Mapping the architecture of secretin receptors with intramolecular fluorescence resonance energy transfer using acousto-optic tunable filter-based spectral imaging.
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.
Lisenbee
Cayle S
CS
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona 85259, USA.
Harikumar
Kaleeckal G
KG
Miller
Laurence J
LJ
D000162:Acoustics; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D031541:Fluorescence Resonance Energy Transfer; D017930:Genes, Reporter; D055095:Optics and Photonics; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011964:Receptors, Gastrointestinal Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D043562:Receptors, G-Protein-Coupled; D011964:Receptors, Gastrointestinal Hormone; C029937:secretin receptor