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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.