Characterization and expression of a heart-selective alternatively spliced variant of alpha II-spectrin, cardi+, during development in the rat.
Spectrin is a large, flexible protein that stabilizes membranes and organizes proteins and lipids into microdomains in intracellular organelles and at the plasma membrane. Alternative splicing occurs in spectrins, but it is not yet clear if these small variations in structure alter spectrin's functions. Three alternative splice sites have been identified previously for alpha II-spectrin. Here we describe a new alternative splice site, a 21-amino acid sequence in the 21st spectrin repeat that is only expressed in significant amounts in cardiac muscle (GenBank GQ502182). The insert, which we term alpha II-cardi+, results in an insertion within the high affinity nucleation site for binding of alpha-spectrins to beta-spectrins. To assess the developmental regulation of the alpha II-cardi+ isoform, we used qRT-PCR and quantitative immunoblotting methods to measure the levels of this form and the alpha II-cardi- form in the cardiac muscles of rats, from embryonic day 16 (E16) through adulthood. The alpha II-cardi+ isoform constituted approximately 26% of the total alpha II-spectrin in E16 hearts but decreased to approximately 6% of the total after 3 weeks of age. We used long-range RT-PCR and Southern blot hybridization to examine possible linkage of the alpha II-cardi+ alternatively spliced sequence with alternatively spliced sequences of alpha II-spectrin that had been previously reported. We identified two new isoforms of alpha II-spectrin containing the cardi+ insert. These were named alpha II Sigma 9 and alpha II Sigma 10 in accordance with the spectrin naming conventions. In vitro studies of recombinant alpha II-spectrin polypeptides representing the two splice variants of alpha II-spectrin, alpha II-cardi+ and alpha II-cardi-, revealed that the alpha II-cardi+ subunit has lower affinity for the complementary site in repeats 1-4 of betaII-spectrin, with a K(D) value of approximately 1 nM, as measured by surface plasmon resonance (SPR). In addition, the alpha II-cardi+ form showed 1.8-fold lower levels of binding to its site on beta II-spectrin than the alpha II-cardi- form, both by SPR and blot overlay. This suggests that the 21-amino acid insert prevented some of the alpha II-cardi+ form from interacting with beta II-spectrin. Fusion proteins expressing the alpha II-cardi+ sequence within the two terminal spectrin repeats of alpha II-spectrin were insoluble in solution and aggregated in neonatal myocytes, consistent with the possibility that this insert removes a significant portion of the protein from the population that can bind beta subunits. Neonatal rat cardiomyocytes infected with adenovirus encoding GFP-fusion proteins of repeats 18-21 of alpha II-spectrin with the cardi+ insert formed many new processes. These processes were only rarely seen in myocytes expressing the fusion protein lacking the insert or in controls expressing only GFP. Our results suggest that the embryonic mammalian heart expresses a significant amount of alpha II-spectrin with a reduced avidity for beta-spectrin and the ability to promote myocyte growth.
Zhang
Yinghua
Y
Department of Physiology, University of Maryland, School of Medicine, 655 W Baltimore Street, Baltimore, MD 21201, USA.
Resneck
Wendy G
WG
Lee
Pervis C
PC
Randall
William R
WR
Bloch
Robert J
RJ
Ursitti
Jeanine A
JA
D017398:Alternative Splicing; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D005786:Gene Expression Regulation; D006321:Heart / Q000196:embryology*; D007700:Kinetics; D008024:Ligands; D009206:Myocardium / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology; D020033:Protein Isoforms; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013049:Spectrin / Q000096:biosynthesis* / Q000378:metabolism*; D020349:Surface Plasmon Resonance
D008024:Ligands; D020033:Protein Isoforms; D013049:Spectrin