tailieunhanh - Báo cáo Y học: Probing intermolecular protein–protein interactions in the calcium-sensing receptor homodimer using bioluminescence resonance energy transfer (BRET)

The calcium-sensing receptor (CaR) belongs to family C of the G-protein coupled receptor superfamily. The receptor is believed to exist as a homodimer due to covalent and non-covalent interactions between the two amino terminal domains (ATDs). It is well established that agonist binding to family C receptors takes place at the ATD and that this causes theATDdimer to , very little isknown about the translationof theATDdimer twist intoG-protein coupling to the 7 transmembrane moieties (7TMs) of these receptor dimers | Eur. J. Biochem. 269 5076-5087 2002 FEBS 2002 doi Probing intermolecular protein-protein interactions in the calcium-sensing receptor homodimer using bioluminescence resonance energy transfer BRET Anders A. Jensen1 Jakob L. Hansen2 Soren P. Sheikh2 and Hans Brauner-Osborne1 lNeuroScience PharmaBiotec Research Centre Department of Medicinal Chemistry The Royal Danish School of Pharmacy Copenhagen Denmark laboratory of Molecular Cardiology Copenhagen University Hospital University of Copenhagen Copenhagen Denmark The calcium-sensing receptor CaR belongs to family C of the G-protein coupled receptor superfamily. The receptor is believed to exist as a homodimer due to covalent and non-covalent interactions between the two amino terminal domains ATDs . It is well established that agonist binding to family C receptors takes place at the ATD and that this causes the ATD dimer to twist. However very little is known about the translation of the ATD dimer twist into G-protein coupling to the 7 transmembrane moieties 7TMs of these receptor dimers. In this study we have attempted to delineate the agonist-induced intermolecular movements in the CaR homodimer using the newbioluminescence resonance energy transfer technique BRET2 which is based on the transference of energy from Renilla luciferase Rluc to the green fluorescent protein mutant GFP2. We tagged CaR with Rluc and GFP2 at different intracellular locations. Stable and highly receptor-specific BRET signals were obtained in tsA cells transfected with Rluc- and GFP2-tagged CaRs under basal conditions indicating that CaR is constitutively dimerized. However the signals were not enhanced by the presence of agonist. These results could indicate that at least parts of the two 7TMs of the CaR homodimer are in close proximity in the inactivated state of the receptor and do not move much relative to one another upon agonist activation. However we cannot exclude the possibility that the BRET .

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