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 [Asp(3)] residue of secretin to [Asn(3)] 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 [Asp(3)] residue: D174, K173 and R166, Mutation of K173 and D174 not only reduced the secretin and [Asn(3)]secretin affinities, but also changed the receptor's selectivity as judged by a decreased secretin and [Asn(3)]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 [Asn(3)]secretin. This suggested that R166, highly conserved in that subgroup of receptor, is a major determinant for the recognition of the [Asp(3)] of the ligand, (C) 1998 Federation of European Biochemical Societies.