Inspection of the amino acid sequence of the human VPAC1and the VPAC2receptors after alignment of the conserved residues indicates that the second extracellular loop (EC2) is one amino acid shorter in the VPAC1receptor due to the lack of a proline residue in position 294. We hypothesized that this could be of importance for receptor structure and/or for ligand recognition. Insertion by directed mutagenesis of a proline in that position (〈Pro〉294 VPAC1) had little consequence on the binding of several agonists but reduced the affinity for the VPAC1antagonist. Coupling of the 〈Pro〉294 VPAC1receptor to adenylate cyclase was improved, as demonstrated by an increased affinity for VIP and other agonists, and by a shift of the VPAC1antagonist to partial agonist behavior. Deletion of the proline 280 (ΔPro280 VPAC2) in the VPAC2receptor markedly reduced the apparent affinity for all the agonists tested. Replacement of the proline by a glycine residue had a smaller effect on the ligands affinities. The proline residue in the VPAC2receptor EC2 is thus essential for the receptor structure, and the EC2 domain is involved in ligand recognition and receptor functionality. © 2001 Elsevier Science Inc. All rights reserved.
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