Photoaffinity labeling of dopamine D1 receptors.
A high-affinity radioiodinated D1 receptor photoaffinity probe, (+/-)-7-[125I]iodo-8-hydroxy-3-methyl-1-(4-azidophenyl)-2,3,4,5-tetra hyd ro- 1H-3-benzazepine ([125I]IMAB), has been synthesized and characterized. In the absence of light, [125I]IMAB bound in a saturable and reversible manner to sites in canine brain striatal membranes with high affinity (KD approximately equal to 220 pM). The binding of [125I]IMAB was stereoselectively and competitively inhibited by dopaminergic agonists and antagonists with an appropriate pharmacological specificity for D1 receptors. The ligand binding subunit of the dopamine D1 receptor was visualized by autoradiography following photoaffinity labeling with [125I]IMAB and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Upon photolysis, [125I]IMAB incorporated into a protein of apparent agents in a stereoselective manner with a potency order typical of dopamine D1 receptors. In addition, smaller subunits of apparent Mr 62,000 and 51,000 were also specifically labeled by [125I]IMAB in these species. Photoaffinity labeling in the absence or presence of multiple protease inhibitors did not alter the migration pattern of [125I]IMAB-labeled subunits upon denaturing electrophoresis in both the absence or presence of urea or thiol reducing/oxidizing reagents. [125I]IMAB should prove to be a useful tool for the subsequent molecular characterization of the D1 receptor from various sources and under differing pathophysiological states.
Niznik
H B
HB
Department of Psychiatry, University of Toronto, Ontario, Canada.
Jarvie
K R
KR
Bzowej
N H
NH
Seeman
P
P
Garlick
R K
RK
Miller
J J
JJ
Baindur
N
N
Neumeyer
J L
JL
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000378:metabolism; D001552:Benzazepines / Q000378:metabolism; D002417:Cattle; D003342:Corpus Striatum / Q000378:metabolism; D004285:Dogs; D007700:Kinetics; D008566:Membranes / Q000378:metabolism; D008970:Molecular Weight; D010777:Photochemistry; D011487:Protein Conformation; D011954:Receptors, Dopamine / Q000378:metabolism*; D013552:Swine
D000345:Affinity Labels; D001386:Azides; D001552:Benzazepines; D011954:Receptors, Dopamine; C058320:7-iodo-8-hydroxy-3-methyl-1-(4-azidophenyl)-2,3,4,5-tetrahydro-1H-3-benzazepine