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• W2078633448 abstract "In this study, we used the regulated secretion/aggregation technology (RPD™) to determine the rate of human muscarinic M 1 (hM 1 ) receptor plasma membrane delivery. hM1 receptors were expressed in CHO cells as C-terminal fusion proteins to a conditional aggregation domain (CAD) consisting of four tandem mutant FKBP12 domains (F m ). The CAD prevented the plasma membrane expression of hM1 receptors by causing the formation and intracellular retention of CAD-fused receptor aggregates as determined using intact cell [ 3 H]NMS binding assays and epi-fluorescence microscopy, respectively. Aggregates of CAD-fused hM 1 receptor could be disrupted in a concentration-dependent manner by the F m -selective ligand AP21998, resulting in an increased hM 1 receptor plasma membrane expression. A furin cleavage site positioned between the CAD and the hM 1 receptor sequence was cleaved by furin once aggregates of fusion protein were disrupted by AP21998, thus ensuring their irreversible dissolution. The plasma membrane delivery of hM 1 receptors begins within 30 min of AP21998 exposure and the rate of delivery was constant for up to eight hours. In the continued presence of AP21998, hM 1 receptor plasma membrane expression continued to increase for up to 18 h, then began to decrease toward basal levels as incubation continued out to 72 h. Using mathematical models, we determined the rate constants for the plasma membrane delivery of hM 1 receptors from these data. Also, hM 1 receptors elicited phosphoinositide hydrolysis to carbachol once expressed at the plasma membrane and the pharmacology of the response varied depending upon the concentration of AP21998 used to cause plasma membrane expression. Overall, our data indicate that the RPD can be used to characterize the kinetics of receptor plasma membrane delivery and to characterize functional responses elicited to different numbers of plasma membrane expressed receptor." @default.
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• W2078633448 date "2006-05-01" @default.
• W2078633448 modified "2023-09-23" @default.
• W2078633448 title "Determination of the rate of muscarinic M1 receptor plasma membrane delivery using a regulated secretion/aggregation system" @default.
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• W2078633448 doi "https://doi.org/10.1016/j.vascn.2005.08.006" @default.
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