Chick ciliary ganglion neurons contain transcripts coding for acetylcholine receptor-associated protein at synapses (rapsyn)

Abstract

A peripheral membrane protein of approximately 43 kDa (rapsyn) clusters muscle nicotinic acetylcholine receptors (AChRs), but molecules relevant to clustering neuronal AChRs have not been identified. Here, we have detected rapsyn transcripts in the chick nervous system, localized rapsyn mRNA in ciliary ganglion (CG) neurons, which are known to cluster AChRs, and identified three rapsyn cDNAs derived from the ganglion. Our initial Northern blots, performed using a mouse probe, revealed rapsyn-like transcripts in chick muscle and brain. To develop species-specific probes, we prepared a chick rapsyn cDNA construct, Ch43K.1, that encodes a protein having extensive homology to mouse rapsyn. Using primers designed to anneal near the 5' and 3' boundaries of Ch43K.1, three prominent cDNAs were amplified from chick muscle templates by reverse transcriptase based-PCR. Products of similar size were also amplified using cDNA prepared from neuronal tissues expected to contain clustered AChRs (CG and brain), whereas none were detected using templates from tissues not displaying clustered AChRs (sensory ganglia and liver). In situ hybridization confirmed that rapsyn mRNA is expressed both in chick muscle fibers and in CG neurons. Sequencing the three cDNAs amplified from CG templates revealed the largest to be Ch43K.1, whereas the smaller two may represent splice variants. These findings suggest that multiple rapsyn-like molecules are involved in clustering the distinct AChRs expressed by muscle fibers and neurons.

Fig. 2.

Isolation and characterization of chicken muscle rapsyn cDNAs. A, Two λgt10 clones isolated by screening a chick muscle library with mouse rapsyn cDNA are depicted. One (8Not 1580) featured a 972 bp open reading frame (black bar) displaying high homology with the 5′ two thirds of M43K.1A, including the position of the putative start ATG at A268 (arrow). A second clone (5.1Not 1750) contained a 1050 bp open reading frame (gray bar) that overlapped in sequence with 8Not 1580 over a 783 bp region and featured an in-frame stop TGA. The cDNA inserts were digested at a sharedBspHI restriction site and the fragments ligated and subcloned into linearized pBluescript to form the plasmid pCh43K.1.B, Nucleotide sequence of chicken muscle cDNA construct Ch43K.1 (top line) and the predicted amino acid sequence of the protein in one letter code (bottom line) are shown. The 1236 bp open reading frame of the cDNA is bounded by a start ATG codon at position 268 and a stop TGA codon at position 1504. Regions of Ch43K.1 corresponding to exon borders in the mouseRapsn gene (Gautam et al., 1994) are shown asinverted Ts separating the appropriate exon numbers. Leucine as well as isoleucine and methionine residues (leucine alternatives) (see Landschulz et al., 1988; Froehner, 1991) within the leucine zipper motif of exon 2 are depicted in boxes. The underlined region (from nucleotides 876 to 1167) represents a cDNA fragment (ChIN) used to prepare probe for detecting PCR products on Southern blots (see Materials and Methods; Fig. 4).Bold arrow segments near the 5′ and 3′ borders indicate regions corresponding to distal (S1 and A1) and nested (S2 and A2) sense–antisense synthetic oligonucleotide primer pairs used in PCR experiments (see Materials and Methods; Results).

Fig. 4.

Amplification of rapsyn-like cDNAs from chick muscle and neuronal tissue templates. A, PCR products detected with ethidium bromide staining. Control template reactions (lanes 2, 3) revealed single products of ≈1470 or 1350 bp after amplification of Ch43K.1 insert using primer pairs S1/A1 or S2/A2, respectively. Subsequent test reactions (lanes 4–9) using only the S1/A1 primer pair reveal products amplified from muscle (Mu), ciliary ganglion (CG), or brain (Br) cDNA templates, synthesized by reverse transcription of cellular RNA (+; lanes 4, 6, 8). Note that multiple products are amplified from muscle cDNA templates (∼1500, 1350, 1150, and 500 bp; lane 4), whereas only a 500 bp product is amplified from brain and ganglionic templates (lanes 6, 8). Negative controls, using “sham” reverse transcriptions from corresponding muscle, brain, and CG reactions lacking RT (−; lanes 5, 7,9), revealed no products. pGEM DNA marker was loaded inlanes 1 and 10, and sizes are labeled atleft. B, Tissue PCR products amplified as in A were detected by Southern blot hybridization using the ^32-P-labeled ChIN probe (see Materials and Methods; Fig. 2B). Autoradiograms exposed for 1 hr from reactions using muscle templates (lane 1) detected the three cDNA products (∼1500, 1350, 1150) resolved on ethidium-stained gels (compare with A) as well as a less intense product at 1000 bp. Subsequent lanes (3–10) depict PCR products amplified from CG, Br, DRG, and liver (Lv) templates exposed for 10–24 hr. Note that the same two product sizes seen for Mu reactions (∼1500 and 1350 bp) are also resolved in CG and Br reactions (lanes 3,5) and that an additional product of 1600 bp is detected in CG. No products are detected in amplifications from DRG (lane 7) or Lv (lane 9) templates, even after exposures >36 hr. As in A, + (lanes 1,3, 5, 7, 9) and − (lanes 2, 4,6, 8, 10) denote presence or absence, respectively, of RT from the initial reverse transcription reaction. Lane 11 depicts the migration of the 292 bp^32-P-labeled ChIN probe. Size markers are pGEM standards (in base pairs) reproduced from the original agarose gel (data not shown).

Fig. 1.

A rodent rapsyn cDNA probe hybridizes with multiple mRNA transcripts in chick muscle and brain. Sources and amounts of chick mRNA transcripts loaded in each lane were liver (A, 10 μg), brain (B, 5 μg;C, 10 μg); muscle (D, 5 μg;E, 10 μg). Markers to left indicate the size (in kilobases) of RNA standards run on the original agarose gel.

Fig. 3.

Homology of the predicted chick rapsyn protein with those from Torpedo, Xenopus, and mouse. Individual amino acid residues were aligned without insertions or gaps, beginning at the start methionine. Identical and similar residues are included within the large box borders; identical residues are shaded. Domains corresponding to fatty acid myristoylation (FA), leucine zipper (LZ1, LZ2), and zinc finger (ZF) motifs of mouse rapsyn, as well as a conserved hydrophobic middle region (HY), are indicated by the dark bars.

Fig. 5.

Localization of rapsyn mRNA transcript in chick muscle and ciliary ganglion. Tissue sections were hybridized with digoxigenin-labeled antisense or sense ChIN riboprobe, treated with anti-digoxigenin Fab conjugated to alkaline phosphatase, and visualized by bright-field (muscle) or fluorescence (ganglia) optics after reaction with a suitable substrate (see Materials and Methods). In muscle sections hybridized with the antisense ChIN riboprobe (A), rapsyn mRNA is evident within extrafusal muscle fibers and spindles (arrow), whereas only background levels are detected after hybridization with the sense riboprobe (C). An unlabeled muscle section stained for hematoxylin and eosin (B) shows the muscle fiber and spindle morphology (arrow) more clearly. In CG sections hybridized with the antisense ChIN riboprobe, rapsyn mRNA is evident in neurons (D), whereas no specific reaction product was detected after hybridization with the sense riboprobe (F). Many of the labeled neurons in D clearly overlap with neuron soma profiles revealed when the same section is viewed with bright-field differential interference contrast optics (e.g., arrows inD, E). Scale bars, 50 μm.

Fig. 6.

Isolation, cloning, and characterization of rapsyn-like cDNAs derived from ciliary ganglia. A, Ganglionic cDNAs were reamplified using the nested primer pair (S2/A2), uncovering three products at 1350, 1250, and 1000 bp (lane 2) on ethidium-stained agarose gels. As in Figure 4, no products were detected when RT was omitted from the initial reverse transcription reaction (lane 3). Three of the cDNAs were subcloned (CG7, CG11, and CG4) and displayed insert sizes appropriate to the corresponding PCR products (lanes 4-6, respectively). B, Sequencing cDNA inserts from each of the ganglionic clones. Only nucleotides within and surrounding exon 2 (boxed region bordered by inverted Ts) are depicted, with dashes indicating nucleotides missing from CG11 or CG4 numbering above CG7 indicating the corresponding position in Ch43K.1 (see Fig.2B). Amino acid residues encoded by CG7 and missing from CG11 or CG4 are shown in one letter code below CG4, with leucine, isoleucine, and methionine (leucine alternatives) (see Landschulz et al., 1988; Froehner, 1991) residues inboxes.