Probing intermolecular protein-protein interactions in the calcium-sensing receptor homodimer using bioluminescence resonance energy transfer (BRET)
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Probing intermolecular protein-protein interactions in the calcium-sensing receptor homodimer using bioluminescence resonance energy transfer (BRET). / Jensen, Anders A.; Hansen, Jakob L; Sheikh, Søren P; Bräuner-Osborne, Hans.
In: European Journal of Biochemistry, Vol. 269, No. 20, 2002, p. 5076-87.Research output: Contribution to journal › Journal article › Research › peer-review
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T1 - Probing intermolecular protein-protein interactions in the calcium-sensing receptor homodimer using bioluminescence resonance energy transfer (BRET)
AU - Jensen, Anders A.
AU - Hansen, Jakob L
AU - Sheikh, Søren P
AU - Bräuner-Osborne, Hans
PY - 2002
Y1 - 2002
N2 - 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 new bioluminescence 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 technology is unable to register putative conformational changes in the CaR homodimer induced by agonist binding because of the bulk sizes of the Rluc and GFP2 molecules.
AB - 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 new bioluminescence 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 technology is unable to register putative conformational changes in the CaR homodimer induced by agonist binding because of the bulk sizes of the Rluc and GFP2 molecules.
KW - Amino Acid Sequence
KW - Biophysics
KW - Cell Line
KW - Cell Membrane
KW - Dimerization
KW - Energy Transfer
KW - Green Fluorescent Proteins
KW - Humans
KW - Inositol Phosphates
KW - Luciferases
KW - Luminescent Measurements
KW - Luminescent Proteins
KW - Molecular Sequence Data
KW - Receptor, Angiotensin, Type 1
KW - Receptors, AMPA
KW - Receptors, Angiotensin
KW - Receptors, Calcium-Sensing
KW - Receptors, Cell Surface
KW - Recombinant Proteins
M3 - Journal article
C2 - 12383267
VL - 269
SP - 5076
EP - 5087
JO - FEBS Journal
JF - FEBS Journal
SN - 1742-464X
IS - 20
ER -
ID: 38485158