C263-6) were able to bind poorly '"I-secretin, and to activate adenylate cyclase with high secretin EC,, values. These results suggest that cysteine residues 24,44, 53, 67, 85 and 101 are necessary for receptor function, and that the two putative disulfide bridges formed by cysteine residues 11, 186, 193 and 263 are functionally relevant, but not essential for receptor expression. Secretin activated the adenylate cyclase through the quadruple mutant (C11,186,193,263-S), the four triple mutants, and through double mutants C186,193-S and C186,263+S with a very high (pM) EC,, value, suggesting that, in the wild-type receptor, disulfide bridges are formed between C11 -C186, and between C193-C263. Prior treatment with dithiothreitol resulted in a marked EC,,, increase of the wild-type receptor and of those receptors with at least the two cysteine residues in positions 11 and 186, suggesting that the Cll-C186 (but not the C193-C263) disulfide bridge was accessible to this reducing agent. Several results nevertheless indicated that, in mutant receptors, alternative disulfide bridges can be formed between cysteine 186 and cysteine 193 or 263, suggesting that these three residues are in close spatial proximity in the wild-type receptor.Keywords: secretin receptor; cysteine ; point-mutation ; disulfide bridge. Despite the low overall sequence similarity in the extracellular part of the secretin receptor family, a conserved pattern of extracellular cysteine residues is one of their primary characteristics; usually, five to seven cysteine residues are present in the amino-terminal extracellular domain, as well as one in each of the first two extracellular loops. Fig. 1 indicates the localisation of the extracellular cysteine residues of the secretin receptor: seven residues in the amino-terminal tail, and three in the extracellular loops. Cysteine residues 44, 53, 67 and 8. 5 in the Nterminal tail and 193 and 263 in the exoloop 1 and 2, respectively, are strictly conserved in the secretin-receptor family. Moreover, Cys193 and Cys263 are highly conserved in all the members of the G-protein-coupled seven-transmembrane-helices family of receptors, with the exception of the human mas oncogene [19] and of the rat cannabinoid receptor [20].In order to determine whether the extracellular cysteine residues of the secretin receptor are involved in disulfide bridges and to define the contribution of these linkages to receptor occupancy and agonist activation, we replaced one or several codons encoding Cys residues by codons for Ser residues and stably expressed the mutant receptors in CHO cells. We assumed that the effect of Cys-Ser mutation is limited to the elimination of the disulfide bond which involved that Cys residue, so that mutation of either or both Cys involved in a given disulfide bond should result in receptor mutants with the same pharmacological properties.The results, based on ligand-binding studies, adenylate cyclase assays, and the effect of a prior treatment with a reducing agent are consistent with the existence o...