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A 629RKLKK633 motif in the hinge region controls the androgen receptor at multiple levels

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Abstract

The androgen receptor protein has specific domains involved in DNA binding, ligand binding, and transactivation, whose activities need to be integrated during transcription activation. The hinge region, more particular a 629RKLKK633 motif, seems to play a crucial role in this process. Indeed, although the motif is not part of the DNA-binding domain, its positive residues are involved in optimal DNA binding and nuclear translocation as shown by mutation analysis. When the mutated ARs are forced into the nucleus, however, the residues seem to play different roles in transactivation. Moreover, we show by FRAP analysis that during activation, the AR is distributed in the nucleus in a mobile and two immobile fractions, and that mutations in the 629RKLKK633 motif affect the distribution of the AR over these three intranuclear fractions. Taken together, the 629RKLKK633 motif is a multifunctional motif that integrates nuclear localization, receptor stability, DNA binding, transactivation potential and intranuclear mobility.

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Acknowledgments

This work was supported by grants from the “Bijzonder Onderzoeksfonds K.U.Leuven” (Onderzoekstoelage and CREA/08/031); the Flanders Research Foundation (F.W.O. grant 1.5.065.05); and the Congressionally Directed Medical Research Program (Prostate Cancer Research Program award DAMD17-02-1-0082). C.H. is holder of a F.W.O. Ph.D. fellowship, A.H. is a postdoctoral F.W.O. fellow.

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Authors and Affiliations

  1. Molecular Endocrinology Laboratory, Department of Molecular Cell Biology, Catholic University of Leuven, Campus Gasthuisberg O&N1, Herestraat 49, Box 901, 3000, Leuven, Belgium

    Tamzin M. Tanner, Sarah Denayer, Nora Van Tilborgh, Stefanie Kerkhofs, Christine Helsen, Lien Spans, Vanessa Dubois, Frank Claessens & Annemie Haelens

  2. Department of Pathology, Josephine Nefkens Institute, Erasmus MC, 3000 CA, Rotterdam, The Netherlands

    Bart Geverts & Adriaan B. Houtsmuller

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  1. Tamzin M. Tanner
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Correspondence to Frank Claessens.

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Supplementary Figure 1.

Functional study of the putative amphipatic helix in the hinge region. (A) A schematic representation of the putative helical structure of the N-terminal part of the hinge region as determined by the structural prediction programme, PredictProtein. (B) Functional analysis of the single asparagine mutants that should disrupt the putative helix. HeLa cells were transfected with the TAT-GRE-luc reporter together with the appropriate AR expression vector. Cells were stimulated for 24 hours with vehicle or 10 nM R1881. Results are shown as induction factors and represent three experiments performed in triplicate, where error bars indicate the SEM. (C) Functional analysis of the proline mutants at the TAT-GRE-luc reporter in HeLa cells (experiments executed as in B above). (D) Expression of the single asparagine mutants (left) and proline point-mutants (right), in HeLa cells after 24 hours stimulation with 10 nM R1881. The expressed proteins were detected using the M2 anti-flag antibody. (PPT 176 kb)

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Tanner, T.M., Denayer, S., Geverts, B. et al. A 629RKLKK633 motif in the hinge region controls the androgen receptor at multiple levels. Cell. Mol. Life Sci. 67, 1919–1927 (2010). https://doi.org/10.1007/s00018-010-0302-1

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