An anti-peptide antibody that recognizes the dopamine D2 receptor from bovine striatum.
The bovine dopamine D2 receptor was purified by wheat-germ-agglutinin-Sepharose chromatography and affinity chromatography, using the D2-receptor-specific agonist N-0434. Purification yields a preparation with a major protein band of 95 kDa. In order to ascertain the identity of this protein, polyclonal antibodies against the dopamine D2 receptor have been raised using synthetic peptides based on the predicted amino acid sequence of the cloned D2 receptor. For the initial screening of these antibodies, three fusion proteins consisting of beta-galactosidase and receptor fragments were constructed. One antiserum reacted strongly with the corresponding D2 receptor fusion protein, both on Western blots and in immunoprecipitation experiments. In each case, recognition was inhibited by competition with free peptide. On Western blots of partially purified receptor preparations from bovine striatum, the antiserum specifically recognized a 95-kDa glycoprotein. From similar preparations, the antiserum precipitated a substantial proportion of active D2 receptor, as determined by a decrease in [3H]spiperone binding in the supernatant. Active receptor could be released from the immunoprecipitate by addition of free peptide. Immunocytochemical analysis of cells transiently transfected with DNA coding for the D2 receptor showed specific staining of transfected cells. The antibody raised against a sequence in the third intracellular loop is able to shift the affinity of the receptor for dopamine from high to low, indicating that the antiserum may be interfering with receptor-GTP-binding-protein interactions.
Department of Medical Biochemistry, Sylvius Laboratory University of Leiden, The Netherlands.
M J
MJ
Plug
J
J
Dijk
J A
JA
Maassen
W
W
Möller
D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D003342:Corpus Striatum / Q000502:physiology*; D004247:DNA / Q000235:genetics; D015259:Dopamine Agents / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007150:Immunohistochemistry; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010627:Phenethylamines / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D011487:Protein Conformation; D011954:Receptors, Dopamine / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D017448:Receptors, Dopamine D2; D011993:Recombinant Fusion Proteins / Q000032:analysis; D013134:Spiperone / Q000378:metabolism; D014162:Transfection
D000906:Antibodies; D015259:Dopamine Agents; D009838:Oligodeoxyribonucleotides; D010627:Phenethylamines; D011954:Receptors, Dopamine; D017448:Receptors, Dopamine D2; D011993:Recombinant Fusion Proteins; D013134:Spiperone; C041369:2-(N-phenethyl-N-propyl)amino-5-hydroxytetralin; D004247:DNA