The Binding of Calcitonin to its Receptor; Studies by Mutagenesis and Molecular Modelling

J Barwell1, J Gingell2, DR Poyner1, DL Hay2. 1School of Life and Health Sciences, Aston University, Birmingham B4 7ET, UK, 2School of Biological Sciences and Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Auckland 1142, New Zealand

The calcitonin (CT) receptor (CTR) is an example of a family B G protein-coupled receptor (GPCR). It is not known how CT binds to its receptor. Here, site-directed mutagenesis and molecular modelling have been used to identify likely receptor-ligand contacts.

Mutagenesis of the human CTR(a), transient transfection into Cos 7 cells, challenge with human CT and measurement of cAMP were as described previously (1). Data were fitted with Graphpad Prism 5 using a logistic equation with fixed Hill slope to obtain pEC50 values. To model the CTR extracellular domain (ECD) (the expected major domain involved in binding), the crystal structure of the CTR-like receptor ECD (PDB 3N7S) was used (2). An homology model was made using Modeller 9v8, (3). Human CT (PDB 2JXZ) was docked using ClusPro 2.0 (4).

The mutagenesis data (Table 1) indicated that residues clustered on the presumed helix 1, loops 2 and 4 and the C-terminus of CTR all influenced binding of CT. These residues define a similar binding pocket to that observed for the binding of certain other family B GPCR ligands to their receptors (5).

Table 1 Effects of mutants on the pEC50 of human CT to stimulate cAMP production

Values are means ± sems of 3-5 determinations. *, P<0.05, Student’s t-test.

Molecular modelling suggests that the residues identified in the mutagenesis form hydrophobic or hydrogen bonds with amino acids along bound CT. This provides a plausible model for the docking of human CT.

References

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