Mutations of aromatic residues in the first transmembrane helix impair signalling by the secretin receptor

The secretin amino terminal residues are essential for high affinity bindin g to the cognate receptor and for the subsequent activation of adenylate cy clase. It has been already established that two basic residues of the recep tor TM 2 are involved in the interaction with aspartate 3 of the ligand. Th e present work investigated the hypothesis that two conserved tyrosine resi dues of the TM 1 (Tyrosines 124 and 128) could also participate to the posi tioning of the amino terminus of the ligand. Tyrosines 124 and 128 were mut ated into alanine and histidine residues, and the properties of the mutant receptors, expressed in CHO cells, were compared with those of the wild-typ e receptor. Mutation of tyrosine 124 to Ala or His decreased the affinity o f the receptor for secretin, [Glu(3)]secretin, [Asn(3)]secretin and the sec retin fragment 2-27, and reduced the intrinsic activity of [Asn(3)]secretin . Mutation of tyrosine 128 to Ala, but not to His reduced 50-fold secretin and [Asn(3)]secretin affinity but only 3-fold that of[Glu(3)]secretin. Secr etin and [Glu(3)]Sn were equipotent in that mutant receptor. These results suggested that tyrosine 128 of the secretin receptor interacted directly wi th the [Asp(3)] residue of secretin and thus that the amino terminal domain of secretin interacts with amino acids buried in both the TM 1 and TM 2 he lices.