White, Michael M. and Mayne, Katharine Mixter and Lester, Henry A. and Davidson, Norman (1985) Mouse-Torpedo Hybrid Acetylcholine Receptors: Functional Homology Does not Equal Sequence Homology. Proceedings of the National Academy of Sciences of the United States of America, 82 (14). pp. 4852-4856. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:WHIpnas85
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The nicotinic acetylcholine (AcCho) receptor (AcChoR) is a multisubunit protein complex of stoichiometry alpha 2ß gamma delta . The several subunits show homology with each other within a given species; in addition, homology is found between analogous subunits between species. We have used the phage SP6 RNA polymerase transcription system to produce single-species RNA in vitro for various AcChoR subunits from cDNAs. Injection of an equimolar mixture of RNA for the alpha, ß, gamma, and delta subunits of Torpedo californica AcChoR into Xenopus oocytes results in the appearance of functional receptors in the oocyte membrane. No response to AcCho is detected when the ß or gamma subunit RNA is omitted, and a small response is seen when the delta subunit RNA is omitted. Replacement of Torpedo delta subunit RNA by the mouse BC3H-1 cell line AcChoR delta subunit RNA leads to the formation of functional receptors that show a 3-4-fold greater response to AcCho than does the full Torpedo complex. No response is seen when the mouse delta RNA replaces Torpedo gamma RNA. By amino acid homology profile comparisons, the mouse delta subunit appears to be moderately but not highly similar to the Torpedo delta subunit; the apparent similarity to the Torpedo gamma subunit is only slightly less. Therefore, the features of the primary sequence that determine the functional delta character of the mouse polypeptide are not revealed by simple homology comparisons.
|Additional Information:||Copyright © 1985 by the National Academy of Sciences Communicated by Norman Davidson, April 5, 1985 We thank Drs. T. Claudio, S. Heinemann, and D. Noonan for kindly providing Torpedo AcChoR cDNA clones, Dr. D. Melton for providing plasmid pSP62-PL and information concerning the SP6 transcription system, Drs. P. Sharp and M. M. Konarska for information concerning the use of GpppG in the transcription reaction, and Dr. N. Dascal for helpful suggestions on the handling of oocytes. We appreciate the counsel of Dr. J.H. Richards about the relation (or lack thereof) of primary sequence to functional domains in proteins. We are especially grateful to Dr. T. Steitz for suggesting the use of helical net projections in the search for significant homologies. This work has been supported in part by research grants GM-10991 (N.D.) and NS-11756 (H.A.L.) from the National Institutes of Health, a research grant from the Muscular Dystrophy Association to N.D., postdoctoral fellowships from the Procter and Gamble Co. and the Muscular Dystrophy Association to M.M.W., and Predoctoral Training Grant GM-07616 from the National Institutes of Health to K.M.M. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.|
|Subject Keywords:||Xenopus oocytes; SP6 RNA polymerase; cDNA clones; Ion channels; in ovo translation|
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|Deposited On:||27 Nov 2006|
|Last Modified:||14 Nov 2014 19:19|
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