Different splice variants of filamin-B affect myogenesis, subcellular distribution, and determine binding to integrin [beta] subunits.
Integrins connect the extracellular matrix with the cell interior, and transduce signals through interactions of their cytoplasmic tails with cytoskeletal and signaling proteins. Using the yeast two-hybrid system, we isolated a novel splice variant (filamin-Bvar-1) of the filamentous actin cross-linking protein, filamin-B, that interacts with the cytoplasmic domain of the integrin beta1A and beta1D subunits. RT-PCR analysis showed weak, but wide, expression of filamin-Bvar-1 and a similar splice variant of filamin-A (filamin-Avar-1) in human tissues. Furthermore, alternative splice variants of filamin-B and filamin-C, from which the flexible hinge-1 region is deleted (DeltaH1), were induced during in vitro differentiation of C2C12 mouse myoblasts. We show that both filamin-Avar-1 and filamin-Bvar-1 bind more strongly than their wild-type isoforms to different integrin beta subunits. The mere presence of the high-affinity binding site for beta1A is not sufficient for targeting the filamin-Bvar-1 construct to focal contacts. Interestingly, the simultaneous deletion of the H1 region is required for the localization of filamin-B at the tips of actin stress fibers. When expressed in C2C12 cells, filamin-Bvar-1(DeltaH1) accelerates their differentiation into myotubes. Furthermore, filamin-B variants lacking the H1 region induce the formation of thinner myotubes than those in cells containing variants with this region. These findings suggest that specific combinations of filamin mRNA splicing events modulate the organization of the actin cytoskeleton and the binding affinity for integrins.
van der Flier
Arjan
A
Netherlands Cancer Institute, Division of Cell Biology, 1066 CX Amsterdams, Netherlands.
Kuikman
Ingrid
I
Kramer
Duco
D
Geerts
Dirk
D
Kreft
Maaike
M
Takafuta
Toshiro
T
Shapiro
Sandor S
SS
Sonnenberg
Arnoud
A
D017398:Alternative Splicing / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002454:Cell Differentiation; D002460:Cell Line; D003285:Contractile Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006224:Cricetinae; D064448:Filamins; D015870:Gene Expression; D006801:Humans; D019012:Integrin beta1 / Q000737:chemistry / Q000378:metabolism*; D008840:Microfilament Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D024510:Muscle Development / Q000235:genetics*; D009132:Muscles / Q000166:cytology; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D020798:Two-Hybrid System Techniques
D003285:Contractile Proteins; D064448:Filamins; D019012:Integrin beta1; D008840:Microfilament Proteins; D020033:Protein Isoforms; D021122:Protein Subunits; D012333:RNA, Messenger