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

1: 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). 2: [(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 nM (57+/-9%) and 160 nM; Y146A, 30 nM (40+/-8%) and 800 nM; Y150A, 4 nM (27+/-8%) and 300 nM). [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. 3: VIP was less potent at the Y130A and Y134A mutated VPAC(2) receptors (EC(50) 200 and 400 nM, respectively) than at the wild-type VPAC(2) receptor (EC(50) 7 nM). 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. 4: 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.