Identification of an interaction between the m-band protein skelemin and beta-integrin subunits. Colocalization of a skelemin-like protein with beta1- and beta3-integrins in non-muscle cells.
Signaling across integrins is regulated by interaction of these receptors with cytoskeletal proteins and signaling molecules. To identify molecules interacting with the cytoplasmic domain of the beta3-integrin subunit (glycoprotein IIIa), a placental cDNA library was screened in the yeast two-hybrid system. Two identical clones coding for a 96-amino acid sequence were identified. This sequence was 100% identical to a sequence in skelemin, a protein identified previously in skeletal muscle. Skelemin is a member of a superfamily of cytoskeletal proteins that contain fibronectin-type III-like motifs and immunoglobulin C2-like motifs and that regulate the organization of myosin filaments in muscle. The amino acid residues in the isolated clones encompassed C2 motifs 4 and 5 of skelemin. A recombinant skelemin protein consisting of C2 motifs 3-7 interacted with beta1- and beta3-integrin cytoplasmic domains expressed as glutathione S-transferase (GST) fusion proteins, but not with GST-beta2-integrin cytoplasmic tail or GST alone. The skelemin-binding region was in the membrane proximal cytoplasmic domains of the integrins. Full-length skelemin interacted with integrin in intact cells as demonstrated by the colocalization of hemagglutinin-tagged skelemin in Chinese hamster ovary (CHO) cells containing alphaIIbbeta3-integrin and by the finding that microinjection of C2 motif 4 of skelemin into C2C12 mouse myoblast cells caused spread cells to round up. A skelemin-like protein was detected in CHO cells, endothelial cells, and platelets, and this protein colocalized with beta1- and beta3-integrins in CHO cells. This study suggests the presence of a skelemin-like protein in non-muscle cells and provides evidence that it may be involved in linking integrins to the cytoskeleton.
Reddy
K B
KB
Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Gascard
P
P
Price
M G
MG
Negrescu
E V
EV
Fox
J E
JE
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D016466:CHO Cells; D048429:Cell Size; D064211:Connectin; D006224:Cricetinae; D003598:Cytoskeletal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D015723:Gene Library; D006801:Humans; D019012:Integrin beta1 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D039661:Integrin beta3; D016023:Integrins / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009124:Muscle Proteins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction
D015703:Antigens, CD; D064211:Connectin; D003598:Cytoskeletal Proteins; D019012:Integrin beta1; D039661:Integrin beta3; D016023:Integrins; C577299:MYOM1 protein, human; D009124:Muscle Proteins; C577300:Myom1 protein, mouse; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D010980:Platelet Membrane Glycoproteins; D011993:Recombinant Fusion Proteins