Calmodulin binding to peptides derived from the i3 loop of muscarinic receptors.
OBJECTIVE
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.


CONCLUSIONS
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.
Department of Biopharmaceutical Sciences, School of Pharmacy, University of California, San Francisco 94143-0446, USA. julie_lucas@berlex.com
Julie L
JL
Lucas
Danxin
D
Wang
Wolfgang
W
Sadée
D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D003619:Dansyl Compounds / Q000737:chemistry; D024202:Electrophoretic Mobility Shift Assay; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction
D002147:Calmodulin; D003619:Dansyl Compounds; D010455:Peptides; D043562:Receptors, G-Protein-Coupled; D011976:Receptors, Muscarinic; D011993:Recombinant Fusion Proteins