Abstract(#br)After stimulation with agonist, G protein coupled receptors (GPCR) undergo conformational changes that allow activation of G proteins to transduce the signal, followed by phosphorylation by kinases and arrestin binding to promote receptor internalization. Actual paradigm, based on a study of GPCR-A/rhodopsin family, suggests that a network of interactions between conserved residues located in transmembrane (TM) domains (mainly TM3, TM6 and TM7) is involved in the molecular switch leading to GPCR activation.(#br)We evaluated in CHO cells expressing the VPAC 1 receptor the role of the third transmembrane helix in agonist signalling by point mutation into Ala of the residues highly conserved in the secretin-family of receptors: Y 224 , N 229 , F 230 , W 232 , E 236 , G 237 , Y... 239 , L 240 . N 229 A VPAC 1 mutant was characterized by a decrease in both potency and efficacy of VIP stimulated adenylate cyclase activity, by the absence of agonist stimulated [Ca 2+ ] i increase, by a preserved receptor recognition of agonists and antagonist and by a preserved sensitivity to GTP suggesting the importance of that residue for efficient G protein activation. N 229 D mutant was not expressed at the membrane, and the N 229 Q with a conserved mutation was less affected than the A mutant. Agonist stimulated phosphorylation and internalization of N 229 A and N 229 Q VPAC 1 were unaffected. However, the re-expression of internalized mutant receptors, but not that of the wild type receptor, was rapidly reversed after VIP washing. Receptor phosphorylation, internalization and re-expression may be thus dissociated from G protein activation and linked to another active conformation that may influence its trafficking.(#br)Mutation of that conserved amino acid in VPAC 2 could be investigated only by a conservative mutation (N 216 Q) and led to a receptor with a low VIP stimulation of adenylate cyclase, receptor phosphorylation and internalization. This indicated the importance of the conserved N residue in the TM3 of that family of receptors.