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.
Di Paolo
E
E
Department of Biochemistry and Nutrition, Faculty of Medicine, Nivelles, Université Libre de Bruxelles, Brussels, Belgium.
De Neef
P
P
Moguilevsky
N
N
Petry
H
H
Bollen
A
A
Waelbroeck
M
M
Robberecht
P
P
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001120:Arginine; D001665:Binding Sites; D004789:Enzyme Activation; D009154:Mutation; D011487:Protein Conformation; D051381:Rats; D043562:Receptors, G-Protein-Coupled; D011964:Receptors, Gastrointestinal Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012633:Secretin / Q000378:metabolism*
D043562:Receptors, G-Protein-Coupled; D011964:Receptors, Gastrointestinal Hormone; C029937:secretin receptor; D012633:Secretin; D001120:Arginine; D000262:Adenylyl Cyclases