Contribution of the second transmembrane helix of the secretin receptor to the positioning of secretin

The secretin amino‐terminal residues are essential for high affinity binding to its cognate receptor and for its biological activity. Mutation of the [Asp3] residue of secretin to [Asn3] decreased the ligand's affinity for the rat wild‐type receptor 100–300‐fold. Receptor mutations in the transmembrane 2 domain and the beginning of the first extracellular loop allowed the identification of three residues involved in recognition of the [Asp3] residue: D174, K173 and R166. Mutation of K173 and D174 not only reduced the secretin and [Asn3]secretin affinities, but also changed the receptor's selectivity as judged by a decreased secretin and [Asn3]secretin potency ratio. The most striking effect was observed when R166 was mutated to Q, D or L. This led to receptors with a very low affinity for secretin but an up to 10‐fold higher affinity than the wild‐type receptor for [Asn3]secretin. This suggested that R166, highly conserved in that subgroup of receptor, is a major determinant for the recognition of the [Asp3] of the ligand.