Direct determination of multiple ligand interactions with the extracellular domain of the calcium-sensing receptor.

Numerous in vivo functional studies have indicated that the dimeric extracellular domain (ECD) of the CaSR plays a crucial role in regulating Ca(2+) homeostasis by sensing Ca(2+) and l-Phe. However, direct interaction of Ca(2+) and Phe with the ECD of the receptor and the resultant impact on its structure and associated conformational changes have been hampered by the large size of the ECD, its high degree of glycosylation, and the lack of biophysical methods to monitor weak interactions in solution. In the present study, we purified the glycosylated extracellular domain of calcium-sensing receptor (CaSR) (ECD) (residues 20-612), containing either complex or high mannose N-glycan structures depending on the host cell line employed for recombinant expression. Both glycosylated forms of the CaSR ECD were purified as dimers and exhibit similar secondary structures with ∼ 50% α-helix, ∼ 20% β-sheet content, and a well buried Trp environment. Using various spectroscopic methods, we have shown that both protein variants bind Ca(2+) with a Kd of 3.0-5.0 mm. The local conformational changes of the proteins induced by their interactions with Ca(2+) were visualized by NMR with specific (15)N Phe-labeled forms of the ECD. Saturation transfer difference NMR approaches demonstrated for the first time a direct interaction between the CaSR ECD and l-Phe. We further demonstrated that l-Phe increases the binding affinity of the CaSR ECD for Ca(2+). Our findings provide new insights into the mechanisms by which Ca(2+) and amino acids regulate the CaSR and may pave the way for exploration of the structural properties of CaSR and other members of family C of the GPCR superfamily.