TY - JOUR
T1 - Mapping the architecture of secretin receptors with intramolecular fluorescence resonance energy transfer using acousto-optic tunable filter-based spectral imaging
AU - Lisenbee, Cayle S.
AU - Harikumar, Kaleeckal G.
AU - Miller, Laurence J.
PY - 2007
Y1 - 2007
N2 - The molecular structure and agonist-induced conformational changes of class II G protein-coupled receptors are poorly understood. In this work, we developed and characterized a series of dual cyan fluorescent protein (CFP)-tagged and yellow fluorescent protein (YFP)-tagged secretin receptor constructs for use in various functional and fluorescence analyses of receptor structural variants. CFP insertions within the first or second intracellular loop domains of this receptor were tolerated poorly or partially, respectively, in receptors tagged with a carboxyl-terminal yellow fluorescent protein that itself had no effect on secretin binding or cAMP production. A similar CFP insertion into the third intracellular loop resulted in a plasma membrane-localized receptor that bound secretin and signaled normally. This fully active third-loop variant exhibited a significant decrease in fluorescence resonance energy transfer signals that were recorded with an acousto-optic tunable filter microscope after exposure to secretin agonist but not to a receptor antagonist. These data demonstrate changes in the relative positions of intracellular structures that support a model for secretin receptor activation.
AB - The molecular structure and agonist-induced conformational changes of class II G protein-coupled receptors are poorly understood. In this work, we developed and characterized a series of dual cyan fluorescent protein (CFP)-tagged and yellow fluorescent protein (YFP)-tagged secretin receptor constructs for use in various functional and fluorescence analyses of receptor structural variants. CFP insertions within the first or second intracellular loop domains of this receptor were tolerated poorly or partially, respectively, in receptors tagged with a carboxyl-terminal yellow fluorescent protein that itself had no effect on secretin binding or cAMP production. A similar CFP insertion into the third intracellular loop resulted in a plasma membrane-localized receptor that bound secretin and signaled normally. This fully active third-loop variant exhibited a significant decrease in fluorescence resonance energy transfer signals that were recorded with an acousto-optic tunable filter microscope after exposure to secretin agonist but not to a receptor antagonist. These data demonstrate changes in the relative positions of intracellular structures that support a model for secretin receptor activation.
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U2 - 10.1210/me.2007-0063
DO - 10.1210/me.2007-0063
M3 - Article
C2 - 17505057
AN - SCOPUS:34547483056
SN - 0888-8809
VL - 21
SP - 1997
EP - 2008
JO - Molecular Endocrinology
JF - Molecular Endocrinology
IS - 8
ER -