Probing Calmodulin Protein–Protein Interactions Using High-Content Protein Arrays

Abstract

The calcium ion (Ca²⁺) is a ubiquitous second messenger that is crucial for the regulation of a wide variety of cellular processes. The diverse transient signals transduced by Ca²⁺ are mediated by intracellular ­Ca²⁺-binding proteins. Calcium ions shuttle into and out of the cytosol, transported across membranes by channels, exchangers, and pumps that regulate flux across the ER, mitochondrial and plasma membranes. Calcium regulates both rapid events, such as cytoskeleton remodelling or release of vesicle contents, and slower ones, such as transcriptional changes. Moreover, sustained cytosolic calcium elevations can lead to unwanted cellular activation or apoptosis. Calmodulin represents the most significant of the Ca²⁺-binding proteins and is an essential regulator of intracellular processes in response to extracellular stimuli mediated by a rise in Ca²⁺ ion concentration. To profile novel protein–protein interactions that calmodulin participates in, we probed a high-content recombinant human protein array with fluorophore-labelled calmodulin in the presence of Ca²⁺. This protein array contains 37,200 redundant proteins, incorporating over 10,000 unique human proteins expressed from a human brain cDNA library. We describe the identification of a high affinity interaction between calmodulin and the single-pass transmembrane proteins STIM1 and STIM2 that localise to the ER. Translocation of STIM1 and STIM2 from the endoplasmic reticulum to the plasma membrane is a key step in store operated calcium entry in the cell.