OBJECTIVES: Two vasoactive intestinal peptide (VIP) receptor subtypes have been cloned. We studied the structure-function relationship of human VIP1 and VIP2 receptors by mutating residues specifically conserved in extracellular domains of these receptors: N-terminal domain (E36, I43, S64, D132 and F138 in VIP1 receptor corresponding to E24, I31, S53, D116 and F122 in VIP2 receptor) and second loop (T288 and S292 in VIP1 receptor corresponding to T274 and S278 in VIP2 receptor). METHODS: Residues were mutated into alanine (A) and the corresponding cDNAs were transfected into Cos cells. Wild-type and mutated receptors were characterized in transfected cells by ligand binding assay using 125I-VIP and cAMP measurements upon VIP challenge. RESULTS: Regarding the VIP1 receptor, no specific binding of 125I-VIP could be detected on Cos cells transfected with the E36A mutant whereas other mutants, with the exception of S64A, exhibited dissociation constants similar to that of the wild-type receptor. The S64A mutant showed a 3-fold increase of its dissociation constant as compared to the wild-type receptor. cAMP experiments showed that the E36A mutant mediated a very weak stimulation by VIP. Regarding the VIP2 receptor, no specific binding of 125I-VIP could be detected on Cos cells transfected with the E24A. I31A and T274A mutants whereas all other mutants exhibited dissociation constants similar to that of the wild-type receptor. cAMP experiments showed that the E24A mutant mediated a very weak stimulation by VIP. Regarding I31A and T274A mutants, the EC50 values were increased 10 and 50 times as compared to the wild-type receptor, respectively. CONCLUSION: a) The conserved glutamate (E) residue in the N-terminal domain of VIP1 and VIP2 receptors is crucial for VIP binding; b) The VIP2 receptor contains two conserved residues isoleucine 31 and threonine 274 which are critical for VIP binding while they can be mutated without loss of function in the VIP1 receptor. This difference in the structure-function relationship should be instrumental for the development of a selective pharmacology of VIP receptor subtypes.
OBJECTIVES: Two vasoactive intestinal peptide (VIP) receptor subtypes have been cloned. We studied the structure-function relationship of humanVIP1 and VIP2 receptors by mutating residues specifically conserved in extracellular domains of these receptors: N-terminal domain (E36, I43, S64, D132 and F138 in VIP1 receptor corresponding to E24, I31, S53, D116 and F122 in VIP2 receptor) and second loop (T288 and S292 in VIP1 receptor corresponding to T274 and S278 in VIP2 receptor). METHODS: Residues were mutated into alanine (A) and the corresponding cDNAs were transfected into Cos cells. Wild-type and mutated receptors were characterized in transfected cells by ligand binding assay using 125I-VIP and cAMP measurements upon VIP challenge. RESULTS: Regarding the VIP1 receptor, no specific binding of 125I-VIP could be detected on Cos cells transfected with the E36A mutant whereas other mutants, with the exception of S64A, exhibited dissociation constants similar to that of the wild-type receptor. The S64A mutant showed a 3-fold increase of its dissociation constant as compared to the wild-type receptor. cAMP experiments showed that the E36A mutant mediated a very weak stimulation by VIP. Regarding the VIP2 receptor, no specific binding of 125I-VIP could be detected on Cos cells transfected with the E24A. I31A and T274A mutants whereas all other mutants exhibited dissociation constants similar to that of the wild-type receptor. cAMP experiments showed that the E24A mutant mediated a very weak stimulation by VIP. Regarding I31A and T274A mutants, the EC50 values were increased 10 and 50 times as compared to the wild-type receptor, respectively. CONCLUSION: a) The conserved glutamate (E) residue in the N-terminal domain of VIP1 and VIP2 receptors is crucial for VIP binding; b) The VIP2 receptor contains two conserved residues isoleucine 31 and threonine 274 which are critical for VIP binding while they can be mutated without loss of function in the VIP1 receptor. This difference in the structure-function relationship should be instrumental for the development of a selective pharmacology of VIP receptor subtypes.