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Abstract
As described previously, receptor dimerization of G protein-coupled receptors may influence signaling, trafficking, and regulation in vivo. Up to now, most studies aiming at the possible role of receptor dimerization in receptor activation and signal transduction are focused on class A GPCRs. In the present work, the dimerization behavior of the corticotropin-releasing factor receptor type 1 (CRF1R), which belongs to class B of GPCRs and plays an important role in coordination of the immune response, stress, and learning behavior, was investigated by using fluorescence resonance energy transfer (FRET). For this purpose, we generated fusion proteins of CRF1R tagged at their C-terminus to a cyan or yellow fluorescent protein, which can be used as a FRET pair. Binding studies verified that the receptor constructs were able to bind their natural ligands in a manner comparable with the wild-type receptor, whereas cAMP accumulation proved the functionality of the constructs. In microscopic studies, a dimerization of the CRF1R was observed, but the addition of either CRF-related agonists or antagonists did not show any dose-related increase of the observed FRET signal, indicating that the dimer-monomer ratio is not changed on addition of ligand.
| Abbreviations | ||
| CFP | = | cyan fluorescent protein |
| CLSM | = | confocal laser scanning microscopy |
| CRF1R | = | corticotropin-releasing factor receptor type 1 |
| FRET | = | fluorescence resonance energy transfer |
| GPCR | = | G protein-coupled receptor |
| HEK | = | human embryonic kidney |
| SDS-PAGE, SDS | = | polyacrylamide gel electrophoresis |
| YFP | = | yellow fluorescent protein. |
| Abbreviations | ||
| CFP | = | cyan fluorescent protein |
| CLSM | = | confocal laser scanning microscopy |
| CRF1R | = | corticotropin-releasing factor receptor type 1 |
| FRET | = | fluorescence resonance energy transfer |
| GPCR | = | G protein-coupled receptor |
| HEK | = | human embryonic kidney |
| SDS-PAGE, SDS | = | polyacrylamide gel electrophoresis |
| YFP | = | yellow fluorescent protein. |