Activation Function 1 of Retinoic Acid Receptor 2 Is an Acidic Activator Resembling VP16

doi:10.1074/jbc.270.40.23552

Activation Function 1 of Retinoic Acid Receptor 2 Is an Acidic Activator Resembling VP16 (*)

From the Hubrecht Laboratory, Netherlands Institute for Developmental Biology, 3584 CT Utrecht, The Netherlands

^¶ To whom correspondence should be addressed:
Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
Tel.: 31-30-51-02-11; Fax: 31-30-51-64-64.

Abstract

The mechanisms underlying transcriptional activation are not very well understood, and knowledge is based on experiments with a small number of mostly viral activators. We have investigated the mechanism underlying transactivation by the activation domain present in the N-terminal part of retinoic acid receptor (RAR) β2 (AF-1). We show that RARβ2 phosphorylation is not crucial for its activity although it may modulate AF-1 activity. Sequential mutation of the negatively charged residues (Asp) resulted in a stepwise decrease in activity, while mutation of all aspartic acid residues resulted in complete loss of activity. Comparison of the critical region for activation with other activators revealed moderate homology with the viral activator VP16. The hydrophobic amino acids surrounding the negatively charged residues reported to be critical for activation by VP16 are all conserved in AF-1. The hydrophobic residues are required for AF-1, since mutation of these residues resulted in a decrease in activity. Furthermore, the activity of this activator, VP16 and TA¹ of RelA, is squelched by overexpression of an AF-1-containing expression construct, indicating that AF-1 is an acidic activator. Squelching experiments further indicate that AF-1 and AF-2 function by different mechanisms. Comparison of activation functions present in the AB region of other members of the steroid/thyroid hormone receptor family: RARα2, RAR2, and GR suggested that also these receptors contain an acidic activation domain. The mechanism underlying activation by AF-1 is discussed.

Footnotes

↵^§ Supported by the Dutch Cancer Society.
↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

DBD

DNA-binding domain

AF

activation function

ER

estrogen receptor

RAR

retinoic acid receptor

GST

glutathione S-transferase

CAT

chloramphenicol acetyltransferase.
↵² G. E. Folkers, E. C. van Heerde, and P. T. van der Saag, unpublished results.
↵³ Folkers, G. E., von der Burg, B., van der Saag, P. T.(1996) J. Steroid Biochem. Mol. Biol., in press.
↵⁴ K. Hård and R. Kaptein, unpublished results.
- Received April 7, 1995.
- Revision received July 12, 1995.