Development and characterization of antibodies against the N terminus of the human dopamine D4 receptor.
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.
Lanau
F
F
Pharmaceutical Research, Preclinical Neuroscience, Hoffmann-La Roche, Basel, Switzerland.
Brockhaus
M
M
Pink
J R
JR
Franchet
C
C
Wildt-Perinic
D
D
Goepfert
C
C
Probst
A
A
Hartman
D S
DS
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001344:Autopsy; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism* / Q000473:pathology; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis* / Q000737:chemistry; D005434:Flow Cytometry; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D051379:Mice; D008961:Models, Structural; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D017433:Protein Structure, Secondary; D051381:Rats; D017448:Receptors, Dopamine D2 / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology*; D050638:Receptors, Dopamine D4; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology; D012016:Reference Values; D012559:Schizophrenia / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D000911:Antibodies, Monoclonal; C493599:DRD4 protein, human; C493600:Drd4 protein, mouse; C493601:Drd4 protein, rat; D000939:Epitopes; D010446:Peptide Fragments; D017448:Receptors, Dopamine D2; D011994:Recombinant Proteins; D050638:Receptors, Dopamine D4; D016244:Guanosine 5'-O-(3-Thiotriphosphate)