Purpose: This study was conducted to identify and characterize the structural requirements of a calmodulin-binding motif identified in the third intracellular (i3) loop of muscarinic acetylcholine receptors (M1-M5), a region important for G protein coupling.
Methods: GST fusion proteins and synthetic peptides derived from the hM1 i3 loop were tested for binding to CaM using a cross-linking gel shift assay and a dansyl-CaM fluorescence assay. Mutagenesis studies further characterized the structural requirements for the interaction and identified critical residues.
Results: 28-Mer peptides from the C terminus of i3, representing the putative calmodulin domains of M1, M2, and M3, were found capable of interacting with CaM. In addition, smaller peptides defined a 5-amino-acid sequence essential for calmodulin binding. Studies performed with M1 peptides derived from GST fusion proteins, representing larger portions of the i3 C terminus, suggested the presence of a second adjacent CaM binding site. Mutagenesis studies identified two mutants that are unable to bind CaM: a point mutation, E360A, and a deletion mutant, delta232-358.
Conclusion: Calmodulin can bind to an M1 region implicated in G protein coupling. This indicates an important role for CaM in the regulation of muscarinic signal transduction.