Unaltered agonist potency upon inducible 5-HT7(a) but not 5-HT4(b) receptor expression indicates agonist-independent association of 5-HT7(a) receptor and Gs
Unaltered agonist potency upon inducible 5-HT7(a) but not 5-HT4(b) receptor expression indicates agonist-independent association of 5-HT7(a) receptor and Gs
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DOI：
10.1080/10606820308245
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发表时间：
2003-03-01
期刊：
影响因子：
--
通讯作者：
Levy, FO
中科院分区：
文献类型：
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作者：
Bruheim, S;Krobert, KA;Levy, FO
我们使用蜕皮激素诱导表达系统比较了G蛋白偶联的人5-羟色胺（5-HT）受体5-HT 4（B）和5-HT 7（a）对腺苷酸环化酶（AC）的激活，该系统允许在克隆HEK 293（EcR 293）细胞系中重复表达增加的受体密度。观察到受体的低组成型表达（2-70 fmol/mg蛋白质），并且可以被蜕皮激素类似物ponasterone A滴定高达50-200倍（类似于400 -7000 fmol/mg蛋白质）。虽然5-HT刺激的AC活性随着受体密度的增加而增加，但克隆间的变化排除了偶联效率的比较。有趣的是，5-HT刺激AC的效力仅在表达5-HT 4（B）受体的克隆中随着受体密度的增加而增加。表达5-HT 7（a）受体的克隆对5-HT的效力没有改变，即使5-HT刺激的AC活性接近渐近水平。这表明5-HT通过5-HT 7（a）受体刺激AC的效力不依赖于受体-G（s）化学计量，并且与其中5-HT 7（a）受体与G蛋白紧密结合的模型一致，不依赖于激动剂结合。这支持了存在的复合物之间的非活性受体和G蛋白，预测的立方三元复合物模型。在这样的系统中，备用受体不会导致激动剂的效力随着受体密度的增加而增加。
We compared adenylyl cyclase (AC) activation by the G protein-coupled human serotonin (5-HT) receptors 5-HT4(b) and 5-HT7(a) using an ecdysone-inducible expression system, which allowed for reproducible expression of increasing receptor densities in clonal HEK293 (EcR293) cell lines. Low constitutive expression of receptors (2-70 fmol/mg protein) was observed and could be titrated up to 50-200-fold (similar to400-7000 fmol/mg protein) by the ecdysone analogue ponasterone A. Although 5-HT-stimulated AC activity increased with receptor density, interclonal variation precluded comparisons of coupling efficiency. Interestingly, the potency of 5-HT to stimulate AC increased with increasing receptor density only in clones expressing 5-HT4(b) receptors. The potency for 5-HT did not change in clones expressing 5-HT7(a) receptors, even though 5-HT-stimulated AC activity approached asymptotic levels. This indicates that potency of 5-HT for stimulation of AC through the 5-HT7(a) receptor is independent of receptor-G(s) stoichiometry and is consistent with a model where the 5-HT7(a) receptors are tightly associated with G protein, independent of agonist binding. This supports the existence of a complex between inactive receptor and G protein, as predicted by the cubic ternary complex model. In such a system, spare receptors do not lead to increased potency of an agonist with increased receptor density.