Dimeric binding of the mouse germ cell nuclear factor.
The mouse germ cell nuclear factor (GCNF), a member of the nuclear receptor superfamily, is highly expressed during spermatogenesis, oogenesis, and during neuronal embryonic differentiation. The in vitro translated receptor binds autonomously to the direct repeat of the sequence 5'-AGGTCA-3'. To gain insights into the determinants necessary for DNA binding, I have generated truncated GCNF molecules by introducing carboxy-terminal deletions into expression constructs. An electrophoretic mobility-shift assay with these polypeptides shows that amino acids in addition to the core DNA-binding domain are important for specific binding. To address the question of whether the protein binds as monomer, homodimer, or heterodimer, I used different approaches. Analysis of the full-length protein was possible with GCNF polypeptides that contain epitopes of six consecutive histidines. Using a monoclonal antibody directed against these epitopes, I demonstrate that two GCNF molecules bind to a direct repeat. Dimerization between wild-type and truncated GCNF is shown by an electrophoretic mobility-shift analysis with a mixture of the proteins. In addition, I show that there is no in vitro interaction of GCNF with the retinoid X receptor, a promiscous partner of many nuclear receptors. The data suggest that GCNF may excert its in vivo function independently of other nuclear receptors.
Borgmeyer
U
U
Zentrum für Molekulare Neurobiologie, Universität Hamburg, Germany.
D000595:Amino Acid Sequence; D000818:Animals; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D006639:Histidine / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D057136:Nuclear Receptor Subfamily 6, Group A, Member 1; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018168:Receptors, Retinoic Acid / Q000502:physiology; D047488:Retinoid X Receptors; D017384:Sequence Deletion; D014157:Transcription Factors / Q000502:physiology
D004268:DNA-Binding Proteins; C539954:Nr6a1 protein, mouse; D057136:Nuclear Receptor Subfamily 6, Group A, Member 1; D018160:Receptors, Cytoplasmic and Nuclear; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D014157:Transcription Factors; D006639:Histidine; D004247:DNA