Production and characterization of monoclonal antibodies sensitive to conformation in the 5HT2c serotonin receptor
- Filippo Mancia*,
- Susan Brenner-Morton†,
- Risa Siegel*,
- Zahra Assur*,
- Yonghua Sun†,
- Ira Schieren†,
- Monica Mendelsohn*,
- Richard Axel*,†, and
- Wayne A. Hendrickson*,†,‡
- *Department of Biochemistry and Molecular Biophysics and
- the †Howard Hughes Medical Institute, Columbia University, New York, NY 10032
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Contributed by Wayne A. Hendrickson, January 12, 2007 (received for review December 15, 2007)
Abstract
mAbs that are sensitive to protein conformation can be helpful in studies of protein structure and function; in particular, mAb fragments are useful reagents in membrane protein crystallization. We immunized mice with the rat 5HT2c serotonin receptor and derived clonal hybridoma cells, which we tested for specific antigen reactivity by using the complementarity of purified protein from bacteria and receptor-embedded mammalian cell membranes. Nine mAbs met our criteria for specificity, affinity, and sensitivity to conformational features. Epitopes were mapped in various additional tests. Five of the nine mAbs have cytoplasmic epitopes, and two of these are sensitive to the ligand state of the receptor. These properties should be useful both for structural analysis and in probes of function.
Footnotes
- ‡To whom correspondence should be addressed. E-mail: wayne{at}convex.hhmi.columbia.edu
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Author contributions: F.M., S.B.-M., I.S., M.M., and W.A.H. designed research; F.M., S.B.-M., R.S., Z.A., Y.S., I.S., and M.M. performed research; F.M., S.B.-M., R.A., and W.A.H. analyzed data; and F.M., R.A., and W.A.H. wrote the paper.
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The authors declare no conflict of interest.
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This article contains supporting information online at www.pnas.org/cgi/content/full/0700301104/DC1.
- Abbreviations:
- 5-HT,
- 5-hydroxytryptamine;
- GPCR,
- G protein-coupled receptor;
- IP,
- immunoprecipitation;
- MBP,
- maltose-binding protein.
- © 2007 by The National Academy of Sciences of the USA
