Differential expression and subcellular distribution of dystrophin Dp71 isoforms during differentiation process.
Dp71 is the major product of the Duchenne muscular dystrophy gene in the brain. In order to study the function of Dp71 in the nervous system we examined the expression of Dp71 isoforms in PC12 rat pheochromocytoma cell line, a well-established system to study neuronal differentiation. We show by reverse transcriptase-polymerase chain reaction and Western blot assays that PC12 cells express two Dp71 isoforms. One isoform lacks exon 71 and the other isoform lacks exons 71 and 78 (Dp71d and Dp71f isoforms respectively). Nerve growth factor-induced neuronal differentiation of PC12 cells results in differential regulation of the expression and subcellular localization of Dp71 isoforms: a) the amount of Dp71f protein increases nine-fold in total extracts while Dp71d increases up to seven-fold in nuclear extracts; b) Dp71f relocates from the cytoplasm to neuritic processes, being prominent at varicosities and the growth cone; c) Dp71d relocates almost entirely to the nucleus and is detected to a lower extent in the cytoplasm and neuritic processes. Dp71f co-localizes with beta-dystroglycan and synaptophysin while Dp71d co-localizes with beta-dystroglycan in the nucleus. Dp71d accumulates at cell-cell contacts where Dp71f is absent. These results suggest that Dp71d and Dp71f associate with different subcellular complexes and therefore may have distinct functions in PC12 cells.
Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del IPN, Avenida Instituto Politécnico Nacional 2508, Apartado Postal 14-740, C.P. 07000, Mexico.
F G
FG
Marquez
B
B
Cisneros
F
F
Garcia
V
V
Ceja
F
F
Velázquez
F
F
Depardón
L
L
Cervantes
A
A
Rendón
D
D
Mornet
H
H
Rosas-vargas
M
M
Mustre
C
C
Montañez
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology*; D003598:Cytoskeletal Proteins / Q000378:metabolism; D049030:Dystroglycans; D016189:Dystrophin / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique / Q000379:methods; D015870:Gene Expression; D008562:Membrane Glycoproteins / Q000378:metabolism; D020932:Nerve Growth Factor / Q000502:physiology; D016716:PC12 Cells / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013347:Subcellular Fractions / Q000378:metabolism; D016708:Synaptophysin / Q000378:metabolism; D013997:Time Factors
D003598:Cytoskeletal Proteins; D016189:Dystrophin; D008562:Membrane Glycoproteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D016708:Synaptophysin; C085339:apo-dystrophin 1; D049030:Dystroglycans; D020932:Nerve Growth Factor