Subunit composition of the heteromeric cytosolic aryl hydrocarbon receptor complex and functional domain mapping ofhsp90

Huey-Shang Chen, Purdue University

Abstract

Cytosolic aryl hydrocarbon receptor (AhR) complex has a heterotetrameric structure with a molecular weight of $\sim$270 kDa. Both cross-linked and $\lbrack \sp{35}$S) -methionine-labeled Hepa 1c1c7 cytosol were used to characterize the subunit composition of the AhR complex by immunoprecipitation with an AhR polyclonal antibody, followed by immunochemical analysis using antibodies against the AhR and 90 kDa heat shock protein (hsp90). Results indicated that the four subunits found in crosslinking experiments were composed of three species; the AhR ligand binding subunit, hsp90, and an unknown 43 kDa protein. Stoichiometry of hsp90 present in each AhR complex was determined in both experiments using cross-linked and $\lbrack \sp{35}$S) -labeled Hepa 1 cytosol by quantitative immunoblotting and phosphor image analysis, respectively and values obtained were 2.4 and 1.72 mol of Hsp90/mol of AhR from each approach. In summary, cytosolic AhR complex contains one ligand binding protein, two hsp90 proteins and a p43 subunit. The identity of the 43 kDa subunit is not known. Functional domains of hsp90 were mapped by utilizing a series of deletion mutants translated in wheat germ lysate containing $\lbrack \sp{35}$S) methionine. Two distinct approaches were used to map the dimerization domain: cross-linking reaction and sucrose gradient analysis. Results revealed that the dimerization region was contained between amino acids 580 and 708. Geldanamycin, an hsp90-binding agent, has been reported to revert the oncogenic transformation induced by tyrosine kinases via disruption of hsp90/kinase complex. We investigated the GA-hsp90 interaction and its effect on other associated proteins by utilizing a solid phase-GA binding assay. Incubation of photoaffinity labeled-Hepa 1 cytosol with GA-coupled beads revealed a stable association of Ah receptor with hsp90 consistent with the data from sedimentation analysis that GA does not disrupt the 9S Ah receptor complex. Deletion of the first 221 amino acids in NH2-terminal domain resulted in loss of binding to solid-phase GA, indicating the location of GA interaction site. The data demonstrated the existence of an antibiotic interaction site in NH2-terminal region of hsp90. Epitope mapping of monoclonal antibodies specific for hsp90 (8D3, 3G3) revealed that these antibodies bind to the NH2-terminal 221 amino acids of hsp90. Monoclonal antibody AC88 was mapped to a region near the carboxyl terminus of hsp90.

Degree

Ph.D.

Advisors

Perdew, Purdue University.

Subject Area

Biochemistry

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