SITE-DIRECTED MUTAGENESIS OF VASOACTIVE-I NTESTINAL-PEPTIDE (VIP) RECEPTOR SUBTYPES VIP1 AND VIP2 - DIFFERENCE IN THEIR STRUCTURE-FUNCTION RELATIONSHIP

Objectives. - Two vasoactive intestinal peptide (VIP) receptor subtype s have been cloned. We studied the structure-function relationship of human VIP1 and VIP2 receptors by mutating residues specifically conser ved in extracellular domains of these receptors: N-terminal domain (E3 6, 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 i n VIP1 receptor corresponding to T274 and S278 in VIP2 receptor). Meth ods. - Residues were mutated into alanine (A) and the corresponding cD NAs were transfected into Cos cells. Wild-type and mutated receptors w ere characterized in transfected cells by ligand binding assay using I -125-VIP and cAMP measurements upon VIP challenge. Results. - Regardin g the VIP1 receptor, no specific binding of I-125-VIP could be detecte d on Cos cells transfected with the E36A mutant whereas other mutants, with the exception of S64A, exhibited dissociation constant as compar ed to the wild-type receptor. cAMP experiments showed that the E36A mu tant mediated a very weak stimulation by VIP. Regarding the VIP2 recep tor, no specific binding of I-125-VIP could be detected on Cos cells t ransfected with the E24A, 131A and T274A mutants whereas all other mut ants exhibited dissociation constants similar to that of the wild-type receptor. cAMP experiments showed that the E24A mutant mediated a ver y weak stimulation by VIP. Regarding I31A and T274A mutants, the EC50 values were increased 10 and 50 times as compared to the wild-type rec eptor, respectively. Conclusion. - a) The conserved glutamate (E) resi due in the N-terminal domain of VIP1 and VIP2 receptors is crucial for VIP binding; b) The VIP2 receptor contains two conserved residues iso leucine 31 and threonine 274 which are critical for VIP binding while they can be mutated without loss of function in the VIP1 receptor. Thi s difference in the structure-function relationship should be instrume ntal for the development of a selective pharmacology of VIP receptor s ubtypes.