Expression of distinct classes of titin isoforms in striated and smooth muscles by alternative splicing, and their conserved interaction with filamins.
While the role of titin as a sarcomeric protein is well established, its potential functional role(s) in smooth muscles and non-muscle tissues are controversial. We used a titin exon array to search for which part(s) of the human titin transcriptional unit encompassing 363 exons is(are) expressed in non-striated muscle tissues. Expression profiling of adult smooth muscle tissues (aorta, bladder, carotid, stomach) identified alternatively spliced titin isoforms, encompassing 80 to about 100 exons. These exons code for parts of the titin Z-disk, I-band and A-band regions, allowing the truncated smooth muscle titin isoform to link Z-disks/dense bodies together with thick filaments. Consistent with the array data, Western blot studies detected the expression of approximately 1 MDa smooth muscle titin in adult smooth muscles, reacting with selected Z-disc, I-band, and A-band titin antibodies. Immunofluorescence with these antibodies located smooth muscle titin in the cytoplasm of cultured human aortic smooth muscle cells and in the tunica media of intact adult bovine aorta. Real time PCR studies suggested that smooth muscle titins are expressed from a promoter located 35 kb or more upstream of the transcription initiation site used for striated muscle titin, driving expression of a bi-cistronic mRNA, coding 5' for the anonymous gene FL39502, followed 3' by titin, respectively. Our work showed that smooth muscle and striated muscle titins share in their conserved amino-terminal regions binding sites for alpha-actinin and filamins: Yeast two-hybrid screens using Z2-Zis1 titin baits identified prey clones coding for alpha-actinin-1 and filamin-A from smooth muscle, and alpha-actinin-2/3, filamin-C, and nebulin from skeletal muscle cDNA libraries, respectively. This suggests that the titin Z2-Zis1 domain can link filamins and alpha-actinin together in the periphery of the Z-line/dense bodies in a fashion that is conserved in smooth and striated muscles.
Labeit
Siegfried
S
Institute for Anaesthesiology and Intensive Care, University Hospital Mannheim, Germany. labeit@embl.de
Lahmers
Sunshine
S
Burkart
Christoph
C
Fong
Chi
C
McNabb
Mark
M
Witt
Stephanie
S
Witt
Christian
C
Labeit
Dietmar
D
Granzier
Henk
H
D000185:Actinin / Q000378:metabolism; D000328:Adult; D017398:Alternative Splicing / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000166:cytology; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D064211:Connectin; D003285:Contractile Proteins / Q000378:metabolism*; D005091:Exons / Q000235:genetics; D064448:Filamins; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism*; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D011494:Protein Kinases / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D013552:Swine; D014158:Transcription, Genetic
D064211:Connectin; D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins; D009124:Muscle Proteins; D020033:Protein Isoforms; C577285:TTN protein, human; D000185:Actinin; D011494:Protein Kinases