The α and β Subunits of the GA-binding Protein Form a Stable Heterodimer in Solution

Abstract

We have studied the assembly of GA-binding protein (GABP) in solution and established the role of DNA in the assembly of the transcriptionally active GABP␣ 2 ␤ 2 heterotetrameric complex. GABP binds DNA containing a single PEA3/Ets-binding site (PEA3/EBS) exclusively as the ␣␤ heterodimer complex, but readily binds as the GABP␣ 2 ␤ 2 heterotetramer complex on DNA containing two PEA3/EBSs. Positioning of the PEA3/EBSs on the same face of the DNA helix stabilizes heterotetramer complex binding. These observations suggest that GABP␣␤ heterodimers are the predominant molecular species in solution and that DNA containing two PEA3/ EBSs promotes formation of the GABP␣ 2 ␤ 2 heterotetrameric complex. We analyzed the assembly of GABP␣ 2 ␤ 2 heteromeric complexes in solution by analytical ultracentrifugation. GABP␣ exists as a monomer in solution while GABP␤ exists in a monomer-dimer equilibrium (K d ‫؍‬ 1.8 ؎ 0.27 M). In equimolar mixtures of the two subunits, GABP␣ and GABP␤ formed a stable heterodimer, with no heterotetramer complex detected. Thus, GABP exists in solution as the heterodimer previously shown to be a weak transcriptional activator. Assembly of the transcriptionally active GABP␣ 2 ␤ 2 heterotetramer complex requires the presence of specific DNA containing at least two PEA3/EBSs.

GA-Binding Protein and p300 Are Essential Components of a Retinoic Acid-Induced Enhanceosome in Myeloid Cells

“…However, the PNT domain of GABP␣ was unable to mediate an interaction with ERK2 when substituted for the corresponding Ets-1 PNT domain (32). Also, GABP␣ is unable to form homodimers in solution, suggesting that the GABP␣ PNT domain cannot self-associate, as is seen with the TEL PNT domain (9). Furthermore, a derivative of the leukemogenic TEL-PDGFR fusion protein, in which the TEL PNT domain was replaced by that of GABP␣, failed to coimmunoprecipitate GABP␣ (16).…”

Identification of a Crucial Site for Synoviolin Expression

“…GABP is composed of two distinct proteins. One is GABP␣, which has an ets domain and not a transactivation domain, and the other is GABP␤1, which is a specific cofactor; together they form complexes for transactivation (6,12,19,41,50). Thus, to ascertain whether GABP can bind EBS-1 of the synoviolin promoter in NIH 3T3 cells, we prepared an EBS-1 probe (Ϫ83/Ϫ68) including EBS-1 (Ϫ76/Ϫ69) and then performed EMSA using this probe.…”

Transcriptional regulatory circuits controlling mitochondrial biogenesis and function

“…an ␣␤ heterodimer but is induced to form the heterotetramer ␣ 2 ␤ 2 by DNA containing two or more binding sites (Chinenov et al 2000). The crystal structure of the heterotetramer bound to DNA has been determined (Batchelor et al 1998).…”