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Agrin is an extracellular synaptic protein that organizes the postsynaptic apparatus, including acetylcholine receptors (AChRs), of the neuromuscular junction. The COOH-terminal portion of agrin has full AChR-aggregating activity in culture, and includes three globular domains, G1, G2, and G3. Portions of the agrin protein containing these domains bind to different cell surface proteins of muscle cells, including alpha-dystroglycan (G1-G2) and heparan sulfate proteoglycans (G2), whereas the G3 domain is sufficient to aggregate AChRs. We sought to determine whether the G1 and G2 domains of agrin potentiate agrin activity in vivo, as they do in culture. Fragments from the COOH-terminal of a neuronal agrin isoform (4,8) containing G3, both G2 and G3, or all three G domains were overexpressed in Xenopus embryos during neuromuscular synapse formation in myotomal muscles. RNA encoding these fragments of rat agrin was injected into one-cell embryos. All three fragments increased the ectopic aggregation of AChRs in noninnervated regions near the center of myotomes. Surprisingly, ectopic aggregation was more pronounced after overexpression of the smallest fragment, which lacks the heparin- and alpha-dystroglycan-binding domains. Synaptic AChR aggregation was decreased in embryos overexpressing the fragments, suggesting a competition between endogenous agrin secreted by nerve terminals and exogenous agrin fragments secreted by muscle cells. These results suggest that binding of the larger agrin fragments to alpha-dystroglycan and/or heparan sulfate proteoglycans may sequester the fragments and inhibit their activity in embryonic muscle. These intermolecular interactions may regulate agrin activity and differentiation of the neuromuscular junction in vivo.
Figure 1 Diagram of agrin fragments overexpressed in the
embryo. RNAs encoding COOH-terminal fragments of rat
agrin were synthesized from cDNA templates encoding one
(T3), two (T2), or three (T1) G-domains (see Materials and
Methods). All fragments used were from a neuronal agrin
isoform containing both y4 and z8 inserts (Ferns et al.,
1993). Epitope tags encoding both FLAG and polyhistidine
sequences (âTâ) were present on the N-terminus of the
fragments, along with a signal sequence (âSâ) to ensure
secretion of the proteins. Predicted molecular mass of the
fragments shown (amino acids only) is about 104.8, 61.3,
and 26.4 kDa for T1-âT3, compared to 101.9 kDa for
âC-agrinâ (Ferns et al., 1993), which is similar to T1 except
that it lacks epitope tags. Location of the minimal fragment
required for AChR aggregating activity and the heparin and
dystroglycan-binding domains are indicated by bold lines at
bottom.
Figure 2 Ectopic acetylcholine receptor (AChR) aggregation induced by overexpression of COOH-terminal agrin fragments. Images of AChR aggregates labeled with rhodamine–a-bungarotoxin
(bright areas) were obtained by confocal microscopy. Projected series of images are shown. Horizontal stripe of AChR aggregates at the top of each panel consisted of synaptic aggregates at the ends of myotomes. Ectopic aggregates were concentrated in a band near the center of myotomes (arrows). (A) Control muscle from embryo injected with 2.8 fmol GFP RNA. (B,C,D) Muscle from embryos injected with T1, T2, or T3 agrin fragment RNA (8.7 fmol). Bar 5 20 mm.
Figure 3 Immunofluorescence for overexpressed agrin fragments is found in nonsynaptic regions
in myotomal muscles of embryos. Rat agrin fragments were labeled with antibody to the FLAG
epitope (C,E) or with a mixture of antibodies against FLAG and rat agrin (A). Immunofluorescence
was most prominent within ectoderm (not shown) and in the central portion of myotomal muscles
(A,C,E), where ectopic AChR aggregates (B,D,F) were induced, but was also seen in the synaptic
region of muscles. Control embryos (injected with GFP RNA; not shown) lacked immunofluorescence.
(A,B) Injected with T1 agrin RNA; (C,D) T2 RNA; (E,F) T3 RNA (all 17.4 fmol). Bar 5 20
mm. A semiquantitative estimate of amount of the overexpressed agrin fragments in the ectopic
region, made by integrating area of immunofluorescent deposits with intensity, gave values of 2.9,
1.2, and 2.9 3 105 for the microscopic fields shown in (A), (T1); (C), (T2); and (E), (T3),
respectively.
Figure 4 Axonal pattern is unaffected by expression of
agrin fragments. Ectopic AChR aggregation was induced by
the T3 fragment containing the COOH-terminal G3 domain
of agrin. Axons were labeled with HNK-1 antibody (green),
and AChR aggregates with rhodamineâa-bungarotoxin
(red). Axons were present in the intermyotomal septal (synaptic)
regions (top and bottom of both panels) in control
embryos injected with GFP RNA (A) and in embryos expressing
the T3 agrin fragment (B). Ectopic AChR aggregates
were focused in a band near the center of the myotome
(arrows, B) and were not contacted by axons detectable with
this antibody. Bar 5 20 mm.
Figure 5 Ectopic AChR aggregation is induced by similar
amounts of injected RNA encoding different agrin fragments.
Agrin fragment RNA (T1, l; T2, n; T3, Œ) was
injected into one-cell stage Xenopus embryos. AChR aggregation
was measured as described in Experimental Methods.
Ectopic aggregation (A) refers to total AChR aggregate area
outside of the innervated (synaptic) region at the ends of
myotomes. Values shown are ratios of ectopic aggregation
in experimental and control embryos (6SEM). Synaptic
AChR aggregation (B) was measured in the same experiment
within the usually innervated region adjacent to the
intermyotomal septum and is presented as a percentage
(6SEM) of control (GFP RNA-injected) synaptic AChR
aggregate area. Synaptic AChR aggregation decreased as
ectopic AChR aggregation increased.
