Analysis of the structure, function and protein-protein interactions of the popeye domain containing proteins

Abstract

The Popeye domain containing (POPDC) proteins are a family of three transmembrane proteins. Dysfunction of these proteins is associated with muscular dystrophy and cardiac dysrhythmias. The Popeye domain, unique to this family, is known to bind to cAMP. The structure of the Popeye domain and the mechanism of cAMP binding has not been directly determined. In addition, there is a lack of pharmacological tools to target the cAMP binding site of the POPDC proteins. Attempts were made to produce a protein construct based on the Popeye domain for structural characterisation and the design of ligand binding assays. A novel construct was produced although, while preliminary structural analysis was initiated, it did not possess robust cAMP binding properties. This meant that the construct was unsuitable for further study. Attempts to develop cell-based assays to detect cAMP and other compound binding to the Popeye domain were also unsuccessful. An interaction between POPDC1 and POPDC2 is thought to occur. Changes in normal subcellular expression of both POPDC1 and POPDC2 within tissue have been found in the presence of mutations in either of these proteins. Here, analysis of skeletal muscle biopsies from patients found this to be the case in two recently identified mutations in POPDC1. Furthermore, it was discovered that plasma membrane localisation of POPDC1 and POPDC2 in HEK293 cells is dependent on the co-expression of both proteins. This finding was exploited to design an assay in HEK293 cells modelling the effect of known mutations on POPDC expression in tissue. The nature of POPDC1-POPDC2 interactions was investigated and a novel interface was proposed to form through a helix-helix interaction within the Popeye domain of each protein. It was found that residues in this putative interface are required for proper POPDC1 and POPDC2 protein subcellular expression.