Two tyrosine residues in the first transmembrane helix of the human vasoactive intestinal peptide receptors play a role in supporting the active conformation

We investigated the human vasoactive intestinal polypeptide (VIP) receptors VPAC 1 and VPAC 2 mutated at conserved tyrosine residues in the first transmembrane helix (VPAC 1 receptor Y146A and Y150A and VPAC 2 receptor Y130A and Y134A). [ 125 I]‐Acetyl‐His 1 [D‐Phe 2 , K 15 , R 16 , L 27 ]‐VIP (1–7)/GRF (8–27) (referred to as [ 125 I]‐VPAC 1 antagonist) labelled VPAC 1 binding sites, that displayed high and low affinities for VIP (IC 50 values and per cent of high affinity binding sites: wild‐type, 1 n M (57±9%) and 160 n M ; Y146A, 30 n M (40±8%) and 800 n M ; Y150A, 4 nM (27±8%) and 300 n M ). [R 16 ]‐VIP behaved as a ‘super agonist’ at both mutated VPAC 1 receptors and the efficacies of VIP analogues modified in positions 1, 3 and 6 were significantly decreased. VIP was less potent at the Y130A and Y134A mutated VPAC 2 receptors (EC 50 200 and 400 n M , respectively) than at the wild‐type VPAC 2 receptor (EC 50 7 n M ). Furthermore, [hexanoyl‐His 1 ]‐VIP behaved as a ‘super agonist’ at the two mutated VPAC 2 receptors, and VIP analogues modified in positions 1, 3 and 6 were less potent and efficient at the mutated than at wild‐type VPAC 2 receptors. However, the Y130A and Y134A mutants could not be studied in binding assays Our results suggest that the conserved tyrosine residues do not interact directly with the VIP His 1 , Asp 3 or Phe 6 residues (that are necessary for receptor activation), but stabilize the correct active receptor conformation.British Journal of Pharmacology (2002) 136 , 1042–1048. doi: 10.1038/sj.bjp.0704802