Short communication The GABA B receptor subunits R1 and R2 interact differentially with the activation transcription factor ATF4 in mouse brain during the postnatal development Barbara Ritter, Jana Zschu ¨ntzsch, Elena Kvachnina, Weiqi Zhang * , Evgeni G. Ponimaskin Abteilung Neuro-und Sinnesphysiologie, Physiologisches Institut, Universita ¨t Go ¨ttingen, Humboldtallee 23, D-37073 Go ¨ttingen, Germany Accepted 17 December 2003 Abstract Gamma-aminobutyric acid type B receptors (GABA B R) belong to the family of G-protein-coupled receptors that mediate synaptic actions by modulation of different ion channels. Here, we demonstrate that the receptor subunits GABA B R1 and GABA B R2 interact directly with the soluble activating transcription factor 4 (ATF4) in different regions of the neonatal mouse brain. We found that about 5–12% of expressed ATF4 protein is involved in the complex formation with GABA B receptors. Confocal fluorescence microscopy showed that GABA B R and ATF4 are co-localized in several well-defined spots in neurons and in glial cells. Co-immunoprecipitation analysis also reveals that the interaction efficiency between GABA B receptors and ATF4 in the mouse brain markedly changed during postnatal development, and such changes in interaction were dependent on the GABA B receptor subtype. D 2004 Elsevier B.V. All rights reserved. Theme: Protein – protein interaction, development, receptors Topic: GABA B receptors and transcription factor ATF4 Keywords: Co-immunoprecipitation; Gamma-aminobutyric acid receptor; Activating transcription factor 4; Western blot; Intracellular distribution Activation of various types of neurotransmitter receptors in the mammalian central nervous system (CNS) causes either direct opening of ion channels or activation of a wide spectrum of intracellular signaling cascades. In many cases, the latter action is followed by the modulation of gene transcription through different regulatory factors. One of such factors is a cAMP-responsive transcription factor Ac- tivating Transcription Factor 4 (ATF4) belonging to the ATF/ CREB family of transcriptional regulators (for review, see Ref. [12]). ATF4 is expressed ubiquitously and needed to be phosphorylated by the cAMP-dependent protein kinase A for the activation of gene transcription [2,17]. Functionally, ATF4 has been found to be involved in regulation of the pituitary gland functions, in spermatogenesis as well as in the molecular modulation of long-term memory [1,7,8]. Gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter in the mammalian CNS, exerts its actions via both ionotropic (GABA A,C ) and metabotropic (GABA B ) receptors [3,4,19]. The GABA B receptors are members of the G-protein-coupled receptors family that modulates mul- tiple signal transduction pathways [3,4,19]. Molecular clon- ing has provided two related receptor subtypes, GABA B R1 and GABA B R2, which are further subdivided in different isoforms [13 – 15,23]. In contrast to other G-protein-coupled receptors, functional GABA B receptor appears to be dimer of GABA B R1 and GABA B R2 [13,15,23]. Although the functional role of this association is not fully understood, dimerization seems to be important for the trafficking and assembly into functional heterodimeric receptor [5,6,9,11, 16,18]. Recent studies have also shown that GABA B R may form complexes with other proteins, including the ATF4. This was assessed by using yeast two-hybrid system as well as pull-down assay and also directly confirmed in cultured cells. Functionally, such complexes have been suggested to be involved in functional regulation and/or in the targeting of GABA B receptors [20,24]. In the present study, we investigate whether the ATF4 protein may interact with both GABA B R1 and GABA B R2 0165-3806/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.devbrainres.2003.12.006 * Corresponding author. Tel.: +49-551-393767; fax: +49-551-396031. E-mail address: Zhang@ukps.gwdg.de (W. Zhang). www.elsevier.com/locate/devbrainres Developmental Brain Research 149 (2004) 73 – 77