Pharmacological properties of native or GFP-tagged receptors
Saturation binding assays were performed using [125I]DOI (5-HT2B receptor) or [125I]GTI (5-HT1B receptor) on membrane fraction of cell homogenate (Membranes) or intact cells (Whole Cells) of the different stable clonal cell lines expressing the 5-HT1B receptor and the 5-HT2B receptor alone with or without tag. Binding data were analyzed using the iterative non-linear fitting software GraphPad Prism 2.0 to calculate dissociation constants (KD) and maximum number of sites (Bmax). Insertion of the various GFPs at the N-terminal part of the receptor did not affect receptor affinity. Intact cell Bmax represents only about 60% of that of binding on membranes. Values are means ± S.E. of three independent determinations in triplicate.
Membrane | Whole Cells | |||||
|---|---|---|---|---|---|---|
| KD | Bmax | KD | Bmax | |||
| nM | fmol / mg protein | nM | fmol / mg protein | |||
| 5-HT1B-YFP clone 9 | 0.82 ± 0.14 | 119 ± 5* | 0.84 ± 0.15 | 73 ± 5 | ||
| 5-HT1B clone 5 | 0.56 ± 0.11 | 72 ± 4 | ||||
| 5-HT1B-PA-GFP clone 3 | 0.83 ± 0.13 | 122 ± 5* | ||||
| 5-HT2B-PA-GFP clone 9 | 26.8 ± 6.3 | 138 ± 8* | 26.3 ± 6.5 | 76 ± 3 | ||
| 5-HT2B clone 17 | 16.5 ± 4.5 | 65 ± 5 | ||||
| 5-HT2B-YFP clone 22 | 28.8 ± 5.8 | 132 ± 8* |
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↵* P < 0.05 versus non-tagged receptors by Student's t test.