Antipeptide antibodies localize N-(4-azido-3-[125I] iodophenethyl)spiperone binding to the carboxyl-terminal portion of the D2 dopamine receptor.
Antibodies against synthetic peptides of the D2 dopamine receptor were used, in combination with photoaffinity labeling, to localize the region of ligand binding in the receptor. Specific antibodies to peptide sequences 221-234 and 259-272 and to the carboxyl-terminal peptide 402-415, all corresponding to cytoplasmic regions in the D2 dopamine receptor, were elicited. After photoaffinity labeling with N-(4-azido-3-[125I]iodophenethyl)spiperone ([125I]NAPS), all three antibodies specifically immunoprecipitated the 90-kDa D2 dopamine receptor. Differential reactivity of the antipeptide antibodies with various proteolytic fragments indicates that [125I]NAPS binds covalently to a 13-kDa fragment of the D2 dopamine receptor. This fragment is immunoprecipitated with anti-peptide 402-415 and not with the other two antipeptide antibodies, indicating that the photoaffinity ligand binds to a fragment that begins beyond amino acid 272 and extends through the carboxyl-terminal end of the receptor.
David
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C
Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
Fuchs
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S
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D002417:Cattle; D010446:Peptide Fragments / Q000276:immunology*; D011233:Precipitin Tests; D011817:Rabbits; D011954:Receptors, Dopamine / Q000276:immunology / Q000378:metabolism*; D017448:Receptors, Dopamine D2; D013134:Spiperone / Q000031:analogs & derivatives* / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology
D000345:Affinity Labels; D000906:Antibodies; D001386:Azides; D010446:Peptide Fragments; D011954:Receptors, Dopamine; D017448:Receptors, Dopamine D2; D013134:Spiperone; C044567:N-(4-azido-3-iodophenethyl)spiperone; D014357:Trypsin