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

Abstract

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.

Binding of smoothelin-like 1 to tropomyosin and calmodulin is mutually exclusive and regulated by phosphorylation

“…Conversely, SMTNL1 phosphorylation during calcium desensitization of smooth muscle could provide enhanced binding to Tpm and restrict binding of CaM in the apo-state, securing phosphorylated SMTNL1 with Tpm on the thin filament. The weak binding modes of SMTNL1 with apo-CaM might not possess enough stability to occur in vivo [12]. While the physiological significance of SMTNL1 association with the contractile filament in situ has not been investigated, we speculate that the protein may act in analogous manners to caldesmon or calponin on actin-myosin cross bridge cycling.…”

“…This agrees with our observation that the rate of PKA-dependent phosphorylation at Ser301 was not influenced when apo- or Ca-CaM was included in the kinase assay (unpublished results). We have previously demonstrated that intramolecular interactions between the C -terminal IQ motif (localization of the apo-CaM binding site, CBD2) and the intrinsically disordered region (localization of the phosphorylation site) influence apo-CaM binding [11, 12]. Furthermore, a portion of the N -terminal intrinsically disordered region forms intramolecular contacts with the globular C -terminal calponin homology (CH) domain [18].…”

“…CaM provides signaling by association with specific binding sites in target proteins, both in its calcium-saturated (Ca-CaM) or its calcium-free (apo-CaM) form [8, 9]. We have previously reported that SMTNL1 possesses two CaM-binding sites: a classic CaM-binding domain (CBD1, amino acids 310–325) in the center of the protein as well as an IQ-motif that serves as an apo-CaM-binding site (CDB2, amino acids 439–457) located in the carboxy-terminal calponin homology domain [10–12]. SMTNL1 was established as a bona fide CaM-binding protein within aortic A7r5 smooth muscle cells using the proximity ligation assay.…”

“…SMTNL1 was established as a bona fide CaM-binding protein within aortic A7r5 smooth muscle cells using the proximity ligation assay. In this regard, CBD1 is thought to provide the majority of CaM-binding in situ; however, CBD2 may contribute cooperatively to binding [12]. …”

“…Previous in vitro studies have defined SMTNL1 as a Tpm- and CaM-binding protein [10–12, 18, 19]. Moreover, the phosphorylation of SMTNL1 was identified during calcium desensitization of smooth muscle [1, 5].…”