Biochemical evidence for interaction between smoothelin and filamentous actin.
The two major isoforms of smoothelin (A and B) contain a calponin homology (CH) domain, colocalize with alpha-smooth muscle actin (alpha-SMA) in stress fibers and are only expressed in contractile smooth muscle cells (SMCs). Based on these findings, we hypothesized that smoothelins are involved in smooth muscle cell contraction, presumably via interaction with actin. The interaction between smoothelins and three different actin isoforms (alpha- and gamma-smooth muscle and alpha-skeletal actin [alpha-SKA]) was investigated using several in vitro assays. Smoothelin-B co-immunoprecipitated with alpha-smooth muscle actin from pig aorta extracts. In rat embryonic fibroblasts, transfected smoothelins-A and -B associated with stress fibers. In vitro dot blot assays, in which immobilized actin was overlaid with radio-labeled smoothelin, showed binding of smoothelin-A to actin filaments, but not to monomeric G-actin. A truncated smoothelin, containing the calponin homology domain, associated with stress fibers when transfected and bound to actin filaments in overlay, but to a lesser extent. ELISA results showed that the binding of smoothelin to actin has no significant isoform specificity. Our results indicate an interaction between smoothelin and actin filaments. Moreover, the calponin homology domain and its surrounding sequences appear to be sufficient to accomplish this interaction, although the presence of other domains is apparently necessary to facilitate and/or strengthen the binding to actin.
Niessen
Petra
P
Department of Molecular Genetics, Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, 6200 MD Maastricht, The Netherlands.
Clément
Sophie
S
Fontao
Lionel
L
Chaponnier
Christine
C
Teunissen
Birgit
B
Rensen
Sander
S
van Eys
Guillaume
G
Gabbiani
Giulio
G
D000199:Actins / Q000378:metabolism*; D000818:Animals; D001011:Aorta / Q000737:chemistry; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D008840:Microfilament Proteins; D009124:Muscle Proteins; D011233:Precipitin Tests; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats / Q000196:embryology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D022502:Stress Fibers / Q000378:metabolism; D013552:Swine; D014162:Transfection; D000096985:Calponins
D000199:Actins; D002135:Calcium-Binding Proteins; D003598:Cytoskeletal Proteins; D005982:Glutathione Transferase; D008840:Microfilament Proteins; D009124:Muscle Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D000096985:Calponins; C491051:Smtn protein, rat