Beukers Margot W, van Oppenraaij Joris, van der Hoorn Patrick P W, Blad Clara C, den Dulk Hans, Brouwer Jaap, IJzerman Adriaan P
Division of Medicinal Chemistry, LACDR, Gorlaeus Laboratories, Leiden, The Netherlands.
Mol Pharmacol. 2004 Mar;65(3):702-10. doi: 10.1124/mol.65.3.702.
To gain insight in spontaneous as well as agonist-induced activation of the human adenosine A2B receptor, we applied a random mutagenesis approach in yeast to create a large number of receptor mutants and selected mutants of interest with a robust screening assay based on growth. The amino acid sequence of 14 mutated receptors was determined. All these mutated receptors displayed constitutive activity. In particular, single-point mutations at T42A, V54L, and F84S and a triple-point mutation at N36S, T42A, and T66A resulted in high constitutive activity. In addition, a C-terminally truncated (after Lys269) mutant, Q214L I230N V240M V250M N254Y T257S K269stop, was highly constitutively active. The T42A, V54L, and F84S mutants showed a considerable decrease, 4.9- to 6.9-fold, in the EC50 value of 5'-N-ethylcarboxamidoadenosine (NECA), an adenosine analog. Combined mutation of I242T, K269R, V284A, and H302Q, as well as F84L together with S95G, resulted in an even greater potency of NECA of 10- and 18-fold, respectively. In fact, all constitutively active mutants had an increased potency for NECA. This suggests that the wild-type (wt) human A2B receptor itself is rather silent, which may explain the low affinity of agonists for this receptor. To verify the ability of the mutant receptors to activate mammalian second messenger systems, cAMP experiments were performed in CHO cells stably expressing the wt and T42A receptors. These experiments confirmed the increased sensitivity of T42A for NECA, because the EC50 values of T42A and the wt receptor were 0.15 +/- 0.04 and 1.3 +/- 0.4 microM, respectively.
为深入了解人腺苷A2B受体的自发激活以及激动剂诱导的激活，我们在酵母中采用随机诱变方法创建了大量受体突变体，并通过基于生长的强大筛选试验筛选出感兴趣的突变体。测定了14个突变受体的氨基酸序列。所有这些突变受体均表现出组成型活性。特别是，T42A、V54L和F84S处的单点突变以及N36S、T42A和T66A处的三点突变导致了高组成型活性。此外，一个C末端截短（在Lys269之后）的突变体Q214L I230N V240M V250M N254Y T257S K269stop具有高度组成型活性。T42A、V54L和F84S突变体显示腺苷类似物5'-N-乙基羧酰胺腺苷（NECA）的EC50值显著降低，降低了4.9至6.9倍。I242T、K269R、V284A和H302Q的联合突变以及F84L与S95G的联合突变分别使NECA的效力提高了10倍和18倍。事实上，所有组成型活性突变体对NECA的效力都有所增加。这表明野生型（wt）人A2B受体本身相当不活跃，这可能解释了激动剂对该受体的低亲和力。为验证突变受体激活哺乳动物第二信使系统的能力，在稳定表达wt和T42A受体的CHO细胞中进行了cAMP实验。这些实验证实了T42A对NECA的敏感性增加，因为T42A和wt受体的EC50值分别为0.15±0.04和1.3±0.4 microM。
