The clustering of acetylcholine receptors (AChRs) in the post-synaptic membrane of skeletal muscle is an early developmental event in the formation of the neuromuscular junction. Several studies show that laminin, as well as neural agrin, can induce AChR clustering in C2C12 myofibers. We recently showed that specific isoforms of the alpha7beta1 integrin (a receptor normally found at neuromuscular junctions) colocalize and physically interact with AChR clusters in a laminin-dependent fashion. In contrast, induction with agrin alone fails to promote localization of the integrin with AChR clusters. Together both agrin and laminin enhance the interaction of the integrin with AChRs and their aggregation into clusters. To further understand this mechanism we investigated cluster formation and the association of the alpha7beta1 integrin and AChR over time following induction with laminin and/or agrin. Our results show that the alpha7beta1 integrin associates with AChRs early during the formation of the post-synaptic membrane and that laminin modulates this recruitment. Laminin induces a rapid stable association of the integrin and AChRs and this association is independent of clustering. In addition to laminin-1, merosin (laminin-2/4) is present both before and after formation of neuromuscular junctions and also promotes AChR clustering and colocalization with the integrin as well as synergism with agrin. Using site directed mutagenesis we demonstrate that a tyrosine residue in the cytoplasmic domain of both (α)7A and (α)7B chains regulates the localization of the integrin with AChR clusters. We also provide evidence that laminin, through its association with the alpha7beta1 integrin, reduces by 20-fold the concentration of agrin required to promote AChR clustering and accelerates the formation of clusters. Thus laminin, agrin and the alpha7beta1 integrin act in a concerted manner early in the development of the post-synaptic membrane, with laminin priming newly formed myofibers to rapidly and vigorously respond to low concentrations of neural agrin produced by innervating motor neurons.

REFERENCES

REFERENCES
Anderson
M. J.
,
Cohen
M. W.
(
1977
).
Nerve-induced and spontaneous redistribution of acetylcholine receptor clusters on cultured muscle cells.
J. Physiol
286
,
757
773
Bayne
E. K.
,
Anderson
M. J.
,
Fambrough
D. M.
(
1984
).
Extracellular matrix organization in developing muscle: correlation with acetylcholine receptor aggregates.
J. Cell Biol
99
,
1486
1501
Bowe
M. A.
,
Fallon
J. R.
(
1995
).
The role of agrin in synapse formation.
Annu. Rev. Neurosci
18
,
443
462
Brown
J. C.
,
Wiedemann
H.
,
Timpl
R.
(
1994
).
Protein binding and cell adhesion properties of two laminin isoforms (AmB1eB2e, AmB1sB2e) from human placenta.
J Cell Sci
107
,
329
338
Burkin
D. J.
,
Gu
M.
,
Hodges
B. L.
,
Campanelli
J. T.
,
Kaufman
S. J.
(
1998
).
A functional role for specific spliced variants of the alpha7 beta1 integrin in acetylcholine receptor clustering.
J. Cell Biol
143
,
1067
1075
Burkin
D. J.
,
Kaufman
S. J.
(
1999
).
The alpha 7 beta 1 integrin in muscle development and disease.
Cell Tissue Res
296
,
183
190
Campanelli
J. T.
,
Roberds
S. L.
,
Campbell
K. P.
,
Scheller
R. H.
(
1994
).
A role for dystrophin-associated glycoproteins and utrophin in agrin-induced AChR clustering.
Cell
77
,
663
674
Collo
G.
,
Starr
L.
,
Quaranta
V.
(
1993
).
A new isoform of the laminin receptor7 1 integrin is developmentally regulated in skeletal muscle.
J. Biol. Chem
268
,
19019
19024
Colognato
H.
,
Yurchenco
P.
(
1999
).
Laminin polymerization is required for laminin-induced acetylcholine receptor clustering.
Mol. Biol. Cell
10
,
68
–.
Colognato
H.
,
Winkelmann
D. A.
,
Yurchenco
P. D.
(
1999
).
Laminin polymerization induces a receptor-cytoskeleton network.
J. Cell Biol
145
,
619
631
DeChiara
T. M.
,
Bowen
D. C.
,
Valenzuela
D. M.
,
Simmons
M. V.
,
Poueymirou
W. T.
,
Thomas
S.
,
Kinetz
E.
,
Compton
D. L.
,
Rojas
E.
,
Park
J. S.
, et al. 
(
1996
).
The receptor tyrosine kinase MuSK is required for neuromuscular junction formation in vivo.
Cell
85
,
501
512
Ehrig
K.
,
Leivo
I.
,
Agraves
W. S.
,
Rhuoslahti
E.
,
Engvall
E.
(
1990
).
Merosin, a tissue-specific basement membrane protein, is a laminin-like protein.
Proc. Nat. Acad. Sci. USA
87
,
3264
3268
Fertuck
H. C.
,
Salpeter
M. M.
(
1976
).
Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscope autoradiography after 125I-bungarotoxin binding at mouse neuromuscular junctions.
J. Cell Biol
69
,
144
148
Frank
E.
,
Fischbach
G. D.
(
1979
).
Early events in neuromuscular junction formation in vitro: Induction of acetylcholine receptor clusters in the postsynaptic membrane and morphology of newly formed synapses.
J. Cell Biol
83
,
143
158
Fuhrer
C.
,
Gautam
M.
,
Sugiyama
J. E.
,
Hall
Z. W.
(
1999
).
Roles of rapsyn and agrin in interaction of postsynaptic proteins with acetylcholine receptors.
J. Neurosci
19
,
6405
6416
Gautam
M.
,
Noakes
P. G.
,
Moscoso
L.
,
Rupp
F.
,
Scheller
R. H.
,
Merlie
J. P.
,
Sanes
J. R.
(
1996
).
Defective neuromuscular synaptogenesis in agrin-deficient mutant mice.
Cell
85
,
525
536
Glass
D. J.
,
Bowen
D. C.
,
Stitt
T. N.
,
Radziejewski
C.
,
Bruno
J.
,
Ryan
T. E.
,
Gies
D. R.
,
Shah
S.
,
Mattsson
K.
,
Burden
S. J.
, et al. 
(
1996
).
Agrin acts via a MuSK receptor complex.
Cell
85
,
513
524
Hayashi
Y. K.
,
Chou
F. L.
,
Engvall
E.
,
Ogawa
M.
,
Matsuda
C.
,
Hirabayashi
S.
,
Yokochi
K.
,
Ziober
B. L.
,
Kramer
R. H.
,
Kaufman
S. J.
, et al. 
(
1998
).
Mutations in the integrin alpha7 gene cause congenital myopathy.
Nature Genet
19
,
94
97
Jaynes
J. B.
,
Chamberlain
J. S.
,
Buskin
J. N.
,
Johnson
J. E.
,
Hauschka
S. D.
(
1986
).
Transcriptional regulation of the muscle creatine kinase gene and related expression in transfected mouse myoblasts.
Mol. Cell. Biol
6
,
2855
2864
Kong
J.
,
Anderson
J. E.
(
1999
).
Dystrophin is required for organizing large acetylcholine receptor aggregates.
Brain Res
839
,
298
304
Kwon
M. S.
,
Park
C. S.
,
Choi
K.-R.
,
Park
C.-S.
,
Ahnn
J.
,
Kim
J. I.
,
Eom
S. H.
,
Kaufman
S. J.
,
Song
W. K.
(
2000
).
Calreticulum couplescalcium release and calcium influx in integrin-mediated calcium signaling.
Mol. Cell Biol
11
,
1433
1443
Martin
P. T.
,
Kaufman
S. J.
,
Kramer
R. H.
,
Sanes
J. R.
(
1996
).
Synaptic integrins in developing, adult, and mutant muscle: selective association of1, and B integrins with the neuromuscular junction.
Dev. Biol
174
,
125
139
Martin
P. T.
,
Sanes
J. R.
(
1997
).
Integrins mediate adhesion to agrin and modulate agrin signaling.
Development
124
,
3909
3917
Mayer
U.
,
Sacher
G.
,
Faessler
R.
,
Boornemann
A.
,
Echtemeyer
F.
,
von der Mark
H.
,
Miosge
N.
,
Poeschl
E.
,
vonder Mark
K.
(
1997
).
Absence of integrin alpha 7 causes a novel muscular dystrophy.
Nature Genet
17
,
318
323
Megeath
L. J.
,
Fallon
J. R.
(
1998
).
Intracellular calcium regulates agrin-induced acetylcholine receptor clustering.
J. Neurosci
18
,
672
678
Montanaro
F.
,
Gee
S. H.
,
Jacobson
C.
,
Lindenbaum
M. H.
,
Froehner
S. C.
,
Carbonetto
S.
(
1998
).
Laminin and alpha-dystroglycan mediated acetylcholine receptor aggregation via a MuSK-independent pathway.
J. Neurosci
18
,
1250
1260
Olek
A. J.
,
Krikorian
J. G.
,
Daniels
M. P.
(
1986
).
Early stages in the formation and stabilization of acetylcholine receptos aggregates on cultures myotubes: sensitivity to temperature and azide.
Dev. Biol
117
,
24
34
Patton
B. L.
,
Miner
J. H.
,
Chiu
A. Y.
,
Sanes
J. R.
(
1997
).
Distribution and function of laminins in the neuromuscular system of developing, adult, and mutant mice.
J. Cell Biol
139
,
1507
1521
Ruegg
M. A.
,
Bixby
J. L.
(
1998
).
Agrin orchestrates synaptic differentiation at the vertebrate neuromuscular junction.
Trends Neurosci
21
,
22
27
Sanes
J. R.
,
Lichtman
J. W.
(
1999
).
Development of the vertebrate neuromuscular junction.
Annu. Rev. Neurosci
22
,
389
442
Schuler
F.
,
Sorokin
L. M.
(
1995
).
Expression of laminin isoforms in mouse myogenic cells in vitro and in vivo.
J. Cell Sci
108
,
3795
3805
Song
W. K.
,
Wang
W.
,
Foster
R. F.
,
Bielser
D. A.
,
Kaufman
S. J.
(
1992
).
H36-7 is a novel integrin chain that is developmentally regulated during skeletal myogenesis.
J. Cell Biol
117
,
643
657
Song
W. K.
,
Wang
W.
,
Sato
H.
,
Bielser
D. A.
,
Kaufman
S. J.
(
1993
).
Expression ofintegrin cytoplasmic domains during skeletal muscle development: alternative forms, conformational change, and homologies with serine/threonine kinases and tyrosine phosphatases.
J. Cell Sci
106
,
1139
1152
Sugiyama
J.
,
Bowen
D. C.
,
Hall
Z. W.
(
1994
).
Dystroglycan binds nerve and muscle agrin.
Neuron
13
,
103
115
Sugiyama
J. E.
,
Glass
D. J.
,
Yancopoulos
G. D.
,
Hall
Z. W.
(
1997
).
Laminin-induced acetylcholine receptor clustering: An alternative pathway.
J. Cell Biol
139
,
181
191
Vachon
P. H.
,
Xu
H.
,
Liu
L.
,
Loechel
F.
,
Hayashi
Y.
,
Arahata
K.
,
Reed
J. C.
,
Wewer
U. M.
,
Engval
E.
(
1997
).
Integrins (in muscle function and survival.
J. Clin. Invest
100
,
1870
1881
Valenzuela
D. M.
,
Stitt
T. N.
,
DiStefano
P. S.
,
Rojas
E.
,
Mattsson
K.
,
Compton
D. L.
,
Nunez.
L.
,
Park
J. S.
,
Stark
J. L.
,
Gies
D. R.
, et al. 
(
1995
).
Receptor tyrosine kinase specific for the skeletal muscle lineage: expression in embryonic muscle, at the neuromuscular junction, and after injury.
Neuron
15
,
573
584
Vogel
Z.
,
Christian
C. N.
,
Vigny
M.
,
Bauer
H. C.
,
Sonderegger
P.
,
Daniels
M. P.
(
1983
).
Laminin induces acetylcholine receptor aggregation on cultured myotubes and enhances the receptor aggregation activity of a neuronal factor.
J Neurosci
3
,
1058
1068
Wallace
B. G.
(
1988
).
Regulation of agrin-induced acetylcholine receptor aggregation by Ca++and phorbol ester.
J. Cell Biol
107
,
267
278
Ziober
B. L.
,
Vu
M. P.
,
Waleh
N.
,
Crawford
J.
,
Lin
C. S.
,
Kramer
R. H.
(
1993
).
Alternative extracellular and cytoplasmic domains of the integrin7 subunit are differentially expressed during development.
J. Biol. Chem
268
,
26773
26783
This content is only available via PDF.