ARIA, a protein that stimulates acetylcholine receptor synthesis, is a member of the neu ligand family

Cell, Vol. 72, 801-815,

March

12, 1993, Copyright

0 1993 by Cell Press

ARIA, a Protein That Stimulates Acetylcholine Receptor Synthesis, Is a Member of the Neu Ligand Family Douglas L. Falls,” Kenneth M. Rosen,” Gabriel Corfas; William S. Lane,t and Gerald D. Fischbach’ l Neurobiology Department Harvard Medical School Boston, Massachusetts 02115 tHarvard Microchemistry Facility Harvard University Cambridge, Massachusetts 02138

Summary Motor neurons stimulate their postsynaptic muscle targets to synthesize neurotransmitter receptors. Polypeptide signaling molecules may mediate this inductive interaction. Here we report the purification of ARIA, a protein that stimulates the synthesis of muscle acetylcholine receptors, and the isolation of AR/A cDNA. Recombinant ARIA increases acetylcholine receptor synthesis greater than 3-fold, and it Induces tyrosine phosphorylation of a 185 kd muscle protein. The ARIA cDNA hybridizes with mRNAs that are expressed in the spinal cord from E4, a time prior to the onset of neuromuscular synapse formation, through adulthood. By E7, hybridizing mRNAs are concentrated in motor neurons. Chicken ARIA is homologous to the rat Neu differentiation factor and human heregulin, ligands for the receptor tyrosine kinase encoded by the neu (c-e&82, HERP) proto-oncogene. Our data suggest that members of the ARIA protein family promote the formation and maintenance of chemical synapses and, furthermore, that receptortyrosine kinases play important roles in this process. Introduction Theefficacyof synaptictransmissiondependsonthe number of neurotransmitter receptors in the postsynaptic membrane. It is important, therefore, to determine how such receptors are regulated during development and how they are maintained throughout adult life. These issues have been studied extensively at thevertebrate neuromuscular junction (for reviews see Schuetze and Role, 1987; Salpeter, 1987; Hall and Sanes, 1993). At this prototypic chemical synapse, the presynaptic motor neurons promote the accumulation of acetylcholine receptors (AChRs). The principal effect of innervation is a local increase in the rate of AChR synthesis and aggregation of AChRs at the point of synaptic contact. These processes increase the concentration of AChRs in the postsynaptic membrane to approximately 20,000/um2, which ensures that acetylcholine released from the nerve terminals depolarizes the muscle membrane sufficiently to trigger a conducted action potential and a rapid contraction. The effect of motor nerve terminals on AChR synthesis is a powerful one. In chick nerve/muscle cultures, the rate

of appearance of newly synthesized receptors is 4-5 times greater at developing junctions than elsewhere along the myotube, and newly synthesized AChRs make up 80%80% of the total number of AChRs at these early synapses (Role et al., 1985). After the initial period of synapse formation, motor axons maintain AChR subunit gene expression by the subsynaptic nuclei despite the down-regulation of AChR synthesis in extrasynaptic regions. Muscle activity, driven by motor neuron impulses, is responsible for the decline in extrasynaptic AChR synthesis (for review see Laufer and Changeux, 1989). Ultimately, a gradient is established with the AChR density in the postsynaptic membrane about three orders of magnitude greater than the receptor density in extrasynaptic regions along the same muscle fiber. The local effect of the nerve on AChR synthesis is clearly evident in the concentration of AChR subunit mRNAs in the subsynaptic cytoplasm (Merlie and Sanes, 1985; Fontaine and Changeux, 1989; Goldman and Staple, 1989) and in the selective expression of AChR subunit genes by synaptic nuclei (Sanes et al., 1991). In some species, motor nerves influence the maturation of AChRs as well as their rate of synthesis. In rodents, the AChR subunit composition changes after birth as a new subunit called E replaces they subunit, so that the composition of the AChR changes from a&8 to a&5 (Mishina et al., 1988; Witzemann et al., 1989). Although thissubunit switch occurs 2 to 3 weeks after the initial burst of synaptic AChR synthesis in utero, it depends on innervation, i.e., it is induced by motor nerve terminals (Brenner and Sakmann, 1978; Brenner et al., 1990; Martinou and Merlie, 1991). Progress has been made in identifying molecules that mediate the local effects of motor axons. Agrin, a protein that is synthesized in motor neuron cell bodies, is a strong candidate for the principal aggregating activity (McMahan, 1990; Reist et al., 1992). It is transported to motor nerve terminals and secreted into the synaptic cleft where it is bound to components of the extracellular matrix. We have focused on identifying the molecules that stimulate the synthesis of AChRs at the synapse. Early experiments suggested that spinal cord explants secreted a factor that increased the number of AChRs in adjacent chick myotubes (Cohen and Fischbach, 1977). Spinal cord and brain extracts were found to stimulate the synthesis of AChRs in uninervated myotubes(Jessell et al., 1979), and this provided a reliable, sensitive assay for AChR-inducing factors. Using this assay, we have purified an -42 kd protein, called ARIA, from chicken brain (Jesse11 et al., 1979; But-Caron et al., 1983; Usdin and Fischbach, 1988; Falls et al., 1990). ARIA has been characterized in partially purified preparations. It increases the rate of AChR synthesis in chick myotubes up to lo-fold, and it also increases the rate of AChR synthesis in mammalian muscle. The increase in synthesis is accompanied by large and selective effects on the level of AChR subunit mRNAs. In chick muscle, the principal effect is on the a subunit mRNA (Harris et al.,

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B

Figure 1. Correlation between 42,000 Protein, AChR-Inducing ~185 Tyrosine-Phosphorylating

-kDi 97-

-97

66-

-66

45-

31-

21l4-

I

an M, 33,000 to Activity, and Activity

(A) C4 reverse-phase column fractions assayed for protein composition by SDS-PAGE (top), for AChR-inducing activity at two dilutions (middle), and for ~185 tyrosine-phosphorylating activity (bottom). A single major silverstained band is seen in the fractions most potent in stimulating AChR synthesis and in promoting ~185 tyrosine phosphorylation. The band seen in fraction 33 at M, 50,000 is an artifact; it was absent in a repeat silver-stained SDS-PAGE analysis of this fraction. For the tyrosine phosphorylation assay, the fractions were tested at 1:250. The tyrosine phosphorylation data extend previously reported experiments correlating AChR-inducing activity with tyrosine phosphorylation activity (Corfas et al., 1993) to our most purified ARIA preparation. (6) SDS-PAGE analysis of the Cl 8 narrowbore reverse-phase column fraction used for generation of tryptic peptides. No bands were seen in any of the other fractions collected from this column run.

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1988,1989). In mouse muscle, an increase in then subunit is the most prominent effect (Martinou et al., 1991), which is significant considering the nerve-dependent subunit switch described above. The actions of ARIA extend beyond AChR gene expression: in cultured chick muscle, ARIA also increases the number of voltage-gated sodium channels, another molecule concentrated at vertebrate endplates (Corfas and Fischbach, 1993). Finally, recent experiments have shown that ARIA causes tyrosine phosphorylation of a 185 kd protein (here referred to as ~185) in both chick and mammalian muscle, and these experiments further suggest that this protein may be an ARIA receptor tyrosine kinase (Corfas et al., 1993). Obtaining direct evidence that the activities attributed to ARIA are due to a single protein and determining the biological relevance of these actions require the molecular identification of ARIA. In previous work, we found that the chicken prion protein was present in highly purified ARIA preparations (Falls et al., 1990; Harris et al., 1991). An altered form of the mammalian prion protein has been implicated in the pathogenesis of several neurodegenerative disorders in sheep, cows, rodents, and humans (for review see Prusiner, 1992); therefore, it seemed possible that the normal cellular form of the protein served atrophic function in the central nervous system. In fact, prion-like immunoreactivity was found in spinal cord motor neurons (Harris et al., 1991) and in other cholinergic neurons in

the brain (F. A. Johnson and G. D. F., unpublished data). However, thechicken prion proteincould not be implicated in regulation of AChRs at the neuromuscular junction. AChR-inducing activity could not be precipitated by antibodies that precipitated the chicken prion protein, and no AChR-inducing activity was recovered following transfection of two different cell types with cDNA encoding the chicken prion protein. Recent experiments have shown that the AChR density and the switch to s-containing AChRs is apparently normal in mice lacking the prion protein (Brenner et al., 1992). The copurification of ARIA and the chicken prion protein must be considered a coincidence. Here we report the purification of ARIA by a protocol that separates ARIA from the chicken prion protein and the use of tryptic peptide sequences to isolate an AR/A cDNA. Expression of this AR/A cDNA produces a protein that promotes tyrosine phosphorylation of ~185 as well as increasing AChR synthesis. The predicted protein is homologous to the Neu differentiation factor (NDF) (Peles et al., 1992; Wen et al., 1992) and human heregulin (HRG) (Holmes et al., 1992) recently discovered ligands for Neu (erbB2, HERP), a 185 kd receptor tyrosine kinase. Our results suggest that ARIA and ARIA-activated tyrosine kinases play important roles in the differentiation of the neuromuscular junction and perhaps of interneuronal synapses as well.

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Regulating

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Synthesis

Figure 2. The h12 cDNA Encodes a Protein That Stimulates AChR Synthesis and pi65 Ty rosine Phosphorylation

180

(A) COS cells were transfected by electroporation with a plasmid containing the 112 insert in the sense orientation (~12.7) or the antisense orientation (~12.6). Medium conditioned for 48 hr was concentrated -25-fold and assayed for its effect on AChR insertion rate (mean f SD, n = 4). (B) Medium from pl2.7-transfected COS cells causes tyrosine phosphorylation of ~165 (arrowhead) in L6 cells, whereas medium from pl2.6-transfected COS cells does not. The effect of purified brain ARIA is shown for comparison.

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Results Purification of ARIA ARIA was purified from an acid extract of 3000 chicken brains by column chromatography as follows: step 1, CM Sepharose cation exchange; step 2, preparative C4 reverse phase; step 3, heparin affinity/ion exchange; step 4, gel filtration; step 5, semipreparative C4 reverse phase; step 6, narrowbore Cl8 reverse phase. After each step, column fractions were selected for further purification based on their ability to stimulate AChR synthesis in chick myotubes. The most important modification of our previously published protocols (Usdin and Fischbach, 1986; Falls et al., 1990; Harris et al., 1991) was the addition of the heparin column. This step allowed the efficient removal of chicken prion protein, the major contaminant of ARIA at later stages of purification. Chicken prion protein eluted earlier than the single peak of AChR-inducing activity, which typically eluted between 450 mM and 650 mM sodium chloride, on the falling limb of the major A280 peak. The fractions pooled for further purification contained greater than 85% of the AChR-inducing activity but less than 1% of the immunoreactive chicken prion protein. Analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of fractions eluted from the second C4 reverse-phase column (step 5 above) showed a single, broad, silver-stained band in each of the fractions most active in stimulating AChR synthesis (Figure 1A). The same fractions were the most active in stimulating the tyrosine phosphorylation of a 185 kd protein (~185) in the rat muscle cell line L6. The silver-stained bands formed a descending staircase pattern that spanned a molecular weight range of 42,000 (top of the band in fraction 31) to 33,000 (bottom of the band in fraction 35). This range is consistent with experiments showing that AChR-inducing activity can be eluted from SDS gel slices corresponding to M, 45,000 to 35,000 (Usdin and Fischbach, 1986; Falls et al., 1990). It is also consistent with estimates of ARIA’s size based on gel filtration. The bands in fractions 31 to 35

12.7

%iF

likely represent a single polypeptide, i.e., a single translation product, with the observed mobility variation due to heterogeneity in glycosylation or other posttranslational modifications. The AChR-inducing protein in these fractions is extremely potent: fraction 33 exhibited a halfmaximal increase in AChR incorporation rate at a concentration of 500 pglml, or approximately 20 pM. Fractions 31-34 from the C4 reverse-phase column of step 5 were pooled and concentrated on a narrowbore Cl8 column. A single 200 pl fraction eluted from this Cl8 column contained all of the protein detectable by silver stain (Figure IB). This fraction, derived from approximately two-thirds of the starting material (see Experimental Procedures), contained 100 pmol of protein, a yield of -0.02 pmol per gram of brain tissue; 70 pmol was digested with trypsin. The resulting peptides were separated by Cl8 reverse-phase chromatography. The peaks on the tryptic peptide map were much lower than expected: all suggested peptide amounts of less than 10 pmol. Three fractions subjected to Edman degradation yielded more than 1 residue at many of the cycles. Unambiguous sequence was obtained from two, CT24 and CT29. The CT24 sequence was NRPENVK and that for CT29 was ATLADAGEYACR. A search of protein data bases using the BLAST program (Altschul et al., 1990) showed that the CT24 and CT29 peptides matched sequences within rat NDF and the HRGs. The CT29 sequence was identical at 8 of 12 residues to the NDF sequence A102 to R113. The CT24 sequence was identical at 5 of 7 residues to the NDF sequence N81 to K87. Like ARIA, NDF and HRG exhibit a molecular massof - 40 kd and induce tyrosine phosphorylation of a 185 kd membrane protein. The similarity in size and phosphorylation activity supports the notion suggested by the peptide sequence match that ARIA is related to NDF and the HRGs. Isolation of an AR/A cDNA Degenerate oligonucleotide primers corresponding to the CT24 and CT29 peptide sequences were used in the poly-

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-s---+ FYK HLG I E F ” ERE ELY Q N Y “MAS QKRV# LTI CAAAACTACG GAACTGTACC AGAAACGGGTGrnACCATA .. . . . . . . . . . . . . . .TAATGGCCAC . . . . . . . . . . .. . . . . CmcTAcARG . . . . . . . .. .. . . . . . CATCTrGGGA . . . . . . . . . . . . . . . . rnAArnAT . . . . . . . . . . . . . . . . GGARGCTGAG ......

Figure

3. ProARIA-

Nucleic

Acid and Amino Acid

Sequence

Amino acid numbers and nucleotide numbers are given in the left and right columns, respectively. The first with an open triangle; the 14 additional nucleotides 5’of this were determined from PCR products. Cysteines and in bold. The immunoglobulin-like domain and the EGF-like domain (first cysteine to last cysteine) are underlined. A potential proteolytic cleavage site is indicated with an arrow. Nucleotides encoding the amino a and 5 forms are indicated by a dotted line. Potential N-glycosylation sites are shown with asterisks. The demarcate areas of sequences represented in the tryptic peptides (see Experimental Procedures).

merase chain reaction (PCR) to amplify cDNAs prepared from embryonic day 19 (E19) chick spinal cord RNA. Based on the NDF/HRG protein sequence to which we had aligned these chick peptides, we predicted an amplified band of 94 bp. Although many bands were evident among the reaction products, a discrete band of about the expected size was present. Four subclones representing this band were sequenced. The sequence between the primers was identical for all, but was 3 bp longer than expected. The amino acid sequence deduced from this

nucleotide of the Al2 insert is indicated in the putative ectodomain are boxed stippled. The hydrophobic segment is acids that vary between the heregulin numbered overlines (solid and dashed)

nucleotide sequence was 70% identical to the NDF/HRG sequence (I88 to A107) after the introduction of a single amino acid gap. The 97 bp fragment was used to probe a once amplified El3 chick brain cDNA library (generously provided by Dr. Barbara Ranscht of the Scripps Institute; see Ranscht, 1988). Five plaques were positive in an initial screen of 800,000 plaques. One phage (X12) that contained the longest S’and longest 3’sequence (relative to the 97 bp probe) was selected for further study.

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Figure 4. Alignment HRG-Pl

coo 93 YRATLADAGEY 10 0 *K:+," ,. s>j,:!lQ; 127 150

------------------A sespirisvstega?tsss

of

ProARIA-

to

Pro-

ProHRG-Pl residuesidentical toproARIA-1 are represented by stippling. Amino acids in proHRG+l that are conservative replacements are shown in uppercase letters; amino acids that are not similar are shown in lowercase letters. Gaps are shown with dashes. The numbersign indicatesastop translationcodon. Residues (except the cysteines) characteristic of the immunoglobulin-like motif are indicated by open circles, and residues characteristic of the EGF-like motif are indicated by closed circles. Other sequence features are coded as in Figure 3. except that stippling is used here to indicate identity. The sequences of proHRGp2, proHRGj33, proHRG-a, and proNDF are shown only in the region where the (t and p forms differ.

ySSKL::DSTKAS"IITDT:---------------.~i~~:"~~~~~~:a~f~Iti~~Es~eiitgmpastegayvs * * IKQKA e@E$iit

309 MTVTQTPSHSWSNGHTESILSESHSVLVSSSVENSRHTSPT-GPRGRLNG 3 5 o t <,:;::i ,f y<, /,l', >1,,'. ~ .j i.l ;, * ,;d, '$ ,~ *h ,,,, :,,,v;. "$ s $$;;j g@g$$~ .~/. <:b II, II' mkO~Llj.gs

408 PKSPPSEMSPPVSSLTISIPSVAVSPFMDEERPLLLVTPPRLRE-KYDNH 4 5 0 a:,~~~~~:,,.':r~I~;;~j:;: M,;V M:,~~M?Ir~~~~l::':Eb!!~~~~,~~~:~~~~k~FtH~

The I.12 cDNA Encodes an AChR-inducing Protein To determine if the cloned cDNA encoded a protein with AChR-inducing activity, we subcloned the 1112 insert into the eukaryotic expression vector pcDNAi/AMP. A piasmid with the insert in the sense orientation (~12.7) and a piasmid with the insert in the antisense orientation (~12.6) were transfected into COS7 cells. Medium conditioned for 46 hr by ceils transfected with the sense construct produced a dose-dependent increase in AChR incorporation rate, whereas medium conditioned by cells transfected with the antisense construct had no effect (Figure 2A). Essentially the same result was observed in two other experiments. The maximal fold increase in AChRs induced by medium conditioned by pl2.7-transfected COS ceils was not further increased by the addition of brain ARIA (recombinant ARIA = 2.5 f 0.4, mean + standard deviation; recombinant ARIA + brain ARIA [amount sufficient to produce a maximal response] = 2.5 f 0.2). Medium conditioned by pl2.7-transfected COS cells also stimulated the tyrosine phosphoryiation of ~165 (Figure 28). The pattern of tyrosine phosphoryiation induced by the recombinant ARIA was the same as the pattern induced by purified brain ARIA. Taken together, these data suggest the 112 cDNA clone encodes a protein that acts by the same mechanism as purified brain ARIA. As discussed below, it is likely that the h12 cDNA en-

codes a protein that is the precursor of the AChR-inducing protein in medium conditioned by transfected COS ceils. We will therefore refer to the hlPencoded protein as proARIA-1 and the released (soluble) form of this protein as ARIA-1 (see Experimental Procedures: Nomenclature Note). The ProARiAcDNA and Deduced Amino Acid Sequences The nucleotide sequence of the h12 cDNA and its predicted amino acid sequence are shown in Figure 3. The first ATG of the 112 insert is a strong translation initiator by sequence context criteria (Kozak, 1969). Beginning with this first ATG, the I.12 cDNA encodes a polypeptide of 632 amino acids. The stop codon is followed by a 520 nt 3’ untranslated sequence ending with an A-rich region that is not preceded by the consensus polyadenylation signal AATAAA. No stop codon was observed upstream of the first ATG. Attempts were made to extend the sequence in the B’direction using PCR. An antisense primer, corresponding to nucieotides 292 to 317 of the sequence shown in Figure 3, and sense primers from the XgtlO arms were used to search for related sequences in aiiquots ?f the El3 chick brain cDNA library. A total of 1.4 x lo6 phage were screened in this manner. A band containing an additional

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14 nt (base pairs 1 through 14 of Figure 3) was amplified repeatedly, but no longer sequences were detected. Attempts to define the length of the 5’untranslated sequence of the mRNA by primer extension have, to date, been inconclusive. Hydropathy analysis revealed that the predicted protein has one string of nonpolar amino acids (V207 to C229) that is of sufficient length and hydrophobicity to traverse the lipid bilayer as an a helix (Kyte and Doolittle, 1982). This raises the possibility that proARIA- is a transmembrane protein. In fact, all of our tryptic peptide sequences (dashed and solid lines in Figure 3) match proARIA- sequences N-terminal to the hydrophobic domain (see Experimental Procedures: Peptide Sequence Determination). This is consistent with the idea that purified brain ARIA is derived from the N-terminal portion (ectodomain) of proARIA- or a proARIA- -like precursor. A pair of basic amino acids (K205-R206) adjacent to the hydrophobic domain might serve as a cleavage site for the release of ARIA-1 from proARIAby a trypsin-like protease. Three potential N-glycosylation sites occur N-terminal to the hydrophobic segment. There are eight cysteines N-terminal to the hydrophobic segment. The spacing of the first two cysteines and the residues surrounding these cysteines fit the consensus for an immunoglobulin-like domain of the C2 type (Williams and Barclay, 1988; Hunkapiller and Hood, 1989; Furley et al., 1990). The spacing of the next six cysteines fits the consensus sequence for an epidermal growth factor (EGF)-like motif (Carpenter and Cohen, 1990; Carpenter and Wahl, 1990). About 60% of the sequence is C-terminal to the hydrophobic segment. This putative cytoplasmic domain is remarkable for its high percentage of proline (10%) and serine+threonine (24%). The C-terminal residue is a valine. This may have significance in regulating the proteolytic release of the ectodomain, as has recently been shown in studies of the transforming growth factor a precursor (Bosenberg et al., 1992). ProARIAIs Homologous to NDF and HRG A protein data base search revealed that proARIA- is, as expected, closely related to NDF and the HRGs. ProARIA-1 is most similar to the 81 form of proHRG (Figure 4). With four gaps in the proARIA- sequence and one in the proHRG-61 sequence, the proteins are identical at 80% of the residues. The overall structural similarity between proARIA-1, the proHRGs, and proNDF are diagramed in Figure 5. However, proARIA- differs strikingly from proHRG-81 and the other published members of the NDFlHRG family in two regions: the N-terminal 27 amino acids and the “spacer” sequence between the immunoglobulin-like and EGF-like domains (Figures 4 and 5). The four reported proHRG forms differ principally in a 19-29 amino acid stretch that begins with the residue following the fifth cysteine of the EGF-like domain (Figure 4). While the sequence of proHRG-a differs from the sequence of the 8 forms throughout this region, the 8 forms differ from each other only near its end. In this region,

proARIA- is 100% identical to proHRG-81. However, despite the differences in sequence between the a and various 8 forms, the spacing of the EGF-like domain cysteines is identical in proARIA-1, the proHRGs, and NDF. This spacing differs from that found in the known ligands of the EGF receptor such as transforming growth factor a and EGF (see Wen et al., 1992). The sequence of the EGF-like domain (including the region of al8 difference) is likely to be important in determining the specific receptor(s) activated by proARIAand its homologs, since this region alone, when expressed as a bacterial fusion protein, can stimulate protein-tyrosine phosphorylation (Holmes et al., 1992; our unpublished data). What inferences can be made with respect to the isoform(s) of the ARIA purified from brain? None of the tryptic peptides correspond to the N-terminal and spacer sequences that are different between proARIAand the proHRGs and proNDF. However, several of the tryptic peptides (dashed bar number 5 in Figure 3) correspond to sequence within the region that differentiates the a form of HRG from the 8 forms. Only @like peptide sequence was seen; there were no a-like peptides. These data demonstrate that the ARIA purified from brain contains a 6 isoform, though they do not exclude the possibility that brain ARIA also contains an a isoform. Since the cDNA we cloned also encodes a protein that has a &type EGF-like domain, 6 forms may predominate in the nervous system. Northern Blot, In Situ Hybridization, and Southern Blot Analyses On Northern blots of spinal cord RNA, the proARIAcDNA hybridized with a major band of - 7.3 kb and a minor band of -3.0 kb (Figure 6A). Judged by the intensity of its signal, the 7.3 kb transcript is not a rare mRNA. Both transcripts were seen at E7, the earliest age examined by Northern blot. The relative intensity of each band remained essentially constant from E7 through postnatal day 1. The 7.3 kb transcript was also prominent in adult spinal cord RNA (data not shown). The significance of the two transcript sizes is not known. Both are larger than needed to encode proARIA-1, suggesting that the proARIA- mRNA may have long 5’ and or 3’ untranslated regions, a feature common to growth factor mRNAs (Kozak, 1991). We also analyzed the expression of proARIA-l-related transcripts in the chick brain, the source of the purified protein. Total RNA isolated after regional dissection of El9 chick brain contained transcripts of the same size and relative abundance as had been found in the spinal cord (Figure 6B). The distribution within E7 spinal cord of proARIA-lrelated mRNAs was assessed by in situ hybridization (Figure 7). mRNAs hybridizing with the proARIA- probe were concentrated in the lateral part of the ventral horn, a localization most consistent with its expression in motor neurons. The number of grains over other areas of the cord and the dorsal root ganglia was always much lower; however, in some sections the accumulation of grains over these areas was above background. No specific signal was detected over the germinal zone surrounding the cen-

Ml;A,

a Protein

Regulating

AChR

Synthesis

neurons. The fact that transcription from the AR/A gene begins before motor neuron axons reach the periphery is consistent with our hypothesis that proARIA-1, or aclosely related molecule, induces the synthesis of AChRs at newly formed neuromuscular synapses. The proARIA- gene is expressed throughout embryonic life and in adults, so ARIAS may play a role in the maturation and maintenance of synapses as well. The ARIA/NDF/HRGIGllal Growth Factor Family The high degree of identity and the similar domain organization between proARIA- and HRG-91 suggest that ARIA is a chicken homolog of the HRGs (Holmes et al., 1992) and NDF (Peles et al., 1992; Wen et al., 1992). HRG and NDF were purified from media conditioned by a human breast tumor cell line and ras-transformed rat fibroblasts,

Figure 5. Diagramatic Representation the ProHRGs and ProNDF

of ProARIA-

Compared

with

The sequence of proARIAis dissimilar to the sequence of the other forms near the N-terminus and in part of the region between the immunoglobulin-like domain and the EGF-like domain. The percentage identity between proARIAand proHRG-51 is computed from the alignment of Figure 4 and excludes gaps. ProNDF aligns to the proHRGs with no gaps but has a shorter cytoplasmic domain; it is 95% identical to proHRG-a. All, except proHRG-63 (which lacks a hydrophobic domain), are diagramed as transmembrane proteins with an N-terminal ectodomain and a C-terminal intracellular domain. Upper hatched box of each, immunoglobulin-like region; lower hatched boxof each, EGFlike region; solid blackarea, hydrophobicdomain; light stippling, alipid bilayer. The length of each box is proportional to the length of sequence represented.

tral canal. In E4 spinal cord, grains clearly above the background level were already evident over the ventral portion of the cord (data not shown). A Southern blot analysis of chicken genomic DNA performed using a 350 bp fragment derived from the 5’ end of the proARIA- cDNA as probe identified a single band of approximately 20 kb in one digest, consistent with the existence of a single copy ARIA gene (data not shown). However, six enzymes (none of which cuts within the probe sequence) generated multiple bands, suggesting that this relatively short sequence is split among multiple exons. Discussion We have determined the amino acid sequence of proARIA-l, a mature form of which stimulates the synthesis of muscle AChRs. The protein is called proARIA-1, because it may be a precursor of a smaller active form and because other protein products of the ARIA gene, generated by alternative splicing, may also have AChR-inducing activity. mRNA that hybridizes with the proARIA- cDNA is present in the chick spinal cord from E4, the earliest time examined. By E7, hybridizing spinal cord mRNA is highly concentrated in the ventrolateral portion of the ventral horn, indicating that it is principally expressed in motor

B Figure 6. Northern and Brain

Analysis

of ProARIA

Transcripts

in Spinal

Cord

Ten micrograms of total RNA was loaded in each lane. Blots were probed using the entire 112 insert under conditions of high stringency. The films shown were exposed for 24 hr at -70°C with two screens. The positions of 26s and 16s ribosomal RNAs are indicated (arrowheads) for reference. The ethidium-stained gels (lower panels) dentonstrate that the lanes were loaded with similar amounts of RNA. (A) Spinal cord RNA from different developmental stages. A major transcript is seen at 7.3 kb and a minortranscript at3.0 kb. The intensity of the signal for both the 7.3 kb and 3.0 kb transcripts did not differ significantly between theagesexamined. E, embryonicday; P, postnatal day. (B) Brain RNA from El9 chick. Brains were dissected into the four regions indicated, and total RNA was prepared from each region. A lane with El9 spinal cord mRNA is included for comparison. The sizes and amounts of hybridizing mRNA are similar in each of the brain regions to what had been found in spinal cord. However, a faint band is also seen immediately above the 7.3 kb band. SC, spinal cord; T, tectum; M, midbrain and diencephalon; H, cerebral hemisphere; C, cerebellum.

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(A) Autoradiographic localization of AR/A mRNA using an antisense RNA probe and dark-field illumination. Grains are concentrated over motor neurons in the ventral horn. No specific signal was detected over the germinal zone. No signal above background was seen with an antisense probe. (6) Nissl stain of a section adjacent to those shown in (A). Scale bar, 500 urn.

Goodearl, personal communication; M. Marchionni, personal communication). Analysis of these clones revealed that some cDNAs corresponded to NDF/HRG sequences while others were novel variants. Of interest relative to our studies of ARIA is the fact that Schwann cells ensheath both motor neuron axons and their terminals. Neu is expressed in Schwann cells during development, and it is up-regulated by nerve injury (Cohen et al., 1992). It is likely that all the known members of this family, including proARIA(chicken), NDF (rat), the HRGs (human), and the glial growth factors (cow and human), are the products of the same gene. While some of the observed protein sequence differences may reflect evolutionarydivergence, the major differences among the forms are likely due to alternative splicing. This idea isconsistent with our Southern blot data suggesting that the AR/A gene is multiexonic. Other factors that activate Neu have been characterized (Yarden and Weinberg, 1989; Lupu et al., 1990; Davisetal., 1991; Dobashietal., 1991; Tarakhovsky et al., 1991; Yarden and Peles, 1991; Huang and Huang, 1992) but their amino acid sequences have not been determined; these too may be products of the ARIA gene. The evidence that there are multiple proteins closely related to proARIAraises a number of questions. Do all of the forms have AChR-inducing activity: and conversely, does proARIAaffect proliferation of Schwann cells or breast cells? Is proARIAthe form expressed in motor neurons; and if so, is it the only ARIA expressed in motor neurons? To understand the biological rolesof these multiple forms, it will be necessary to determine where and when each is expressed and to characterize the specific functional properties of each.

respectively, based on their ability to stimulate the phosphorylation of tyrosine residues in Neu, a 185 kd receptor tyrosine kinase. Neu, the product of the proto-oncogene neu (also known as c-e&B2 and HER2) (Coussens et al., 1985; Bargmann et al., 1986; Yamamoto et al., 1986) was first identified in central nervous system tumors (Padhy et al., 1982). It is overproduced in human breast cancers and in several other adenocarcinomas (Singleton and Strickler, 1992). Therefore, one interest in identifying Neu ligands has been to study their role in the pathogenesis and therapy of these carcinomas. The role of the NDFlHRG proteins in normal cell growth and differentiation has received less attention. NDF- and HRG-related mRNAs are present in several adult tissues including the central nervous system. In fact, NDF-related message is most abundant in the spinal cord (Wen et al., 1992) and the spinal cord mRNAs labeled are approximately the same size as those detected in chick spinal cord RNA by the proARIAprobe. Recently, glial growth factors, purified from bovine pituitaries on the basis of their ability to stimulate DNA synthesis in rat Schwann cells, have been found to be members of this family. Amino acid sequences of peptides derived from each of three forms were used to isolate several cDNA clones from bovine and human libraries (A.

Tyrosine Phosphorylstion and ARIA Action The homology between ARIA and the NDFlHRG group of proteins leads directly to the suggestion that Neu or a Neu-related receptor tyrosine kinase is ARIA’s receptor in skeletal muscle. Neu is closely related to the EGF receptor (for review see Prigent and Lemoine, 1992), but ARIA, HRG, and NDF are not ligands for the EGF receptor. The only other reported member of this receptor family is the more distantly related erbB3 (HERS) (Kraus et al., 1989; Plowman et al., 1990); no ligands are known for this 170 kd protein. ARIA does induce tyrosine phosphorylation of a 185 kd protein in the Neu-expressing human breast tumor cell line MDA-MB-453 (ATCC HTB 131), suggesting that ARIA is capable of promoting Neu tyrosine phosphorylation, and we have detected Neu by immunoprecipitation and immunoblot in L6 cells (unpublished data). However, it is not yet clear whether Neu is the major receptor for ARIA in muscle. As is the case for the neurotrophins(Chao, 1992; Meakin and Shooter, 1992) and fibroblast growth factor (Yayon and Givol, 1992) there may be several related receptor tyrosine kinases that can be activated by ARIA. Other factors and agents known to regulate AChR synthesis have not been demonstrated to act through tyrosine kinases. Ascorbic acid can increase AChRs in cultured muscle (Horovitz et al., 1989a, 1989b), but the pathway

B I Figure 7. Localization Situ Hybridization

of ProARIA-

1 mRNA

in E7 Spinal Cord by In

;:;A,

a Protein

Regulating

AChR

Synthesis

mediating this effect is unknown. CGRP, a peptide present in motor neurons, increases AChR synthesis in cultured muscle through activation of CAMP-dependent protein kinase (for review see Changeux, 1991). Electrical activity may down-regulate the synthesis of AChRs through activation of protein kinase C (Klarsfeld et al., 1989; Huang et al., 1992). It will be important to understand how these various pathways interact in the regulation of AChR synthesis with the tyrosine phosphorylation pathway activated by ARIA and to determine the specific roles of each pathway in synapse formation and maintenance. Interestingly, tyrosine kinases have also been implicated in the aggregation of AChRs by agrin (Wallace et al., 1991) and by several other agents, including basic fibroblast growth factor-coated beads (Peng et al., 1991), native polystyrene beads (Baker et al., 1992; Baker and Peng, 1993), and electric fields (Peng et al., 1993). In particular, evidence has been presented that agrin-induced clustering of AChRs requires protein-tyrosine phosphorylation of muscle proteins and that tyrosine phosphotylation of the AChR itself may be important in agrin’s action (Wallace, 1992). The receptor for agrin is unknown, but agrin does not induce tyrosine phosphorylation of ~185 (Corfas et al., 1993) nor does it stimulate AChR synthesis(Godfrey et al., 1984). Conversely, ARIA preparations have little or no clustering activity. Thus, it appears that tyrosine kinases are important in the regulation of both the synthesis and the clustering of AChRs at the synapse, but that two distinct pathways are involved. Is ProARIAa Transmembrane ARIA Precursot? Perhaps the best argument that proARIA- is a transmembrane protein is by analogy to the EGF and transforming growth factor a precursors. ProARIAis similar to these molecules in that all havean EGF-likedomain immediately amino-terminal of a strongly hydrophobic segment 24 to 29 amino acids long. Mature transforming growth factor a and EGF are released from their transmembrane precursors by proteolytic cleavage (MassaguB, 1990). A similar proposal has been made with regard to the biogenesis of soluble NDF (Wen et al., 1992) and HRG (Holmes et al., 1992) from transmembrane precursors. More generally, it appears that the ligands for a number of receptor tyrosine kinases, including the kit ligand, colony-stimulating factor 1, and bride of sevenless, in addition to the ligands for the EGF receptor, are synthesized as transmembrane proteins (reviewed by Massague, 1990; Gordon, 1991). With the exception of bride of sevenless (Cagan et al., 1992), soluble forms of these ligands are released by proteolytic cleavage adjacent to the membrane. The regulation of this proteolytic event may serve to modulate the activity of these ligands and their cognate receptors. While we propose that proARIAis a transmembrane protein, proARIA- lacks the N-terminal signal peptide typical of membrane and secreted proteins (von Heijne, 1988). ProNDF and the proHRGs also lack a signal peptide, even though their N-terminal sequences are different than that of proARIA-1. A number of transmembrane and secreted proteins are known that do not have a classical cleaved signal peptide (Perin et al., 1991; Meek et al., 1982; von

Heijne, 1988), but the possibility that recombinant ARIA is released from COS cells by an alternative pathway (Kandel et al., 1991) independent of the endoplasmic reticulum and Golgi must be considered. Regardless of the release pathway followed byrecombinantARIA, it seemsclearthat the ARIA purified from brain matures in the endoplasmic reticulum and Golgi since it is glycosylated and loses its AChR-inducing activity when treated with reducing agents (Falls et al., 1990; see also Peles et al., 1992). The idea that soluble ARIA-1 corresponds to an N-terminal fragment of proARIAis consistent with our estimates of the molecular mass of purified brain ARIA. The calculated molecular mass of the 802 amino acid proARIA-1 polypeptide is 87 kd, whereas the apparent molecular mass of the ARIA purified from brain is approximately 33-42 kd. For proARIA- the longest possible polypeptide (Ml-R208) produced by cleavage N-terminal of the hydrophobic domain would have a molecular mass of 23 kd. Glycosylation might increase the apparent molecular mass to that observed for purified ARIA. The fact that the tryptic peptide sequences of brain ARIA represent 38% of the proARIAresidues between L45 and Al86 but no residues of the putative hydrophobic or cytoplasmic domains (see Figure 3) also further supports the hypothesis that purified brain ARIA corresponds to the putati\je ectodomain of proARIA-1. It should be noted that only the N-terminal portion of proARIA- would be available for extracellular release if proARIAis, in fact, a transmembrane protein.

Active Forms of ARIA Evidence for a diffusible form of ARIA has been obtained in vitro and in vivo. In culture, chick myotubes located near a slice of spinal cord exhibit more AChRs than do myotubes located farther away from the neural explant (Cohen and Fischbach, 1977). In addition, medium conditioned byculturesof motorneuronsstimulatessynthesisof AChRs (Kirilovsky et al., 1989), increases AChR a subunit mRNA (Bursztajn et al., 1990), and promotes the tyrosine phosphorylation of ~185 (Corfae et al., 1993). In intact embryos, many muscle AChR dusters are found 50-100 pm away from the nearest growth cone when motor axons first invade the limb mesenchyrne (Morgan, 1990; Dahm and Landmesser, 1991). Our observation that ARIA binds with moderate affinity to heparin suggests that ARIA released from the motor nerve terminal may bind to the extracellular matrix of the synaptic cleft. Such extracellular matrix binding could serve to limit the diffusion of ARIA as well as to create an ARIA reservoir. In fact, evidence for an AChR-inducing activity attached to the extracellular matrix in the synaptic cleft has recently been obtained: when deneNated and damaged muscle was allowed to regenerate into the vacated basal lamina sheaths, local synthesis of AChRs was restored at the site of the original synapse even though reinnervation was prevented (Goldman et al., 1991; Jo and Burden, 1992). A stable form of ARIA in the basement membrane non

reported

might

also

explain

the

“imprinting”

phenome-

by Brenner et al. (1990), who found that de-

Cell 810

nervation of muscle at postnatal day 1 did not prevent the subsequent accumulation of E subunits at the endplate. ProARIA- itself may be an active transmembrane AChRinducing molecule as well as a precursor of soluble ARIA. Active transmembrane forms of ligands for several receptor tyrosine kinases are known; and in the case of the kit ligand, the membrane-bound species is apparently necessary for normal development in vivo (Flanagan et al., 1991). Of course, these mechanisms are not exclusive: ARIA may act in a freely diffusible form at one stage, as an integral membrane protein at another, and as a molecule tethered to a basal lamina component at still another. At the first nerve-muscle contacts the gap is only about 20 nm and the basal lamina is not well developed (Takahashi et al., 1987). This morphology would allow cell-cell interaction via membrane-bound ligand and receptor. At a later stage, when the cleft has widened to about 50 nm and the basal lamina is uniformly thick, a released form of ARIA and binding to the extracellular matrix may be more important. Roles of ARIA in the Nervous System ARIA, like other polypeptide differentiation and growth factors, is likely to be a multifunctional protein whose biological activities are context dependent. Evidence presented here and previously suggests that ARIA plays an important role in the regulation of AChRs at the neuromuscular synapse. This hypothesis can now be directly tested. ARIA also increases the number of voltage-gated sodium channels in skeletal muscle (Corfas et al., 1993), and it will be of interest to define other actions of ARIA based on the known specializations at the neuromuscular junction. For example, it is possible that the cytoplasmic region of the molecule, which is so highly conserved across species, plays a role in the differentiation of transmitter release mechanisms or in the target-dependent trophic support necessary for motor neuron survival. It will be important to investigate the effects of ARIA on ligand-gated and voltage-gated ion channels in peripheral ganglia and in the brain, the source of the purified protein. Nicotinic AChRs and excitatory and inhibitory amino acid receptors are concentrated beneath synaptic boutons at interneuronal synapses on cell bodies and throughout the dendritic tree. Although mechanisms of receptor regulation at these sites may not be identical to those at the neuromuscular junction, it is likely that lessons learned at the neuromuscular junction will have bearing on inductive interactions between neurons. Changes in channel density will certainly influence overall neuronal activity, and this, in turn, might have important long-term consequences for synaptic function and survival. Experimental

Procedures

Muscle Cultures Mononucleated cells were dissociated from pectoral muscles of El 1 chicks as previously described (Buc-Caron et al., 1983) and plated in 98-well tissue culture plates (Falcon) at a density of 20,OOO/well in 100 ~1 of Eagle’s minimal essential medium (GIBCO) supplemented with horse serum (10% v/v), chick embryo extract (2% v/v), L-glutamine (1 mM), penicillin (50 U/ml), and streptomycin (50 wg/ml). Cultures were usually fed with the same medium on day 4 and again immediately

prior to bioassay on day 7 to 8 after platmg. Myocytes were plated in wells coated with Matrigel (1:20; Collaborative) plus gelatin (0.1% v/v; Sigma) because the cultures performed better in the assay when plated on this substrate as opposed to gelatin alone. Matrigel and gelatin were diluted in minimal essential medium. L6 rat muscle cells were grown in 48-well plates in Dulbecco’s modified Eagle’s medium supplemented with fetal calf serum (10% v/v), L-glutamine (1 mM), penicillin (100 U/ml), and streptomycin (100 pg/ ml). L6 cultures were confluent at the time of assay. AChR Synthesis Assay The rate of AChR synthesis was estimated by measuring the rate of AChR incorporation into the surface membrane of chick myotubes as previously described (Usdin and Fischbach, 1986; Devreotes and Fambrough, 1975). In brief, myotubes were treated with column fractions (or conditioned medium) for 20 to 24 hr beginning on day 7 or 8 after plating. AChRs exposed on the surface membrane at the end of this treatment period were then blocked by incubation with unlabeled a-bungarotoxin (a-ETX) (10-l M). After washing out unbound toxin, fresh medium containing 1251-a-BTX (5 nM) was added, and the plates were returned to the incubator for 5 hr. The wells were then washed to remove unbound Y-a-BTX, and the well contents were solubilized in a solution of sodium hydroxide (1.0 M) and sodium deoxycholate (0.5 mglml) and counted in a y counter. Samples were assayed in quadruplicate. Bioassay results were normalized by dividing the sample cpm by the mean cpm for untreated cultures. ARIA does not alter the 18-24 hr half-life of surface AChFls (But-Caron 81 al., 1983), so the net number of Y-a-BTX-binding sites present at the end of a 5 hr period following block of preexisting surface bindingsitesislinearlyproportional to therateofappearanceofAChRs in the surface membrane. ‘251-a-BTX was prepared by methods modified from those described by Wang and Schmidt (1980). a-BTX was iodinated by the chloramineT-catalyzed reaction, and the reaction was quenched with tyrosine. Monoiodo-a-BTX was purified by spin chromatography on Sephadex G15 (Tuszynski et al., 1980) and by ion-exchange chromatography on CM Sepharose. Our preparations had a specific activity of approximately 1000 cpm/fmol. Tyrosine Phosphorylation Assay After incubation for 1 hr with column fractions or conditioned medium, rat L6 cells were washed and solubilized in SDS sample buffer plus dithiothreitol, transferred to an Eppendorf tube, and immediately heated to 95OC for 10 min. Samples were electrophoresed in SDSpolyacrylamide (5%) minigels, and the proteins were electroblotted to a polyvinylidene difluoride membrane (Immobilon, Millipore). The membranes were blocked by incubation in 3% nonfat dry milk-phosphate-buffered saline and incubated with an anti-phosphotyrosine monoclonal antibody (4GlO; generously provided by Drs. B. Druker and T. Roberts of the Dana Farber Cancer Institute). Bound antiphosphotyrosine monoclonal antibody was visualized with a horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G antibody (Boehringer Mannheim) and an enhanced chemiluminescence horseradish peroxidase substrate (Amersham). Autoluminograms were prepared by exposing the filters toXAR-5 film (Kodak) for intervals ranging from 5 min to 4 hr. Purlflcation of ARIA The experiments reported here began with 3000 adult chicken brains (Pelfreeze, AK), approximately 9 kg wet weight. The brains were powdered and delipidated at -15OC with acetone (6 ml per brain) and then with ether (4 ml per brain). The delipidated tissue was homogenized at 4oC in a solution (10 ml per brain) containing trifluoroacetic acid (20 ml/l), formic acid (50 ml of 88% w/w stock per liter), HCI (1 N), sodium chloride (100 mM), EDTA (2 mM), thiodiglycol (100 PI/I), pepstatin (1 mg/l), leupeptin (1 mgll), and phenylmethylsulfonyl fluoride (0.5 mM). The extract was clarified by centrifugation and filtration and then desalted by adsorption to Vydac Cl8 packed in a Buchner funnel (60 g; extract from 200-250 brains loaded per batch). The Cl8 was equilibrated in 0.1% trifluoroacetic acid and eluted with 0.1% trifluoroacetic acid, 60% acetonitrile). The eluate was then purified by the following protocol: Step 1, ion-exchange chromatography on CM Sepharose (500 ml bed; eluted with a gradient from 100 mM sodium chloride to

;rttA,

a Protein

Regulating

AChR

Synthesis

1000 mM sodium chloride in 25 mM MES [pH 6.01). Step 2, reversephase chromatography on Vydac C4 (22 x 250 mm column; eluted with a linear gradient from 15% to 26% isopropyl alcohol in 0.1% trifluoroacetic acid). Step 3, affinity/ion-exchange chromatography on Heparin-SPW (TosoHaas glass; 6 x 75 mm; eluted with a linear gradient of 150 to 1500 mM sodium chloride in sodium/potassium phosphate buffer [pH 7.01). Step 4, gel filtration chromatography on Superdex 75 (Pharmacia; 16 x 600 mm; equilibrated in 150 mM sodium chloride in sodium/potassium phosphate buffer [pH 7.01). Step 5, reverse-phase chromatography on Vydac C4 (10 x 250 mm column; eluted with a linear gradient from 30% to 45% isopropyl alcohol in 0.13% heptafluorobutyric acid). Step 6, reverse-phase chromatography on Vydac Cl6 (2.1 x 250 mm column). This step was used to concentrate the pooled fractions from step 5 prior to tryptic digestion. The column was equilibrated in 16% acetonitrile, 0.1% trifluoroacetic acid and was eluted by stepping the programed acetonitrile concentration from 16% to 95%. Owing to the dead space of the system and low flow rate, a steep gradient of acetonitrile is produced by this programed step. In this manner, 20 ml was concentrated to 200 ul. To avoid column overloading, we loaded one-third of the acid extract (representing 1000 brains starting material) on the CM Sepharose column in each of three runs. Similarly, the material was processed in three batches for the first Vydac C4 column step (step 2, above) and in four batches for the heparin chromatography step. Two-thirds of the bioactive heparin fraction pool (representing -2000 brains starting material) was concentrated by ultrafiltration (Centriprep 10, Amicon), and one-half of the concentrated material was loaded in each of two runs on the Superdex 75 column (step 4). Pooled active Superdex 75 fractions were further purified in one run of the Vydac C4 column (step 5) then concentrated in one run on the Vydac Cl6 column (step 6). The single fraction from this column that contained protein detected by silver stain was used for the preparation of tryptic peptides. The remaining one-third of the pooled bioactive heparin fractions was further purified by reverse-phase chromatography and SDS-PAGE for N-terminal sequence analysis. These results were ambiguous and are not presented here. For bioassay, reverse-phase fractions were dried by vacuum centrifugation and redissolved in culture medium. Heparin-5PW fractions were diluted directly into culture medium. Protein concentrations were determined by amino acid analysis. Calculations of molar concentration assume mature ARIA to have a polypeptide molecular mass of 23 kd. SDS gel analysis was performed by standard methods. Gels were 13% polyacrylamide, and the samples were prepared in a buffer that did not contain reductant. Peptfde Sequence Detemlnatlon ARIA was reduced, S-carboxyamidomethylated, alkylated, and digested with sequencing grade trypsin (Boehringer Mannheim). The substrate-trypsin (25:l [w/w]) mixture was maintained at 37OC for 1620 hr (Stone et al., 1969). Tryptic ARIA peptides were separated by narrowbore reverse-phase chromatography on a Hewlett-Packard 1090 high pressure liquid chromatographer equipped with a 1040 diode array detector, using a Vydac Cl6 column (2.1 x 150 mm). Peptides were eluted with a gradient of acetonitrile in 0.06% trifluoroacetic acid at a flow rate of 150 ullmin. Peaks, defined by absorbance at 210 nm, were manually collected into 1.5 ml microcentrifuge tubes. Strategies for the selection of peptide fractions and sequencing by Edman degradation were as previously described (Lane et al., 1991). N-terminal sequence analysis data of the peak 24 and 29 fractions showed a single unambiguous amino acid signal at each cycle (numbered bars 2 and 3 of Figure 3, respectively). For the peak 25,44, and 46fractions signals representing more than 1 amino acid were present in some cycles, indicating that these fractions might contain more than one peptide. The signal amplitudes were very low (initial yields 2.2 to 0.3 pmol), making assignment of residues to primary, secondary, or tertiary sequence difficult. Only the unambiguous sequences were useful for the cloning of proARIA-1. However, after the sequence of proARIAhad been determined, it was possible to infer the peptides contained in the three fractions with ambiguous sequences. This was done by comparing the chemically determined amino acid sequence data with the proARIAsequence and assigning residues to peptide sequences to maximize

identity. In no case were residues moved to different cycles. (Numbers in square brackets following the peptides indicate the numbered bar of Figure 3 bounding this peptide sequence.) For peak 25. the inferred peptides are: LVL--T [l], ETTSEYPALR [l], and -PENV [2]. For peak 44, the inferred peptides are: -FCV 141. VLRCET [l], and -NEFT [S]. For peak 46, the peptides are CPNEFTG-GNW [5] and CQNYV-A [5]. As would be expected for tryptic peptides, these peptides all align with proARIAsequence immediately C-terminal to a basic residue. The sequences inferred for these peptides allowed us to conclude that purified brain ARIA contains a component with a P-type EGF-like domain. Isolation of cDNA clones PCR Oligonucleotides were synthesized based upon the sequences NRPENVK and ATLADAGEYACR, which were obtained from the peptides in peaks 24 and 29. A pair of sense oligonucleotides, differing at their 3’ ends (5”GICCIGARAAYGTNAAG-3’ and 5’-GICCIGARAAYGTNAAA-3’) corresponding to the peptide RPENVK, and a nested pair of antisense oligonucleotides corresponding to the peptides ADAGEY (5’-TAYTCICCIGCRTCNGC-3’) and GEYACR (B’-CKRCAIGCRTAYTCNCC-3’) were obtained from Genosys (The Woodlands, TX). Single stranded cDNA was synthesized in a 50 ul reaction from 2 ug of El9 chick spinal cord total RNA using Superscript H- Moloney murine leukemiavirus reverse transcriptase (GIBCO BRL, Gaithersburg. MD). Five microliters of cDNA synthesis reaction was used for PCR with 1 ug of each of the sense and antisense oligonucleotides in a 100 ul final volume containing 400 uM dNTPs, 1 x Taq buffer, and 5 U of Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN). The reaction was heated to 94°C for 5 min followed by 40 cycles of 94OC for 1 min, 44OC for 1 min, and 72OC for 1 min. PCR products were purified from an agarose gel and subcloned into the vector pCR-II using the TA cloning kit (Invitrogen, San Diego, CA). Library Screening and Clone Isolation After verification of the identity of cloned PCR products by DNA sequencing analysis, the 97 bp PCR-generated cDNA was labeled by random oligonucleotide priming and used to screen an El3 chicken brain cDNA library in bacteriophage 5gtlO (kindly provided by Dr. B. Ranscht). Positive clones were isolated by repetitive screening, and the insert size was estimated by PCR using phage-derived primers to amplify the insert. The insert of the largest clone (112) was subcloned into the plasmid pGEM7Zf(+) (Promega). DNA Sequence Analysis Both the entire 112 insert subcloned into pGEMJZf(+) and specific restriction fragments subcloned into bacteriophage Ml3 were subjected to DNA sequence analysis. Nucleotide sequences were determined by the dideoxy chain termination method (Sanger et al., 1977) using an Applied Biosystems 373A automated DNA sequencer and Applied Biosystems Taq DyeDeoxy Terminator cycle sequencing kits and/or [a-=P]dATP (New England Nuclear, Boston, MA) and Sequenase (US Biochemicals). Both strands of the 512 insert were fully sequenced. Nucleic acid and amino acid sequence analysis was done using the MacVector (IBI) and GCG (Genetics Computer Group, Madison, WI) sequence analysis software packages. Sequence alignments were performed with the GCG program GAP (Needleman and Wunsch, 1970) with a gap weight of 3 and a length weight of 0.1. Searches of a nonredundant protein sequence data base (SwissProt+PIR+GenPept+GPUpdate) were done through the BLAST network service with computation at the National Center for Biotechnology Information using a local alignment algorithm (Altschul et al., 1990). Expression of ProARIAThe entire 112 insert was subcloned into the eukaryotic expression vector pcDNAl/AMP (Invitrogen, San Diego, CA). No signal sequence or other protein coding sequence is provided by the vector. Orientation was determined by restriction digest analysis and confirmed by sequencing. Plasmid DNA with the insert in the sense (~12.7) or antisense (~12.6) orientation was prepared for transfection by purification on Qiagen columns. COS7 cells (Gluzman, 1961) were obtained from the American Type Culture Collection (ATCC CRL 1651). Two protocols were used for the

Cdl 812

preparation of conditioned media. For earlier experiments (such as that shown in Figure 28) COS cells in 60 mm dishes were transfected using the cationic lipid preparation DOTAP (Boehringer Mannheim) according to the directions of the manufacturer. After 16 hr, the medium was changed to the medium used for chick myotube cultures and AChR incorporation rate bioassay. Following 48 hr of conditioning, the medium was centrifuged at 1000 x g for 10 min to remove debris, then filtered through a 0.45 urn filter to remove debris. In some cases the medium was stored at -20°C prior to use in the assay. While conditioned medium prepared as described above was suitable for the tyrosine phosphorylation assay (which required only 1 hr incubations), it was toxic to chick myotubes when applied at concentrations effective in increasing the AChR incorporation rate for the 18 to 24 hr required for this assay. The toxicity was manifested by morphological changes and, in many cases, by a depression of the AChR insertion rate in cells treated with medium from pl2.8-transfected COS cells. Therefore, for later experiments (such as that shown in Figure 2A), COS cells were transfected by electroporation, plated in T75 flasks, and after 24 hr transferred to 1% fetal calf serum-Dulbecco’s modified Eagle’s medium. Following 48 hr of conditioning, this medium was prepared for assay by centrifugation and filtering (as above) and concentrated using a centrifugal ultrafiltration device with a nominal molecular weight cutoff of 10,000 (Centriprep 10; Amicon) (see Wen et al., 1992). In the experiment shown in Figure 2A, the ~12.6 medium was concentrated 23-fold and the ~12.7 medium was concentrated 27-fold. Medium prepared by this protocol from ~12.7 (sense)transfected COS cells increased the AChR incorporation rate at concentrations that had no evident morphological effect. Further, medium prepared by this protocol from ~12.6 (antisense)-transfected COS cells did not significantly alter the AChR insertion rate compared with that in untreated controls. Northern Blot Analysis Total RNA was isolated (Chomczynski and Sacchi, 1987) from brain and spinal cord tissue pooled from several animals. For Northern blots (Rosen et al., 1990) 10 ug of total RNA was fractionated on 1.3% agarose-2.2 M formaldehyde surface tension gels and transferred to charged nylon membranes (MagnaGraph, MSI). Filters were hybridized overnight at 45OC in buffer containing 50% formamide, 6x SSC (1 x SSC = 0.15 M sodium chloride, 0.015 M sodium citrate), 2x Denhardt’s solution (1 x Denhardt’s = 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone, 0.02% ficoll), 5% dextran sulfate, 100 pgl ml salmon sperm DNA, and labeled probe at 2 x 108 cpmlml. Probes were prepared by random oligonucleotide priming (Feinberg and Vogelstein, 1983) using [a-“P)dCTP and a commercially available labeling kit (Stratagene, La Jolla, CA). Following hybridization, the filters were washed to a final stringency of 0.1 x SSC, 0.1% SDS at 50°C wrapped in plastic wrap, and exposed to Kodak XAR-5 film with intensifying screens (DuPont Lightning Plus) at -8OOC. Transcript size was estimated by comparison with a series of RNA standards (GIBCO BRL). Southern Blot Analysis Chicken genomic DNA was isolated by the procedure of Bell et al. (1981). Southern blotting was performed essentially as described by Southern (1975). Following digestion and electrophoresis, the samples were transferred to a charged nylon membrane (MagnaGraph, MSI). Probe preparation and hybridization were performed as described for Northern blots. In Situ Hybridization Spinal cords from E7chick embryos were dissected and fixed for 16 hr in freshly prepared cold 4% paraformaldehyde in phosphate-buffered saline. Tissue was then slowly dehydrated and embedded in paraffin (Sassoon et al., 1988). Serial sections (7 urn) were collected individuallyon glass microscope “subbed” slides (Gall and Pardue, 1971). The procedures used for section treatment, hybridization, and washings were as described by Sassoon et al. (1988). Hybridization was carried out at 52OC for approximately 16 hr in 50% deionized formamide, 0.3 M sodium chloride, 20 mM Tris-HCI (pH 7.4) 5 mM EDTA, 10 mM NaP04 (pH 8) 10% dextran sulfate, 1 x Denhardt’s solution, 50 uglml total yeast RNA with 3 x 10’ cpmlul =S-labeled RNA probe under siliconized coverslips. Following hybridizations, coverslips were

floated off in 5 x SSC, 10 mM dithiothreitol at 50°C, and washed in 50% formamide. 2x SSC, 0.1 M dithiothreitol at 65’C. Slides were then rinsed in washing buffer, treated with RNAase A (20 ug/ml; Sigma), and washed at 37OC for 15 min in 2x SSC and then for 15 min in 0.1 x SSC. Sections were dehydrated rapidly, processed for autoradiography using NTB-2 Kodak emulsion, exposed for 4-l 4 days at 4OC, and examined using both light- and dark-field optics on a Zeiss microscope. The template for probe transcription was a plasmid containing base pairs 15 to 344 of the sequence shown in Figure 3 in the vector pGEM7Zf(+) (Promega). Sense and antisense [a-“S]UTP (>lOOO Cilmmol, New England Nuclear)-labeled RNA probes were generated by runoff transcription of the restriction digested plasmid using T7 or SP6 RNA polymerase. Nomenclature Note In past publications, we have used the acronym ARIA (for acetylcholine receptor-inducing activity) to refer to an - 42 kd protein purified from acid extracts of chicken brain that stimulates synthesis of AChRs. Here we use ARIA to refer specifically to the protein encoded by the proARIAcDNA reported in this paper and to other protein products of the same gene (and its homologs in other species) that stimulate the synthesis of AChR. We assume that the purified brain protein from which we obtained tryptic peptides is a product of the AR/A gene. Acknowledgments We thank Randy Brenner. Richmond Hung, Renee Robinson, and Mary Gordy for expert technical assistance. We also thank Kathleen M. Buckley, Jeffrey B. Miller, and Jonathan B. Cohen for helpful criticism of the manuscript, Julie Nardone for her advice on transfection techniques, Giovanna Marazzi for her advice on the in situ hybridization, and Sylvia Colard-Keene for her assistance in the preparation of figures. This work was supported by a grant from the National Institute of Neurological Diseases and Stroke (G. D. F.), a Physician Scientist Award from the National Institute of Child Health and Human Development (D. L. F.), and fellowships from the Harvard Mahoney Neuroscience Institute (K. M. R.) and the Muscular Dystrophy Association (G. C.). Received

February

5, 1993; revised

February

16, 1993.

References Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990). Basic local alignment search tool. J. Mol. Biol. 275, 403-410. Baker, L. P., and Peng, H. B. (1993). Tyrosine phosphorylation and acetylcholine receptor cluster formation in cultured Xenopus muscle cells. J. Cell Biol. 120, 185-195. Baker, L. P., Chen, Q., and Peng, H. B. (1992). Induction of acetylcholine receptor clustering by native polystyrene beads: implication of an endogenous muscle-derived signaling system. J. Cell. Sci. 702, 543555. Bargmann, C. I., Hung, M. C., and Weinberg, R. A. (1986). The neu oncogene encodes an epidermal growth factor receptor-related protein. Nature 319, 228-230. Bell, G. I., Karam, J. H., and Rutter, W. J. (1981). Polymorphic region adjacent to the 5’ end of the human insulin gene. Proc. Acad. Sci. USA 78, 5759-5763.

DNA Natl.

Bosenberg, M. W., Pandiella, A., and Massague, J. (1992). The cytoplasmic carboxy-terminal amino acid specifies cleavage of membrane TGFa into soluble growth factor. Cell 71, 1157-l 165. Brenner, H. R., and Sakmann, B. (1978). Gating choline receptor in newly formed neuromuscular 277, 366-368.

properties of acetylsynapses. Nature

Brenner, H. R., Witzemann, V., and Sakmann, B. (1990). Imprinting of acetylcholine receptor messenger RNA accumulation in mammalian neuromuscular synapses. Nature 344, 544-547. Brenner, H. R., Herczeg, A., and Oesch, B. (1992). Normal develop ment of nerve-muscle synapses in mice lacking the prion protein gene. Proc. R. Sot. Lond. (6) 250, 151-155.

ARIA, 813

a Protein

Regulating

AChR

Synthesis

But-Caron, M. H., Nystrom, P., and Fischbach, G. D. (1983). Induction of acetylcholine receptor synthesis and aggregation: partial purification of low-molecular-weight activity. Dev. Biol. 95, 378-386. Bursztajn, S., Berman, S. A., and Gilbert, W. (1990). Factors released byciliary neurons and spinal cord explants induce acetylcholine receptor mRNA expression in cultured muscle cells. J. Neurobiol. 21,387399. Cagan, R. L., Kramer, H., Hart, A. C., and Zipursky, S. L. (1992). The bride of sevenless and sevenless interaction: internalization of a transmembrane ligand. Cell 69, 393-399. Carpenter, G., and Cohen, Chem. 265, 7709-7712.

S. (1990).

Epidermal

growth

factor.

J. Biol.

Furley, A. J., Morton, S. B.. Manalo, D., Karagogeos, D., Dodd, J., and Jessell, T. M. (1990). The axonal glycoprotein TAG-l is an immunoglobulin superfamily member with neurite outgrowth-promoting activity. Cell 87, 157-170. Gall, J. G., and Pardue, cytological preparations.

M. L. (1971). Nucleic acid hybridization Meth. Enzymol. 27, 470-480.

Gluzman, Y. (1981). SV40-transformed simian cells support cation of early SV40 mutants. Cell 23, 175-182.

in

the repli-

Godfrey, E. W., Nitkin, R. M., Wallace, B. G., Rubin, L. L., and McMahan, U. J. (1984). Components of Torpedo electric organ and muscle that cause aggregation of acetylcholine receptors on cultured muscle cells. J. Cell Biol. 99, 615-627.

Carpenter, G., and Wahl, M. I. (1990). The epidermal growth factor family. In Peptide Growth Factors and Their Receptors, M. B. Sporn and A. B. Roberts, eds. (New York: Springer-Verlag), pp. 69-171.

Goldman, D., and Staple, J. (1989). Spatial and temporal expression of acetylcholine receptor RNAs in innervated and denervated rat soleus muscle. Neuron 3, 219-228.

Changeux, J. P. (1991). Compartmentalized line receptor genes during motor endplate 413-429.

transcription epigenesis.

Goldman, D.. Carlson, B. M., and Staple, J. (1991). type nicotinicacetylcholine receptor geneexpression regenerating muscle. Neuron 7, 649-658.

Chao, M. V. (1992). Neurotrophin receptors: differentiation. Neuron 9, 583-593.

a window

of acetylchoNew Biol. 3, into neuronal

Chomczynski, P., and Sacchi, N. (1987). Single-step method isolation by acidic guanidinium thiocyanate-phenol-chloroform tion. Anal. Biochem. 162, 156-159.

of RNA extrac-

Induction of adultin noninnervated

Gordon, M. Y. (1991). bound and free. Cancer

Hemopoietic growth Cells 3, 127-l 33.

factors

and receptors:

Hall, Z. W., and Sanes, ment: the neuromuscular 121.

J. R. (1993). Synaptic structure and develop junction. Cell 72/Neuron 70 (Suppl.), 99-

Cohen, J. A., Yachnis, A. T., Arai, M., Davis, J. G., and Scherer, S. S. (1992). Expression of the neu proto-oncogene by Schwann cells during peripheral nerve development and Wallerian degeneration. J. Neurosci. Res. 37, 622-634.

Harris, D. A., Falls, D. L., Dill-Devor, R. M., and Fischbach, G. D. (1988). Acetylcholine receptor-inducing factor from chicken brain increases the level of mRNA encoding the receptor a-su bunit. Proc. Natl. Acad. Sci. USA 85, 1893-1897.

Cohen, S. A., and Fischbach, G. D. (1977). receptors located at identified nerve-muscle Biol. 59, 2436.

Harris, D. A., Falls, D. L., and Fischbach, G. D. (1969). Differential activation of myotube nuclei following exposure to an acetylcholine receptor-inducing factor. Nature 337, 173-176.

Clusters of acetylcholine synapses in vitro. Dev.

Corfas, G., and Fischbach, G. D. (1993). The number of Na’channels in cultured chick muscle is increased by ARIA, an acetylcholine receptor-inducing activity. J. Neurosci. 13, in press.

Harris, D.A., Falls, D. L., Johnson, F. A., and Fischbach, G. D. (1991). A prion-like protein from chicken brain copurifies with an acetylcholine receptor-inducing activity. Proc. Natl. Acad. Sci. USA 88,7664-7668.

Corfas, G., Falls, D. L., and Fischbach, G. D. (1993). ARIA, a protein that stimulates acetylcholine receptor synthesis, also induces tyrosine phosphorylation of a 185kDa muscle transmembrane protein. Proc. Natl. Acad. Sci. USA 90, 1624-1628. Coussens, L.. Yang-Feng, T. L.. Liao. Y. C., Chen, E., Gray, A., McGrath, J., Seeburg, P. H., Libermann, T. A., Schlessinger, J., Francke, U., Levinson, A., and Ullrich, S. A. (1965). Tyrosine kinase receptor with extensive homology to EGF receptor shares chromoSomal location with neu oncogene. Science 230, 1132-1139.

Holmes, W. E.. Sliwkowski, M. X., Akita. R. W., Henzel, W. J., Lee, J., Park, J. W., Yansura, D., Abadi, N., Raab, H., Lewis, G. D., Shepard, H. M., Kuang, W.-J., Wood, W. I., Goeddel, D. V., and Vandlen, R. L. (1992). Identification of heregulin, a specific activator of p185erbB2. Science 256, 1205-1210.

Dahm, L. M., and Landmesser, L. T. (1991). The regulation of synaptogenesis during normal development and following activity blockade. J. Neurosci. 77, 238-255.

Horovitz, O., Spitsberg, V., and Salpeter, M. M. (1989b). acetylcholine receptor synthesis at the level of translation muscle cells. J. Cell Biol. 708, 1817-1822.

Davis, J. G., Hamuro, J., Shim, C. Y., Samanta, A., Greene, M. I., and Dobashi, K. (1991). Isolation and characterization of a neu proteinspecific activating factor from human ATL-2 cell conditioned medium. Biochem. Biophys. Res. Commun. 179, 1536-1542.

Huang, C.-F., Tong, J., and Schmidt, J. (1992). Protein kinase C couples membrane excitation to acetylcholine receptor gene inactivation in chick skeletal muscle. Neuron 9, 671-678.

Devreotes, P. N.. and Fambrough, D. M. (1975). Acetylcholine receptor turnover in membranes of developing muscle fibers. J. Cell Biol. 65, 335-358. Dobashi, K., Davis, J. G., Mikami, Y., Freeman, J. K., Hamuro, J., and Greene, M. I. (1991). Characterization of a neulc-erbB-2 proteinspecific activating factor. Proc. Natl. Acad. Sci. USA 88, 8582-8586. Falls, D. L., Harris, D. A., Johnson, F. A., Morgan, M. M., Corfas, G., and Fischbach, G. D. (1990). Mr 42,000 ARIA: a protein that may regulate the accumulation of acetylcholine receptors at developing chick neuromuscularjunctions. Cold Spring Harbor Symp. Quant. Biol. 55, 397-406. Feinberg, A. P., and Vogelstein, ing DNA restriction endonuclease Anal. Biochem. 732, 6-13.

B. (1983). A technique for radiolabelfragments to high specific activity.

Flanagan, J. G., Chan, D., and Leder, P. (1991). Transmembrane form of the kit ligand growth factor can be regulated by alternative splicing and is deleted in the SP mutant. Cell 64, 1025-1035. Fontaine, B., and Changeux, J.-P. (1969). Localization of nicotinic acetylcholine receptor a-subunit transcripts during myogenesis and motorendplatedevelopment in the chick. J. Cell Biol. 708,1025-1037.

Horovitz, O., Knaack, D., Podleski, T. R., and Salpeter, M. M. (1989a). Acetylcholine receptor a-subunit mRNA is increased by ascorbic acid in cloned L5 muscle cells: northern blot analysis and in situ hybridization. J. Cell Biol. 708, 1823-1832. Regulation of in rat primary

Huang, S. S., and Huang, J. S. (1992). Purification and characterization of the neulerb 82 ligand-growth factor from bovine kidney. J. Biol. Chem. 287, 11508-11512. Hunkapiller, T., and Hood, L. (1969). Diversity gene superfamily. Adv. Immunol. 44, l-63.

of the immunoglobulin

Jessell, T. M., Siegel, R. E., and Fischbach, G. D. (1979). Induction of acetylcholine receptors on cultured skeletal muscle by a factor extracted from brain and spinal cord. Proc. Natl. Acad. Sci. USA 76, 5397-5401. Jo, S. A., and Burden, S. J. (1992). Synaptic basal lamina contains a signal for synapse-specific transcription. Development 7 75, 673-680. Kandel, J., Bossy-Wetzel, E., Radvanyi, F., Klagsbrun, M., Folkman, J., and Hanahan, D. (1991). Neovascularization is associated with a switch to the export of bFGF in the multistep development of fibrosarcoma. Cell 88, 1095-l 104. Kirilovsky, J., Duclert, A., Fontaine, B., Devillers, T. A., Osterlund, M., and Changeux, J. P. (1969). Acetylcholine receptor expression in primary cultures of embryonic chick myotubes. II. Comparison between the effects of spinal cord cells and calcitonin gene-related peptide. Neuroscience 32, 289-296.