Protein biochemistry of Popdc1 and -2 in heart and skeletal muscle

Abstract

Popeye domain containing (Popdc) proteins are a novel class of cAMP-binding proteins, which are highly expressed in heart and skeletal muscle. Genetic inactivation of Popdc1 and -2 genes in mice revealed a role in regulating heart rhythm in response to β-adrenergic signalling. Furthermore, it was shown that Popdc1 is involved in muscle regeneration. This thesis aims at shedding light on the function of Popdc1 and -2 in the heart and in skeletal muscle tissue on the protein level in order to get a deeper insight into the molecular mechanisms underlying the observed phenotypes in striated muscle tissue of model organisms. The two-pore domain potassium channel TREK-1 and the caveolae-associated protein Caveolin-3 are the first identified cardiac interaction partners of Popdc proteins. In this thesis, domains have been mapped that are involved in protein-protein interaction with TREK-1 and Caveolin-3. Towards the goal to identify additional interaction partners for Popdc proteins in striated muscle tissue a co-immunoprecipitation (CoIP) assay followed by protein mass spectrometry was devised. Among the list of interacting proteins were the adaptor proteins Ankyrin-B, and -G, and the Na+/Ca2+ exchanger 1 (NCX-1). Their interaction with Popdc1 and/or -2 was confirmed by co-transfection analysis in Cos-7 cells and CoIP experiments using heart tissue. Furthermore, physical interaction with the Na+ channel α-subunit SCN5A, as well as with Dysferlin and Dystrophin could be established. It has been known for about a decade that Popdc proteins are able to form homodimers. Here, interaction of different members of the Popdc protein family with each other and the formation of heteromeric complexes are reported. A familial case of AV-block and muscular dystrophy has recently been associated with a point mutation in POPDC1. This point mutation was introduced into the human POPDC1 cDNA. Since the mutation was localised to the cyclic nucleotide binding-site, cAMP binding was tested by affinity pull-down. A significant reduction in cAMP-binding capacity was observed. Furthermore, the role of Popdc proteins in skeletal muscle regeneration was studied using a Cardiotoxin-induced injury mouse model. Differences in cross-sectional area of regenerating fibres of wildtype and Popdc2 null mutant muscles point to a different time course of regeneration after loss of Popdc2. In addition, subcellular localisation of Popdc1 and Popdc2 proteins in myoblasts was examined. Although being established as integral membrane proteins with three hydrophobic helices, the Popdc1 and -2 proteins were found to be present in the nucleus of primary myoblasts and also in established cell lines. These data suggest that Popdc proteins, in addition to their membrane function, also have a role as nuclear proteins in different cell types. A region in the Popdc1 protein, which is involved in nuclear localisation, was determined by deletion analysis. Taken together, experiments performed for this thesis have expanded the understanding of the protein interaction network Popdc proteins are acting in. The identification of several novel protein interaction partners and the definition of domains involved in some of these interactions may help to explain the observed striated muscle pathologies caused by mutations of Popdc genes in model organisms and in patients.