-coupled receptor
KW - Oligomerization
KW - Scanning fluorometry
KW - Yeast
KW - Yellow fluorescent protein
UR - http://www.scopus.com/inward/record.url?scp=0036384365&partnerID=8YFLogxK
U2 - 10.1016/S1046-2023(02)00090-7
DO - 10.1016/S1046-2023(02)00090-7
M3 - Article
C2 - 12217648
AN - SCOPUS:0036384365
SN - 1046-2023
VL - 27
SP - 324
EP - 332
JO - Methods
JF - Methods
IS - 4
ER -