ABSTRACT
The human dopamine D4 receptor (hD4R), which has been implicated in human diseases such as schizophrenia and in a personality trait called "novelty seeking," has not yet been characterized at the protein level. Following epitope scanning of the hD4R, we have produced a highly specific monoclonal antibody named DFR1 raised against an amino-terminal peptide in a predicted extracellular region of the receptor. DFR1 decorated recombinant hD4Rs on the surface of intact Chinese hamster ovary (CHO) cells by flow cytometry and fluorescence microscopy and also recognized recombinant hD4.2, hD4.4, and hD4.7 receptor isoforms by western blot analysis. When expressed stably in CHO cells, all three hD4R isoforms contained N-linked glycosylation and showed apparent molecular masses of 48, 55, and 67 kDa for hD4.2, hD4.4, and hD4.7, respectively. DFR1 immunoreactivity representing hD4R protein or dopamine D4 receptor-like antigens was observed in crude membrane extracts of postmortem human brain tissue by immunoblotting. The DFR1 antibody provides a new immunological tool with the potential to further our understanding of the human dopamine D4 receptor protein.
Subject(s)
Antibodies, Monoclonal , Brain/metabolism , Epitopes/analysis , Receptors, Dopamine D2/chemistry , Receptors, Dopamine D2/immunology , Amino Acid Sequence , Animals , Antibody Specificity , Autopsy , Blotting, Western , Brain/pathology , CHO Cells , Cell Membrane/metabolism , Cricetinae , Enzyme-Linked Immunosorbent Assay , Epitopes/chemistry , Flow Cytometry , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Humans , Mice , Models, Structural , Molecular Sequence Data , Organ Specificity , Peptide Fragments/chemistry , Peptide Fragments/immunology , Protein Structure, Secondary , Rats , Receptors, Dopamine D2/biosynthesis , Receptors, Dopamine D4 , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Reference Values , Schizophrenia/drug therapy , Schizophrenia/metabolism , Schizophrenia/pathology , Sequence Alignment , Sequence Homology, Amino Acid , TransfectionABSTRACT
We describe here a novel homeobox gene, denoted TGIF (5"TG3' interacting factor), which belongs to an expanding TALE (three amino acid loop extension) superclass of atypical homeodomains. The TGIF homeodomain binds to a previously characterized retinoid X receptor (RXR) responsive element from the cellular retinol-binding protein II promoter (CRBPII-RXRE), which contains an unusual DNA target for a homeobox. The interactions of both the homeprotein TGIF and receptor RXR alpha with the CREBPII-RXRE DNA motif occur on overlapping areas and generate a mutually exclusive binding in vitro. Transient cellular transfections demonstrate that TGIF inhibits the 9-cis-retinoic acid-dependent RXR alpha transcription activation of the retinoic acid responsive element. TGIF transcripts were detected in a restricted number of tissues. The canonical binding site of TGIF is conserved and is an integral part of several responsive elements which are organized like the CRBPII-RXRE. Hence, a novel auxiliary factor to the steroid receptor superfamily may participate in the transmission of nuclear signals during development and in the adult, as illustrated by the down-modulation of the RXR alpha activities.
