Download Evidence for a utrophin-glycoprotein complex in cultured cell lines

398s Biochemical Society Transactions ( 1 995) 23
Evidence for a utrophin-glycoprotein complex in cultured cell
lines and a possible role in cell adhesion.
Marian JAMESI. Catherine SIMMONSI, Clare J. WISE2, Gareth
E. J O N E S ~and Glenn E. MORRIS^.
1 MRlC Biotechnology Group, N.E. Wales Institute, Deeside,
Clwyd, CH5 4BR.
2 The Randall Institute, King's College, London, WC2B 5RL.
Dystrophin, the defective protein in patients with the X-linked
muscle wasting disorders Duchenne and Becker muscular
dystrophies [ 11, is attached to an oligomeric complex which spans
the sarcolemma of skeletal muscle 121. The complex is divided
into two groups of proteins. The dystroglycan complex is
comprised ofthe 43DAG and 156DAG proteins and is found in all
tissues. The sarcoglycan complex is found only in skeletal muscle
and is comprised of three proteins, SODAG, A3b and 35DAG.
Only thc 43DAG protein binds directly to dystrophin. In addition,
two cytoplasmic proteins 59DAP (syntrophin [ 3 ] ) and A0 also
bind to dystrophin but appear not to be attached to the sarcoglycan
and dystroglyean complexes 141.
The autosomal homologue of dystrophin, utrophin, has becn
shown to be closely related to dystrophin in sequence and
structure. The homology is especially great in the dystroglycan
binding region, the cysteine-rich and C-terminal (CRCT) domains
151. Utrophin is more widely distributed than dystrophin and is
found in most tissues [6] and i n many cell lines which lack
dystrophin where it is associated with the membrane [71. I n
normal skeletal muscle, utrophin is found primarily at the
neuromuscular and myotendinous junctions. However, in
dystrophic muscle, elevated levels o l utrophin are found
throughout the sarcolemma 18.91. In md.r mouse muscle [ l o ] and
rabbit sciatic nerve [ 1 I] i t has been demonstrated that utrophin
associates with a "dystrophin-glycoprotein"4kecomplex.
The N-terminal region of dystrophin binds actin at the
intracellular surface of the sarcolemma [ 121, while the 156kd
glycoprotein binds laminin in the muscle fibre basal lamina
[ 13,141. This link is thought to be essential for the maintenance of
membrane integrity and in preventing the disruption of the
sarcolemma seen in DMD patients. Most cultured cells, when
grown on extracellular matrix (ECM) proteins, form structures
called focal contacts [15,16] where bundles of actin filaments
terminate and interact with the ECM via the plasma membrane
[ 151. Dystrophin has been identified at focal contacts in cultured
Xenopus muscle [ 171, and has the same distribution as the focal
contact proteins, vinculin and a-actinin in skeletal muscle [ 181,
but not in smooth muscle [ 191.
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Figure 1. Analysis of 43DAG and syntrophin content of cultured
cell lines. Protein concentration was measured using densitometer
readings from the photograph negatives of the Western blots. The
cell lines are rat C6 glioma (G), SWA rat Schwannoma cells (S),
rat L6 myoblast cell line (L), human skin fibroblasts (F), HeLa
cells (H), a mouse monocyte-macrophage cell line P388D1 (P)
and S p y 0 mouse myeloma cells (Sp).
In figure 1 we demonstrate for the first time that cultured cell
lines produce the dystrophin-glycoprotein complex components
43DAG and syntrophin. The presence of 43DAG and syntrophin
means there is the potential for utrophin 10 form part of an
oligomeric complex similar to the dystrophin-glycoprotein seen in
skeletal muscle [4].
In preliminary experiments using confocal microscopy we
determined that both utrophin and 43DAG were located in the
same optical "section" of human skin fibroblasts. Both utrophin
and 43DAG were seen at the level or the cell membrane, and not
at the level of the nucleus. Using methods described previously
[20], we isolated HeLa cell membranes and separated the
membrane proteins on sucrose density gradients. Utrophin.
43DAG and syntrophin from HeLa cclls, all sedimented in the
same region of the sucrose gradients as dystrophin and 43DAG
from rabbit skeletal muscle, suggesting the presence of a utrophinglycoprotein complex.
In astrocytes, it has been demonstrated that utrophin is associated
with the extracellular matrix protein, laminin, although the
mechanism which attaches these two proteins across the
membrane has not been determined [21]. Our results suggest that
a dystroglyean-like complex forms the link across the membrane
between utrophin and the basal lamina and that this association
may play a role in cell adhesion. As shown in figure I. all cell
lines studied produce substantial amounts of 43DAG. but Sp2/0
cells produced little syntrophin compared with the othcr cell lines.
Furthermore, Sp2/0 cells do not produce utrophin [71 and they do
not attach to the substatum when grown in culture. This may be
due to lack of an utrophin-glycoprotein complex. HeLa cells,
which can be grown in suspension, continue to produce utrophin
but, unlike Sp2/0 myeloma cells, they retain the ability to attach to
the substratum.
This work was supported by the Muscular Dystrophy Group of
Great Britain and Northern Ireland, the Medical Rcsaereh Council
and the Royal Society. We thank Drs R. Sealock and S.C.
Froehner (Universityof North Carolina) for supplying mAb
I35 1SYN.
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