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 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.
Jensen
Anders A
AA
NeuroScience PharmaBiotec Research Centre, Department of Medicinal Chemistry, The Royal Danish School of Pharmacy, Copenhagen, Denmark. aaj@dfh.dk
Hansen
Jakob L
JL
Sheikh
Søren P
SP
Bräuner-Osborne
Hans
H
D000595:Amino Acid Sequence; D001703:Biophysics / Q000379:methods*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D019281:Dimerization; D004735:Energy Transfer; D049452:Green Fluorescent Proteins; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D044140:Receptor, Angiotensin, Type 1; D018091:Receptors, AMPA / Q000235:genetics / Q000378:metabolism; D011945:Receptors, Angiotensin / Q000235:genetics / Q000378:metabolism; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism
D049452:Green Fluorescent Proteins; D007295:Inositol Phosphates; D008156:Luciferases; D008164:Luminescent Proteins; D044140:Receptor, Angiotensin, Type 1; D018091:Receptors, AMPA; D011945:Receptors, Angiotensin; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins