Acetylcholine receptor biosynthesis: from kinetics to molecular mechanism

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Acetylcholine receptor biosynthesis: from kinetics to molecular mechanism David J. Anderson The study o f acetylcholine receptor (A ChR) biosynthesis is currently in a transition period. New approaches and reagents have made it possible to explore previously inaccessible aspects o f this problem. Formerly, studies o f A ChR synthesis treated the receptor as a single entity, defined solely by its ability to bind ligands such as a-bungarotoxin. Thus, our understanding o f this complex process was inferred exclusively from measurements o f the kinetics with which this entity moved through the cell and was inserted into the plasma membrane. However, as the result o f extensive efforts at biochemical characterization o f A ChR, we now know that the protein is a tightly associated non-covalent complex o f four large (approximate mol. wt 50 000) glycoprotein subunits, in stoichiometry a2fl'y8 (Ref. 13). The problem o f AChR biogenesis must therefore be entirely reconsidered in the context o f this new perspective. Questions have been raised relating to the synthesis o f the individual subunits, their insertion into the membrane, and their assembly into a functional oligomeric complex. To approach these questions, it is necessary to examine directly the synthesis o f the constituent AChR polypeptides. This has only recently become possible with the advent o f A ChR subunit-specific antibodies, both polyclonal ~4and monoclonal 2~. In this review, I shall describe how the use o f these new reagents has allowed us to begin to think about the synthesis o f AChR in terms o f discrete, molecular events. However, it should become clear that the biogenesis o f this important membrane protein is a much more complicated process than we originally thought. Cell biology of AChR synthesis The kinetics of AChR synthesis have been studied extensively in cultured embryonic muscle cells, where AChR is a trace protein. However, it can be measured precisely and sensitively using high specific-activity [125I]-ct- bungarotoxin (for review, see Ref. 8). Briefly, it was found that new synthesis of AChR occurs in less than 30 minutes, but that transport of this material to the cell surface requires 2-3 hours 5. This transport was both energy- and temperature-dependent, and could occur in the absence of ongoing protein synthesis. In fact, almost 20% of the total cellular AChR content consists of a pool of 'preassembled' intracellular receptors en route to the plasma membrane s . At least some of these receptors were localized, by EM autoradiography, to the Golgi region9. This was an important early demonstration that an integral membrane protein followed the same intracellular transport pathway as that proposed for secretory proteins by Palade 21. (Although this has been disputed recently24, more extensive investigations have questioned these findings 2~ and confh-med ~) 1983. Elsevie, Science Publishers B.V., Amsterdam

the original contention.) While providing crucial, new information, the studies mentioned above were ultimately limited by the fact that a-bungarotoxin cannot detect AChR at its earliest stages in biosynthesis (see below). The study of these early events thus required direct measurement of AChR subunit synthesis. Immunological probes for AChR subunits have recently allowed determination of some of the discrete molecular events underlying the kinetics measured previously. One of the earliest of these events is the synthesis of the subunits, from separate mRNAs, by membrane-bound ribosomes of the rough endoplasmic reticulum (RER). This has been demonstrated by two independent approaches, both utilizing antisubunit antibodies to detect the in vitro translation products of AChR mRNA (Ref. 15). Merlie and co-workers have demonstrated synthesis of the a subunit by membrane-bound polysomes isolated from the BC3H-1 muscle-like tumor cell line ~G. Work from Gunter Blobel's laboratory has established that all four subunits of the Torpedo AChR are, like secretory pro-

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teins, co-translationally transferred into rough microsomal membranes in vitro ". In other words, the synthesis of the subunits was examined by a cell-free reconstruction of the events which, in the cell, normally occur in the RER. It was further established in this system that each subunit is translated from a separate mRNA (Ref. 1); this has subsequently been confirmed by 'Northern blot' experiments using cloned AChR cDNA probes ~.2°.

Insertion of AChR subunits into the membrane The independent synthesis of each of the four AChR subunits from membranebound ribosomes implies that these subunits, like other membrane and secretory proteins, are synthesized with 'signal peptide' extensions at their NH2-termini4. This was directly demonstrated in the case of the 8 subunit by partial radiosequence analysis of the primary translation product synthesized in vitro ~. Subsequently, such signal sequences have been detected for the o~ (Ref. 20) and 3' (Ref. 3) subunits by sequence analysis of cDNA clones. How are the AChR subunits inserted into the lipid bilayer? It is the signal sequence that directs the asymmetric integration of the polypeptide into the RER membrane, by a co-translational mechanism that utilizes protein-protein 4, rather than protein-lipid 7, interactions. The information contained in the signal sequence is decoded by a soluble, 11S ribonucleoprotein complex, termed signal recognition particle (SRP), which consists of six polypeptide subunits and one molecule of 7S-L RNA (Ref. 27). SRP forms a high-affinity 'bridge' between polysomes whose nascent proteins contain signal sequences, and an integral membrane protein receptor in the RER~°'xL The complex then initiates translocation of the NH2-terminal portion of the nascent polypeptide across the lipid bilayer. In the case of membrane proteins, this transfer process is at some point interrupted, presumably due to the emergence of appropriate hydrophobic amino acid residues from the ribosome 4. In the case of the AChR subunits, synthesis of the polypeptide continues on the cytoplasmic surface of the RER, leaving in each case ~ a fairly large (mol. wt 5-20 000) untranslocated domain. (In the case of the 8 subunit, this domain 2s contains a phosphorylation site.) The precise means whereby the subunits achieve their topology in the lipid bilayer is, however, unknown. Post-translational transport and assembly of the AChR complex The four AChR subunits are not assem-

17U bled with one another into a functional complex immediately upon insertion into the RER membrane. Rather, multimeric assembly o f AChR appears to be a lengthy, post-translational process. Thus, the invitro-synthesized a subunit does not exhibit high-affinity binding to ~-bungarotoxin t, and in cultured cells does not acquire this capacity until some 30 rain post-synthesis~L This finding suggests that assembly requires transport of the individual subunits out of the RER, to the Golgi apparatus ~7, by analogy to the case of viral membrane glycoproteins H. Although lack of high-affinity toxin-binding by the early biosynthetic form of the a subunit is correlated with lack of inter-subunit associations, this should not be taken to imply that acquisition of the toxin-binding site requires multimeric assembly. Unfortunately, there is no direct evidence regarding the relative order of these two events. Interestingly, however, X e n o p u s oocytes were found to be capable of complete intracellular transport and assembly of a functional a° AChR when injected with a total mRNA fraction from Torpedo 2~. This finding argues that the capacity to assemble AChRs is not a unique property of nerve or muscle cells. It has not yet been possible to directly determine the intracellular compartment in which AChR assembly occurs. Part of the reason for this is the inherent technical difficulty of fractionating tissue-cultured cells. Another reason is the lack o f an appropriate cell system in which the synthesis of each of the four AChR subunits can be followed, at the earliest stages, with subunit-specific antibodies. Detection o f early biosynthetic forms o f the y and ~ chains in the BC3H- 1 cell line, for example, has been frustrated by both rapid endogenous proteolysis and lack of appropriate antibodies rsvp8. Tissue culture of dissociated Torpedo electrocytes is unfortunately not possible on a routine basis at this time. There is hope, however, that the introduction of recombinant DNA technology to the study of AChR will provide new approaches to this problem. All four of the AChR subunits receive Asn-linked 'core' oligosaccharide side chains during their integration into the RER membrane The c~ and /3 chains each receive one core group ~'~s, while 8 receives three, and 3/ at least three and perhaps four ~2. The exact function of these carbohydrates in mature AChR is not known; however it has been suggested22 that they may play a role in controlling the degradation rate of the molecule. The core oligosaccharides are presumably trimmed and/or modified during subsequent transport through the Golgi apparatus, by analogy to cases of viral glycoproteins TM studied

earlier. However, there is minimal evidence as to the precise nature of these changes in the case of AChR. Studies in cultured chick embryonic myotubes imply that the c~ subunit, at least, does not undergo terminal glycosylation in contrast to the previous cases (D. Anderson, unpublished observations). It is likely that the subunits 'also receive 'O-linked' carbohydrates in the Golgi as do the viral glycoproteins TM. The function of these transitory glycosylation events is not known; studies using inhibitor)' drugs such as tunicamycin ~s.22 have yielded conflicting results in different systems. The confusion regarding this question in large part reflects our more basic ignorance of the fundamental role of these carbohydrate modifications during glycoprotein biosynthesis. Another unsolved problem concerns the stoichiometry of AChR subunit synthesis. Merlie et al. ts have found that the o~ subunit is synthesized in excess of the amount of functional AChR that is made. This suggests the other subunit may exist in limiting quantities, but this has not been shown. The assembly of a hydrophilic ion channel through the membrane from multiple, independent subunits poses a novel problem. Structural studies of mature AChR suggest that each subunit borders the ionophore 29 and must therefore have a hydrophilic surface embedded in the lipid bilayer. It might be energetically unfavorable for these surfaces to contact the hydrocarbon interior of the membrane prior to oligomeric assembly. One solution to this problem would require that the subunits

adopt a different transmembrane conlbnnation prior to quaternary assembly. However, this seems not to be the case, based on a comparison of the major proteolytic sub. fragments that can be generated from the RER Iorm of the subunits and from their mature counterparts 28 (Anderson et al., J. Neurosci., in press). Another solution to the problem would be the lbrmation of homo-oligomers by each subunit, resulting in the sequestration of the hydrophilic ionophore surfaces in the interior of these aggregates. The mature complex would then have to be formed by a series of ordered exchange reactions between these homo-oligomers. An evolutionary 'precedent' exists for such structures, in that the contemporary AChR subunits are thought to have evolved by gene duplication, implying that the (ancestral) AChR was a homooligomer. Furthermore, in the contemporary case, synthesis of multiple copies of the same subunit by the multiple ribosomes on its mRNA would provide a high local concentration of the same chain, even if the four different mRNAs were quite distant from one another. Evidence tbr such homologous associations has recently come from in-vitro systems (Anderson and Blobel, Proc. Nail Acad. Sci. U S A , in press); it remains to be seen whether such homo-oligomeric forms of the newly synthesized subunits exist in vivo.

Perspective The post-translational assembly of a receptor-ionophore complex is a problem without precedent in the field of membrane

TABLE1. Summaryof acetylcholinereceptorbiosynthesis 1 Acetylcholinereceptor(AChR) is a non-covalentcomplexof fourdistinctintegralmembraneglycoprotein subanits. Recentwork has shed light on the mechanismof its synthesisand post-translationalassemblyl 2 AChRsubunitsare translatedindependently,ti'omseparatemRNAs,on membrane-boundribosomesof the rough endoplasmicreticulum (RER). 3 Insertionof these subunitsinto the membraneis a co-translationalprocess, it utilizestwo recentlypurified proteincomponentsof the RER. These proteinmediatorsact upon informationcontainedin the signalpeptide extensionswhichare presenton each of the nascentAChRpolypeptidechains. The signalsequencesare proteolyticallyremovedfollowingmembraneinsertion.'Core' glycosylationof the subunitsalso occursduring this process. 4 AChR subunitsappear not to he assembledwith one anotherin the RER. The high-affinitytoxin-binding activity is also not displayedat this stage. 5 2-3 hoursare requiredto transportnewlysynthesizedAChR subunitsto the cell surface.High-affinity toxin-bindingactivityappearsduringthe first30 minutesof thisperiod.Heterologoussubunitassemblyappears to take at least as long, and perhapslonger. It has been suggestedthat assemblymay requiretransportto the Golgi apparatus. 6 Furtherglycosylationof AChRis thoughtto occurduring this periodof transport,but its significanceis not known. 7 Evidencefromcell-freesystemssuggeststhe AChR subanitsmay serf-associate,prior to complexformation. Theseassociationscould serveto stabilizehydrophiliesurfacesof the subuhits,buriedin the membrane,which will eventuallyform the ion channel in the mature complex.There is no evidencethat such homologous associationsoccurin vivo. 8 Recentadvancesin cloningAChR subunitgenes shouldmake it possibleto analysein turn the synthesisand processingof AChR mRNA.

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TINS-May 1983 protein biogenesis. Thus, the case of AChR ous regulatory influences. Recently, a is particularly exciting in that its solution number of laboratories have announced the may provide general principles applicable cloning of genes coding for the various to a wide range of systems. None the less, it AChR subunits3.2°. These cloned genes can should be remembered that AChR shares themselves be used as specific reagents to many aspects of its post-translational pro- measure the synthesis, processing and decessing in common with simple, single- gradation of AChR mRNA. This technosubunit transmembrane glycoproteins of logical advance raises the exciting prospect the type found in enveloped animal viruses. that it will soon be possible to analyse the Because of their simplicity and experimen- molecular basis of these regulatory tal accessibility, these viral systems have phenomena, down to the transcriptional historically been the first to reveal the basic level. Thus, AChR biosynthesis, once the events in the process of membrane protein exclusive domain of physiologists and cell synthesis. Thus, many of our notions about biologists, has now moved into the realm of AChR biosynthesis are founded on molecular biology. analogies to the viral proteins 11,~. Fortunately, direct experimental evidence has Reading list 1 Anderson, D. J. and Blobel, G. (1981 ) Proc. Nad shown this analogy to be valid in certain Acad. Sci. U.S.A. 78, 5598-5602 respects, such as those concerning the ini2 Anderson, D. J., Walter, P. and Blobel, G. (1982) tial membrane insertions and subsequent J. Cell Biol. 93,501-506 transport to the Golgi apparatus 9. However, 3 Ballivet, M., Patrick, J., Lee, J. and Heinemann, it is important to realize that many fundaS. (1982) Proc. Natl Acad. Sci. U.S.A. 79, mental problems in membrane biogenesis 4466--4470 4 Blobel, G. (1980) Proc. Natl Acad. Sci. U.S.A. have yet to be solved, even in the most sim77, 1496-1500 ple viral systems. Those who maintain an 5 Devreotes, P . N . , Gardner, J . M . and Famactive interest in the synthesis of AChR brough, D. M. (1977)Cell 10, 365-373 would thus be well-advised to keep abreast 6 Dunphy, W. G., Fries, E., Urbani, L. J. and of contemporary developments in the viral Rothman, J. E. (1981) Proc. Natl Acad. Sci. field (see, for example, Ref. 6). With the U.S.A. 78, 7453-7457 application of recombinant-DNA technol7 Engleman, D. M. and Steitz, T. A. (1981) Cell 23, 411-422 ogy, this area is likely to yield important 8 Fambrough, D. M. (1979) Physiol. Rev. 59, and profound results within the next few

Elsevier/North-Holland Biomedical Press, New York 14 Lindstrom, J., Einarson, B. and Merlie, J. (1978) Proc. Natl Acad. Sci. U.S.A. 75, 76%773 15 Mendez, B., Valenzuela, P., Martial, J. A. and Baxter, J. D. (1980)Science 209, 695-697 16 Merlie, J. P., Hoffler, J. and Sebbane, R. (1981) J. Biol. Chem. 256, 6995-6999 17 Merlie, J. P. and Sebbane, R. (1981) J. Biol. Chem. 256, 3605-3608 18 Merlie, J. P., Sebbane, R., Tzartos, S. and Lindstrom, J. (1982)J. Biol. Chem. 257, 2694--2701 19 Meyer, D. 1., Krause, E. and Dobberstein, B. (1982) Nature (London) 297,647-650 20 Noda, M., Takahashi, M., Tanaka, T., Toyosato, M., Furatani, Y., Mirose, T., Asai, M., lnayama, S., Miyata, T. and Numa, S. (1982) Nature (London) 299,793-797 21 Palade, G. E. (1975)Science 189, 347-358 22 Prives, J. and Bar-Sagi, D. ( 1982)J. Cell Biol. 95, 416a 23 Rotundo, R. L. and Fambrough, D. M. (1980) Cell 22,595-602 24 Smilowitz, H. (1980) Cell 19, 237-244 25 Sumikawa, K., Houghton, M., Smith, J. C., Bell, L., Richards, B. M. and Bamard, E. A. (1981) Nature (London) 292, 862-864 26 Tzartos, S. J. and Lindstrom, J. (1980)Proc. Natl Acad. Sci. U.S.A. 77,755-759 27 Walter, P. and Blobel, G. (1982) Nature (London) 299, 691-698 28 Wennogle, L. P., Oswald, R., Saitoh, T. and Changeux, J.-P. (1981) Biochemistry 220, 2492-2497 29 Wise, D. S., Wall, J. and Karlin, A. (1981) J. Biol. Chem. 256, 12624-12627 Rcfecmc~a~k.dfiapc~f

years.

30 Barnard, E. A. et al. (1982) Proc. R. Soc. London, Set. B 215,241

The problem of AChR gene expression is perhaps of more profound interest to the neuroscientist, in that it concerns issues of central importance to developmental neurobiology: the regulation of synaptic protein synthesis by neurotrophic factors, and by synaptic activity. Both factors have been shown to affect AChR synthesis. Thus, AChR is a good model system for studying the mechanistic bases of these vari-

165-227 9 Fambrough, D. M. and Devreotes, P. N. (1978) J. Cell Biol. 76, 237-244 10 Gilmore, R., Walter, P. and Blobel, G. (1982) J. Cell Biol. 95,470-477 11 Green, J., Griffiths, G., Louvard, D., Quinn, P. and Warren, G. (1981)J. Mol. Biol. 152, 663~98 12 Hunt, P. W. and Summers, D. F. (1976)J. Virol. 20, 646--657 13 Karlin, A. (1980) in The Cell Surface and Neuronal Function (Cotman, C. W., Poste, G. and Nicolson, G . L . , eds), pp. 191-260,

David J. Anderson was at the Laboratory o.1"('ell Biology, The Rockefeller University, New York, N Y 10021, U.S.A. Current Address: The Institute of Cancer Research, Columbia University, College o f Physicians and Surgeons, 70l West 168th Street, New York, N Y 10032, U.S.A.