Cardiac muscle cell cytoskeletal protein 4.1: Analysis of transcripts and subcellular location?relevance to membrane integrity, microstructure, and possible role in heart failure

Abstract

The spectrin-based cytoskeleton assembly has emerged as a major player in heart functioning; however, cardiac protein 4.1, a key constituent, is uncharacterized. Protein 4.1 evolved to protect cell membranes against mechanical stresses and to organize membrane microstructure. 4.1 Proteins are multifunctional and, among other activities, link integral/signaling proteins on the plasma and internal membranes with the spectrin-based cytoskeleton. Four genes, EPB41, EPB41L1, EPB41L2, and EPB41L3 encode proteins 4.1R, 4.1N, 4.1G, and 4.1B, respectively. All are extensively spliced. Different isoforms are expressed according to tissue and developmental state, individual function being controlled through inclusion/exclusion of interactive domains. We have defined mouse and human cardiac 4.1 transcripts; other than 4. 1B in humans, all genes show activity. Cardiac transcripts constitutively include conserved FERM and C-terminal domains; both interact with membrane-bound signaling/transport/cell adhesion molecules. Variable splicing within and adjacent to the central spectrin/actin-binding domain enables regulation of cytoskeleton-binding activity. A novel heart-specific exon occurs in human 4.1G, but not in mouse. Immunofluorescence reveals 4.1 staining within mouse cardiomyocytes; thus, both at the plasma membrane and, interdigitated with sarcomeric myosin, across myofibrils in regions close to the sarcoplasmic reticulum. These are all regions to which spectrin locates. 4.1R in human heart shows similar distribution; however, there is limited plasma membrane staining. We conclude that cardiac 4.1s are highly regulated in their ability to crosslink plasma/integral cell membranes with the spectrin-actin cytoskeleton. We speculate that over the repetitive cycles of heart muscle contraction and relaxation, 4.1s are likely to locate, support, and coordinate functioning of key membrane-bound macromolecular assemblies.

Lack of Protein 4.1G Causes Altered Expression and Localization of the Cell Adhesion Molecule Nectin-Like 4 in Testis and Can Cause Male Infertility

“…These data confirm that the gene trap cassette has resulted in a functional knockout of 4.1G. The detection of multiple bands in testis and brain indicates the presence of differentially spliced isoforms as described previously (43). No 4.1G protein was detected in liver, prostate, small intestine, kidney, and skeletal muscle under the same experimental conditions.…”

Correlation between protein 4.1R and the progression of heart failure in vivo

“…On the contrary, the highest expression of 4.1R was observed in the HF group increasingly along the zones of connective tissue as well as in areas of myocardial necrosis. Meanwhile, the mRNA expression levels of 4.1R were upregulated in the HF group, which is in accordance with the results of a previous study (Taylor-Harris et al, 2005). Therefore, we speculate that the upregulation of 4.1R both at the transcriptional and protein level might contribute to the progression of HF.…”

Comprehensive characterization of expression patterns of protein 4.1 family members in mouse adrenal gland: implications for functions

“…While 4.1R does not contain U3 region, the U3 region of 4.1G, 4.1B and 4.1N are encoded by exon 17D, exon 17C and exon 17 D-E-F, respectively. Similar phenomena were also observed in kidney and cardiac 4.1 proteins (Ramez et al 2003;Taylor-Harris et al 2005). These Wndings suggest Immunolocalization of 4.1B in mouse adrenal gland.…”

“…Surprisingly, 4.1G does not contain either exon 16 or 17 and thus cannot bind to either spectrin or actin. It is important to note that of all 4.1G isoforms characterized to date (Ramez et al 2003;Taylor-Harris et al 2005), the adrenal gland 4.1G is the only isoform that does not contain any part of the SAB domain. Even though 4.1G cannot link transmembrane proteins to the cytoskeleton, it may still participate in membrane protein targeting because the presence of both FERM and CTD would allow 4.1G to cross-link proteins (e.g.…”