Mapping two functional domains of clathrin light chains with monoclonal antibodies

D. S. Kohtz, V. Georgieva-Hanson, J. D. Kohtz

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10 Scopus citations

Abstract

The two forms of clathrin light chains (LC(A)) and LC(B)) or clathrin-associated proteins (CAP1 and CAP2) have presented an immunochemical paradox. Biochemically similar, both possess two known functional parameters: binding the clathrin heavy chain and mediating the action of an uncoating ATPase. All previously reported anti-CAP mAbs, however, react specifically with only CAP1 (Brodsky, F.M., 1985, J. Cell Biol., 101:2047-2054; Kirchhausen, T., S.C. Harrison, P. Praham, and F.M. Brodsky, 1983, Proc. Natl. Acad. Sci. USA, 80:2481-2485). Four new anti-CAP mAbs are reported here: two, C-7H12 and C-6C1, react with both forms; two others, C-10B2 and C-4E5, react only with the lower form. Sandwich ELISAs indicated that C-10B2, C-4E5, C-6C1, and C-7H12 react with distinct epitopes. Monoclonal antibodies C-10B2 and C-4E5 immunoprecipitate clathrin-coated vesicles (CCVs) and react with CAP2 epitopes accessible to chymotrypsin on the vesicle. These mAbs inhibit phosphorylation of CAP2 by endogenous CCV casein kinase II. In contrast, C-6C1 and C-7H12 react with epitopes that are relatively insensitive to chymotrypsin. CAP peptide fragments containing these epitopes remain bound to reassembled cages or CCVs after digestion. Immunoprecipitation and ELISAs demonstrate that C-7H12 and C-6C1 react with unbound CAPs but not with CAPs bound to triskelions or CCVs. The data indicate that the CAPs consist of at least two discernible structural domains: a nonconserved, accessible domain that is relevant to the phosphorylation of CAP2 and a conserved, inaccessible domain that mediates the binding of CAPs to CCVs.

Original languageEnglish (US)
Pages (from-to)891-903
Number of pages13
JournalJournal of Cell Biology
Volume104
Issue number4
DOIs
StatePublished - 1987

ASJC Scopus subject areas

  • Cell Biology

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