In situ Analysis of Smoothelin‐like 1 and Calmodulin Interactions in Smooth Muscle Cells by Proximity Ligation

The smoothelin‐like 1 (SMTNL1) protein is the newest member of the smoothelin family of muscle proteins. Two calmodulin (CaM)‐binding domains (CBD1 for Ca‐CaM; CBD2 for apo‐CaM) have been described for the SMTNL1 protein using in vitro assays. We now demonstrate in situ associations of SMTNL1 and CaM in A7r5 smooth muscle cells using the proximity ligation assay (PLA). We quantified CaM‐SMTNL1 proximity events accurately after taking into account variations in protein expression levels. The refined method allows quantification of in situ proximity after transient transfection with an associated error of <10%. The proximity of SMTNL1 and CaM in A7r5 cells could be reduced by scrambling the amino acid sequence and mutation of large hydrophobic amino acids of CBD1. The truncation of CBD2 did not influence SMTNL1 proximity to CaM. Ultimately, we conclude that SMTNL1 forms complex interactions with CaM in smooth muscle cells, with a role for CBD1 and possibly the intrinsically disordered region. J. Cell. Biochem. 116: 2667–2675, 2015. © 2015 Wiley Periodicals, Inc.