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Regulation of transcript encoding the 43K subsynaptic protein during development and after denervation.
Baldwin TJ
,
Theriot JA
,
Yoshihara CM
,
Burden SJ
.
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The postsynaptic membrane of vertebrate neuromuscular synapses is enriched in the four subunits of the acetylcholine receptor (AChR) and in a peripheral membrane protein of Mr = 43 x 10(3) (43K). Although AChRs are virtually restricted to the postsynaptic membrane of innervated adult muscle, developing and denervated adult muscle contain AChRs at nonsynaptic regions. These nonsynaptic AChRs accumulate because the level of mRNA encoding AChR subunits increases in response to a loss of muscle cell electrical activity. We have determined the level of mRNA encoding the 43K subsynaptic protein in developing muscle and in innervated and denervated adult muscle. We isolated a cDNA that encodes the entire protein-coding region of the 43K subsynaptic protein from Torpedo electric organ and used this cDNA to isolate a cDNA that encodes the 43K subsynaptic protein from Xenopus laevis. We used the Xenopus cDNA to measure the level of transcript encoding the 43K protein in embryonic muscle and in innervated and denervated adult muscle by RNase protection. The level of transcript encoding the 43K protein is low in innervated adult muscle and increases 25- to 30-fold after denervation. The level of transcript encoding the alpha subunit of the AChR increases to a similar extent after denervation. Moreover, during development, transcripts encoding the 43K protein and the alpha subunit are expressed initially at late gastrula and are present in similar quantities in embryonic muscle. These results demonstrate that transcripts encoding the 43K protein and AChR subunits appear coordinately during embryonic development and that the level of mRNA encoding the 43K protein is regulated by denervation.
Fig. 1. Nucleotide sequence and
deduced amino acid sequence of the
43K subsynaptic protein of Xenopus
laevis. Nucleotide 1 indicates the first
nucleotide of the codon encoding the
amino terminal residue and
nucleotides to the 5' side of this
amino terminal residue are indicated
with negative numbers. The number
of the nucleotide residue at the end
of each line is provided. The
predicted amino acid sequence is
shown above the nucleotide
sequence. Amino acid residues are
numbered beginning with the amino
terminal residue. The 5' and 3' ends
of the Xen 43.1 cDNA is bordered
by EcoRI linkers which were
introduced during cloning.
Fig. 2. Alignment of the amino acid sequences of the Xenopus laevis and Torpedo californica 43K proteins. The
Xenopus (XN 43) sequence is shown by the one-letter amino acid notation. Identical residues in the Torpedo (TOR 43)
sequence are indicated with a dash (â) and amino acid substitutions are shown by the one-letter amino acid notation.
71 % (282/399) of the amino acid residues in the two sequences are identical.
Fig. 3. cDNA encoding the 43K
protein hybridizes to transcripts of
4-0 kb and 2-0 kb in Northern blots
of Xenopus embryo RNA. 1 n% of
poly(A)+ RNA from stage-41
Xenopus embryos was fractionated
by electrophoresis in a formaldehyde
agarose (1%) gel, transferred to
Zetabind, and hybridized to 32Plabelled
Xen 43.1 RNA probe
(Materials and methods). The RNAs
that hybridize migrate at 40 and
2-0kb (arrowheads). The filter was
exposed to X-ray film with an
intensifying screen at â70°C for
lday.
Fig. 4. Transcript encoding the 43K protein and
transcript encoding the AChR alpha subunit are 25- to
35-fold more abundant in denervated than in innervated
adult muscle. The triceps femoris muscle of adult
Xenopus was denervated for 10 days and AChR alpha
subunit and 43K protein transcript levels were measured
by RNase protection. 5 fig of RNA from innervated
(INN) and denervated (DEN) muscle were included in
hybridizations with 32P-labelled probes. The amount of
alpha subunit and 43K protein transcript is low in
innervated muscle and increases 25- to 35-fold in
denervated muscle. Denervation results in no change in
either the amount of total RNA or the level of transcript
encoding the translation elongation factor Ef-1-alpha
(Baldwin et al. 1988). The arrowheads mark the positions
of the protected fragments at 403 nucleotides (43K
protein) and 429 nucleotides (AChR alpha subunit).
Fig. 5. Transcript encoding the 43K protein is first expressed at late gastrula. 43K protein transcript levels were
measured in Xenopus embryos by RNase protection. RNA from 30 eggs (E) and 30 embryos at stages 10, 12 and 14
were included in hybridizations with 32P-labelled Xen 43 probe. Hybridization reactions with RNA from embryos at
later stages (20 and 41) included RNA from 10 embryos. The gel was exposed to X-ray film with an intensifying screen
at —70°C for 4 days to analyse expression at early stages (egg, 10, 12 and 14) and for 3 days to analyse later stages (20
and 41). The stages are indicated at the top of each lane; the arrowhead marks the position of the protected fragment at
403 nucleotides.
